Non-Coding RNAs: New Players in Skin Wound Healing.
Herter Eva K,Xu Landén Ning
Advances in wound care
Wound healing is a basic physiological process that is utilized to keep the integrity of the skin. Impaired wound repair, such as chronic wounds and pathological scars, presents a major health and economic burden worldwide. To date, efficient targeted treatment for these wound disorders is still lacking, which is largely due to our limited understanding of the biological mechanisms underlying these diseases. Research driven around discovering new therapies for these complications is, therefore, an urgent need. The vast majority of the human genome is transcribed to RNAs that lack protein-coding capacity. Intensive research in the recent decade has revealed that these non-coding RNAs (ncRNAs) function as important regulators of cellular physiology and pathology, which makes them promising therapeutic and diagnostic entities. A class of short ncRNAs, microRNAs, has been found to be indispensable for all the phases of skin wound healing and plays important roles in the pathogenesis of wound complications. The role of long ncRNAs (lncRNA) in skin wound healing remains largely unexplored. Recent studies revealed the essential role of lncRNAs in epidermal differentiation and stress response, indicating their potential importance for skin wound healing, which warrants future research. An investigation of ncRNAs will add new layers of complexity to our understanding of normal skin wound healing as well as to the pathogenesis of wound disorders. Development of ncRNA-based biomarkers and treatments is an interesting and important avenue for future research on wound healing.
Extracellular vesicle-enclosed miR-486-5p mediates wound healing with adipose-derived stem cells by promoting angiogenesis.
Lu Yingjie,Wen Huicai,Huang Jinjun,Liao Peng,Liao Huaiwei,Tu Jun,Zeng Yuanlin
Journal of cellular and molecular medicine
Adipose-derived stem cells (ASC) are said to have a pivotal role in wound healing. Specifically, ASC-secreted extracellular vesicles (EV) carry diverse cargos such as microRNAs (miRNAs) to participate in the ASC-based therapies. Considering its effects, we aimed to investigate the role of ASC-EVs in the cutaneous wound healing accompanied with the study on the specific cargo-medicated effects on wound healing. Two full-thickness excisional skin wounds were created on mouse dorsum, and wound healing was recorded at the indicated time points followed by histological analysis and immunofluorescence staining for CD31 and α-SMA. Human skin fibroblasts (HSFs) and human microvascular endothelial cells (HMECs) were co-cultured with EVs isolated from ASC (ASC-EVs), respectively, followed by the evaluation of their viability and mobility using CCK-8, scratch test and transwell migration assays. Matrigel-based angiogenesis assays were performed to evaluate vessel-like tube formation by HMECs in vitro. ASC-EVs accelerated the healing of full-thickness skin wounds, increased re-epithelialization and reduced scar thickness whilst enhanced collagen synthesis and angiogenesis in murine models. However, miR-486-5p antagomir abrogated the ASC-EVs-induced effects. Intriguingly, miR-486-5p was found to be highly enriched in ASC-EVs, exhibiting an increase in viability and mobility of HSFs and HMECs and enhanced the angiogenic activities of HMECs. Notably, we also demonstrated that ASC-EVs-secreted miR-486-5p achieved the aforesaid effects through its target gene Sp5. Hence, our results suggest that miR-486-5p released by ASC-EVs could be a critical mediator to develop an ASC-based therapeutic strategy for wound healing.
Extracellular Vesicles Derived From Mesenchymal Stem Cells (MSC) in Regenerative Medicine: Applications in Skin Wound Healing.
Casado-Díaz Antonio,Quesada-Gómez José Manuel,Dorado Gabriel
Frontiers in bioengineering and biotechnology
The cells secrete extracellular vesicles (EV) that may have an endosomal origin, or from evaginations of the plasma membrane. The former are usually called exosomes, with sizes ranging from 50 to 100 nm. These EV contain a lipid bilayer associated to membrane proteins. Molecules such as nucleic acids (DNA, mRNA, miRNA, lncRNA, etc.) and proteins may be stored inside. The EV composition depends on the producer cell type and its physiological conditions. Through them, the cells modify their microenvironment and the behavior of neighboring cells. That is accomplished by transferring factors that modulate different metabolic and signaling pathways. Due to their properties, EV can be applied as a diagnostic and therapeutic tool in medicine. The mesenchymal stromal cells (MSC) have immunomodulatory properties and a high regenerative capacity. These features are linked to their paracrine activity and EV secretion. Therefore, research on exosomes produced by MSC has been intensified for use in cell-free regenerative medicine. In this area, the use of EV for the treatment of chronic skin ulcers (CSU) has been proposed. Such sores occur when normal healing does not resolve properly. That is usually due to excessive prolongation of the inflammatory phase. These ulcers are associated with aging and diseases, such as diabetes, so their prevalence is increasing with the one of such latter disease, mainly in developed countries. This has very important socio-economic repercussions. In this review, we show that the application of MSC-derived EV for the treatment of CSU has positive effects, including accelerating healing and decreasing scar formation. This is because the EV have immunosuppressive and immunomodulatory properties. Likewise, they have the ability to activate the angiogenesis, proliferation, migration, and differentiation of the main cell types involved in skin regeneration. They include endothelial cells, fibroblasts, and keratinocytes. Most of the studies carried out so far are preclinical. Therefore, there is a need to advance more in the knowledge about the conditions of production, isolation, and action mechanisms of EV. Interestingly, their potential application in the treatment of CSU opens the door for the design of new highly effective therapeutic strategies.
Porcine acellular dermal matrix accelerates wound healing through miR-124-3p.1 and miR-139-5p.
Chen Xiaodong,Yang Ronghua,Wang Jingru,Ruan Shubin,Lin Zepeng,Xin Qi,Yang Ridong,Xie Julin
BACKGROUND AIMS:Cutaneous wound management is a major health problem and imposes a huge economic burden worldwide. Previous studies have demonstrated that wound healing is a highly coordinated process including epithelialization, angiogenesis, remodeling and scarring. This progression requires self-renewal, preservation and repair properties of stem cells. However, our understanding of the detailed internal regulatory mechanism following injury and the means to accelerate wound healing are limited. METHODS:Our previous research revealed that porcine acellular dermal matrix (ADM) effectively promotes wound healing and scar formation through epidermal stem cells (ESCs), and this process is relevant to the alteration of internal miRNA levels. In this study, we investigated the regulatory function of porcine ADM treatment on miRNAs in ESCs. RESULTS:We report that the treatment of porcine ADM reduced the levels of miR-124-3p.1 and miR-139-5p in wounds. MiR-124-3p.1 and miR-139-5p inhibited the expression of JAG1 and Notch1, respectively, by directly targeting miRNAs in ESCs. CONCLUSIONS:This work demonstrates that porcine ADM induced down-regulation of miR-124-3p.1/139-5p in wounds and up-regulation of JAG1/Notch1 in ESCs, thus enhancing cutaneous wound healing.
Expression profile and bioinformatics analyses of circular RNAs in keloid and normal dermal fibroblasts.
Zhang Zhibin,Yu Kaihui,Liu Ougen,Xiong Yifeng,Yang Xinyue,Wang Shuhua,Zhang Shulan,Feng Yueying,Peng Yating
Experimental cell research
Increasing evidence indicates that circular RNAs (circRNAs) play a crucial regulatory role in the pathogenesis of multiple diseases. However, no study has examined the potential biological function and expression profile of circRNAs in keloid dermal fibroblasts (KDFs). Therefore, the aim of this study to investigate the expression profile of circRNAs and analyze their role in KDFs. Bioinformatic analyses and high-throughput RNA sequencing technology were applied to explore the expression profile of circRNAs in 3 human KDFs and normal dermal fibroblasts (NDFs). The differentially expressed circRNAs were verified by reverse transcription PCR (RT-PCR), quantitative real-time-PCR (qRT-PCR) and Sanger sequencing. A circRNA-microRNA (miRNA)-mRNA interaction network was created using bioinformatics tools. Hsa_circ_0008259, was selected to confirm its function by qRT-PCR and Western blot. Collectively, 411 circRNAs, of which 206 were upregulated and 205 decreased, were found to be differentially expressed in KDFs and could bind to 2532 miRNA response elements (MREs). GO and KEGG pathways enrichment analyses showed that differentially expressed circRNAs were mainly involved in apoptosis, focal adhesion, PI3K-Akt and metabolic pathway, and may regulate the pathogenesis and development of keloid. Two candidate circRNAs (hsa_circRNA_0008259, hsa_circRNA_0005480) were verified to be significantly reduced in KDFs, and one candidate circRNA (hsa_circRNA_0002198) was significantly elevated in accordance with RNA-Seq data analysis. Overexpression of hsa_circRNA_0008259 inhibited type I and Ⅲ collagen expression. Taken together, our study demonstrates for the first time that circRNAs exhibits differential expression in KDFs, and may be key players in the pathogenesis of keloid, or act as biomarkers of keloid.
Expression Profiles of lncRNAs and circRNAs in Keloid.
Wang Jie,Wu Hao,Xiao Zhibo,Dong Xiaoqun
Plastic and reconstructive surgery. Global open
Background:We hypothesized that crosstalk between noncoding RNAs, including microRNA (miRNA), lncRNA, and circRNA, might play a critical role in keloids development and physiology. To reveal the molecular mechanisms involved in the pathogenesis of keloids, we compared their gene expression profiles and differential expressions in keloid and normal skin tissues. Methods:Expression profiles of mRNAs and lncRNAs and circRNAs in 2 pairs (identification set) of keloid and matched normal skin tissues were analyzed through sequencing. Real-time quantitative PCR was performed to validate the sequencing results using 5 pairs (validation set) of keloid and matched normal skin tissues. Presumed targets of differentially expressed lncRNAs and circRNAs were functionally annotated by bioinformatics approaches. Results:The differential expression of mRNAs in keloid and normal skin by high-throughput sequencing was 2,528, of which 1,271 were downregulated, whereas 1,257 were upregulated. In the meantime, sequencing identified 2,227 differentially expressed lncRNAs, including 1,224 upregulated and 1,003 downregulated in keloid tissue compared with normal skin tissue. Additionally, 154 differentially expressed circRNAs were identified, including 81 upregulated and 73 downregulated in keloid tissue compared with normal skin tissue. Functional annotations of differentially expressed circRNA targets revealed their enrichment in several signaling pathways important for scar wound healing. Conclusions:Expression profiles of mRNAs, lncRNAs, and circRNAs were altered in keloid tissue, which may partly contribute to the etiology of keloids by affecting several signaling pathways relevant to scar wound healing. A better understanding of keloids pathogenesis may identify new therapeutic targets for keloids.
Extracellular vesicles as modulators of wound healing.
Cabral Joana,Ryan Aideen E,Griffin Matthew D,Ritter Thomas
Advanced drug delivery reviews
Impaired healing of cutaneous wounds and ulcers continues to have a major impact on the quality of life of millions of people. In recent years, the capacity for stem and progenitor cells to promote wound repair has been investigated with evidence that secreted factors are responsible for the observed therapeutic benefits. This review addresses current evidence in support of stem/progenitor cell-derived extracellular vesicles (EVs) as a regenerative therapy for acceleration of wound healing. Encouraging results for local or systemic administration of EVs have been reported in a range of clinically-relevant animal models of cutaneous wounds. Furthermore, a number of plausible mechanisms involving EV-mediated transfer of proteins and RNAs that trigger pro-repair pathways in target cells have been demonstrated experimentally. However, for successful clinical translation in the coming years, further emphasis on standardized experimental protocols, detailed methodological reporting and clear definition of EV-based therapeutic products will be required.
Keloid research: current status and future directions.
Tsai Chia-Hsuan,Ogawa Rei
Scars, burns & healing
Introduction:Keloids and hypertrophic scars are fibroproliferative disorders of the skin that result from abnormal healing of injured or irritated skin. Multiple studies suggest that genetic, systemic and local factors may contribute to the development and/or growth of keloids and hypertrophic scars. A key local factor may be mechanical stimuli. Here, we provide an up-to-date review of the studies on the roles that genetic variation, epigenetic modifications and mechanotransduction play in keloidogenesis. Methods:An English literature review was performed by searching the PubMed, Embase and Web of Science databases with the following keywords: genome-wide association study; epigenetics; non-coding RNA; microRNA; long non-coding RNA (lncRNA); DNA methylation; mechanobiology; and keloid. The searches targeted the time period between the date of database inception and July 2018. Results:Genetic studies identified several single-nucleotide polymorphisms and gene linkages that may contribute to keloid pathogenesis. Epigenetic modifications caused by non-coding RNAs (e.g. microRNAs and lncRNAs) and DNA methylation may also play important roles by inducing the persistent activation of keloidal fibroblasts. Mechanical forces and the ensuing cellular mechanotransduction may also influence the degree of scar formation, scar contracture and the formation/progression of keloids and hypertrophic scars. Conclusions:Recent research indicates that the formation/growth of keloids and hypertrophic scars associate clearly with genetic, epigenetic, systemic and local risk factors, particularly skin tension around scars. Further research into scar-related genetics, epigenetics and mechanobiology may reveal molecular, cellular or tissue-level targets that could lead to the development of more effective prophylactic and therapeutic strategies for wounds/scars in the future.
Molecular Changes in Diabetic Wound Healing following Administration of Vitamin D and Ginger Supplements: Biochemical and Molecular Experimental Study.
Al-Rawaf Hadeel A,Gabr Sami A,Alghadir Ahmad H
Evidence-based complementary and alternative medicine : eCAM
Background:Circulating micro-RNAs are differentially expressed in various tissues and could be considered as potential regulatory biomarkers for T2DM and related complications, such as chronic wounds. Aim:In the current study, we investigated whether ginger extract enriched with -gingerol-fractions either alone or in combination with vitamin D accelerates diabetic wound healing and explores underlying molecular changes in the expression of miRNA and their predicted role in diabetic wound healing. Methods:Diabetic wounded mice were treated with -gingerol-fractions (GF) (25 mg/kg of body weight) either alone or in combination with vitamin D (100 ng/kg per day) for two weeks. Circulating miRNA profile, fibrogenesis markers, hydroxyproline (HPX), fibronectin (FN), and collagen deposition, diabetic control variables, FBS, HbA1c, C-peptide, and insulin, and wound closure rate and histomorphometric analyses were, respectively, measured at days 3, 6, 9, and 15 by RT-PCR and immunoassay analysis. Results:Treatment of diabetic wounds with GF and vitamin D showed significant improvement in wound healing as measured by higher expression levels of HPX, FN, collagen, accelerated wound closure, complete epithelialization, and scar formation in short periods (11-13 days, ( < 0.01). On a molecular level, three circulating miRNAs, miR-155, miR-146a, and miR-15a, were identified in diabetic and nondiabetic skin wounds by PCR analysis. Lower expression in miR-155 levels and higher expression of miR-146a and miR-15a levels were observed in diabetic skin wounds following treatment with gingerols fractions and vitamin D for 15 days. The data showed that miRNAs, miR-146a, miR-155, and miR-15a, correlated positively with the expression levels of HPX, FN, and collagen and negatively with FBS, HbA1c, C-peptide, and insulin in diabetic wounds following treatment with GF and /or vitamin D, respectively. Conclusion:Treatment with gingerols fractions (GF) and vitamin D for two weeks significantly improves delayed diabetic wound healing. The data showed that vitamin D and gingerol activate vascularization, fibrin deposition (HPX, FN, and collagen), and myofibroblasts in such manner to synthesize new tissues and help in the scar formation. Accordingly, three miRNAs, miR-155, miR-146a, and miR-15, as molecular targets, were identified and significantly evaluated in wound healing process. It showed significant association with fibrin deposition, vascularization, and reepithelialization process following treatment with GF and vitamin D. It proposed having anti-inflammatory action and promoting new tissue formation via vascularization process during the wound healing. Therefore, it is very interesting to consider miRNAs as molecular targets for evaluating the efficiency of nondrug therapy in the regulation of wound healing process.
MicroRNA-205-5p regulates extracellular matrix production in hyperplastic scars by targeting Smad2.
Qi Jun,Liu Yifei,Hu Kesu,Zhang Yi,Wu Yangyang,Zhang Xia
Experimental and therapeutic medicine
Hypertrophic scar (HS) formation is the result of poor skin-wound healing. At present, the pathogenesis of HS formation is largely unclear. Micro (miR)RNAs have important effects on a variety of biological and pathological processes. The role of miRNA in HS formation remains largely unclear. The present study aimed to investigate the role of miR-205-5p in HS, and explore the underlying molecular mechanism. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine the expression of miR-205-5p in HS. Western blot assay and RT-qPCR were performed to assess the expression of associated proteins and genes, respectively. TargetScan was performed to predict the target gene of miR-205-5p, and the luciferase reporter assay was applied to verify the prediction. The function of miR-205-5p on cell proliferation was detected using Cell Counting Kit-8 assay, and cell apoptosis was detected via flow cytometry. miR-205-5p expression was decreased in HS tissues and human hypertrophic scar fibroblasts (hHSFs). Mothers against decapentaplegic homolog (Smad)2 was significantly increased in HS tissues and HSFs, and it was directly targeted by miR-205-5p. Restoration of miR-205-5p suppressed HSF cell proliferation and induced cell apoptosis. It was also demonstrated that RAC-Alpha Serine/Threonine-Protein Kinase (AKT) phosphorylation and the expression of α-smooth muscle actin, collagen I and collagen III were inhibited by miR-205-5p. In addition, Smad2 weakened the effects of miR-205-5p on HSFs. In conclusion, miR-205-5p exhibited an important role in HS by targeting smad2 and suppressing the AKT pathway. These findings provide a clearer understanding of the mechanism for HS that may be used to develop novel treatments for HS.
Identification of Differentially Expressed Circular RNAs in Keloid and Normal Skin Tissue by High-throughput Sequencing.
Zhang Jing,Liu Ninghua,Wu Xiufa,Wu Peixuan,Song Nan,Ma Jing
Keloid is a kind of pathological skin scar with unclear molecular pathology. Circular RNAs (circRNAs) are involved in the occurrence and development of many diseases; however, their relationship with keloid is not well understood. To investigate the involvement of dysregulated circRNAs in keloid. Thirty-seven keloids and 37 normal skin tissues were collected, and the changes of circRNAs, microRNAs(miRNAs) and mRNAs in 3 keloids and 3 normal samples by high-throughput sequencing were detected firstly. Based on the circRNA-miRNA-mRNA interaction network construction, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis combining several signaling pathways associated with keloid formation and progression, the circRNAs required further verification were screened out. The expression levels of the selected circRNAs were verified in 37 keloids and 37 normal skin tissues using quantitative real-time polymerase chain reaction (QPCR). The interaction of candidate circRNAand its predicted binding microRNA was tested by dual luciferase reporter gene experiment. Compared with normal controls, there were an average of 120 and 12 circRNAs, 44 and 63 miRNAs, 656 and 156 mRNAs were up-regulated and down-regulated, respectively, in keloids. According to the analysis of bioinformation, six circRNAs were picked out. The QPCR validation results of two upregulated circRNAs (hsa_circ_0001320 and circCOL5A1) were consistent with previous sequencing results. The interaction between hsa_circ_0001320 and miR-574-5p was confirmed. This study makes it clear that the abnormal expression of circRNAs may be related to the pathological process of keloid.
[In vitro study of effects and mechanism of rapamycin-induced autophagy in keloid fibroblasts].
Gao Jing,Jiao Hu,Cao Rui,Yang Zhigang,Sun Xuejian,Zhuang Qiang,Xiao Zuo,Yan Zuo
Zhonghua zheng xing wai ke za zhi = Zhonghua zhengxing waike zazhi = Chinese journal of plastic surgery
Objective:To investigate the effect of rapamycin on biological characteristics and autophagy of keloid fibroblasts, and the regulation of rapamycin in mTOR (mammalian target of rapamycin) signaling pathway and autophagy-related non-coding RNAs in keloid fibroblasts. Methods:After Keloid fibroblasts were treated with rapamycin (10、50、100 nmol/L), and MTS assay was used to test the cell proliferation. The apoptosis of cells was tested by the flow cytometry analysis. The formation of autophagy was observed by TEM, and the Western Blot was used to detect the expression of autophagy-related protein LC3.Real-time PCR was used to detect the expression of genes of involued in mTOR pathway and autophagy-related non-coding RNAs. Statistical significance was determined using Paired-Samples t Test,P value less than 0.05 was considered statistically significant. Results:The ratio of 490nm was decreased significantly in rapamycin-treated keloid fibroblasts compared with that in untreated cells (P ＜ 0.05).Meanwhile the mRNA expressions of extracellular matrix (ECM) genes, including collagen-1 、α-SMA and Fibronectin, were inhibited by rapamycin (P ＜ 0.05).The flow cytometry analysis showed that the percent of apoptosis cells was not increased in rapamycin-induced cells (P ＞ O.05). The double-layer membrane structure of autophagosomes could be observed under the TEM in rapamycin-treated fibroblasts, accompanied by the increased expression of autophagy-related protein LC3.The mRNA expressions of downstream genes of mTOR pathway,4EBP1 and p70S6K,were down-regulated in rapamycin-treated fibroblasts, while the expressions of autophagy-related miRNAs, including miR-885-3p,miR-204,miR-101,miR-376b and lncRNA FLJ11812 were enhanced, and miR-30a,lncRNA HULC5 was decreased in rapamycin-treated fibroblasts (P ＜ 0.05). Conclusions:Rapamycin could inhibit the proliferation of keloid fibroblasts, and could not affect the apoptosis of cells.However, rapamycin induced the autophagy of keloid fibroblasts through regulating the expression of autophagy-related non-coding RNAs and genes in the mTOR signaling pathway.
Development of microRNA-21 mimic nanocarriers for the treatment of cutaneous wounds.
Wang Sun Young,Kim Hyosuk,Kwak Gijung,Jo Sung Duk,Cho Daeho,Yang Yoosoo,Kwon Ick Chan,Jeong Ji Hoon,Kim Sun Hwa
: Of the regulatory microRNAs expressed in the wounded skin, microRNA-21 (miR21) plays a pivotal role in wound repair by stimulating re-epithelialization, an essential feature to facilitate healing and reduce scar formation. Despite their crucial roles in wound healing, synthetic exogenous microRNAs have limited applications owing to the lack of an appropriate delivery system. Herein, we designed an miR21 mimic nanocarrier system using facial amphipathic bile acid-conjugated polyethyleneimines (BA-PEI) for the intracellular and transdermal delivery of synthetic miR21 molecules to accelerate wound repair. : To design miR21 mimic nanocarriers, BA-conjugated PEIs prepared from three different types of BA at molar feed ratios of 1 and 3 were synthesized. The intracellular uptake efficiency of synthetic miR21 mimics was studied using confocal laser scanning microscopy and flow cytometry analysis. The optimized miR21/BA nanocarrier system was used to evaluate the wound healing effects induced by miR21 mimics in human HaCaT keratinocytes and a murine excisional acute wound model . : The cell uptake efficiency of miR21 complexed with BA-conjugated PEI was dramatically higher than that of miR21 complexed with PEI alone. Deoxycholic acid (DA)-modified PEI at a molar feed ratio of 3:1 (DA3-PEI) showed the highest transfection efficiency for miR21 without any increase in toxicity. After effective transdermal and intracellular delivery of miR21/DA3 nanocarriers, miR21 mimics promoted cell migration and proliferation through the post-transcriptional regulation of programmed cell death protein 4 (PDCD4) and matrix metalloproteinases. Thus, miR21 mimic nanocarriers improved both the rate and quality of wound healing, as evident from enhanced collagen synthesis and accelerated wound re-epithelialization. : Our miRNA nanocarrier systems developed using DA3-PEI conjugates may be potentially useful for the delivery of synthetic exogenous miRNAs in various fields.
Impact of MiR-21 on the expression of FasL in the presence of TGF-β1.
Wang Xiaoxue,Liu Ying,Chen Xi,Zhang Miaobo,Xiao Zhibo
Aesthetic surgery journal
BACKGROUND:Micro-ribonucleic acids (miR) are small, noncoding RNA molecules 19 to 25 nucleotides in length that typically function as negative regulators of expression for many target genes involved in cell proliferation, differentiation, and apoptosis. However, the effects of miR-21 on keloid fibroblasts are currently unknown. OBJECTIVES:The authors investigate whether miR-21, a specific miR implicated in multiple aspects of keloid fibroblasts, affects the expression of Fas ligand (FasL) in the presence of transforming growth factor (TGF)-β1. METHODS:The relationship between TGF-β1 and miR-21 expression was investigated by TaqMan quantitative real-time polymerase chain reaction (Life Technologies, Grand Island, New York). FasL protein was determined by Western blotting, and regulation of cell proliferation/migration/apoptosis ability by TGF-β1 inhibitor or plasmid was evaluated respectively by EdU incorporation, Transwell assay, and flow cytometry analysis. RESULTS:Fibroblasts from keloid tissue were confirmed to express high levels of TGF-β1 and miR-21 compared with normal skin fibroblasts. Expression of TGF-β1 and miR-21 was positively correlated in fibroblasts. In addition, cells transfected with TGF-β1 inhibitor or miR-21 inhibitor showed significant increases in FasL protein levels and number of apoptotic cells compared with control cells, whereas cell growth and migration significantly decreased. The opposite results could also be confirmed when TGF-β1 was upregulated in normal skin fibroblasts. CONCLUSIONS:TGF-β1 could effectively influence cell proliferation, apoptosis, and migration via its control of miR-21. These findings also identify a novel mechanism of interaction between TGF-β1 and miR-21 in the regulation of FasL protein, which is involved in keloid formation.
Knockdown of lncRNA-ATB suppresses autocrine secretion of TGF-β2 by targeting ZNF217 via miR-200c in keloid fibroblasts.
Zhu Hua-Yu,Bai Wen-Dong,Li Chao,Zheng Zhao,Guan Hao,Liu Jia-Qi,Yang Xue-Kang,Han Shi-Chao,Gao Jian-Xin,Wang Hong-Tao,Hu Da-Hai
Abnormally high activation of transforming growth factor-β (TGF-β) signaling has been demonstrated to be involved in the initiation and progression of keloids. However, the functional role of long non-coding RNA (lncRNA)-activated by TGF-β (lncRNA-ATB) in keloids has not been documented. Here we investigated the role of lncRNA-ATB in the autocrine secretion of TGF-β in keloid fibroblasts (KFs) and explored the underlying molecular mechanism. Using immunohistochemistry and quantitative RT-PCR analysis, we showed that lncRNA-ATB and ZNF217, a transcriptional activator of TGF-β, were overexpressed and miR-200c, which targets ZNF217, was under-expressed in keloid tissue and keloid fibroblasts. Through gain- and loss-of-function studies, we demonstrated that knockdown of lncRNA-ATB decreased autocrine secretion of TGF-β2 and ZNF217 expression but upregulated expression of miR-200c in KFs. Stable downregulation of ZNF217 expression decreased the autocrine secretion of TGF-β2. miR-200c was endogenously associated with lncRNA-ATB, and inhibition of miR-200c overcame the decrease in ZNF217 expression in KFs. Taken together, these findings indicate that lncRNA-ATB governs the autocrine secretion of TGF-β2 in KFs, at least in part, by downregulating the expression level of ZNF217 via miR-200c, suggesting a signaling axis consisting of lncRNA-ATB/miR-200c/ZNF217/TGF-β2. These findings may provide potential biomarkers and targets for novel diagnostic and therapeutic approaches for keloids.
Wang Peng-Hui,Huang Ben-Shian,Horng Huann-Cheng,Yeh Chang-Ching,Chen Yi-Jen
Journal of the Chinese Medical Association : JCMA
Wound healing is an important physiological process to maintain the integrity of skin after trauma, either by accident or by intent procedure. The normal wound healing involves three successive but overlapping phases, including hemostasis/inflammatory phase, proliferative phase, and remodeling phase. Aberration of wound healing, such as excessive wound healing (hypertrophic scar and keloid) or chronic wound (ulcer) impairs the normal physical function. A large number of sophisticated experimental studies have provided insights into wound healing. This article highlights the information after 2010, and the main text includes (i) wound healing; (ii) wound healing in fetus and adult; (iii) prostaglandins and wound healing; (iv) the pathogenesis of excessive wound healing; (v) the epidemiology of excessive wound healing; (vi) in vitro and in vivo studies for excessive wound healing; (vii) stem cell therapy for excessive wound healing; and (viii) the prevention strategy for excessive wound healing.
miR-141-3p inhibits fibroblast proliferation and migration by targeting GAB1 in keloids.
Feng Jingjuan,Xue Siliang,Pang Qiuyu,Rang Zhen,Cui Fan
Biochemical and biophysical research communications
Keloids are benign dermal fibroproliferative tumors that develop as a result of several dysregulated processes. Emerging evidence has revealed that miRNAs contribute to keloid formation. However, the molecular mechanisms of keloid pathogenesis remain unclear. In our study, we found that miR-141-3p in keloid tissues and keloid fibroblasts was significantly decreased compared with the levels in normal tissues and normal skin fibroblasts, respectively. miR-141-3p overexpression resulted in significantly decreased proliferation and migration and the promotion of apoptosis in keloid fibroblasts, whereas miR-141-3p knockdown in keloid fibroblasts yielded the opposite results. Growth factor receptor binding 2-associated binding protein 1 (GAB1) was identified and confirmed as a direct target of miR-141-3p. The expression of GAB1 was up-regulated in keloid tissues, and the restoration of GAB1 partially reversed the inhibitory effects of miR-141-3p on the proliferation and migration of keloid fibroblasts. All data suggested that miR-141-3p decreased the proliferation and migration of keloid fibroblasts by repressing GAB1 expression, providing a useful target for keloid management.
Epidermal stem cell-derived exosomes promote skin regeneration by downregulating transforming growth factor-β1 in wound healing.
Duan Mengna,Zhang Yan,Zhang Haiyang,Meng Yupeng,Qian Ming,Zhang Guokun
Stem cell research & therapy
BACKGROUND:Scar formation, which may be caused by myofibroblast aggregations, is the greatest challenge during skin wound healing in the clinical setting. Studies have indicated that epidermal stem cells (EPSC) improve wound healing and reduce scar formation. METHODS:We investigated the therapeutic effects of EPSC-derived exosomes (EPSC-Exos) on skin wound healing in a skin-defect rat model. We also examined the roles of EPSC-Exos-specific microRNAs in inhibiting the differentiation of human dermal fibroblasts (HDF) into myofibroblasts. RESULTS:We found that EPSC-Exos increased the wound healing rate and reduced scar formation in rats. Also, EPSC-Exos improved the regeneration levels of skin appendages, nerves and vessels, as well as the natural distribution of collagen. Furthermore, we found these functions may be achieved by inhibiting the activity of transforming growth factor-β1 (TGF-β1) and its downstream genes. The results showed that some specific microRNAs, including miR-16, let-7a, miR-425-5p and miR-142-3p, were enriched in EPSC-Exos. EPSC-Exos-specific microRNAs, especially miR-425-5p and miR-142-3p, played vital roles in inhibiting myofibroblast differentiation via reducing the TGF-β1 expression in dermal fibroblasts. CONCLUSION:We found a novel function of EPSC-Exos-specific microRNAs, suggesting that EPSC-Exos might represent a strategy to prevent scar formation during wound healing in the clinical setting.
MicroRNA-21 affects proliferation and apoptosis by regulating expression of PTEN in human keloid fibroblasts.
Liu Ying,Wang Xiaoxue,Yang Daping,Xiao Zhibo,Chen Xi
Plastic and reconstructive surgery
BACKGROUND:MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate gene expression in the progress of proliferation, differentiation, and apoptosis. A keloid is considered to be a type of benign tumor. The exact contribution of miRNAs in keloid fibroblasts remains largely unknown. METHODS:Loss and gain of function was used by transfecting the keloid fibroblast cells with chemically synthesized oligonucleotides complementary to microRNA-21 (miR-21). Expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in keloid tissues and adjacent normal skin tissues were investigated by quantitative real-time polymerase chain reaction and Western blot assay. Moreover, cell apoptosis and proliferation were checked in keloid cells, and related proteins were determined by Western blot assay. RESULTS:MiR-21 inhibitor and mimic transfection changed the apoptosis and DNA synthesis. Of the 23 paired samples analyzed, expression of PTEN was low in keloid tissues relative to adjacent normal skin tissues. Cells showed an inverse correlation between miR-21 and PTEN protein after transfection. In addition, cell proliferation was increased when normal skin fibroblasts were transfected with miR-21 mimics. It is worth noting that the expression of phosphorylated AKT decreased to relatively low levels after miR-21 inhibitor treatment. CONCLUSIONS:This in vitro study demonstrates important interactions among miR-21 expression, keloid fibroblast apoptosis, cell proliferation, and some related proteins. These findings may provide some hints toward effective applications of miR-21 as a therapy target for keloids.
MiR-21 regulates the apoptosis of keloid fibroblasts by caspase-8 and the mitochondria-mediated apoptotic signaling pathway via targeting FasL.
Liu Ying,Ren Lihong,Liu Wenjing,Xiao Zhibo
Biochemistry and cell biology = Biochimie et biologie cellulaire
MicroRNA-21 (miR-21) has been found to be upregulated in keloid tissue and to affect the proliferation and apoptosis of keloid fibroblasts; however, the possible mechanisms remain unclear. In this study, we aimed to evaluate the role of miR-21 in FasL-induced caspase-8 activation and the mitochondria-mediated apoptotic signaling pathway in keloid fibroblasts. Our study found that the protein level of FasL was decreased by miR-21 over-expression, while being enhanced by miR-21 inhibition in keloid fibroblasts. Subsequently, the mitochondria-mediated apoptosis of keloid fibroblasts was restrained by miR-21 over-expression, as evidenced by enhanced mitochondrial membrane potential and decreased production of mitochondrial ROS. Moreover, over-expression of miR-21 inhibited the activation of the caspase-8 and the mitochondria-mediated apoptotic signaling pathway. As expected, inhibition of miR-21 had the opposite effects. Finally, silencing of FasL suppressed miR-21 inhibition-induced apoptosis by inactivation of caspase-8 and the mitochondria-mediated apoptotic signaling pathway, which was comparable to Z-IETD-FMK, a caspase-8 inhibitor. Taken together, these results suggest that miR-21 regulates the apoptosis of keloid fibroblasts via targeting FasL, and caspase-8 and the mitochondria-mediated apoptotic signaling pathway is involved in this process. Our findings provide evidence that miR-21 may be considered to be a therapeutic target for keloids.
MiR-203 regulates keloid fibroblast proliferation, invasion, and extracellular matrix expression by targeting EGR1 and FGF2.
Shi Kai,Qiu Xiang,Zheng Wei,Yan Dongmei,Peng Weihai
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Keloid is a fibrous benign tumor of the skin caused by increased fibroblast proliferation and overproduction of extracellular matrix (ECM) in the dermis. Several miRNAs exhibit critical roles in regulating keloid development. This is study aimed to investigate the effects and mechanisms of miR-203 in keloid fibroblasts. The miR-203 expression was detected by qRT-PCR; The cell viability was measured by MTT assay; The cell proliferation was measured by BrdU assay; The cell invasion was measured by Transwell assay; The protein expression was detected by Western blot; The target relationship between miR-203 and mRNA was measured by dual-luciferase assay. We found that miR-203 was significantly downregulated in both keloid tissues and keloid fibroblasts from keloid patients. MiR-203 overexpression in vitro led to a significant decrease of proliferation, invasion, and ECM production in keloid fibroblasts, whereas miR-203 inhibition induced the opposite results. A dual-luciferase reporter assay identified early growth response 1 (EGR1) and fibroblast growth factor 2 (FGF2) as targets of miR-203. EGR1 and FGF2 were overexpressed in keloid fibroblasts and negatively regulated by miR-203. Furthermore, overexpression of EGR1 and FGF2 partially attenuated the suppressive effect of miR-203 on the proliferation, invasion, and ECM production of keloid fibroblasts. In conclusion, we demonstrated for the first time that miR-203 decreased the proliferation, invasion, and ECM production of keloid fibroblasts by repressing EGR1 and FGF2 expression, suggesting a potential role of miR-203 in preventing and treating keloids.
miR-21 promotes collagen production in keloid via Smad7.
Zhou Renpeng,Wang Chen,Wen Congji,Wang Danru
Burns : journal of the International Society for Burn Injuries
OBJECTIVE:To explore the biological function of miR-21 in the formation of keloid. METHODS:Normal skin and keloid tissue samples underwent histopathologic study and qPCR analysis. The expression of miR-21 and mRNA expression of Smad7, Col1A1, Col3A1 in fibroblasts derived from keloid tissue and normal skin tissue samples were detected by qPCR. Normal and keloid fibroblasts were transfected with miR-21 mimics or inhibitor respectively, and expression of Smad7, Col1A1 and Col3A1 were examined. After the normal fibroblasts were transfected with Smad7 siRNA, expression of Col1A1 and Col3A1 were detected by Western blot and qPCR analysis. RESULTS:Collagen was obviously thick and disorganized in keloid tissue. The expression of miR-21, Col1A1 and Col3A1 in keloid tissue and keloid-derived fibroblasts were higher than that of normal counterparts, while the expression of Smad7 in keloid tissue and keloid-derived fibroblasts was lower. miR-21 mimics attenuated expression of Smad7, and enhanced the expression of Col1A1, Col3A1. Furthermore, the Smad7 siRNA increased expression of Col1A1and Col3A1. CONCLUSIONS:miR-21 promoted collagen production in keloid by negatively regulating the expression of the Smad7.
MiR-21 Regulates Keloid Formation by Downregulating Smad7 via the TGF-β/Smad Signaling Pathway.
Wu Junliang,Fang Lu,Cen Ying,Qing Yong,Chen Junjie,Li Zhengyong
Journal of burn care & research : official publication of the American Burn Association
A keloid is a benign fibroproliferative skin tumor that results from abnormal wound healing after injury and tends to grow beyond the boundary of the original wound; the mechanism of keloid formation is still unclear. MicroRNA-21 (MiR-21) is a representative microRNA that plays a key role in a variety of fibrotic diseases via the transforming growth factor-β/Smad signaling pathway. The aim of our study was to explore the mechanism of keloid formation. First, we found that the expression of miR-21 in keloids and keloid fibroblasts was significantly upregulated by microRNA microarray and real-time polymerase chain reaction. Additionally, at the protein level, our study confirmed that the overexpression of miR-21 could promote the process of keloid fibrosis to some extent and also indicated that a low expression of miR-21 could inhibit the process of keloid fibrosis. Finally, the results proved that miR-21 could participate in the keloid fibrosis process through negative regulation of its downstream target gene Smad7 via the transforming growth factor-β/Smad signaling pathway, which provides a guiding framework for further studies and new theoretical support for keloid clinical treatment.
BTXA regulates the epithelial-mesenchymal transition and autophagy of keloid fibroblasts via modulating miR-1587/miR-2392 targeted ZEB2.
Hou Zhanying,Fan Feixiang,Liu Po
Keloids are very resistant to treatment in dermatology and plastic surgical practice. The present study aimed to explore the underlying mechanism of botulinum toxin A (BTXA) treated human skin keloid fibroblasts (HSFBs) proving some new insights into keloids treatment. Expression of miR-1587 and miR-2392 were significantly down-regulated in keloid tissues and HSFBs, while the ZEB2 was a target of both and up-regulated in keloid tissues and HSFBs compared with the normal controls. BTXA could significantly increase the expression of miR-1587 and miR-2392 but decrease the expression of ZEB2. BTXA could significantly inhibit the proliferation, cell cycle, and migration and promote apoptosis and autophagy of HSFBs; however, miR-1587 and miR-2392 inhibitors could reverse these effects of BTXA on HSFBs. Silencing ZEB2 could significantly attenuate the effects of miR-1587 and miR-2392 inhibitors in promoting cell proliferation and migration and suppressing apoptosis and autophagy of HSFBs after treating with BTXA. BTXA could suppress the proliferation and migration and promote apoptosis and autophagy of HSFBs via modulating miR-1587/miR-2392 targeted ZEB2.
Long non-coding RNA HOXA11-AS induces type I collagen synthesis to stimulate keloid formation via sponging miR-124-3p and activation of Smad5 signaling.
Jin Jun,Zhai Hong-Feng,Jia Zhen-Hua,Luo Xiao-Hua
American journal of physiology. Cell physiology
Keloid, characterized by exuberant collagen deposition and invasive growth beyond original wound margins, results from abnormal wound healing. A recent microarray analysis identified homeobox (HOX) A11 antisense (HOXA11-AS) as a keloid-specific long non-coding RNA, although its potential role in keloid formation remains elusive. In this study, hematoxylin-eosin, Masson, and immunohistochemical staining of type I collagen (ColI) revealed abnormal arrangement and hyperplasia of fibers in keloid tissues along with increased ColI level. qRT-PCR and Western blot showed that HOXA11-AS and ColI were significantly upregulated, while miR-124-3p was decreased in both keloid tissues and human keloid fibroblasts (HKFs). Knockdown of HOXA11-AS inhibited cell proliferation (by CCK-8 and immunofluorescence staining of Ki67) and cell migration (by wound healing and transwell assays). Mechanistic experiments verified that HOXA11-AS acted as a sponge of micro-RNA (miR)-124-3p and Smad5 was a target of miR-124-3p. miR-124-3p sufficiently reversed the regulatory effects of HOXA11-AS, and Smad5 was involved in miR-124-3p-mediated biological functions. Furthermore, HOXA11-AS induced ColI synthesis via sponging miR-124-3p-mediated Smad5 signaling, thus promoting keloid formation. Overall, our study implied that HOXA11-AS induces ColI synthesis to promoted keloid formation via sponging miR-124-3p-mediated Smad5 signaling, which might offer a novel target for developing the therapy of keloid formation.
MiR-152-3p regulates cell proliferation, invasion and extracellular matrix expression through by targeting FOXF1 in keloid fibroblasts.
Wang Rui,Bai Zhuanli,Wen Xiulin,Du Huicong,Zhou Lin,Tang Zhishui,Yang Zhuangqun,Ma Wei
Emerging evidence has revealed that microRNAs (miRNAs) play critical roles in keloid pathogenesis. However, potential molecular mechanism of keloid formation remains unclear. In the present study, our findings showed that miR-152-3p mRNA expression level was notably up-regulated in keloid tissues and keloid fibroblasts compared with that of normal skin tissues and normal skin fibroblasts, respectively. Furthermore, miR-152-3p inhibition remarkably suppressed cell proliferation, which was increased by miR-152-3p overexpression. Cell invasion was also significantly decreased by miR-152-3p inhibition, whereas was increased by miR-152-3p overexpression. The mRNA and protein expression levels of extracellular matrix components including type I collagen, type III collagen and fibronectin were decreased by miR-152-3p inhibition, but were increased by miR-152-3p overexpression. In addition, results of dual-luciferase reporter assay indicated that FOXF1 is a direct target of miR-152-3p. FOXF1 overexpression significantly inhibits cell proliferation, invasion, and extracellular matrix in keloid fibroblasts, and the suppressive effects of miR-152-3p mimic on these functions were notably partly reversed by FOXF1 overexpression. Taken together, these findings indicated that miR-152-3p regulates cell proliferation, invasion and extracellular matrix expression through targeting FOXF1 in keloid fibroblasts, suggesting that miR-152-3p is a novel and promising molecular target for keloid treatment.
MiR-21-5p Links Epithelial-Mesenchymal Transition Phenotype with Stem-Like Cell Signatures via AKT Signaling in Keloid Keratinocytes.
Yan Li,Cao Rui,Liu YuanBo,Wang LianZhao,Pan Bo,Lv XiaoYan,Jiao Hu,Zhuang Qiang,Sun XueJian,Xiao Ran
Keloid is the abnormal wound healing puzzled by the aggressive growth and high recurrence rate due to its unrevealed key pathogenic mechanism. MicroRNAs contribute to a series of biological processes including epithelial-mesenchymal transition (EMT) and cells stemness involved in fibrotic disease. Here, using microRNAs microarray analysis we found mir-21-5p was significantly up-regulated in keloid epidermis. To investigate the role of miR-21-5p in keloid pathogenesis, we transfected miR-21-5p mimic or inhibitor in keloid keratinocytes and examined the abilities of cell proliferation, apoptosis, migration and invasion, the expressions of EMT-related markers vimentin and E-cadherin and stem-like cells-associated markers CD44 and ALDH1, and the involvement of PTEN and the signaling of AKT and ERK. Our results demonstrated that up-regulation or knockdown of miR-21-5p significantly increased or decreased the migration, invasion and sphere-forming abilities of keloid keratinocytes, and the phenotype of EMT and cells stemness were enhanced or reduced as well. Furthermore, PTEN and p-AKT were shown to participate in the regulation of miR-21-5p on EMT phenotypes and stemness signatures of keloid keratinocytes, which might account for the invasion and recurrence of keloids. This molecular mechanism of miR-21-5p on keloid keratinocytes linked EMT with cells stemness and implicated novel therapeutic targets for keloids.
Downregulation of microRNA-31 inhibits proliferation and induces apoptosis by targeting in human keloid.
Zhang Juan,Xu Dan,Li Na,Li Yan,He Yongjing,Hu Xingbo,Lyu Lechun,He Li
microRNAs (miRNAs) play a pivotal role in the regulation of cell proliferation and apoptosis in keloid scarring. Integrative analysis of the previous miRNA microarray revealed miRNA-31 was among the most frequently altered miRNAs in keloid and hypertrophic scar. Using qRT-PCR, we further validated miRNA-31 was increased in keloid tissues and keloid-derived fibroblasts. Moreover, downregulation of miRNA-31 inhibited the cell proliferation, induced the cell apoptosis and disturbed the cell cycle progression by targeting , a negative modulator of hypoxia inducible factor 1. Through the luciferase reporter assay, was confirmed to be a target of miRNA-31. Further studies demonstrated that miRNA-31 regulated proliferation, apoptosis and cell cycle of keloid-derived fibroblasts by mediating HIF1AN/VEGF signaling pathway. Overall, our findings shed new light on miRNA-31 as a promising therapeutic target in keloid scarring.
Inhibition of microRNA-21-5p reduces keloid fibroblast autophagy and migration by targeting PTEN after electron beam irradiation.
Yan Li,Wang Lian-Zhao,Xiao Ran,Cao Rui,Pan Bo,Lv Xiao-Yan,Jiao Hu,Zhuang Qiang,Sun Xue-Jian,Liu Yuan-Bo
Laboratory investigation; a journal of technical methods and pathology
Electron beam (EB) irradiation is useful to reduce the recurrence of keloids; however, the underlying mechanism remains unknown. MicroRNA-21 (miR-21), which regulates autophagy during cancer radiation therapy, was identified as a potential therapeutic target for keloids. Here, we investigate the regulatory mechanism(s) of miR-21-5p on keloid fibroblast autophagy and migration after EB irradiation. The microRNA expression profile of the keloid dermis was examined by performing a microRNA microarray. Levels of LC3B and Beclin-1 were detected by immunohistochemical and western blot analysis in the keloid dermis and fibroblasts. Autophagy and apoptosis were tested in keloid fibroblasts after EB irradiation or transfection with an miR-21-5p inhibitor using electron microscopy, a Cyto-ID Green Autophagy Detection Kit, and an Annexin V PE Apoptosis Detection Kit. Migration was analyzed by an in vitro scratch-wound healing assay. Mechanistic tests were performed using small interfering RNAs to phosphatase and tensin homolog (siPTEN). Levels of miR-21-5p, PTEN, programmed cell death 4 (PDCD4), p-AKT, and apoptosis- and autophagy-associated genes were examined by qRT-PCR and western blotting. LC3B expression and migration ability were enhanced in fibroblasts and the keloid margin dermis compared with those in the adjacent normal skin. Both EB irradiation and an miR-21-5p inhibitor reduced keloid fibroblast autophagy, which was accompanied by decreased expression of miR-21-5p, p-AKT, and LC3B-II and increased expression of PTEN, PDCD4, and apoptosis-related genes. MiR-21-5p downregulation inhibited migration and suppressed LC3B expression and this was reversed by PTEN reduction. In conclusion, with increasing apoptosis, EB irradiation inhibits autophagy in keloid fibroblasts by reducing miR-21-5p, which regulates migration and LC3B expression via PTEN/AKT signaling. These data suggest a potential mechanism wherein miR-21-5p inhibition regulates autophagy and migration in EB-irradiated keloid fibroblasts, effectively preventing local invasion and recurrence. Therefore, miR-21-5p could be a new therapeutic target, to replace EB irradiation, and control keloid relapse.
Comprehensive Analysis of Long Noncoding RNAs and Messenger RNAs Expression Profiles in Patients with Marjolin Ulcer.
Liu Zan,Ren Licheng,Tian Jing,Liu Ning,Hu Yanke,Zhang Pihong
Medical science monitor : international medical journal of experimental and clinical research
BACKGROUND Marjolin ulcer (MU) is an aggressive cutaneous malignancy. Typically, MU occurs over a period of time in post-burn and/or post-traumatic lesions and scars. However, the pathogenesis of scar carcinogenesis and MU development remains to be elucidated. The present study aimed to investigate the long noncoding RNA (lncRNA) and messenger RNA (mRNA) expression profiling in MU, which could provide new information on the potential molecular mechanisms of MU development. MATERIAL AND METHODS The lncRNA microarray analysis was conducted in normal skin, scar, and MU tissue, and quantitative real-time PCR experiment was carried out to validate the reliability of the microarray data. Furthermore, a series of integrative bioinformatic approaches were applied to decipher the function of differentially expressed lncRNAs. RESULTS A total of 7130 lncRNAs and 9867 mRNAs were differentially expressed among normal skin, scar, and MU tissues. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that these aberrantly expressed transcripts were mainly involved in cell cycle, immune response, and the p53 signaling pathway. Series Test of Cluster analysis indicated certain dysregulated lncRNAs were expressed with a gradually increasing or decreasing trend and might participated in malignant transformation of scar tissue postburn. Co-expression analysis showed 5 selected lncRNAs might regulate cell proliferation through the p53 signaling pathway. Finally, the competing endogenous RNA (ceRNA) network indicated that lncRNA uc001oou.3 might be implicated in ceRNA mechanism during MU development. CONCLUSIONS Taken together, our study implied the aberrant expression of lncRNAs may play an important role in the pathogenesis and development of MU, and the exact mechanism warrants further investigation.
A MicroRNA-29 Mimic (Remlarsen) Represses Extracellular Matrix Expression and Fibroplasia in the Skin.
Gallant-Behm Corrie L,Piper Joseph,Lynch Joshua M,Seto Anita G,Hong Seok Jong,Mustoe Thomas A,Maari Catherine,Pestano Linda A,Dalby Christina M,Jackson Aimee L,Rubin Paul,Marshall William S
The Journal of investigative dermatology
MicroRNA-29 (miR-29) negatively regulates fibrosis and is downregulated in multiple fibrotic organs and tissues, including in the skin. miR-29 mimics prevent pulmonary fibrosis in mouse models but have not previously been tested in the skin. This study aimed to identify pharmacodynamic biomarkers of miR-29 in mouse skin, to translate those biomarkers across multiple species, and to assess the pharmacodynamic activity of a miR-29b mimic (remlarsen) in a clinical trial. miR-29 biomarkers were selected based on gene function and mRNA expression using quantitative reverse transcriptase polymerase chain reaction. Those biomarkers comprised multiple collagens and other miR-29 direct and indirect targets and were conserved across species; remlarsen regulated their expression in mouse, rat, and rabbit skin wounds and in human skin fibroblasts in culture, while a miR-29 inhibitor reciprocally regulated their expression. Biomarker expression translated to clinical proof-of-mechanism; in a double-blinded, placebo-randomized, within-subject controlled clinical trial of single and multiple ascending doses of remlarsen in normal healthy volunteers, remlarsen repressed collagen expression and the development of fibroplasia in incisional skin wounds. These results suggest that remlarsen may be an effective therapeutic to prevent formation of a fibrotic scar (hypertrophic scar or keloid) or to prevent cutaneous fibrosis, such as scleroderma.
Swainsonine inhibits proliferation and collagen synthesis of NIH-3T3 cells by declining miR-21.
Li Chao,Wang Peipei,Fu Ziyang,Li Yongtao,Li Shouju
Artificial cells, nanomedicine, and biotechnology
Swainsonine (SW) is an indolizidine alkaloid first discovered in . This study explored the effects of SW on mouse embryo fibroblast NIH-3T3 cell proliferation and collagen synthesis, as well as potential molecule mechanisms. We discovered that SW exposure lowered the viability and proliferation of NIH-3T3 cells. The collagen synthesis was reduced after SW exposure, as evidenced by declines of the mRNA and protein levels of collagen I (CoI I), collagen III (CoI III) and α-smooth muscle actin (α-SMA) in NIH-3T3 cells, as well as reduction of collagen concentration in the culture supernatant of NIH-3T3 cells. Mechanically, transforming growth factor β1 (TGF-β1) stimulation elevated the microRNA-21 (miR-21) expression in NIH-3T3 cells. SW reversed the TGF-β1-caused elevation of miR-21. Up-regulation of miR-21 attenuated the inhibitory influences of SW on NIH-3T3 cell viability, proliferation and collagen synthesis. Silence of miR-21 had converse influence. Besides, SW inactivated PI3K/AKT and NF-κB pathways via declining miR-21. Altogether, SW inhibited the proliferation and collagen synthesis of fibroblast NIH-3T3 might be through declining miR-21 and then suppressing PI3K/AKT and NF-κB pathways. SW may be an effective therapeutic medicine for scar hyperplasia.
Scarless integumentary wound healing in the mammalian fetus: molecular basis and therapeutic implications.
Kathju Sandeep,Gallo Phillip H,Satish Latha
Birth defects research. Part C, Embryo today : reviews
Adult mammals respond to injury of their skin/integument by forming scar tissue. Scar is useful in rapidly sealing an injured area, but can also lead to significant morbidity. Mammals in fetal life retain the ability to heal integumentary wounds regeneratively, without scar. The critical molecular mechanisms governing this remarkable phenomenon have been a subject of great interest, in the hopes that these could be dissected and recapitulated in the healing adult wound, with the goal of inducing scarless healing in injured patients. Multiple lines of investigation spanning decades have implicated a number of factors in distinguishing scarless from fibrotic wound healing, including most prominently transforming growth factor-β and interleukin-10, among others. Therapeutic interventions to try to mitigate scarring in adult wounds have been developed out of these studies, and have reached the level of clinical trials in humans, although as yet no FDA-approved treatment exists. More recent expressomic studies have revealed many more genes that are differentially expressed in scarlessly healing fetal wounds compared with adult, and microRNAs have also been identified as participating in the fetal wound healing response. These represent an even greater range of potential therapeutics (or targets for therapy) to translate the promise of scarless fetal wound healing to the injured adult patient.
A novel regulatory function for miR-29a in keloid fibrogenesis.
Zhang G-Y,Wu L-C,Liao T,Chen G-C,Chen Y-H,Zhao Y-X,Chen S-Y,Wang A-Y,Lin K,Lin D-M,Yang J-Q,Gao W-Y,Li Q-F
Clinical and experimental dermatology
BACKGROUND:A growing body of evidence has shown that microRNA-29 (miR-29) plays a central role in the progression of fibrosis. However, the mechanisms underlying the role of miR-29 in keloid fibrogenesis remain unknown. AIM:To investigate the roles of miR-29 in dermal fibroblasts in the pathogenesis of keloids. METHODS:Primary fibroblasts from 9 patients with keloid and 6 healthy controls (HCs) were cultured and pretreated with transforming growth factor (TGF)-β1. Next, fibroblasts were transfected with precursor miRNA and anti-miR-29a miRNA. TGF-β1-associated miR-29 alterations were investigated by quantitative real-time PCR. Collagen I and collagen III protein levels were analysed by western blotting. RESULTS:miR-29a, miR-29b and miR-29c levels were significantly lower in keloid compared with healthy fibroblasts (P < 0.05), and in particular, miR-29a was especially markedly reduced (P < 0.001). Type I and type III collagen mRNA and protein levels were decreased in keloid fibroblasts transfected with pre-miR-29a (P < 0.05), whereas knockdown with anti-miR-29a increased type I and type III collagen mRNA and protein expression (P < 0.05) in the fibroblasts. Interestingly, pretreatment of fibroblasts with TGF-β1 significantly decreased miR-29a (P < 0.05), whereas miR-29b and miR-29c were reduced to a lesser extent, which was not significant. CONCLUSIONS:These findings show that miR-29a exerts as a novel regulator in the fibrogenesis of keloid, suggesting that miR-29a might be a novel marker for keloid.
High‑throughput sequencing reveals differentially expressed lncRNAs and circRNAs, and their associated functional network, in human hypertrophic scars.
Li Min,Wang Jian,Liu Dewu,Huang Heping
Molecular medicine reports
Growing evidence suggests that long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) are involved in the occurrence and development of tumors and fibrotic diseases. However, the integrated analysis of lncRNA and circRNA expression, alongside associated co‑expression and competing endogenous RNA (ceRNA) networks, has not yet been performed in human hypertrophic scars (HS). The present study compared the expression levels of lncRNAs, circRNAs and mRNAs in human HS and normal skin tissues by high‑throughput RNA sequencing. Numerous differentially expressed lncRNAs, circRNAs and mRNAs were detected. Subsequently, five aberrantly expressed lncRNAs and mRNAs, and six circRNAs were measured to verify the RNA sequencing results by reverse transcription‑quantitative polymerase chain reaction. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed for the dysregulated genes, in order to elucidate their principal functions. In addition, a coding‑noncoding gene co‑expression (CNC) network and ceRNA network were constructed for specific significantly altered genes. The CNC network analysis suggested that AC048380.1 and LINC00299 were associated with metastasis‑related genes, including inhibin subunit βA (INHBA), SMAD family member 7 (SMAD7), collagen type I α1 chain (COL1A1), transforming growth factor β3 (TGFβ3) and MYC proto‑oncogene, bHLH transcription factor (MYC). Inhibitor of DNA binding 2 was associated with the lncRNAs cancer susceptibility 11, TGFβ3‑antisense RNA 1 (AS1), INHBA‑AS1, AC048380.1, LINC00299 and LINC01969. Circ‑Chr17:50187014_50195976_‑, circ‑Chr17:50189167_50194626_‑, circ‑Chr17:50189167_ 50198002_‑ and circ‑Chr17:50189858_50195330_‑ were also associated with INHBA, SMAD7, COL1A1, TGFβ3 and MYC. COL1A1 and TGFβ3 were associated with circ‑Chr9:125337017_125337591_+ and circ‑Chr12:120782654_120784593_‑. The ceRNA network indicated that INHBA‑AS1 and circ‑Chr9:125337017_125337591_+ were ceRNAs of microRNA‑182‑5p targeting potassium voltage‑gated channel subfamily J member 6, ADAM metallopeptidase with thrombospondin type 1 motif 18, SRY‑box 11, MAGE family member L2, matrix metallopeptidase 16, thrombospondin 2, phosphodiesterase 11A and collagen type V a1 chain. These findings suggested that lncRNAs and circRNAs may act as ceRNAs, which are implicated in the pathophysiology and development of human HS, and lay a foundation for further insight into the novel regulatory mechanism of lncRNAs and circRNAs in hypertrophic scarring.
MicroRNA-22 may promote apoptosis and inhibit the proliferation of hypertrophic scar fibroblasts by regulating the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase/p21 pathway.
Dong Shihua,Sun Yanfeng
Experimental and therapeutic medicine
Hypertrophic scarring (HS) is a common skin disorder that occurs during the wound healing process, and the pathogenesis of HS remains unclear. Increasing evidence indicated that specific microRNAs (miRs) may be involved in the onset and progression of HS. In the present study, the association between miR-22 and HS was investigated. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to examine the expression of miR-22 in 30 HS and matched normal skin tissues. In addition, human hypertrophic scar fibroblasts (HSFBs) were cultured and transfected with miR-22 mimics, and MTT and Annexin V apoptosis assays were performed to investigate the role of miR-22 in the proliferation and apoptosis of the human HSFBs. Next, RT-qPCR and western blot assays were performed to compare the expression levels of mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK) and p21 in untransfected and miR-22 mimic-transfected skin fibroblasts. The results identified that miR-22 was significantly downregulated in HS tissues as compared with the normal skin. Furthermore, transfection with miR-22 mimics in human HSFBs led to inhibited cell proliferation, increased apoptosis, as well as to decreased MEK expression and ERK1/2 phosphorylation, and increased expression of p21. In conclusion, the present study was the first to prove that aberrant expression of miR-22 may serve an important role in the pathogenesis of HS by regulating the MEK/ERK/p21 pathway, thus suggesting that miR-22 has the potential to become a therapeutic target for the treatment of HS.
Baicalein inhibits proliferation and collagen synthesis of mice fibroblast cell line NIH/3T3 by regulation of miR-9/insulin-like growth factor-1 axis.
Yang Li,Li Xueli,Zhang Shoumin,Song Jinghui,Zhu Tingting
Artificial cells, nanomedicine, and biotechnology
The aberrant scar is a challenging problem. Baicalein has effects in attenuating hypertrophic scar formation. Herein, the roles of baicalein in NIH-3T3 were obtained. Cells were treated by baicalein. CCK-8 method and western blot were used to detect cell viability and proliferation-related factors. Furthermore, collagen 1, collagen 3 and α-SMA expression were detected by qRT-PCR and western blot. Besides, total soluble collagen was detected by Sircol assay. In addition, the levels of NF-κB and Wnt/β-catenin signal pathways related factors were determined by western blot. The relationship between miR-9 and insulin-like growth factor (IGF)-1 were tested by luciferase reporter assay, qRT-PCR and western blot. We found baicalein cell restrained proliferation and collagen production. Besides, baicalein increased expression of miR-9 and further experiments validated that transfection with miR-9 inhibitor reversed the results led by baicalein. Moreover, baicalein decreased the phosphorylation of pathway-related proteins while transfection with miR-9 inhibitor revised this result. Otherwise, IGF-1 was authenticated as a target of miR-9 and si-IGF-1 reversed the miR-9 inhibitor-induced change in cell proliferation and collagen production. In conclusion, baicalein restrained cell proliferation and collagen production by regulating miR-9/IGF-1 axis through NF-κB and Wnt/β-catenin signal pathways. Highlights: Baicalein restrains NIH/3T3 cell proliferation and collagen production. Baicalein restrains NF-κB and Wnt/β-catenin signal pathways. IGF-1 is a target of miR-9. Baicalein exerts its functions by regulating miR-9/IGF-1 axis.
Exploring the role of microRNAs in axolotl regeneration.
Abo-Al-Ela Haitham G,Burgos-Aceves Mario A
Journal of cellular physiology
The axolotl, Ambystoma mexicanum, is used extensively for research in developmental biology, particularly for its ability to regenerate and restore lost organs, including in the nervous system, to full functionality. Regeneration in mammals typically depends on the healing process and scar formation with limited replacement of lost tissue. Other organisms, such as spiny mice (Acomys cahirinus), salamanders, and zebrafish, are able to regenerate some damaged body components. Blastema is a tissue that is formed after tissue injury in such organisms and is composed of progenitor cells or dedifferentiated cells that differentiate into various cell types during regeneration. Thus, identifying the molecules responsible for initiation of blastema formation is an important aspect for understanding regeneration. Introns, a major source of noncoding RNAs (ncRNAs), have characteristic sizes in the axolotl, particularly in genes associated with development. These ncRNAs, particularly microRNAs (miRNAs), exhibit dynamic regulation during regeneration. These miRNAs play an essential role in timing and control of gene expression to order and organize processes necessary for blastema creation. Master keys or molecules that underlie the remarkable regenerative abilities of the axolotl remain to be fully explored and exploited. Further and ongoing research on regeneration promises new knowledge that may allow improved repair and renewal of human tissues.
miR-196a downregulation increases the expression of type I and III collagens in keloid fibroblasts.
Kashiyama Kazuya,Mitsutake Norisato,Matsuse Michiko,Ogi Tomoo,Saenko Vladimir A,Ujifuku Kenta,Utani Atsushi,Hirano Akiyoshi,Yamashita Shunichi
The Journal of investigative dermatology
Keloids are a fibroproliferative disease due to abnormal wound healing process after skin injury. They are characterized by overproduction of extracellular matrix (ECM) such as collagens. MicroRNAs (miRNAs) are noncoding small RNAs and negatively regulate protein expression. Several miRNAs that have critical roles in tissue fibrosis and ECM metabolism have been reported. However, regulation and function of miRNAs in keloid remain to be explored. The purpose of this study was to identify miRNAs involved in keloid pathogenesis. We performed miRNA microarray analysis to compare miRNA expression profiles between keloid-derived fibroblasts (KFs) and normal fibroblasts (NFs). In all, 7 upregulated and 20 downregulated miRNAs were identified. Among these, we focused on miR-196a, which showed the highest fold change. Overexpression or knockdown of miR-196a led to a decreased or increased level of secreted type I/III collagens, respectively. Reporter analysis showed direct binding of miR-196a to the 3' untranslated region (UTR) of COL1A1 and COL3A1. In conclusion, we demonstrate for the first time that miRNA expression profile is altered in KFs compared with NFs. Downregulation of miR-196a may be one of the mechanisms by which collagens are highly deposited in keloid tissues. Our findings suggest that miR-196a could be a new therapeutic target for keloid lesions.
miR-188-5p regulates proliferation and invasion via PI3K/Akt/MMP-2/9 signaling in keloids.
Zhu Wenyan,Wu Xiaoyan,Yang Bo,Yao Xiaodong,Cui Xiaomei,Xu Pan,Chen Xiaodong
Acta biochimica et biophysica Sinica
Keloids (KDs) and hypertrophic scars (HSs), two forms of pathological scars, seriously affect the physical and psychological health of patients. Despite many similarities with HSs, KDs are characterized by invasion and a high rate of recurrence after surgery, features they share in common with tumors. The underlying molecular mechanisms of this phenomenon have not been fully elucidated. In this study, we used microRNA (miRNA) array analysis to search for invasion-associated miRNAs in KDs. The expression of miR-188-5p in KDs, HSs, normal skin (NS) tissues, and cell lines was measured by quantitative real-time polymerase chain reaction. Furthermore, cell proliferation, migration, and invasion were detected in KD fibroblasts (KFs) and HS fibroblasts (HSFs), and interrelated proteins were ascertained by western blot analysis. It was found that miR-188-5p was significantly decreased in KD tissue compared with HS and NS tissues. Upregulated expression of miR-188-5p suppressed KF proliferation, migration, and invasion; and decreased expression of miR-188-5p also promoted HSF proliferation, migration, and invasion. The protein levels of MMP-2, MMP-9, PI3K, and p-Akt in miR-188-5p mimic-transfected KFs were repressed. In contrast, after transfection with miR-188-5p inhibitor, the protein levels of MMP-2, MMP-9, PI3K, and p-Akt were higher than the control in HSFs. Treatment with PI3K/Akt inhibitor LY294002 in KFs with miR-188-5p inhibitor did not further reduce their proliferation, migration, and invasion. The upregulation of MMP-2 and MMP-9 by miR-188-5p inhibitor could be abolished by LY294002. These findings together demonstrate a tumor-suppressive role of miR-188-5p in KD proliferation and invasion via PI3K/Akt/MMP-2/9 signaling, indicating that miR-188-5p may be a potential prognostic marker and therapeutic target for KDs.
MiR-181a Targets PHLPP2 to Augment AKT Signaling and Regulate Proliferation and Apoptosis in Human Keloid Fibroblasts.
Rang Zhen,Wang Zong-Yang,Pang Qiu-Yu,Wang You-Wei,Yang Ge,Cui Fan
Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology
BACKGROUND/AIMS:Keloids are fibrous overgrowths induced by cutaneous injury. MicroRNAs (miRNAs) have recently emerged as post-transcriptional gene repressors and participants in a diverse array of pathophysiological processes leading to skin disease. The purpose of the current study was to explore the precise functions of miR-181a in human keloid development and the underlying mechanisms. METHODS:A miRNA microarray analysis was performed to compare expression profiles between keloid and normal skin tissues. Quantitative real-time PCR was conducted to estimate miR-181a expression. Cell proliferation was determined using the cell counting kit-8 (CCK-8) and 5-ethynyl-2-deoxyuridine (EdU) assays, and cell cycle and apoptosis were detected with flow cytometry. Direct targets of miR-181a were identified using the luciferase reporter assay. RESULTS:miR-181a was significantly upregulated in human keloid tissues and fibroblasts, compared with their control counterparts. Overexpression of miR-181a enhanced keloid fibroblast DNA synthesis and proliferation and inhibited apoptosis, whereas miR-181a suppression triggered the opposite effects. Moreover, miR-181a suppressed the expression of PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) through direct interactions with its 3'UTR region and subsequently enhanced AKT activation. Overexpression of PHLPP2 without its 3'UTR attenuated the effects of miR-181a on cell proliferation and apoptosis in keloid fibroblast cells. Furthermore, miR-181a mimics increased normal skin fibroblast proliferation. CONCLUSIONS:Our results highlight a novel pathway mediated by miR-181a, which may be effectively used as a therapeutic target for treatment of keloids.
Preparation of complex microcapsules of soluble polysaccharide from Glycyrrhiza uralensis and its application in wound repair and scar inhibition.
Hao Baocheng,Wang Xinjian,Ma Xueqing,Jin Yongming,Fan Wenguang,Laba Cidan,Wujin Cuomu,Wang Yonggang,Liang Jianping
International journal of biological macromolecules
The extraction process of Glycyrrhiza soluble polysaccharide (GP) was optimized by RSM, a rat trauma model was established via longitudinal incision on the back skin. The effects of GP combined with microcapsule collagen on the repair of rat injury model were discussed at different levels, Based on the content of hydroxyproline at the whole animal level, the proliferation of granulation tissue stained by HE, the number of microvessels labeled by CD34, the production of collagen fibers stained by Masson, the level of phosphorylation of STAT3 protein and that of VEGF at protein level were investigated. The results showed that after the administration of GP combined with microcapsules, the content of hydroxyproline in granulation tissue increased, the proliferation of capillaries and fibroblasts in granulation tissue became active, and the number of microvessels in wound increased. The formation density of collagen fibers was uniform and orderly. GP combined with microcapsules could activate the expression of p-STAT3 and VEGF proteins and up-regulate the transcription level of VEGF mRNA and miRNA-21 genes. Furthermore, GP combined with microcapsules could accelerate wound healing and promote neovascularization.
The Scar-in-a-Jar: studying potential antifibrotic compounds from the epigenetic to extracellular level in a single well.
Chen C Z C,Peng Y X,Wang Z B,Fish P V,Kaar J L,Koepsel R R,Russell A J,Lareu R R,Raghunath M
British journal of pharmacology
BACKGROUND AND PURPOSE:Fibrosis, a pathological accumulation of collagen in tissues, represents a major global disease burden. Effective characterization of potential antifibrotic drugs has been constrained by poor formation of the extracellular matrix in vitro, due to tardy procollagen processing by collagen C-proteinase/BMP-1, and difficulties in relating this matrix to cell numbers in experimental samples. EXPERIMENTAL APPROACH:The Scar-in-a-Jar model provided, in vitro, the complete biosynthetic cascade of collagen matrix formation including complete conversion of procollagen by C-proteinase/BMP-1, its subsequent extracellular deposition and lysyl oxidase-mediated cross-linking, achieved by applying the biophysical principle of macromolecular 'crowding'. Collagen matrix deposition, velocity and morphology can be controlled using negatively charged 'crowders' in a rapid (2 days) mode or a mixture of neutral 'crowders' in an accelerated (6 days) mode. Combined with quantitative optical bioimaging, this novel system allows for in situ assessment of the area of deposited collagen(s) per cell. KEY RESULTS:Optical evaluation of known and novel antifibrotic compounds effective at the epigenetic, post-transcriptional/translational/secretional level correlated excellently with corresponding biochemical analyses. Focusing on quantitation of deposited collagen, the Scar-in-a-Jar was most effective in assessing novel inhibitors that may have multiple targets, such as microRNA29c, found to be a promising antifibrotic agent. CONCLUSIONS AND IMPLICATIONS:This novel screening system supersedes current in vitro fibroplasia models, as a fast, quantitative and non-destructive technique. This method distinguishes a reduction in collagen I deposition, excluding collagen cross-linking, and allows full evaluation of inhibitors of C-proteinase/BMP-1 and other matrix metalloproteinases.
MicroRNA-152-5p inhibits proliferation and migration and promotes apoptosis by regulating expression of Smad3 in human keloid fibroblasts.
Pang Qianqian,Wang Yuming,Xu Mingyuan,Xu Jiachao,Xu Shengquan,Shen Yichen,Xu Jinghong,Lei Rui
Keloids are the most common pathological form of trauma healing, with features that seriously affect appearance and body function, are difficult to treat and have a high recurrence rate. Emerging evidence suggests that miRNAs are involved in a variety of pathological processes and play an important role in the process of fibrosis. In this study, we investigated the function and regulatory network of miR-152-5p in keloids. The miRNA miR-152-5p is frequently downregulated in keloid tissue and primary cells compared to normal skin tissue and fibroblasts. In addition, the downregulation of miR-152-5p is significantly associated with the proliferation, migration and apoptosis of keloid cells. Overexpression of miR-152-5p significantly inhibits the progression of fibrosis in keloids. Smad3 is a direct target of miR-152-5p, and knockdown of Smad3 also inhibits fibrosis progression, consistent with the overexpression of miR-152-5p. The interaction between miR-152-5p and Smad3 occurs through the Erk1/2 and Akt pathways and regulates collagen3 production. In summary, our study demonstrates that miR-152-5p/Smad3 regulatory pathways involved in fibrotic progression may be a potential therapeutic target of keloids. [BMB Reports 2019; 52(3): 202-207].
miR-34 modulates apoptotic gene expression in Ingenol mebutate treated keloid fibroblasts.
De Felice Bruna,Manfellotto Francesco,Garbi Corrado,Santoriello Margherita,Nacca Massimo
Molecular medicine reports
Keloids are benign skin tumors that develop in individuals who have a positive family history of keloid disorders. Keloids are characterized by a deregulated wound‑healing process, atypical fibroblasts with extreme deposition of extracellular matrix components, particularly collagen, increased cell proliferation and associated failure of apoptosis. Recently ingenol‑mebutate has been used as a novel agent with anti‑proliferative activity on human keloids as an alternative treatment option in patients, once conventional therapies have failed. We hypothesized that microRNAs (miR/miRNA) may be involved in the balance between lesion formation and repair. A comprehensive understanding of the molecular mechanism underlying the Ingenol‑mebutate response in keloid fibroblast following Ingenol‑mebutate exposure has been established previously. Therefore, the present study analyzed changes in miRNAs and apoptotic gene regulation in Ingenol‑mebutate treated keloid fibroblast, by reverse transcription‑quantitative polymerase chain reaction and a DNA fragmentation assay. The range of upregulated miRNAs and downregulated genes encoding cell death appeared to be associated with the degree of the morphological alterations in Ingenol‑mebutate treated keloids. In particular, the upregulation of miR‑34a was detected in keloid fibroblasts during and following Ingenol‑mebutate exposure. Keloid fibroblasts that overexpressed miR‑34a showed differential expression of genes involved in the apoptotic signaling pathway such as p53. In conclusion, the Ingenol‑mebutate treatment used here was effective in reducing keloid fibroblast growth in cell culture experiments and the expression of particular miRNAs modulated the pro‑apoptotic gene expression following Ingenol-mebutate treatment.
Keloid microRNA expression analysis and the influence of miR-199a-5p on the proliferation of keloid fibroblasts.
Wu Z-Y,Lu L,Liang J,Guo X-R,Zhang P H,Luo S-J
Genetics and molecular research : GMR
The purpose of this study was to identify microRNAs (miRNAs) involved in keloid formation and determine their influence on the proliferation of keloid fibroblasts (KFs). Eight specimens each of resected keloid tissue and normal skin tissue were collected. miRNAs that are differentially expressed in keloid tissue and normal skin were detected using an miRNA microarray and verified by quantitative real-time polymerase chain reaction (RT-PCR). Seventeen differentially expressed miRNAs, including miR-199a-5p, were identified by microarray hybridization. qRT-PCR analysis confirmed the decrease in miR-199a-5p expression in keloid vs normal tissue that was detected by the microarray analysis. Mimics of differentially expressed miRNAs were then transfected into a KF cell line, and the effect of miRNA overexpression on the proliferation of KFs was assayed using the EdU assay. Compared with mock-transfected cells, KFs transfected with a miR-199a-5p mimic showed significantly lower cell proliferation and an altered cell cycle, with cells having significantly longer S and G2/M phases. The significantly lower expression of miRNA-199a-5p in keloids likely influences the cell cycle of KFs and restrains their proliferation, suggesting that miR-199a-5p probably plays a role in the regulation of KF proliferation.
Tumor suppressive role of miR-1224-5p in keloid proliferation, apoptosis and invasion via the TGF-β1/Smad3 signaling pathway.
Yao Xiaodong,Cui Xiaomei,Wu Xiaoyan,Xu Pan,Zhu Wenyan,Chen Xiaodong,Zhao Tianlan
Biochemical and biophysical research communications
The molecular mechanism of the pathogenesis of keloids is still not known and the clinical management of keloids remains challenging. MiRNA (microRNA) is a novel class of small regulatory RNAs that has emerged as key post-transcriptional regulators of gene expression. MiRNAs participate in diverse biological processes of various skin diseases and function as key regulators in the occurrence and development of tumors. The purpose of this study was to investigate the involvement of miRNAs in keloid pathogenesis. We performed miRNA microarray analysis to compare miRNA expression between keloid and normal skin samples. We found that 46 miRNAs were upregulated and 28 miRNAs were downregulated in keloid compared with normal skin samples. We focused on miR-1224-5p, which has been reported to function in cancers, although the expression and mechanism of miR-1224-5p in keloids remain to be explored. Overexpression of miR-1224-5p led to inhibition of keloid fibroblast proliferation, promotion of apoptosis and decrease of migration and invasion. Our results suggest that downregulation of miR-1224-5p may be one of the mechanisms involved in the occurrence and development of keloids.
Upregulation of microRNA-205 suppresses vascular endothelial growth factor expression-mediated PI3K/Akt signaling transduction in human keloid fibroblasts.
An Gang,Liang Shuzeng,Sheng Chunhong,Liu Yan,Yao Wei
Experimental biology and medicine (Maywood, N.J.)
Keloid is one of the most frustrating problems related to wounding healing and presents a great challenge in clinic. MicroRNAs (miRs) have shown their potential as a novel therapy for the prevention and treatment of keloid. Vascular endothelial growth factor (VEGF) plays a critical role in the regulation of scar development. In the current study, it was hypothesized that miR-205-5p was capable of suppressing keloid formation by inhibiting the VEGF-mediated wound healing cascade. The expression statuses of miR-205-5p and VEGF in clinical keloid tissues and keloid cell line human keloid fibroblasts (HKF) were detected. Then the direct action of miR-205-5p on VEGF gene was assessed using dual-luciferase assay. Thereafter, orchestrated administrations on HKF with miR-205-5p mimic, specific VEGF siRNA, PI3K agonist (740 Y-P), and PI3K inhibitor (LY294002) were performed to reveal the roles of miR-205-5p and VEGF in keloid formation and further explain the mechanism through which miR-205-5p affected the VEGF-mediated signaling transductions. Our results showed that there was significant low expression of miR-205-5p in keloid tissue specimens and the cell line while the expression of VEGF in keloid tissues was augmented. Moreover, miR-205-5p overexpression dramatically impaired the cell viability, induced the cell apoptosis, and inhibited the cell invasion and migration ability in HKF. Based on the detection of dual luciferase assay and detection at protein level, miR-205-5p antagonized the keloids by directly targeting VEGF expression and subsequently inhibiting PI3K/Akt pathway. The current study is the first one demonstrating that miR-205-5p inhibits the pathogenesis of keloids, indicating the potential of miR-205-5p in the development of therapies for prevention and treatment of keloids.
microRNA deregulation in keloids: an opportunity for clinical intervention?
Yu Xin,Li Zheng,Chan Matthew T V,Wu William K K
Keloids are defined as benign dermal scars invading adjacent healthy tissue, characterized by aberrant fibroblast dynamics and overproduction of extracellular matrix. However, the aetiology and molecular mechanism of keloid production remain poorly understood. Recent discoveries have shed new light on the involvement of a class of non-coding RNAs known as microRNAs (miRNA), in keloid formation. A number of miRNAs have differential expression in keloid tissues and keloid-derived fibroblasts. These miRNAs have been characterized as novel regulators of cellular processes pertinent to wound healing, including extracellular matrix deposition and fibroblast proliferation. Delineating the functional significance of miRNA deregulation may help us better understand pathogenesis of keloids, and promote development of miRNA-directed therapeutics against this condition.
Exosomes Derived from Bone Mesenchymal Stem Cells with the Stimulation of FeO Nanoparticles and Static Magnetic Field Enhance Wound Healing Through Upregulated miR-21-5p.
Wu Di,Kang Lin,Tian Jingjing,Wu Yuanhao,Liu Jieying,Li Zhengyao,Wu Xiangdong,Huang Yue,Gao Bo,Wang Hai,Wu Zhihong,Qiu Guixing
International journal of nanomedicine
Background:Both magnetic nanoparticles (MNPs) and exosomes derived from bone mesenchymal stem cells (BMSC-Exos) have been reported to improve wound healing. In this study, novel exosomes (mag-BMSC-Exos) would be fabricated from BMSCs with the stimulation of MNPs and a static magnetic field (SMF) to further enhance wound repair. Methods:Mag-BMSC-Exos, namely, exosomes derived from BMSCs preconditioned with FeO nanoparticles and a SMF, together with BMSC-Exos were both first isolated by ultracentrifugation, respectively. Afterwards, we conducted in vitro experiments, including scratch wound assays, transwell assays, and tube formation assays, and established an in vivo wound healing model. The miRNA expression profiles were compared between BMSC-Exos and mag-BMSC-Exos to detect the potential mechanism of improving wound healing. At last, the function of exosomal miR-21-5p during wound healing was confirmed by utilizing a series of gain- and loss-of-function experiments in vitro. Results:The optimal working magnetic condition was 50 µg/mL FeO nanoparticles combined with 100 mT SMF. In vitro, mag-BMSC-Exo administration promoted proliferation, migration and angiogenesis to a greater extent than BMSC-Exo administration. Local transplantation of mag-BMSC-Exos into rat skin wounds resulted in accelerated wound closure, narrower scar widths and enhanced angiogenesis compared with BMSC-Exo transplantation. Notably, miR-21-5p was found to be highly enriched in mag-BMSC-Exos and served as a critical mediator in mag-BMSC-Exo-induced regulatory effects through inhibition of SPRY2 and activation of the PI3K/AKT and ERK1/2 signaling pathways. Conclusion:Mag-BMSC-Exos can further enhance wound healing than BMSC-Exos by improving angiogenesis and fibroblast function, and miR-21-5p upregulation in mag-BMSC-Exos might be the potential mechanism. This work offers an effective and promising protocol to improve wound healing in clinic.
MicroRNA-26a inhibits hyperplastic scar formation by targeting Smad2.
Qi Jun,Liu Yifei,Hu Kesu,Zhang Yi,Wu Yangyang,Zhang Xia
Experimental and therapeutic medicine
Hypertrophic scar (HS) is a fibrotic disease in which excessive extracellular matrix forms due to the response of fibroblasts to tissue damage. Novel evidence suggests that microRNAs (miRNAs or miRs) may contribute to hypertrophic scarring; however, the role of miRNAs in HS formation remains unclear. In the present study, miR-26a was significantly downregulated in HS tissues and human HS fibroblasts (hHSFs) was detected by reverse transcription-quantitative analysis. TargetScan was used to predict that mothers against decapentaplegic homolog 2 (Smad2) is a potential target gene of miR-26a and a dual-luciferase reporter assay confirmed that Smad2 was a target gene of miR-26a. The expression of Smad2 was upregulated in HS tissues and hHSFs. Cell Counting Kit-8 and flow cytometry analyses demonstrated that the overexpression of miR-26a significantly suppressed the proliferation ability of hHSFs and the apoptotic rate of hHSFs was significantly upregulated in response to miR-26a mimic transfection. Furthermore, the expression of B-cell lymphoma-2 (Bcl-2)-associated X protein was increased and Bcl-2 expression was decreased following miR-26a mimic transfection. The expression of collagens I and III was significantly inhibited following treatment with miR-26a mimics in hHSF cells. Conversely, miR-26a inhibitors served an opposing role in hHSFs. Furthermore, Smad2 overexpression enhanced the expression of collagens I and c III; however, Smad2 silencing inhibited the expression of collagens I and c III. In conclusion, the results of the present study indicate that miR-26a inhibits HS formation by modulating proliferation and apoptosis ad well as inhibiting the expression of extracellular matrix-associated proteins by targeting Smad2.
TM4SF1 involves in miR-1-3p/miR-214-5p-mediated inhibition of the migration and proliferation in keloid by regulating AKT/ERK signaling.
Xu Mingyuan,Sun Jiaqi,Yu Yijia,Pang Qianqian,Lin Xiaohu,Barakat May,Lei Rui,Xu Jinghong
AIMS:Transmembrane 4 L six family member 1 (TM4SF1) is a small plasma membrane glycoprotein that is highly expressed in cancers. However, the role of TM4SF1 that plays in keloids remains unknown. We investigated the expression, function and the microRNA (miRNA) regulatory network of TM4SF1 in keloids. MAIN METHODS:Small interfering RNAs and lentivirus were used to alter the expression of TM4SF1 in fibroblasts. Dual-luciferase reporter assays were applied to determine the miRNA targets. Immunohistochemistry, western blotting, qRT-PCR, wound healing assays, Transwell assays, cell count kit-8 assays and flow cytometry were also employed in this study. KEY FINDINGS:TM4SF1 was frequently upregulated in human keloid fibroblasts (HKFs) compared with human normal skin fibroblasts (HSFs). The downregulation of TM4SF1 significantly inhibited proliferation and migration, and induced apoptosis in HKFs. Furthermore, si-TM4SF1 inhibited the AKT/ERK signaling. Meanwhile, the upregulation of TM4SF1 promoted proliferation, migration and the activation of AKT/ERK signaling in human foreskin fibroblasts (HFF-1). Moreover, TM4SF1 can be regulated by miRNAs, which have been validated to play important roles in keloids by posttranscriptional regulation of gene expression. After screening, we found miR-1-3p and miR-214-5p targeted TM4SF1, inhibited TM4SF1 expression, cell proliferation, migration, and induced apoptosis in HKFs. And the level of miR-1-3p and miR-214-5p were found lower in HKFs than in HSFs. SIGNIFICANCE:Our study demonstrates a novel regulatory mechanism by which miR-1-3p, miR-214-5p, and TM4SF1 are involved in proliferation, cell motility, and apoptosis, suggesting that they may be potential targets in therapies for keloids.
Inhibition of miR-31a-5p decreases inflammation by down-regulating IL-25 expression in human dermal fibroblast cells (CC-2511 cells) under hyperthermic stress via Wnt/β-catenin pathway.
Jiang Lei,Xue Wenjun,Wang Yibing
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
BACKGROUND:Hypertrophic scar is a skin disorder, which occurs following damage to deep dermis by burns. MicroRNAs (miRNAs) are involved in various skin disorders, including hypertrophic scar. The present study evaluated the effects of miR-31a-5p on human dermal fibroblast cells (CC-2511 cells) under hyperthermic stress. METHODS:CC-2511 cells were incubated at high temperature conditions (41 °C-43 °C for 1-3 h). The expression levels of miR-31a-5p, interleukin (IL)-25, and the productions of other pro-inflammatory mediators in CC-2511 cells under hyperthermic stress were examined by using qRT-PCR and ELISA assay. Luciferase reporter assay was used to identify the relationship between miR-31a-5p and IL-25. Effects of heat shock protein 70 (HSP70) on miR-31a-5p and cell viability were then examined under hyperthermic stress. Furthermore, the effects of IL-25 on pro-inflammatory mediators, cell proliferation and Wnt/β-catenin pathway were finally studied. RESULTS:Hyperthermic treatment increased miR-31a-5p expression, enhanced IL-1β, IL-6, IL-8 and TNF-α productions, as well as decreased proliferation and induced apoptosis in CC-2511 cells. MiR-31a-5p suppression decreased IL-25 expression, inhibited IL-1β, IL-6, IL-8, and TNF-α secretions, and decreased proliferation in CC-2511 cells under hyperthermic stress. Additionally, miR-31a-5p expression was increased by HSP70, and HSP70 knockdown promoted cell viability in CC-2511 cells under hyperthermic stress. Further, we observed that IL-25 promoted the expression of inflammatory mediators and regulated cell survival by activating Wnt/β-catenin signal pathway in CC-2511 cells. CONCLUSIONS:These results demonstrated that miR-31a-5p inhibition could decrease inflammation and improve cell survival of CC-2511 cells under hyperthermic stress via regulation Wnt/β-catenin signal pathway.
The Differential Expression of miRNAs and a Preliminary Study on the Mechanism of miR-194-3p in Keloids.
Xu Zhishan,Guo Bingyu,Chang Peng,Hui Qiang,Li Wei,Tao Kai
BioMed research international
The aim of this study was to detect abnormally expressed microRNA (miRNA) in keloids and to study their functions. The differential expression of miRNAs in keloids and normal tissue was detected by gene microarray. MiRNA expression was verified by real-time PCR. A luciferase reporter gene assay, western blot, and real-time PCR were used to detect the effect of miR-194-3p on RUNX2. An MTT assay and a transwell assay were used to detect the effect of miR-194-3p in both primary cultured fibroblasts and HKF cells. Related proteins were analysed by western blot and real-time PCR. The expression of miR-194-3p was lower in keloids, and MiR-194-3p was shown to target RUNX2 directly. MiR-194-3p inhibited the proliferation and migration of fibroblasts through the inhibition of CDK4 and MMP2. MiR-194-3p and RUNX2 may become new targets for the prevention and treatment of keloids.
Human Novel MicroRNA Seq-915_x4024 in Keratinocytes Contributes to Skin Regeneration by Suppressing Scar Formation.
Zhao Feng,Lang Hongxin,Wang Zhe,Zhang Tao,Zhang Dianbao,Wang Rui,Lin Xuewen,Liu Xiaoyu,Shi Ping,Pang Xining
Molecular therapy. Nucleic acids
Early in gestation, wounds in fetal skin heal by regeneration, in which microRNAs play key roles. Seq-915_x4024 is a novel microRNA candidate confirmed by deep sequencing and mirTools 2.0. It is highly expressed in fetal keratinocytes during early gestation. Using an in vitro wound-healing assay, Transwell cell migration assay, and MTS proliferation assay, we demonstrated that keratinocytes overexpressing seq-915_x4024 exhibited higher proliferative activity and the ability to promote fibroblast migration and fibroblast proliferation. These characteristics of keratinocytes are the same biological behaviors as those of fetal keratinocytes, which contribute to skin regeneration. In addition, seq-915_x4024 suppressed the expression of the pro-inflammatory markers TNF-α, IL-6, and IL-8 and the pro-inflammatory chemokines CXCL1 and CXCL5. We also demonstrated that seq-915_x4024 regulates TGF-β isoforms and the extracellular matrix. Moreover, using an in vivo wound-healing model, we demonstrated that overexpression of seq-915_x4024 in keratinocytes suppresses inflammatory cell infiltration and scar formation. Using bioinformatics analyses, luciferase reporter assays, and western blotting, we further demonstrated that Sar1A, Smad2, TNF-α, and IL-8 are direct targets of seq-915_x4024. Furthermore, the expression of phosphorylated Smad2 and Smad3 was reduced by seq-915_x4024. Seq-915_x4024 could be used as an anti-fibrotic factor for the treatment of wound healing.
MicroRNA profiling in denatured dermis of deep burn patients.
Liang Pengfei,Lv Chunliu,Jiang Bimei,Long Xiao,Zhang Pihong,Zhang Minghua,Xie Tinghong,Huang Xiaoyuan
Burns : journal of the International Society for Burn Injuries
BACKGROUND:Denatured dermis is a part of the dermis in deep burn wound and has the ability to restore normal morphology and function. Skin grafting with the preservation of denatured dermis is a new kind of surgical procedure and has reported satisfactory clinical effects, such as lessened scar contracture and a better restoration of the appearance and function. However, the underlying mechanism of the recovery of denatured dermal function remains unclear. MicroRNAs (miRNAs) are a new class of regulatory noncoding single-stranded RNAs, which play a key role in normal development and physiology, as well as in disease development. This study analysed the profile of miRNAs in denatured dermis from patients and further investigated the possible roles of miRNAs played in the functional recovery of denatured dermis by prediction of the potential target genes of differentially expressed miRNAs. METHODS:The denatured dermis and paired normal skin were collected and analysed by miRNA array. The miRNA profiling results were validated by real-time reverse transcriptase polymerase chain reaction (RT-PCR), and bioinformatics' analysis was employed to further predict the miRNA targets. RESULTS:A total of 66 miRNAs were differentially expressed in denatured dermis compared with those in normal skin, among which 34 were down-regulated while 32 are up-regulated. The most significantly up-regulated miRNA was miR-663, and the most significantly down-regulated one was miR-203. Differentially expressed miRNAs were predicted to be related with several signalling pathways in wound healing. CONCLUSION:The differential miRNA expression identified in this study supplies experimental basis for further understanding the mechanisms of functional recovery of the denatured dermis.
Exosomes from human umbilical cord blood accelerate cutaneous wound healing through miR-21-3p-mediated promotion of angiogenesis and fibroblast function.
Hu Yin,Rao Shan-Shan,Wang Zhen-Xing,Cao Jia,Tan Yi-Juan,Luo Juan,Li Hong-Ming,Zhang Wei-She,Chen Chun-Yuan,Xie Hui
The application of blood plasma for soft tissue wound healing is receiving much more attention recently. Exosomes are critical paracrine mediators that can be obtained from biological fluids including plasma and be able to induce regenerative effects by transferring bioactive molecules such as microRNAs (miRNAs). This study aimed to investigate the effects of exosomes from human umbilical cord blood plasma (UCB-Exos) on wound healing and to elucidate the underlying mechanism. UCB-Exos were isolated by ultracentrifugation and subcutaneously injected into full-thickness skin wounds in mice. The efficacy of UCB-Exos on wound healing was evaluated by measuring wound closure rates, histological analysis and immunofluorescence examinations. , quantitative real-time PCR (qRT-PCR) analysis was performed to detect the expression levels of a class of miRNAs that have positive roles in regulating wound healing. The scratch wound assay, transwell assay and cell counting kit-8 analysis were conducted to assess the effects of UCB-Exos on migration and proliferation of human skin fibroblasts and endothelial cells. Tube formation assay was carried out to test the impact of UCB-Exos on angiogenic tube formation ability of endothelial cells. Meanwhile, by using specific RNA inhibitors or siRNAs, the roles of the candidate miRNA and its target genes in UCB-Exos-induced regulation of function of fibroblasts and endothelial cells were assessed. The local transplantation of UCB-Exos into mouse skin wounds resulted in accelerated re-epithelialization, reduced scar widths, and enhanced angiogenesis. , UCB-Exos could promote the proliferation and migration of fibroblasts, and enhance the angiogenic activities of endothelial cells. Notably, miR-21-3p was found to be highly enriched in UCB-Exos and served as a critical mediator in UCB-Exos -induced regulatory effects through inhibition of phosphatase and tensin homolog (PTEN) and sprouty homolog 1 (SPRY1). Our results suggest that UCB-Exos are important effectors of plasma activity and can be used as a novel promising strategy for soft tissue wound healing.
Circulating Hypoxia Responsive microRNAs (HRMs) and Wound Healing Potentials of Green Tea in Diabetic and Nondiabetic Rat Models.
Al-Rawaf Hadeel A,Gabr Sami A,Alghadir Ahmad H
Evidence-based complementary and alternative medicine : eCAM
Green tea () has many biological activities and may promote diabetic wound healing by regulation of circulating hypoxia responsive microRNAs (HRMs) which triggers the wound repairing process in diabetic and nondiabetic wounds. Thus, in this study, the potential effects of green tea extract (GTE) on the expression of miRNAs; miR-424, miR-199a, miR-210, miR-21, and fibrogenitic markers; hydroxyproline (HPX), fibronectin (FN), and nitric oxide (NO) were evaluated in wounds of diabetic and nondiabetic rats. The animals were topically treated with vaseline, 0.6% GTE, and 5%w/w povidone iodine (standard control). HPX, FN, and NO levels and microRNAs, miR-424, miR-210, miR-199a, and miR-21, were estimated in wound tissues using colorimetric, immunoassay, and molecular PCR analysis. In vitro analysis was performed to estimate active constituents and their antioxidant activities in methanolic green teat extract (GTE). Wounds treated with green tea, a dose of 0.6, healed significantly earlier than those treated with standard vehicle and vaseline treated diabetic wounds. Higher expressions of HRMs, miR-199a, and miR-21, and lower expression of HRMs, miR-424 and miR-210, were significantly reported in tissues following treatment with green tea extract compared to standard control vehicle. The tissues also contained more collagen expressed as measures of HPX, FN, and NO and more angiogenesis, compared to wounds treated with standard control vehicle. Diabetic and nondiabetic wounds treated with green tea (0.6%) for three weeks had lesser scar width and greater re-epithelialization in shorter periods when compared to standard control vehicle. Expression of HRMs, miR-199a, miR-21, and HRMs and miR-424 and miR-210 correlated positively with HPX, fibronectin, NO, better scar formation, and tensile strength and negatively with diabetes. In addition to antidiabetic and antioxidant activities of green tea components, GTE showed angiogenesis promoting activity in diabetic wound healing. In conclusion, Camellia sinensis extracts in a dose of 0.6% significantly promote more collagen and fibronectin deposition with higher expression of NO, promoting angiogenesis process via molecular controlling of circulating hypoxia responsive microRNAs: miR-424, miR-210, miR-199a, and miR-21 in diabetic and nondiabetic wounds. Our results support a functional role of circulating hypoxia responsive microRNAs: miR-424, miR-210, miR-199a, and miR-21 as potential therapeutic targets in angiogenesis and vascular remodeling in diabetic wound healing.
Overexpression of miR-29b reduces collagen biosynthesis by inhibiting heat shock protein 47 during skin wound healing.
Zhu Yumin,Li Zhongwu,Wang Yanling,Li Lin,Wang Dongmiao,Zhang Wei,Liu Laikui,Jiang Hongbing,Yang Jianrong,Cheng Jie
Translational research : the journal of laboratory and clinical medicine
Skin scar formation is characterized by excessive synthesis and aberrant deposition of collagens during wound healing. MicroRNAs are endogenous gene regulators critically involved in diverse biological events including skin scar formation and hold considerable promise as therapeutic targets. However, the detailed molecular mechanisms responsible for collagen production during skin wound repair and scar formation remain incompletely known. Here our data revealed that significant downregulation of miR-29b and upregulation of heat shock protein 47 (HSP47) were observed during wound healing in both excisional and burn wound models and also detected in facial skin scar as compared to adjacent healthy skin. HSP47, a specific chaperon for collagen production and secretion, was identified as a novel and direct post-transcriptional target of miR-29b in skin fibroblasts via bioinformatics prediction and experimental validation. Moreover, the regulatory functions of miR-29b in collagen biosynthesis are partially achieved through modulating HSP47 expression in skin fibroblasts. Furthermore, the profibrotic growth factor TGF-β1 inhibited miR-29b transcription by activating TGF-β/Smads signaling and in turn depressed HSP47 and enhanced collagen 1 production. In contrast, the proinflammatory cytokines IL-1β and TNF-α significantly induced miR-29b transcription via activating NF-κB signaling but had no significant effect on HSP47 and collagen production in skin fibroblasts. Importantly, local delivery of miR-29b lentiviral particles inhibited HSP47 expression and collagen biosynthesis as well as suppressed angiogenesis, thus reducing scar formation in an excisional wound splinting model. Collectively, our data reveal that miR-29b can reduce collagen biosynthesis during skin wound healing likely via post-transcriptional inhibition of HSP47 expression. These findings also suggest that therapeutic targeting of miR-29b/HSP47 might be a viable alternative strategy to prevent or reduce scar formation.
MiR-10a and miR-181c regulate collagen type I generation in hypertrophic scars by targeting PAI-1 and uPA.
Li Chao,Zhu Hua-Yu,Bai Wen-Dong,Su Lin-Lin,Liu Jia-Qi,Cai Wei-Xia,Zhao Bin,Gao Jian-Xin,Han Shi-Chao,Li Jun,Hu Da-Hai
Urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) have been proposed to play key roles in extracellular matrix (ECM) deposition in hypertrophic scars (HS). Here, we found that in HS fibroblasts (HFs) miR-181c and miR-10a were differentially-expressed and targeted uPA and PAI-1, respectively. The production of Type 1 collagen (Col1) was inhibited by miR-181c knockdown or miR-10a overexpression in HFs, and this resulted in increased levels of metalloproteinase 1 (MMP1). These results suggest that the miR-181c-uPA and miR-10a-PAI-1 regulatory pathways have an integral role in HS pathogenesis.
MicroRNAs as diagnostic and prognostic biomarkers of age-related macular degeneration: advances and limitations.
Martinez Bridget,Peplow Philip V
Neural regeneration research
A main cause of vision loss in the elderly is age-related macular degeneration (AMD). Among the cellular, biochemical, and molecular changes linked to this disease, inflammation and angiogenesis appear as being crucial in AMD pathogenesis and progression. There are two forms of the disease: dry AMD, accounting for 80-90% of cases, and wet AMD. The disease usually begins as dry AMD associated with retinal pigment epithelium and photoreceptor degeneration, whereas wet AMD is associated with choroidal neovascularization resulting in severe vision impairment. The new vessels are largely malformed, leading to blood and fluid leakage within the disrupted tissue, which provokes inflammation and scar formation and results in retinal damage and detachment. MicroRNAs are dysregulated in AMD and may facilitate the early detection of the disease and monitoring disease progression. Two recent reviews of microRNAs in AMD had indicated weaknesses or limitations in four earlier investigations. Studies in the last three years have shown considerable progress in overcoming some of these concerns and identifying specific microRNAs as biomarkers for AMD. Further large-scale studies are warranted using appropriate statistical methods to take into account gender and age disparity in the study populations and confounding factors such as smoking status.
Clinical and molecular effects on mature burn scars after treatment with a fractional CO(2) laser.
Qu Le,Liu Austin,Zhou Li,He Chundi,Grossman Peter H,Moy Ronald L,Mi Qing-Sheng,Ozog David
Lasers in surgery and medicine
BACKGROUND AND OBJECTIVE:There have been several case reports of improvement in the appearance of mature burn scars following treatment with fractional CO(2) lasers. However, the biochemical mechanisms responsible for these improvements have not been elucidated. MATERIALS AND METHODS:Ten patients with mature, full-thickness, hypertrophic burn scars received initial treatment with a fractional CO(2) laser. Clinical improvement was measured with Vancouver Scar Scale as well as Patient and Observer Scar Assessment Scale. Fresh tissue samples were obtained before the initial treatment and 48 hours after the first treatment for TaqMan Real-time RT-PCR analyses. Expressions of several scar-related biological markers, including types I and III procollagen, matrix metalloproteinase (MMP)-1, -13, transforming growth factor (TGF)-β1, β2, β3, and basic fibroblast growth factor (bFGF), as well as microRNA miR-17-92 cluster, were investigated. RESULTS:There were significant improvements in both observer and subject ratings in all scales. Both types I and III procollagen mRNA levels were dramatically down-regulated after treatment, but the ratio of types I/III procollagen mRNA was not different. The expression of MMP-1 was significantly up-regulated after treatment, while TGF-β2, -β3, and bFGF levels were significantly down-regulated. Expression of miR-18a and miR-19a were dramatically up-regulated (P < 0.05) after treatment. CONCLUSIONS:Our study indicated that fractional CO(2) resulted in clinical improvement of mature burn scar. Alteration of types I and III procollagen, MMP-1, TGF-β2, -β3, bFGF, as well as miRNAs miR-18a and miR-19a expression may be responsible for the clinical improvement after treatment. Our finding may have implications for novel treatments and further our understanding of fractional CO(2) laser treatment.
miR-145 Contributes to Hypertrophic Scarring of the Skin by Inducing Myofibroblast Activity.
Gras Christiane,Ratuszny Dominica,Hadamitzky Catarina,Zhang Haijiao,Blasczyk Rainer,Figueiredo Constança
Molecular medicine (Cambridge, Mass.)
Hyperthrophic scarring of the skin is caused by excessive activity of skin myofibroblasts after wound healing and often leads to functional and/or aesthetic disturbance with significant impairment of patient quality of life. MicroRNA (miRNA) gene therapies have recently been proposed for complex processes such as fibrosis and scarring. In this study, we focused on the role of miR-145 in skin scarring and its influence in myofibroblast function. Our data showed not only a threefold increase of miR-145 levels in skin hypertrophic scar tissue but also in transforming growth factor β1 (TGF-β1)-induced skin myofibroblasts compared with healthy skin or nontreated fibroblasts (p < 0.001). Consistent with the upregulation of miR-145 induced by TGF-β1 stimulation of fibroblasts, the expression of Kruppel-like factor 4 (KLF4) was decreased by 50% and α-smooth muscle actin (α-SMA) protein expression showed a threefold increase. Both could be reversed by miR-145 inhibition (p < 0.05). Restoration of KLF4 levels equally abrogated TGF-β1-induced α-SMA expression. These data demonstrate that TGF-β1 induces miR-145 expression in fibroblasts, which in turn inhibits KLF4, a known inhibitor of α-SMA, hence upregulating α-SMA expression. Furthermore, treatment of myofibroblasts with a miR-145 inhibitor strongly decreased their α-1 type I collagen expression, TGF-β1 secretion, contractile force generation and migration. These data demonstrate that upregulation of miR-145 plays an important role in the differentiation and function of skin myofibroblasts. Additionally, inhibition of miR-145 significantly reduces skin myofibroblast activity. Taken together, these results suggest that miR-145 is a promising therapeutic target to prevent or reduce hypertrophic scarring of the skin.
10‑Hydroxycamptothecin induces apoptosis in human fibroblasts by regulating miRNA‑23b‑3p expression.
Zeng Lingrong,Sun Yu,Li Xiaolei,Wang Jingcheng,Yan Lianqi
Molecular medicine reports
10‑Hydroxycamptothecin (HCPT) effectively controls epidural fibrosis, but the exact underlying mechanisms remain ambiguous. Abnormal microRNA (miR)‑23b‑3p expression has been detected in various types of fibrotic tissues that are present in different diseases. The aim of the present study was to elucidate the mechanisms through which HCPT induces fibroblast apoptosis. Reverse transcription‑quantitative polymerase chain reactions were performed on six traumatic scar samples and matched normal skin samples; traumatic scar formation was revealed to be significantly inversely associated with miR‑23b‑3p expression. In addition, the miR‑23b‑3p expression level in human fibroblasts was examined following HCPT treatment. The effects of HCPT and miR‑23b‑3p on fibroblast apoptosis were assessed using terminal deoxynucleotidyl‑transferase‑mediated dUTP nick‑end labeling, flow cytometry and western blot analysis. The results demonstrated that HCPT treatment notably increased miR‑23b‑3p expression levels and accelerated fibroblast apoptosis. Therefore, upregulation of miR‑23b‑3p expression was demonstrated to promote fibroblast apoptosis, consistently with the effects of HCPT. The results of the present study indicated that HCPT may induce fibroblast apoptosis by regulating miR‑23b‑3p expression.
MicroRNA-21 in Skin Fibrosis: Potential for Diagnosis and Treatment.
Li Yan,Zhang Juan,Lei Yuying,Lyu Lechun,Zuo Ruiling,Chen Ting
Molecular diagnosis & therapy
Skin fibrosis is a common pathological process characterized by fibroblast proliferation and excessive deposition of extracellular matrix. However, the pathogenesis of the disease is still not clear. Previous studies have shown that microRNA-21 may play pivotal roles in the regulation of a variety of skin fibrosis, including keloid, scleroderma, and hypertrophic scar. In this review, we outline the structure, expression, and regulation of microRNA-21 and its role in fibrotic skin diseases. In future, it may be useful as a prognostic or diagnostic marker. However, there is a significant amount of work required to increase our current understanding of the role of microRNA-21 in skin fibrosis.
Decreased expression of microRNA-145 promotes the biological functions of fibroblasts in hypertrophic scar tissues by upregulating the expression of transcription factor SOX-9.
Wang Shoujie,Li Caiyun,Yu Yijia,Qiao Jianjun
Experimental and therapeutic medicine
The present study aimed to determine the expression of microRNA (miRNA or miR)-145 in hypertrophic scars at the tissue and cellular levels, and to investigate its biological functions and mechanism of action. A total of 36 patients who were diagnosed with hypertrophic scar were included in the present study. Reverse transcription-quantitative polymerase chain reaction was used to determine the expression of miR-145 in tissues and fibroblasts. Primary fibroblasts were transfected with negative control miRNA, miR-145 mimics or inhibitor. A Cell Counting Kit-8 assay was performed to determine the level of proliferation of fibroblasts. Flow cytometry was employed for cell cycles determination and apoptosis in fibroblasts. A Matrigel assay was used to evaluate the invasion ability of fibroblasts. Western blotting was used to determine the expression of the transcription factor SOX-9 (SOX-9) protein in fibroblasts. Rescue experiments were performed to examine the effect of SOX-9 on the regulation of fibroblasts by miR-145. The dual luciferase reporter assay was performed to identify the direct interaction between SOX-9 and miR-145. The expression of miR-145 was reduced in hypertrophic tissues and fibroblasts. Overexpression of miR-145 inhibited the proliferation, G1/S phase transition and invasion of fibroblasts, and promoted the apoptosis of fibroblasts. In addition, overexpression of miR-145 inhibited SOX-9 protein expression. By contrast, the expression of SOX-9 reversed the effects of miR-145 on the proliferation, cell cycle, apoptosis and invasion of fibroblasts. The miR-145 seed region was able to bind with the 3'-untranslated region of the SOX-9 mRNA to regulate its expression. The present study demonstrated that miR-145 expression is reduced in hypertrophic scar tissues and negatively associated with SOX-9 expression. In addition, miR-145 inhibits the proliferation, cell cycle and invasion, and promotes the apoptosis of fibroblasts by down-regulating the expression of SOX-9. The current study provides a potential target for the clinical diagnosis and treatment of hypertrophic scars.
Trichostatin A‑induced miR‑30a‑5p regulates apoptosis and proliferation of keloid fibroblasts via targeting BCL2.
Jian Xiaoqing,Qu Le,Wang Yunlin,Zou Qianlei,Zhao Qing,Chen Shuang,Gao Xinghua,Chen Hongduo,He Chundi
Molecular medicine reports
Keloids are benign fibrous overgrowths that occur as a result of abnormal wound healing following cutaneous injury. MicroRNAs (miRNAs/miRs) are short non‑coding RNAs that serve critical roles in numerous important biological processes, such as cell proliferation, differentiation and apoptosis. However, their role in keloid development remains largely unknown. In the present study, the role of miR‑30a‑5p, a miRNA regulated by Trichostatin A (TSA), in apoptosis within cultured keloid fibroblasts was investigated. An MTT assay was used to detect the proliferation of cultured keloid fibroblasts treated with TSA. Cell apoptosis and cell cycle phases were analyzed using flow cytometry. In addition, an miRNA microarray was performed to compare expression profiles between cultured keloid fibroblasts treated with or without 1,000 nM TSA. Reverse transcription‑quantitative polymerase chain reaction analysis was conducted to estimate miRNA expression levels. The direct target of miR‑30a‑5p was identified using a dual‑luciferase reporter assay. Western blotting was employed to assess protein expression levels in keloid fibroblasts. The results demonstrated that TSA inhibited the proliferation of keloid fibroblasts in a time‑ and dose‑dependent manner. The miRNA microarray revealed alterations in the expression of numerous miRNA sequences in response to TSA when compared with controls. Notably, the expression of miR‑30a‑5p was downregulated in keloid tissues. In addition, overexpression of miR‑30a‑5p induced apoptosis by targeting B‑cell lymphoma 2, which was similar to that observed in response to TSA. These results provide important information regarding a novel miR‑30a‑5p‑mediated signaling pathway induced by TSA treatment, and suggest a potential use for TSA and miR‑30a‑5p as effective therapeutic strategies for keloids.
An updated review of mechanotransduction in skin disorders: transcriptional regulators, ion channels, and microRNAs.
Wang Jing,Zhang Yifan,Zhang Ning,Wang Chuandong,Herrler Tanja,Li Qingfeng
Cellular and molecular life sciences : CMLS
INTRODUCTION:The skin is constantly exposed and responds to a wide range of biomechanical cues. The mechanobiology of skin has already been known and applied by clinicians long before the fundamental molecular mechanisms of mechanotransduction are elucidated. MATERIALS AND METHODS:Despite increasing knowledge on the mediators of biomechanical signaling such as mitogen-associated protein kinases, Rho GTPases or FAK-ERK pathways, the key elements of mechano-responses transcription factors, and mechano-sensors remain unclear. Recently, canonical biochemical components of Hippo and Wnt signaling pathway YAP and β-catenin were found to exhibit undefined mechanical sensitivity. Mechanical forces were identified to be the dominant regulators of YAP/TAZ activity in a multicellular context. Furthermore, different voltage or ligand sensitive ion channels in the cell membrane exhibited their mechanical sensitivity as mechano-sensors. Additionally, a large number of microRNAs have been confirmed to regulate cellular behavior and contribute to various skin disorders under mechanical stimuli. Mechanosensitive (MS) microRNAs could not only be activated by distinct mechanical force pattern, but also responsively target MS sensors such as e-cadherin and cytoskeleton constituent RhoA. CONCLUSION:Thus, a comprehensive understanding of this regulatory network of cutaneous mechanotransduction will facilitate the development of novel approaches to wound healing, hypertrophic scar formation, skin regeneration, and the progression or initiation of skin diseases.
Serum miRNAs Signature Plays an Important Role in Keloid Disease.
Luan Y,Liu Y,Liu C,Lin Q,He F,Dong X,Xiao Z
Current molecular medicine
The molecular mechanism underlying the pathogenesis of keloid is largely unknown. MicroRNA (miRNA) is a class of small regulatory RNA that has emerged as a group of posttranscriptional gene repressors, participating in diverse pathophysiological processes of skin diseases. We investigated the expression profiles of miRNAs in the sera of patients to decipher the complicated factors involved in the development of keloid disease. MiRNA expression profiling in the sera from 9 keloid patients and 7 normal controls were characterized using a miRNA microarray containing established human mature and precursor miRNA sequences. Quantitative real-time PCR was performed to confirm the expression of miRNAs. The putative targets of differentially expressed miRNAs were functionally annotated by bioinformatics. MiRNA microarray analysis identified 37 differentially expressed miRNAs (17 upregulated and 20 downregulated) in keloid patients, compared to the healthy controls. Functional annotations revealed that the targets of those differentially expressed miRNAs were enriched in signaling pathways essential for scar formation and wound healing. The expression profiling of miRNAs is altered in the keloid, providing a clue for the molecular mechanisms underlying its initiation and progression. MiRNAs may partly contribute to the etiology of keloids by affecting the critical signaling pathways relevant to keloid pathogenesis.
The role of microRNAs in skin fibrosis.
Babalola Olubukola,Mamalis Andrew,Lev-Tov Hadar,Jagdeo Jared
Archives of dermatological research
Fibrotic skin disorders may be debilitating and impair quality of life. There are few effective treatment options for cutaneous fibrotic diseases. In this review, we discuss our current understanding of the role of microRNAs (miRNAs) in skin fibrosis. miRNAs are a class of small, non-coding RNAs involved in skin fibrosis. These small RNAs range from 18 to 25 nucleotides in length and modify gene expression by binding to target messenger RNA (mRNA), causing degradation of the target mRNA or inhibiting the translation into proteins. We present an overview of the biogenesis, maturation and function of miRNAs. We highlight miRNA’s role in key skin fibrotic processes including: transforming growth factor-beta signaling, extracellular matrix deposition, and fibroblast proliferation and differentiation. Some miRNAs are profibrotic and their upregulation favors these processes contributing to fibrosis, while anti-fibrotic miRNAs inhibit these processes and may be reduced in fibrosis. Finally, we describe the diagnostic and therapeutic significance of miRNAs in the management of skin fibrosis. The discovery that miRNAs are detectable in serum, plasma, and other bodily fluids, and are relatively stable, suggests that miRNAs may serve as valuable biomarkers to monitor disease progression and response to treatment. In the treatment of skin fibrosis, antifibrotic miRNAs may be upregulated using mimics and viral vectors. Conversely, profibrotic miRNAs may be downregulated by employing anti-miRNAs, sponges, erasers and masks. We believe that miRNA-based therapies hold promise as important treatments and may transform the management of fibrotic skin diseases by physicians.
Downregulation of miR‑637 promotes proliferation and metastasis by targeting Smad3 in keloids.
Zhang Ye,Guo Bingyu,Hui Qiang,Li Wei,Chang Peng,Tao Kai
Molecular medicine reports
Keloids are a type of abnormal scar tissue. MicroRNAs (miRNAs) exhibit a pivotal role in the regulation of cell proliferation and metastasis of keloids. miRNA microarray revealed that miR‑637 was one of the most frequently altered miRNAs in keloids. Furthermore, up-regulation of miR‑637 inhibited cell proliferation and metastasis by targeting mothers against decapentaplegic homolog (Smad)3, one of the important proteins that affects the formation of keloids. Further studies demonstrated that miR‑637 regulated the proliferation and metastasis of human keloid fibroblast (HKF) cells by mediating the Smad3 signaling pathway. Overall, the present findings suggest that miR‑637 may be a promising therapeutic target in keloids.
Identification and integrated analysis of microRNA expression profiles in keloid.
Zhong Lianmei,Bian Ligong,Lyu Jing,Jin Huiyan,Liu Zijie,Lyu Lechun,Lu Di
Journal of cosmetic dermatology
BACKGROUND:Keloid is a common abnormal cutaneous fibroproliferative disorder. However, the process underlying keloid pathogenesis still remains to be unclear. OBJECTIVE:To integrated analyse the miRNA expression profiles of keloid. METHODS:We performed miRNA expression profiles analysis of 3 paired keloid and normal tissue samples by miRNA microarray. Differentially expressed miRNAs were further performed integrative analysed and validated using quantitative reverse transcription polymerase chain reaction (qRT-PCR). After predicting target genes, we constructed the miRNA-target genes interaction network and carried out bioinformatics analysis. RESULTS:The results revealed that 264 miRNAs were significantly altered. The expression of high frequency miRNAs which included miRNA-199a-5p, miRNA-21-5p, miRNA-214-5p, miRNA-424-5p, and miRNA-205-5p was further evaluated. The gene ontology (GO) analyses and the top enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed these target genes were associated with MAPK signaling pathway and HIF-1 signaling pathway. CONCLUSION:This study revealed the profiling of miRNAs in keloid that are potentially implicated in the development of this disease and could serve as novel diagnostic and therapeutic target of keloid.
Baicalein retards proliferation and collagen deposition by activating p38MAPK-JNK via microRNA-29.
Yang Xiaoliang,Zhang Chunyan,Jiang Jinjie,Li Yinghao
Journal of cellular biochemistry
Immoderate proliferation and deposition of collagen generally result in hypertrophic scars and even keloids. microRNA-29 (miR-29) has been proved as a crucial regulator in these pathological processes. Although mounting evidence have proved baicalein (BAI) impairs scar formation, it is still incompletely understood whether miR-29 participated in the underlying mechanism. In the present study, NIH-3T3 cells were stimulated with BAI, and then cell viability was analyzed by cell counting kit-8 (CCK-8) and Western blot. We further analyzed total soluble collagen, collagen 1, and alpha-smooth muscle actin (α-SMA) in NIH-3T3 cells, which were exposed to transforming growth factor beta 1 (TGF-β1)/BAI, using a Sircol assay kit, quantitative reverse transcription-PCR (qRT-PCR) and Western blot, respectively. Besides, the miR-29 inhibitor was transduced and its transfection efficiency was verified by qRT-PCR. Finally, the phosphorylated p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) were examined by Western blot. BAI effectively retarded NIH-3T3 proliferation in a dose-dependent manner. Besides, TGF-β1-induced deposition of total soluble collagen and synthesis of collagen 1 and α-SMA were repressed by BAI at mRNA and protein levels. However, miR-29 inhibitor reversed the effects of BAI. Remarkably, BAI promoted phosphorylated expression of p38MAPK and JNK while miR-29 inhibitor reversed its effects on the phosphorylated expression of p38MAPK and JNK. BAI effectively weakened the cell viability and repressed TGF-β1-induced total soluble collagen as well as collagen 1 and α-SMA by upregulating miR-29. Mechanically, BAI activates the p38MAPK/JNK pathway by promoting miR-29.
MiR-3613-3p inhibits hypertrophic scar formation by down-regulating arginine and glutamate-rich 1.
Li Lisha,Han Weiqiang,Chen Yun,Chen Yuhua
Molecular and cellular biochemistry
Hypertrophic scar (HS) is a severe skin disorder characterized by excessive extracellular matrix production and abnormal function of fibroblasts. Recent studies have demonstrated that microRNAs (miRNAs) play critical roles in HS formation. This study aims to investigate the role of miR-3613-3p in the formation of HS. The mRNA and miRNA levels were measured by quantitative RT-PCR analysis. The protein levels were examined by Western blot assay. Cell proliferation was determined by Cell Counting Kit-8 assay. The Caspase-3 and Caspase-9 activities were measured using flow cytometry assay. Dual-luciferase activity reporter assay and mRNA-miRNA pulldown assay were conducted to validate the target of miR-3613-3p. miR-3613-3p was downregulated, while arginine and glutamate-rich 1 (ARGLU1) was upregulated in HS fibroblasts (HSFs) and tissues. Overexpression of miR-3613-3p or knockdown of ARGLU1 markedly inhibited the expression of extracellular matrix (ECM) production-associated proteins and promoted Caspase-3 and Caspase-9 activations in HSFs. ARGLU1 was further identified as a direct target of miR-3613-3p. Restoration of ARGLU1 abrogated the suppressive effect of miR-3613-3p on cell proliferation and ECM protein expression of HSFs. Our results demonstrated that miR-3613-3p inhibited HS formation via targeting ARGLU1, which may provide potential therapeutic targets for the management of HS.
MicroRNA profiling in mid- and late-gestational fetal skin: implication for scarless wound healing.
Cheng Jie,Yu Hongbo,Deng Simin,Shen Guofang
The Tohoku journal of experimental medicine
Mid-gestational mammalian skin has unique capacity to heal without scar. Fetal skin undergoes phenotypic transition from scarless healing to scar repairing during embryonic development. However, the molecular mechanisms underlying the scarless phenotype and phenotypic transition remain largely unknown. MicroRNAs (miRNAs) are a novel class of small regulatory RNAs emerged as post-transcriptional gene repressors and play essential roles in diverse pathophysiological processes including skin morphogenesis and pathogenesis. Here, we performed a genome-wide miRNA profiling to identify the differentially expressed miRNAs between mid-gestational (E16 day) and late-gestational (E19 day) mouse skin, corresponding to scarless and scarring phenotype, respectively. Two miRNAs (miR-29b and miR-29c) with highest fold changes were further validated independently by real-time RT-PCR. Functional annotations of putative targets of differentially expressed miRNAs via bioinformatics approaches revealed that these predicted targets, including Smads, beta-catenin and Ras, were significantly enriched and involved in several signaling pathways important for scarless wound healing. In addition, Dicer, one of the key RNase III responsible for miRNA biogenesis and functions, was found to be up-regulated in the E19 fetal skin as compared with the E16 counterpart. Taken together, our results identified differentially expressed miRNAs between mid-and late-gestational fetal skin that correlated with phenotypic transition from scarless to scarring repair during skin development. Our bioinformatics' analysis suggests that miRNAs might contribute to this phenotypic transition probably by affecting multiple target genes and signaling pathways.
MicroRNA-149 contributes to scarless wound healing by attenuating inflammatory response.
Lang Hongxin,Zhao Feng,Zhang Tao,Liu Xiaoyu,Wang Zhe,Wang Rui,Shi Ping,Pang Xining
Molecular medicine reports
A fibrotic or pathological scar is an undesired consequence of skin wound healing and may trigger a series of problems. An attenuated inflammatory response is a significant characteristic of fetal skin wound healing, which can contribute to the scarless healing of fetal skin. According to deep sequencing data, microRNA‑149 (miR‑149) expression was increased in mid-gestational compared with that in late‑gestational fetal skin keratinocytes. It was demonstrated that overexpression of miR‑149 in HaCaT cells can downregulate the expression of pro‑inflammatory cytokines interleukin (IL)‑1α, IL‑1β, and IL‑6 at basal levels and in inflammatory conditions. Furthermore, miR‑149 was revealed to indirectly accelerate transforming growth factor‑β3 and collagen type III expression in fibroblasts, which are essential cells in extracellular matrix remodeling. In a rat skin wound model, miR‑149 improved the quality of the arrangement of collagen bundles and reduced inflammatory cell infiltration during skin wound healing. These results indicate that miR‑149 may be a potential regulator in improving the quality of skin wound healing.
Differentially expressed miRNAs in acute wound healing of the skin: a pilot study.
Li Ping,He Quanyong,Luo Chengqun,Qian Liyuan
The aim of the present study was to compare expression of microRNAs (miRNAs) from scar and normal skin areas in patients who suffered acute injuries in the skin. A total of 9 patients with acute injuries in the skin who received surgical treatment from December 2012 to March 2013 were included in this pilot study. Specimens from the hypertrophic scar and normal skin areas were obtained from the same patient during surgery. To screen for differentially expressed miRNAs, we applied 3 statistical methods, namely the traditional t test, the false discovery rate (FDR), and a novel sure independence screening procedure based on the distance correlation (DC-SIS). We examined the functional trends and metabolic and regulatory pathways for the target genes of the identified miRNAs, and explored interaction of these miRNAs in the implication of scar healing using Ingenuity Pathway Analysis. DC-SIS identified 18 differentially expressed miRNAs, 4 of which (miR-149, miR-203a, miR-222, miR-122) were also identified by FDR. The target genes of the 4 miRNAs exhibit a variety of biological functions, and are involved in various pathways such as mitogen-activated protein kinase, Wnt signaling, and focal adhesion. We identified 1 network in which 14 out of the 18 differentially expressed miRNAs were involved. Many of the miRNAs in the network target genes were involved in cell proliferation and apoptosis.In this pilot study, we identified several miRNAs exhibiting differential expression in patients who suffered acute injuries in the skin. Further studies on these miRNAs are needed to validate our findings and explore their roles in the wound healing process of the skin.
Aberrant miR-21 and miR-200b expression and its pro-fibrotic potential in hypertrophic scars.
Zhou Renpeng,Zhang Qi,Zhang Yun,Fu Shibo,Wang Chen
Experimental cell research
The post-traumatic hypertrophic scar (HS) is a fibrotic disease with excessive extracellular matrix (ECM) production by fibroblasts in response to tissue injury. Although dysregulation of miRNAs is known to be involved in a variety of pathophysiologic processes, the role of miRNA in hypertrophic scar formation is unclear. Abnormal expression of miRNA in fibrosis has been investigated in several studies. The transforming growth factor β1 (TGF-β1) promotes fibroblasts proliferation, the synthesis of collagen and other extracellular matrix, and ultimately leads to the formation of the HS by inducing excessive deposition of ECM. We identified two miRNAs whose expression was correlated with fibrotic diseases: miR-21 and miR-200b. This study further confirmed that after stimulation with TGF-β1, the expression of miR-21 was increased, whereas the mRNA level of SMAD7 was decreased in fibroblasts. TGF-β1 reduced the expression of miR-200b, while it augmented that of Zinc finger E-box-binding homeobox 1(Zeb1). Our experiments demonstrated that the expression of miR-21 and miR-200b are related to a disorder, and the TGF-β/miR-21/Smad7 and TGF-β/miR200b/Zeb1 pathways might participate in the pathogenesis of HS. Thus, a novel, beyond the traditional methods, approach for HS treatment via miRNA therapeutics could have been provided.
[Identification of differently expressed microRNAs in keloid and pilot study on biological function of miR-199a-5p].
Wu Zhi-Yuan,Lu Ling,Guo Xiao-Rui,Zhang Pei-Hua
Zhonghua zheng xing wai ke za zhi = Zhonghua zhengxing waike zazhi = Chinese journal of plastic surgery
OBJECTIVE:To screen out related microRNAs in keloid tissue, and identify their effect on the proliferation of keloid fibroblasts. METHODS:8 cases of keloid tissue and 8 cases of normal skin tissue were collected as specimens. The differently expressed miRNA in keloid tissue from normal skin tissue were screened out with gene chip( Exiqon company), which was validated with quantitative real-time PCR. Then miRNA mimics was transfected into keloid fibroblasts line to stimulate high expression of mature miRNA in cells. The effect on the proliferation of fibroblasts in keloid was tested by Edu. RESULTS:(1) A total of 17 differently expressed microRNAs were found, including miR-199a-5p. (2) The expression of miR-199a-5p had been verified by qRT-PCR to be down-regulated in keloid, which was consistent with the result of array. (3) The positive rate of EdU in miR-199a-5p mimics transfected group and negative control group was (20.72 +/- 2.50)% and (27.68 +/- 4.92)%, respectively. The proliferative rate of keloid fibroblasts turned down in miR-199a-5p-transfected group (t = 2.183, P = 0.047). Besides that, the cell cycle changed after transfection. The percentage of S and G2/M phase in miR-199a-5p mimics transfected group was 33.93 +/- 1.30 and 10.87 +/- 0.80, respectively, while it was 31.39 +/- 0.79 and 9.27 +/- 0.46 in negative control group, and the difference was statistically significant. CONCLUSIONS:(1) The miRNA expression profile is different between keloid and normal skin; (2) The expression of miR-199a-5p is down-regulated in keloid and miR-199a-5p can affect the cell cycle and suppress proliferation of keloid fibroblasts. It indicateds that miR-199a-5p may be involved in regulating fibroblastic proliferation.
MicroRNA expression signature and the therapeutic effect of the microRNA‑21 antagomir in hypertrophic scarring.
Guo Liang,Xu Kai,Yan Hongbo,Feng Haifeng,Wang Tao,Chai Linlin,Xu Guozheng
Molecular medicine reports
Hypertrophic scars (HS) area fibroproliferative disorder of the skin, which causes aesthetic and functional impairment. However, the molecular pathogenesis of this disease remains largely unknown and currently no efficient treatment exists. MicroRNAs (miRNAs) are involved in a variety of pathophysiological processes, however the role of miRNAs in HS development remains unclear. To investigate the miRNA expression signature of HS, microarray analysis was performed and 152 miRNAs were observed to be differentially expressed in HS tissue compared with normal skin tissues. Of the miRNAs identified, miRNA‑21 (miR‑21) was significantly increased in HS tissues and hypertrophic scar fibroblasts (HSFBs) as determined by reverse transcription‑quantitative polymerase chain reaction analysis. It was also observed that, when miR‑21 in HSFBs was blocked through use of an antagomir, the phenotype of fibrotic fibroblasts in vitro was reversed, as demonstrated by growth inhibition, induction of apoptosis and suppressed expression of fibrosis‑associated genes collagen type I α 1 chain (COL1A1), COL1A2 and fibronectin. Furthermore, miR‑21 antagomir administration significantly reduced the severity of HS formation and decreased collagen deposition in a rabbit ear HS model. The total scar area and scar elevation index were calculated and were demonstrated to be significantly decreased in the treatment group compared with control rabbits. These results indicated that the miR‑21 antagomir has a therapeutic effect on HS and suggests that targeting miRNAs may be a successful and novel therapeutic strategy in the treatment of fibrotic diseases that are difficult to treat with existing methods.
TGF-β1 promotes scar fibroblasts proliferation and transdifferentiation via up-regulating MicroRNA-21.
Liu Ying,Li Yue,Li Ning,Teng Wen,Wang Min,Zhang Yingbo,Xiao Zhibo
TGF-β1, upregulated in keloid tissue, promotes the proliferation, collagen formation and differentiation of dermal fibroblasts. miR-21 is one of microRNAs first found in human genome. The aim of our study is to explore the mechanisms of miR-21 in TGF-β1-induced scar fibroblasts proliferation and transdifferentiation. In the present study, first we found that TGF-β1 promoted scar fibroblasts proliferation and transdifferentiation via up-regulating miR-21 expression, which could be attenuated when miR-21 was inhibited. Overexpression of miR-21 had similar effect as TGF-β1 on proliferation and transdifferentiation. Additionally, TGF-β1 increased the expressions and activities of MMP2 and MMP9 in keloid fibroblasts, which was suppressed by miR-21 inhibition. Finally, the results demonstrated that PTEN/AKT signaling pathway played important role in TGF-β1-induced transdifferentiation. In conclusion, our study suggests that TGF-β1 promotes keloid fibroblasts proliferation and transdifferentiation via up-regulation of miR-21 and PTEN/AKT signalling pathway plays important role in this process, which provides a potential theoretical basis for clinical treatment of skin scars.
The integrative regulatory network of circRNA and microRNA in keloid scarring.
Shi Jingpei,Yao Shuluan,Chen Pan,Yang Yuan,Qian Mengjiao,Han Yi,Wang Nan,Zhao Yu,He Yongjing,Lyu Lechun,Lu Di
Molecular biology reports
Circular RNA (circRNA), a novel type of non-coding RNA that consists of a circular loop, has been demonstrated to act as a "sponge" for microRNAs (miRNAs). However, the role of circRNAs in keloid remains unknown. In this study, we investigated circRNA expression profiles in keloid to identify potential diagnostic and therapeutic circRNAs. We performed a circRNA microarray assay to determine circRNA expression in keloid and paired normal skin tissues. Quantitative reverse transcription polymerase chain reaction was used to evaluate the expression levels of candidate circRNAs. The most significantly over-expressed circRNA was used to predict putative miRNA targets and the binding sites of miRNAs with this circRNA. Finally, we constructed a circRNA-miRNA interaction network and carried out gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. We found 52 significantly upregulated and 24 downregulated circRNAs in keloid compared with normal skin tissue. We confirmed that hsa_circ_0057452, hsa_circ_0007482, hsa_circ_0020792, hsa_circ_0057342, and hsa_circ_0043688 were significantly upregulated in keloid tissues. Analysis of the circRNA-miRNA interaction network revealed that circRNAs could interact with miRNAs, including miRNA-29a, miRNA-23a-5p and miRNA-1976. GO and KEGG analyses indicated that these target genes were involved in biological functions and signaling pathways that may play vital roles in the pathogenesis of keloid. This study revealed that circRNAs are potentially implicated in the development of keloid and could serve as novel diagnostic and therapeutic targets.
[microRNA-222 regulates proliferation and apoptosis of fibroblasts in hypertrophic scar via matrix metalloproteinase 1].
Zhang Yi,Zhang Li,Zhang Qiyu,Hong Weilong,Lin Xiaohua
Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences
<b>Objective:</b> To explore the effect of microRNA(miR)-222 on cell proliferation and apoptosis of fibroblasts in hypertrophic scar (HS) and the underlying mechanisms. <b>Methods:</b> The expression of miR-222 in the HS and the normal skin tissues was detected by real-time RT-PCR. The HS fibroblasts were transfected with miR-222 mimic and miR-222 inhibitor respectively. The cell viability was tested with MTT assay, cell cycle distribution and apoptosis were detected with flow cytometry and the expression levels of proliferation, apoptosis and cell cycle related proteins were determined with Western blot. Direct target of miR-222 was evaluated by dual-luciferase reporter assay. <b>Results:</b> miR-222 was significantly up-regulated in HS tissues compared with normal skin tissues(<i>P</i><0.05). Overexpression of miR-222 enhanced the cell viability of HS fibroblasts; increased mRNA and protein expressions of proliferating cell nuclear antigen (PCNA), collagen alpha-1 (Ⅰ) chain (Col1A1) and collagen alpha-1 (Ⅲ) chain (Col3A1); increased cell population in S phase and protein expressions of cyclin D1, cyclin E1 and cyclin-dependent kinases 1 (CDK1); inhibited cell apoptosis and reduced protein expressions of caspase-3/9. Overexpression of MMP1 attenuated the effects of miR-222 on the cell viability and apoptosis in fibroblasts, reduced expression levels of PCNA, cyclin D1 and the expression of caspase-3 was increased. <b>Conclusion:</b> miR-222 enhances cell proliferation and inhibits cell apoptosis of HS fibroblasts through negative regulation of MMP1, which suggests that miR-222 and MMP1 might be used as novel biomarkers and targets in diagnostic and therapeutic approaches for HS.
Comparative study of microRNA profiling in keloid fibroblast and annotation of differential expressed microRNAs.
Li Chao,Bai Yuehong,Liu Hua,Zuo Xuemei,Yao Haichang,Xu Yiming,Cao Manlin
Acta biochimica et biophysica Sinica
Keloids are tumor-like skin scars that grow as a result of the aberrant healing of skin injuries, with no effective treatment. The molecular mechanism underlying keloid pathogenesis is still largely unknown. In this study, we compared microRNA (miRNA) expression profiles between keloid-derived fibroblasts and normal fibroblasts (including fetal and adult dermal fibroblasts) by miRNA microarray analysis. We found that the miRNA profiles in keloid-derived fibroblasts are different with those in normal fibroblasts. Nine miRNAs were differentially expressed, six of which were significantly up-regulated in keloid fibroblasts (KFs), including miR-152, miR-23b-3p, miR-31-5p, miR-320c, miR-30a-5p, and hsv1-miR-H7, and three of which were significantly down-regulated, including miR-4328, miR-145-5p, and miR-143-3p. Functional annotations of differentially expressed miRNA targets revealed that they were enriched in several signaling pathways important for scar wound healing. In conclusion, we demonstrate that the miRNA expression profile is altered in KFs compared with in fetal and adult dermal fibroblasts, and the expression profile may provide a useful clue for exploring the pathogenesis of keloids. miRNAs might partially contribute to the etiology of keloids by affecting several signaling pathways relevant to scar wound healing.
miRNA expression profiles in keloid tissue and corresponding normal skin tissue.
Liu Ying,Yang Daping,Xiao Zhibo,Zhang Miaobo
Aesthetic plastic surgery
BACKGROUND:Because the molecular mechanism behind keloid pathogenesis is still largely unknown, the clinical management of keloids remains problematic. miRNA (microRNA) is a novel class of small regulatory RNA that has emerged as post-transcriptional gene repressors and participants in diverse pathophysiological processes of skin disease. In the present study we aimed to investigate expression profiles of miRNA in keloid tissue and to develop a further understanding of the molecular mechanism involved in the pathogenesis of keloids. METHODS:miRNA expression profiles in 12 pairs of keloid tissue and corresponding normal skin tissue were analyzed through a mammalian miRNA microarray containing established whole human mature and precursor miRNA sequences. Real-Time quantitative PCR was performed to confirm the array results. The putative targets of differentially expressed miRNA were functionally annotated by bioinformatics approaches. RESULTS:miRNA microarray analysis identified 32 differentially expressed miRNAs, and a total of 23 miRNAs exhibited higher expression, while 9 miRNAs demonstrated lower expression in keloid tissue than in normal skin tissue. Functional annotations of differentially expressed miRNA targets revealed that they were enriched in several signaling pathways important for scar wound healing. CONCLUSION:This study showed that the expressions of many miRNAs were altered in keloid tissue, and their expression profiling may provide a useful clue for exploring the pathogenesis of keloids. miRNAs might partly contribute to the etiology of keloids by affecting several signaling pathways relevant to scar wound healing.
Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist inhibits collagen synthesis in human hypertrophic scar fibroblasts by targeting Smad3 via miR-145.
Zhu Hua-Yu,Li Chao,Zheng Zhao,Zhou Qin,Guan Hao,Su Lin-Lin,Han Jun-Tao,Zhu Xiong-Xiang,Wang Shu-yue,Li Jun,Hu Da-Hai
Biochemical and biophysical research communications
The transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ) functions to regulate cell differentiation and lipid metabolism. Recently, its agonist has been documented to regulate extracellular matrix production in human dermal fibroblasts. This study explored the underlying molecular mechanisms and gene interactions in hypertrophic scar fibroblasts (HSFBs) in vitro. HSFBs were cultured and treated with or without PPAR-γ agonist or antagonist for gene expression. Bioinformatical analysis predicted that miR-145 could target Smad3 expression. Luciferase assay was used to confirm such an interaction. The data showed that PPAR-γ agonist troglitazone suppressed expression of Smad3 and Col1 in HSFBs. PPAR-γ agonist induced miR-145 at the gene transcriptional level, which in turn inhibited Smad3 expression and Col1 level in HSFBs. Furthermore, ELISA data showed that Col1 level in HSFBs was controlled by a feedback regulation mechanism involved in PPAR-γ agonist and antagonist-regulated expression of miR-145 and Smad3 in HSFBs. These findings indicate that PPAR-γ-miR-145-Smad3 axis plays a role in regulation of collagen synthesis in HSFBs.
Suppressive effects of human fetal keratinocytes on the proliferation, differentiation and extracellular matrix synthesis of human hypertrophic scar fibroblasts in vitro.
Wang Zhe,Song Qibin,Li Hongqiu
Molecular medicine reports
A hypertrophic scar is characterized by fibroblast proliferation and excessive extracellular matrix deposition. Emerging evidence has revealed that fetal keratinocytes (KCs) contribute to scarless wound healing. However, the association between fetal keratinocytesand hypertrophic scarring remains unclear. In the present study, human KCs of different gestational ages were isolated and co‑cultured with human hypertrophic scar fibroblasts (HSFbs) or normal skin fibroblasts. Gene expression andprotein levels of fibronectin, collagen 1and α‑smooth muscle actinin thefibroblasts were measured by reverse transcription‑quantitative polymerase chain reaction and western blot analyses. It was observed that fetal KCs significantly inhibited the proliferation of HSFbs in vitro. Fetal keratinocytes also affected the expression of fibronectin, collagen 1and α‑smooth muscle actin in HSFbs. In addition, miR‑940 may modulate the suppressive effects of fetal KCson the cell proliferation, differentiation and extracellular matrix synthesis of HSFbs by directly targeting transforming growth factor‑β1. Taken together, the results of the present studyprovide evidence to support the potential use of fetal KCsfor cell‑based therapeutic grafting in the prevention of hypertrophic scarring.
miR-495 inhibits the growth of fibroblasts in hypertrophic scars.
Guo Bingyu,Hui Qiang,Xu Zhishan,Chang Peng,Tao Kai
Noncoding RNAs are known to be importantly involved in a variety physiological and pathophysiolgical processes. Their role in the pathogenesis of hypertrophic scars remains unclear, however. After preliminary screening of the microRNA (miRNA) gene expression profiles, we explored the role of miR-495 in the development of hypertrophic scar by comparing expression of miR-495 and focal adhesion kinase (FAK) between hypertrophic scar and normal skin tissue. We also used 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and annexin V-fluorescein isothiocyanate/propidium iodide assays to assess the effect of miR-495 on the proliferation and apoptosis in human hypertrophic scar fibroblasts. Western blotting and real-time polymerase chain reaction were used to evaluate expression of miR-495, FAK, and related proteins in the FAK pathway. Our findings show that miR-495 inhibits FAK and its downstream mediators in vitro and vivo, and suggest that miR-495 may be a useful therapeutic target for the treatment of hypertrophic scar.
[Advances in the research of effects of competing endogenous RNAs and their regulatory networks in pathological scars of skin].
Li M,Liu D W,Lei W
Zhonghua shao shang za zhi = Zhonghua shaoshang zazhi = Chinese journal of burns
The skin pathologic scar is a skin fibrous proliferative disease characterized by abnormal proliferation of fibroblasts and overdeposition of extracellular matrix. Unclarity of genesis and development mechanism is the main reason that restricts its diagnosis and treatment. In recent years, it has been found that microRNAs play important roles in the regulation mechanism of pathological scars. The competing endogenous RNAs (ceRNAs) have microRNA response elements which can be competitively combined with microRNAs through sponge adsorption. Through the mutual regulation of RNAs, ceRNAs regulate the expression of target gene and participate in the development of disease. Based on the ceRNA hypothesis, this paper systematically reviews the biological functions and clinical significance of ceRNAs in pathological scars of skin, and discusses the role of ceRNAs and " RNA-microRNA-RNA" regulation network in pathologic scars. The ceRNA therapy may become a new model therapy for skin scars in the future.
MiR-6836-3p promotes proliferation of hypertrophic scar fibroblasts by targeting CTGF.
Liu F,Chen W-W,Li Y,Zhang J-Q,Zheng Q-B
European review for medical and pharmacological sciences
OBJECTIVE:To explore the role of micro ribonucleic acid (miR)-6836-3p in regulating hypertrophic scar (HS) and its potential mechanism. PATIENTS AND METHODS:The level of miR-6836-3p in HS or normal skin was determined by Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Cell counting kit-8 (CCK8) and flow cytometry were applied to explore the effect of miR-98 on the growth and apoptosis of HS fibroblasts (HSFBs). Luciferase assay was employed to acknowledge whether connective tissue growth factor (CTGF) is a target of miR-6836-3p. Western blotting was used to detect the protein expression of CTGF after transfection with miR-6836-3p mimic or inhibitor. RESULTS:MiR-6836-3p expression was much higher in HS than that in normal skin. MiR-6836-3p mimic promoted fibroblast growth, and CTGF was confirmed to be a direct target of miR-6836-3p. The results of Western blotting clarified that miR-6836-3p mimic raised the level of CTGF, and its expression was positively correlated with that of CTGF. CONCLUSIONS:MiR-6836-3p promoted the development of HS by increasing the expression of CTGF. MiR-6836-3p may be a potential novel molecular target for the treatment of HS.
MicroRNA‑185 regulates transforming growth factor‑β1 and collagen‑1 in hypertrophic scar fibroblasts.
Xiao Kaiyan,Luo Xusong,Wang Xiuxia,Gao Zhen
Molecular medicine reports
Transforming growth factor-β1 (TGF-β1) and collagen type I (Col-1) serve a critical role in the development and progression of hypertrophic scarring (HS). The present study hypothesized that a post‑translational mechanism of microRNAs (miR) regulated the expression of TGF‑β1 and Col‑1 in HS fibroblasts (HSFBs). A collection of 20 HS tissues was compared with corresponding normal tissues from clinical patients, and the expression of miR‑185 was measured. Using PicTar, TargetScan and miRBase databases, it was identified that miR‑185 may be a regulator of TGF‑β1 and Col‑1 in humans. Based on these hypotheses, the expression of miR‑185, TGF‑β1 and Col‑1 in HS tissues was investigated. The results demonstrated that the expression of miR‑185 was markedly suppressed, and TGF‑β1 and Col‑1 levels were increased, in HS tissues. The expression levels of endogenous miR‑185 negatively correlated with the TGF‑β1 and Col‑1 mRNA levels (Pearson's correlation coefficient r=‑0.674, P<0.01 and r=‑0.590, P<0.01, respectively). In vitro, miR‑185 can regulate TGF‑β1 and Col‑1 through the predicted binding sites in its 3'‑untranslated region. miR‑185 had an effect on cell proliferation and apoptosis, thereby regulating HSFBs growth. In addition, miR‑185 gain‑of‑function decreased TGF‑β1 and Col‑1 protein expression, and miR‑185 loss‑of‑function increased TGF‑β1 and Col‑1 protein expression in HSFBs. In conclusion, overexpressed miR‑185 could inhibit HSFBs growth, and the underlying mechanism was mediated, at least partly, through the suppression of TGF‑β1 and Col‑1 expression. However, above all, miR‑185 might serve as a potential therapeutic approach for the treatment of HS.
MicroRNA-192 regulates hypertrophic scar fibrosis by targeting SIP1.
Li Yan,Zhang Julei,Zhang Wei,Liu Yang,Li Yuehua,Wang Kejia,Zhang Yijie,Yang Chen,Li Xiaoqiang,Shi Jihong,Su Linlin,Hu Dahai
Journal of molecular histology
Hypertrophic scar (HS) is a fibro-proliferative disorder which is characterized by excessive deposition of collagen and accumulative activity of myofibroblasts. Increasing evidences have demonstrated miRNAs play a pivotal role in the pathogenesis of HS. MiR-192 is closely associated with renal fibrosis, but its effect on HS formation and skin fibrosis remains unknown. In the study, we presented that miR-192 was up-regulated in HS and HS derived fibroblasts (HSFs) compared to normal skin (NS) and NS derived fibroblasts (NSFs), accompanied by the reduction of smad interacting protein 1 (SIP1) expression and the increase of Col1, Col3 and α-SMA levels. Furthermore, we confirmed SIP1 was a direct target of miR-192 by using luciferase reporter assays. Meanwhile, the overexpression of miR-192 increased the levels of Col1, Col3 and α-SMA. The synthesis of collagen and more positive α-SMA staining were also observed in bleomycin-induced dermal fibrosis model of BALB/c mice treated with subcutaneous miR-192 mimics injection, whereas the inhibition of miR-192 decreased the expression of Col1, Col3 and α-SMA. Moreover, SIP1 siRNA could enhance the levels of Col1, Col3 and α-SMA, showing that the effect of knockdown SIP1 was similar to miR-192 mimics, and the phenomenon manifested miR-192 regulated HS fibrosis by targeting SIP1. Together, our results indicated that miR-192 was a critical factor of HS formation and facilitated skin fibrosis by targeting directly SIP1.
Mitomycin C induces fibroblast apoptosis and reduces intra-articular scar adhesion by regulating miR-21 and its target Programmed cell death 4.
Chen Hui,Wang Shuguang,Sun Yu,Wang Jingcheng
Previous studies have shown that mitomycin C (MMC) can prevent scar adhesion after joint surgery, but the specific mechanism underlying this effect remains unclear. The purpose of this study was to explore the specific mechanism by which MMC promotes fibroblast apoptosis and prevents joint adhesion. The effect of MMC on fibroblasts was assessed using cell counting kit-8 (CCK-8) assays, western blotting, and TUNEL staining. We used qRT-PCR to measure the expression of miR-21 in fibroblasts treated with MMC. Luciferase activity assays were used to determine the relationships between miR-21 and Programmed cell death 4 (PDCD4). The effects of miR-21 and PDCD4 on fibroblast apoptosis were assessed using flow cytometry and western blotting. HE staining was used to determine the role of miR-21 in scar tissue formation in a model of joint adhesion. The results showed that MMC induced apoptosis of fibroblasts and decreased the expression of miR-21. Moreover, miR-21 down-regulation also induced apoptosis of fibroblasts. PDCD4 was confirmed to be a direct target of miR-21 by luciferase activity assay. The results from the animal model indicated that miR-21 attenuated the effect of MMC on reducing the number of fibroblasts. Our study shows that MMC can induce fibroblast apoptosis and prevent joint adhesion by regulating the expression of miR-21 and its target PDCD4.
Fibroproliferative effect of microRNA-21 in hypertrophic scar derived fibroblasts.
Li Guangzao,Zhou Renpeng,Zhang Qi,Jiang Banghong,Wu Qingkai,Wang Chen
Experimental cell research
Hypertrophic scar (HS) is a fibroproliferative disorder caused by abnormal wound healing, which is characterized by excessive deposition of extracellular matrix (ECM) secreted by fibroblasts. We previous have found that expression of microRNA-21(miR-21) was increased in tissues and fibroblasts of HS. However, the underlying molecular mechanism remains to be further elucidated. In this study, we identified the miR-21 was a marker for the phenotype of HS fibroblasts, as anti-miR-21 reduced expression of fibrosis markers such as Col1A1, Col3A1, Fn and α-SMA in fibroblasts and overexpression of miR-21 promoted fibroproliferative expression in fibroblasts. Furthermore, we also found that miR-21 promoted TGF-β1 induced fibroproliferative expression by repressing Smad7 expression in vitro. In addition, the miR-21 inhibitor inhibited the growth of hypertrophic scar tissue in vivo (nude mice experimental model). These results indicated that miR-21 was a critical regulator for HS formation and TGF- β1/miR-21/Smad7 pathway could be a useful therapeutic target for the treatment of HS.
MicroRNA 181b regulates decorin production by dermal fibroblasts and may be a potential therapy for hypertrophic scar.
Kwan Peter,Ding Jie,Tredget Edward E
Hypertrophic scarring is a frequent fibroproliferative complication following deep dermal burns leading to impaired function and lifelong disfigurement. Decorin reduces fibrosis and induces regeneration in many tissues, and is significantly downregulated in hypertrophic scar and normal deep dermal fibroblasts. It was hypothesized that microRNAs in these fibroblasts downregulate decorin and blocking them would increase decorin and may prevent hypertrophic scarring. Lower decorin levels were found in hypertrophic scar as compared to normal skin, and in deep as compared to superficial dermis. A decorin 3' un-translated region reporter assay demonstrated microRNA decreased decorin in deep dermal fibroblasts, and microRNA screening predicted miR- 24, 181b, 421, 526b, or 543 as candidates. After finding increased levels of mir-181b in deep dermal fibroblasts, it was demonstrated that TGF-β1 stimulation decreased miR-24 but increased miR-181b and that hypertrophic scar and deep dermis contained increased levels of miR-181b. By blocking miR-181b with an antagomiR, it was possible to increase decorin protein expression in dermal fibroblasts. This suggests miR-181b is involved in the differential expression of decorin in skin and wound healing. Furthermore, blocking miR-181b reversed TGF-β1 induced decorin downregulation and myofibroblast differentiation in hypertrophic scar fibroblasts, suggesting a potential therapy for hypertrophic scar.
MiR-138/peroxisome proliferator-activated receptor β signaling regulates human hypertrophic scar fibroblast proliferation and movement in vitro.
Xiao Ying-ying,Fan Peng-ju,Lei Shao-rong,Qi Min,Yang Xing-hua
The Journal of dermatology
Excessive scars affect a patient's quality of life, both physically and psychologically, by causing pruritus, pain and contractures. Because there is a poor understanding of the complex mechanisms underlying the processes of hypertrophic scar formation, most therapeutic approaches remain clinically unsatisfactory. In this study, we found that miR-138 was downregulated and peroxisome proliferator-activated receptor (PPARβ) was inversely upregulated in hypertrophic scar tissues compared to in paired normal skin tissues. Using a dual-luciferase assay, we validated that miR138 directly targets PPARβ and regulates its expression at the transcriptional and translational levels. In gain-and-loss experiments, we found that miR-138/PPARβ signaling regulated human hypertrophic scar fibroblast proliferation and movement, and affected scarring-related protein expression, which suggests that miR-138/PPARβ signaling is important for hypertrophic scarring. Thus, our study provides evidence to help determine whether miR-138/PPARβ signaling may be a potential target for hypertrophic scarring management.
MicroRNA-130a has pro-fibroproliferative potential in hypertrophic scar by targeting CYLD.
Zhang Jian,Zhou Qin,Wang Hongtao,Huang Meiling,Shi Jihong,Han Fu,Cai Weixia,Li Yan,He Ting,Hu Dahai
Archives of biochemistry and biophysics
Hypertrophic scars are dermal fibrosis diseases that protrude from the surface of the skin and irregularly extend to the periphery, seriously affecting the appearance and limb function of the patient. In this study, we found that microRNA-130a (miR-130a) was increased in hypertrophic scar tissues and derived primary fibroblasts, accompanied by up-regulation of collagen1/3 and α-SMA. Inhibition of miR-130a in hypertrophic scars fibroblasts suppressed the expression of collagen1/3 and α-SMA as well as the cell proliferation. Bioinformatics analysis combined with luciferase reporter gene assay results indicated that CYLD was a target gene of miR-130a, and the miR-130a mimic could reduce the level of CYLD. In contrast to miR-130a, the expression of CYLD was downregulated in hypertrophic scars and their derived fibroblasts. Overexpressing CYLD inhibited the expression of collagen 1/3 and α-SMA, slowed cell proliferation, and inhibited Akt activity. As expected, further study showed that the overexpression of CYLD could prevent the pro-fibroproliferative effects of miR-130a. Consistent with the in vitro results, the inhibitor of miR-130a effectively ameliorated excessive collagen deposition in bleomycin-induced skin fibrosis mouse model. Taken together, our results indicate that miR-130a promotes collagen secretion, myofibroblast transformation and cell proliferation by targeting CYLD and enhancing Akt activity. Therefore, the miR-130a/CYLD/Akt pathway may serve as a novel entry point for future skin fibrosis research.
MicroRNA-519d inhibits proliferation and induces apoptosis of human hypertrophic scar fibroblasts through targeting Sirtuin 7.
Zhou Xiaoqian,Xie Yidun,Xiao Houan,Deng Xudong,Wang Yu,Jiang Liyuan,Liu Chen,Zhou Rui
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
MicroRNAs (miRNAs) play critical roles in various pathological processes, including hypertrophic scar (HS) formation. However, the precise role of miRNAs in HS formation remains largely unknown. In this study, we aimed to investigate the role of miR-519d in HS formation. We found that miR-519d expression was significantly downregulated in HS tissues and fibroblasts. Overexpression of miR-519d inhibited the expression of type I collagen (Col I), type III collagen (Col III) and α-smooth muscle actin (α-SMA) in HS fibroblasts. Moreover, overexpression of miR-519d reduced the proliferation and induced the apoptosis of HS fibroblasts. In contrast, suppression of miR-519d showed the opposite effects. Interestingly, Sirtuin 7 (SIRT7) was identified as a target gene of miR-519d. The results showed that miR-519d directly targeted the 3'-untranslated region of SIRT7 and negatively regulated its expression. Furthermore, miR-519d regulated the expression of TGF-β type I receptor (TGFBRI) and the phosphorylation of Smad2. Knockdown of SIRT7 by siRNA inhibited the expression of Col I, Col III and α-SMA, and reduced the proliferation and induced the apoptosis of HS fibroblasts. Overexpression of SIRT7 abrogated the effects mediated by miR-519d overexpression in HS fibroblasts. Overall, these results suggest that miR-519d inhibits the expression of extracellular matrix-associated genes, reduces the proliferation and induces the apoptosis of HS fibroblasts by targeting SIRT7, implying a suppressive role of miR-519d in HS formation. This study suggests that miR-519d may serve as a promising therapeutic target for treatment of human HS.
Dynamic Expression of Novel MiRNA Candidates and MiRNA-34 Family Members in Early- to Mid-Gestational Fetal Keratinocytes Contributes to Scarless Wound Healing by Targeting the TGF-β Pathway.
Zhao Feng,Wang Zhe,Lang Hongxin,Liu Xiaoyu,Zhang Dianbao,Wang Xiliang,Zhang Tao,Wang Rui,Shi Ping,Pang Xining
BACKGROUND:Early- to mid-gestational fetal mammalian skin wounds heal rapidly and without scarring. Keratinocytes (KCs) have been found to exert important effects on the regulation of fibroblasts. There may be significant differences of gestational fetal KCs at different ages. The advantages in early- to mid-gestational fetal KCs could lead to fetal scarless wound healing. METHODS:KCs from six human fetal skin samples were divided into two groups: a mid-gestation group (less than 28 weeks of gestational age) and a late-gestation group (more than 28 weeks of gestational age). RNA extracted from KCs was used to prepare a library of small RNAs for next-generation sequencing (NGS). To uncover potential novel microRNA (miRNAs), the mirTools 2.0 web server was used to identify candidate novel human miRNAs from the NGS data. Other bioinformatical analyses were used to further validate the novel miRNAs. The expression levels of the miRNAs were further confirmed by real-time quantitative RT-PCR. RESULTS:A total of 61.59 million reads were mapped to 1,170 known human miRNAs in miRBase. Among a total of 202 potential novel miRNAs uncovered, 106 candidates have a higher probability of being novel human miRNAs. A total of 110 miRNAs, including 22 novel miRNA candidates, were significantly differently expressed between mid- and late-gestational fetal KCs. Thirty-three differentially expressed miRNAs and miR-34 family members are correlated with the transforming growth factor-β (TGF-β) pathway. CONCLUSIONS:Taken together, our results provide compelling evidence supporting the existence of 106 novel miRNAs and the dynamic expression of miRNAs that extensively targets the TGF-β pathway at different gestational ages in fetal KCs. MiRNAs showing altered expression at different gestational ages in fetal KCs may contribute to scarless wound healing in early- to mid-gestational fetal KCs, and thus may be new targets for potential scar prevention and reduction therapies.
miR-137 Inhibits Proliferation and Metastasis of Hypertrophic Scar Fibroblasts via Targeting Pleiotrophin.
Zhang Qian,Guo Bingyu,Hui Qiang,Chang Peng,Tao Kai
Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology
BACKGROUND/AIMS:There are few effective treatment options for hypertrophic scars. MircoRNAs are a class of small, noncoding RNAs involved in multiple biological functions. METHODS:Gene chip screening was used to screen out the differential expression of miRNAs in hypertrophic scars and normal tissues. Western blot and real-time PCR were used to confirm the expression of pleiotrophin (PTN) in hypertrophic scars. After analyze the correlation between PTN and miR-137 using correlation analysis, we used miRDB software to analyze the binding sites of miR-137 and PTN. Luciferase reporter gene, western blot and real-time PCR experiments were used to detect the regulatory effect of miR-137 on PTN. MTT and transwell assay were used to detect the effect of miR-137 on proliferation and metastasis. Western blot and real-time PCR were used to detect the regulatory effects of miR-137 on cyclin B1, matrix metalloproteinase 9 (MMP9), α-smooth muscle actin (α-SMA), vimentin, and type-I collagen (COL1A). Finally, miR-137 inhibitor was transfected into fibroblasts which was silent PTN, and the proliferation and migration of cells were detected by MTT and transwell. Western blot and real-time PCR were used to detect the expression of related proteins. RESULTS:Various miRNAs was abnormal expressed in hypertrophic scars. miR-137 was decreased in hypertrophic scar, however PTN was up regulated in hypertrophic scars. miR-137 induced proliferation and metastasis in fibroblasts. This effect was accompanied by decreased expression of cyclin B1, MMP9, α-SMA, vimentin, and COL1A mediated via the target of PTN. CONCLUSION:Modulation of miR-137 expression in fibroblasts could provide an important therapeutic strategy for hypertrophic scars.
MicroRNA-98 inhibits the cell proliferation of human hypertrophic scar fibroblasts via targeting Col1A1.
Bi Sheng,Chai Linlin,Yuan Xi,Cao Chuan,Li Shirong
BACKGROUND:Hypertrophic scarring (HS) is a severe disease, and results from unusual wound healing. Col1A1 could promote the hypertrophic scar formation, and the expression of Col1A1 in HS tissue was markedly higher than that in the normal. In present study, we aimed to identify miRNAs as post-transcriptional regulators of Col1A1 in HS. METHODS:MicroRNA-98 was selected as the key miRNA comprised in HS. The mRNA levels of miR-98 in HS tissues and the matched normal skin tissues were determined by qRT-PCR. MTT and flow cytometry were used to determine the influence of miR-98 on cell proliferation and apoptosis of HSFBs, respectively. Col1A1 was found to be the target gene of miR-98 using luciferase reporter assay. Luciferase assay was performed to determine the relative luciferase activity in mimic NC, miR-98 mimic, inhibitor NC and miR-98 inhibitor with Col1A13'-UTR wt or Col1A13'-UTR mt reporter plasmids. The protein expression of Col1A1 in HSFBs after transfection with mimic NC, miR-98 mimic, inhibitor NC and miR-98 inhibitor were determined by western blotting. RESULTS:The mRNA level of miR-98 in HS tissues was much higher than that in the control. Transfection of HSFBs with a miR-98 mimic reduced the cell viability of HSFBs and increased the apoptosis portion of HSFBs, while inhibition of miR-98 increased cell viability and decreased apoptosis portion of HSFBs. miR-98 inhibitor increased the relative luciferase activity significantly when cotransfected with the Col1A1-UTR reporter plasmid, while the mutant reporter plasmid abolished the miR-98 inhibitor-mediated increase in luciferase activity. Western blotting revealed that overexpression of miR-98 decreased the expression of Col1A1. CONCLUSIONS:Overexpression of miR-98 repressed the proliferation of HSFBs by targeting Col1A1.
MicroRNA-21 regulates hTERT via PTEN in hypertrophic scar fibroblasts.
Zhu Hua-Yu,Li Chao,Bai Wen-Dong,Su Lin-Lin,Liu Jia-Qi,Li Yan,Shi Ji-Hong,Cai Wei-Xia,Bai Xiao-Zhi,Jia Yan-Hui,Zhao Bin,Wu Xue,Li Jun,Hu Da-Hai
BACKGROUND:As an important oncogenic miRNA, microRNA-21 (miR-21) is associated with various malignant diseases. However, the precise biological function of miR-21 and its molecular mechanism in hypertrophic scar fibroblast cells has not been fully elucidated. METHODOLOGY/PRINCIPAL FINDINGS:Quantitative Real-Time PCR (qRT-PCR) analysis revealed significant upregulation of miR-21 in hypertrophic scar fibroblast cells compared with that in normal skin fibroblast cells. The effects of miR-21 were then assessed in MTT and apoptosis assays through in vitro transfection with a miR-21 mimic or inhibitor. Next, PTEN (phosphatase and tensin homologue deleted on chromosome ten) was identified as a target gene of miR-21 in hypertrophic scar fibroblast cells. Furthermore, Western-blot and qRT-PCR analyses revealed that miR-21 increased the expression of human telomerase reverse transcriptase (hTERT) via the PTEN/PI3K/AKT pathway. Introduction of PTEN cDNA led to a remarkable depletion of hTERT and PI3K/AKT at the protein level as well as inhibition of miR-21-induced proliferation. In addition, Western-blot and qRT-PCR analyses confirmed that hTERT was the downstream target of PTEN. Finally, miR-21 and PTEN RNA expression levels in hypertrophic scar tissue samples were examined. Immunohistochemistry assays revealed an inverse correlation between PTEN and hTERT levels in high miR-21 RNA expressing-hypertrophic scar tissues. CONCLUSIONS/SIGNIFICANCE:These data indicate that miR-21 regulates hTERT expression via the PTEN/PI3K/AKT signaling pathway by directly targeting PTEN, therefore controlling hypertrophic scar fibroblast cell growth. MiR-21 may be a potential novel molecular target for the treatment of hypertrophic scarring.
MicroRNA‑486‑5p inhibits the growth of human hypertrophic scar fibroblasts by regulating Smad2 expression.
Shi Yingying,Wang Luping,Yu Pijun,Liu Yi,Chen Wei
Molecular medicine reports
The aim of the current study was to investigate the expression and role of microRNA‑486‑5p (miR‑486‑5p) in hypertrophic scar (HS) formation, and to examine the associated mechanisms. First, miR‑486‑5p expression was detected in HS tissues and human hypertrophic scar fibroblasts (hHSFs) by reverse transcription‑quantitative polymerase chain reaction. Target genes of miR‑486‑5p were predicted using TargetScan and verified by dual‑luciferase reporter assays. To investigate the role of miR‑486‑5p in HS formation, miR‑486‑5p was overexpressed in hHSFs through transfection with miR‑486‑5p mimics. MTT, cell apoptosis and cell cycle assays were preformed to investigate the proliferation, cell apoptosis and cell cycle distribution of hHSFs, respectively. Additionally, protein expression was measured by western blot analysis. The results demonstrated that miR‑486‑5p expression was significantly decreased in HS tissues and cells. Mothers against decapentaplegic homolog (Smad)2 was a target gene of miR‑486‑5p, and it was negatively regulated by miR‑486‑5p. It was also found that Smad2 expression was significantly increased in HS tissues and cells. Further analysis indicated that miR‑486‑5p mimic transfection inhibited the proliferation, induced cell apoptosis and increased G1/S phase arrest in hHSFs. Furthermore, the expression of cyclin‑dependent kinase (CDK)2, CDK4 and apoptosis regulator Bcl‑2 was repressed, while apoptosis regulator BAX expression was enhanced by miR‑486‑5p mimic transfection. Notably, the effects of miR‑486‑5p mimic on hHSFs were significantly eliminated by Smad2 plasmid transfection. Taken together, these results demonstrated that miR‑486‑5p inhibited the proliferation, induced apoptosis and increased G1/S phase arrest of hHSFs by targeting Smad2. miR‑486‑5p may be a promising therapeutic target for HS management.
MiR-564 promotes hypertrophic scar formation through TGF-β1 upregulation.
Xiao Long,Tang Tianyi,Huang Yu,Guo Jianxing
Giornale italiano di dermatologia e venereologia : organo ufficiale, Societa italiana di dermatologia e sifilografia
BACKGROUND:To evaluate the role of microRNA564 (miR-564) in the development of hypertrophic scar and investigate the possible mechanism of this process. METHODS:The hypertrophic scar (HS) tissues and adjacent normal skin (NS) tissues were selected from 10 patients. The fibrosis-related proteins were detected via hematoxylin eosin (HE) staining, Masson staining and immunohistochemical detection. The relative expression difference of miR-564 in NS tissues and HS tissues was detected via reverse transcription-polymerase chain reaction (RT-PCR). The protein expression difference of transforming growth factor-β1 (TGF-β1) in NS tissues and HS tissues was detected via Western blotting. Moreover, the hypertrophic scar fibroblast (HSF) cells were isolated from HS tissues and divided into four groups according to different treatment methods: blank control group, scramble-transfected negative control group, miR-564 inhibitor-transfected miR-564 inhibitor group, and miR-564 plasmid-transfected miR-564 mimic group. The expressions of TGF-β1 in blank control group, scramble group, miR-21 mimic group and miR-21 inhibitor group were detected via RT-PCR and Western blotting. RESULTS:MiR-564 was highly expressed in HSF cells. Compared with that of blank control group, the expression of TGF-β1 was down-regulated through inhibiting the miR-564 expression, thus inhibiting the activation and proliferation of HSF cells. However, the overexpression of miR-564 achieved the opposite results. CONCLUSIONS:Up-regulated mir-564 promoted the development of hypertrophic scar via enhancing the expression of TGF-β1. MiR-564 may be a potential novel molecular target for the treatment of hypertrophic scarring.
[Differential expression profile of microRNA between hyperplastic scar and normal skin].
Ning Pu,Liu De-wu,Mao Yuan-gui,Peng Yan,Lin Zun-wen,Liu De-ming
Zhonghua yi xue za zhi
OBJECTIVE:To explore the miRNA differential expression profiles of hyperplastic scar and normal skin so as to further elucidate the pathogenesis of hyperplastic scar and search for new therapeutic targets. METHODS:The total RNA was extracted from 5 human hyperplastic scar and normal skin tissues by Trizol. The specimens were collected from the First Affiliated Hospital of Nanchang University from November 2010 to May 2011, and purified by mirVana(TM) miRNA Isolation Kit and then labeled and hybridized by miRNA Complete Labeling and Hyb Kit. The images of hybridization were analyzed by the Feature Extraction (v10.7) software and the microarray results confirmed by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS:In hyperplastic scar, 92 miRNA genes were up-regulated and 13 down-regulated. The most significantly up-regulated miRNAs were hsa-miR-564 and hsa-miR-936, etc. while hsa-miR-451, hsa-miR-223, hsa-miR-363 and hsa-miR-29b-1* became significantly down-regulated. The findings of RT-PCR on hsa-miR-21 and hsa-miR-451 of regulation were in a high concordance with the microarray results. CONCLUSION:Distinct differences of miRNA expression between human hyperplastic scar and normal skin, it may be closely correlated with the formation, development and evolution of hyperplastic scar.
Regulatory mechanism of miR-29 over TGF-β1 and COL1 in scar cells.
Bi S,Cao C,Chai L-L,Li S-R,Yang D-Y
European review for medical and pharmacological sciences
OBJECTIVE:To study the regulatory mechanism of miR-29 over TGF-β1 and COL1 in scar cells. PATIENTS AND METHODS:5 clinical cases of hypertrophic scar (HS) skin and adjacent normal skin tissues were separated into fibroblast for primary culture and subculture before being observed morphologically and standard HE staining under an ordinary optical microscope. RT-PCR method was applied to test the expression level of miR-29, TGF-β1, and COL1 mRNA. ELISA method was applied to test the expression level of extracellular matrix COL1, fibronectin (FN) and α-SMA. The miR-29 overexpression vector was built and transfected in vitro. RT-PCR method was applied to test related genes and ELISA method was applied to test the expression level of the extracellular matrix. RESULTS:The color of karyon and cytoplasm of normal fibroblast were both light red, with little ECM. The color of karyon of scar fibroblast was blue. The cytoplasm was red of different degrees, with relatively much ECM, in deep blue color. Compared with that in the normal fibroblast group, the miR-29 mRNA in fibroblast in the scar group significantly decreased (p<0.05). The TGF-β1 and COL1 mRNA significantly increased (p<0.05). The COL1, FN and α-SMA level were significantly higher (p<0.05) than that in the normal group. These mRNAs levels in miR-29 overexpression group were lower than scar group but higher than the normal group. CONCLUSIONS:The expression of miR-29 which regulates the expression of TGF-β1 and COL1 and increases the level of ECM significantly decreases in scar cells. This one suggests a mechanism of the formation of the scars through TGF-β1 and COL1.
Integrated Interaction Network of MicroRNA Target Genes in Keloid Scarring.
Lyu Lechun,Zhao Yu,Lu Hongquan,Liu Zijie,Guo Jiazhi,Lu Di,Li Xiang
Molecular diagnosis & therapy
Keloids are a common dermal pathological disorder characterized by the excessive deposition of extracellular matrix components; however, the exact pathogenesis of the disease is still not clear. Studies increasingly suggest that microRNAs (miRNAs) can play a key role in the process of keloid scarring. In this study, the valuable miRNAs and target genes were screened and the interaction network was constructed. We also predicted target genes of reported miRNAs using TargetScan and miRTarBase software. Cytoscape 3.0.1 further showed the interaction network of miRNA and target genes. Among the various miRNAs involved in keloid pathogenesis, miRNA-21, miRNA-141-3p, miRNA-181a, and miRNA-205 were thought to up-regulate the proliferation and decrease apoptosis of keloid-derived fibroblasts through the PI3K/Akt/mammalian target of rapamycin (mTOR) signaling pathway. miRNA-637 and miRNA-1224 inhibited keloid fibroblasts proliferation and promoted apoptosis via the transforming growth factor (TGF)-β1/Smad3 signaling pathway. miRNA-21 was also involved in mitochondrial-mediated apoptosis and miRNA-31 targeted vascular endothelial growth factor (VEGF) signaling pathway. miRNA-199a may be one key factor in the cell cycle checkpoint signal pathway of keloid-derived fibroblasts. It was also found that miRNA-29a and miRNA-196a mediated collagen metabolism. These pivotal miRNAs and regulatory processes further improve the data on the epigenetic mechanisms of keloids and provide hope for the use of small molecules in the treatment of keloids.
miR-145-5p attenuates hypertrophic scar via reducing Smad2/Smad3 expression.
Shen Weichang,Wang Yingqian,Wang Dewei,Zhou Hong,Zhang Huafeng,Li Lingqiao
Biochemical and biophysical research communications
The study was designed to explore the underlying mechanism of micro ribonucleic acids (miR)-145-5p in the process of hypertrophic scar (HS). The difference in the relative content of miR-145-5p between HS and adjacent normal skin collected from 5 patients was detected via RT-PCR. Expressions of Smad2 and Smad3 with or without TGF-β1 was detected by western blotting. Fibroblasts apoptosis rate was examined by Annexin V/Propidium Iodide double staining. HS fibroblasts (HSFs) were isolated from HS tissues, cultured and then divided into control group, miR-145-5p inhibitor group (transfected with miR-145-5p inhibitor) and miR-145-5p mimic group (transfected with miR-145-5p plasmid) based on different treatment methods. Next, CCK-8 was employed to examine the function of miR-145-5p in HSF proliferation. Luciferase assay was conducted to confirm whether Smad2/3 were direct targets of miR-145-5p, and RT-PCR was done to measure the expression of miR-145-5p, Smad2/Smad3 and fibrosis-related genes of fibroblasts in three groups. Wound injury mice model was established to determine the function of miR-145-5p in regulating scar formation. miR-145-5p was found lowly expressed in HS tissues. Compared with Control group, miR-145-5p mimic decreased the levels of Smad2/3, arrested the activation and proliferation of HSFs and induced HSFs apoptosis. Overexpressing miR-145-5p achieved the contrary results. Smad2/3 was confirmed as the target of miR-145-5p. Moreover, miR-145-5p mimic decreased the recruitment of fibroblasts in vivo and decreased the expression of fibrosis-related genes after wound injury. In conclusion, miR-145-5p arrests the development of fibrogenesis and decreases HS formation by reducing the expression of Smad2/3. miR-145-5p may be an optional novel molecular target for treating HS.
MicroRNA-143-3p inhibits hyperplastic scar formation by targeting connective tissue growth factor CTGF/CCN2 via the Akt/mTOR pathway.
Mu Shengzhi,Kang Bei,Zeng Weihui,Sun Yaowen,Yang Fan
Molecular and cellular biochemistry
Post-traumatic hypertrophic scar (HS) is a fibrotic disease with excessive extracellular matrix (ECM) production, which is a response to tissue injury by fibroblasts. Although emerging evidence has indicated that miRNA contributes to hypertrophic scarring, the role of miRNA in HS formation remains unclear. In this study, we found that miR-143-3p was markedly downregulated in HS tissues and fibroblasts (HSFs) using qRT-PCR. The expression of connective tissue growth factor (CTGF/CCN2) was upregulated both in HS tissues and HSFs, which is proposed to play a key role in ECM deposition in HS. The protein expression of collagen I (Col I), collagen III (Col III), and α-smooth muscle actin (α-SMA) was obviously inhibited after treatment with miR-143-3p in HSFs. The CCK-8 assay showed that miR-143-3p transfection reduced the proliferation ability of HSFs, and flow cytometry showed that either early or late apoptosis of HSFs was upregulated by miR-143-3p. In addition, the activity of caspase 3 and caspase 9 was increased after miR-143-3p transfection. On the contrary, the miR-143-3p inhibitor was demonstrated to increase cell proliferation and inhibit apoptosis of HSFs. Moreover, miR-143-3p targeted the 3'-UTR of CTGF and caused a significant decrease of CTGF. Western blot demonstrated that Akt/mTOR phosphorylation and the expression of CTGF, Col I, Col III, and α-SMA were inhibited by miR-143-3p, but increased by CTGF overexpression. In conclusion, we found that miR-143-3p inhibits hypertrophic scarring by regulating the proliferation and apoptosis of human HSFs, inhibiting ECM production-associated protein expression by targeting CTGF, and restraining the Akt/mTOR pathway.
Overexpression of miR-200b inhibits the cell proliferation and promotes apoptosis of human hypertrophic scar fibroblasts in vitro.
Li Ping,He Quan-Yong,Luo Cheng-Qun
The Journal of dermatology
Hypertrophic scarring leads to a deformed appearance and contracted neogenetic tissue, resulting in physiological and psychological problems for patients. Millions of people suffer these discomforts each year. Emerging evidence has reported that miRNA contributed to hypertrophic scarring or keloid formation. In this study, nine hypertrophic scar samples and the matched normal skin tissues were used to perform a miRNA microarray. The results of miRNA array showed that miR-200b was downregulated by more than 2-fold, validated by qPCR in hypertrophic scar tissues and human hypertrophic scar fibroblasts, suggesting that there was an important correlation between miR-200b and hypertrophic scarring. We also found that miR-200b affected hypertrophic scarring through regulating the cell proliferation and apoptosis of human hypertrophic scar fibroblasts by affecting the collagen I and III synthesis, fibronectin expression and TGF-β1/α-SMA signaling. Thus, our study provides evidence to support that miR-200b may be a useful target for hypertrophic scarring management.
miR-29b promotes skin wound healing and reduces excessive scar formation by inhibition of the TGF-β1/Smad/CTGF signaling pathway.
Guo Jingdong,Lin Quan,Shao Ying,Rong Li,Zhang Duo
Canadian journal of physiology and pharmacology
The hypertrophic scar is a medical difficulty of humans, which has caused great pain to patients. Here, we investigated the inhibitory effect of miR-29b on scar formation. The scalded model was established in mice and miR-29b mimics or a negative control was subcutaneously injected into the injury skin. Then various molecular biological experiments were performed to assess the effect of miR-29b on scar formation. According to our present study, first, the results demonstrated that miR-29b was down-regulated in thermal injury tissue and miR-29b treatment could promote wound healing, inhibit scar formation, and alleviate histopathological morphologic alteration in scald tissues. Additionally, miR-29b treatment suppressed collagen deposition and fibrotic gene expression in scar tissues. Finally, we found that miR-29b treatment inhibited the TGF-β1/Smad/CTGF signaling pathway. Taken together, our data suggest that miR-29b treatment has an inhibitory effect against scar formation via inhibition of the TGF-β1/Smad/CTGF signaling pathway and may provide a potential molecular basis for future treatments for hypertrophic scars.
Identification of collagen 1 as a post-transcriptional target of miR-29b in skin fibroblasts: therapeutic implication for scar reduction.
Cheng Jie,Wang Yanling,Wang Dongmiao,Wu Yunong
The American journal of the medical sciences
BACKGROUND:Excessive collagen synthesis and deposit during skin wound healing results in scar formation. MicroRNAs (miRNAs) are endogenous noncoding RNA regulators that mediate diverse biological functions through repressing target genes and hold great potentials for clinical therapeutic applications. The aim of the present study was to identify miRNAs as post-transcriptional regulators of collagen 1 in skin fibroblasts. METHODS:miRNA candidates that potentially target collagen 1 were predicted by computational algorithms PicTar and TargetScan. The expression changes of collagen subunits 1α1 and 1α2 were measured by real-time reverse transcription-polymerase chain reaction and western blot after the primary skin fibroblasts were transfected with miR-29b mimics or inhibitor, respectively. A luciferase reporter assay was performed to further determine whether both collagen 1 subunits were probably direct targets of miR-29b. RESULTS:Computational predictions identified several miRNAs as possible regulators for collagen 1 synthesis, including miR-29b. Enforced overexpression of miR-29b resulted in remarkable decrease of collagen 1α1 and 1α2, whereas knockdown of endogenous miR-29b induced pronounced increase of collagen 1α1 and 1α2 at both the messenger RNA and the protein levels. The luciferase activities were significantly inhibited when cells were cotransfected with reporter constructs and miR-29 mimics in vitro. Moreover, miR-29b transcriptional abundance inversely related to the levels of both collagen 1 subunits in skin scar as compared with normal skin. CONCLUSIONS:Our data indicate that miR-29b is a potent post-transcriptional repressor of collagen 1 in skin fibroblasts and its deregulation might be implicated in scar formation, suggesting that miR-29b might represent a potential therapeutic target for scar reduction.
Non-Coding RNAs: The New Insight on Hypertrophic Scar.
Chen Ling,Li Jingyun,Li Qian,Yan Hui,Zhou Bei,Gao Yanli,Li Jun
Journal of cellular biochemistry
Hypertrophic scarring (HS), a fibroproliferative disorder caused by abnormal wound healing after skin injury, is characterized by excessive deposition of extracellular matrix and invasive growth of fibroblasts. Numerous studies have reported that non-coding RNAs (ncRNAs) including microRNAs (miRNAs, miRs) and long non-coding RNAs (lncRNAs) play important roles in HS formation. Exploring non-coding RNA-based methods to treat scar would be instrumental for devising new effective therapies against scar. J. Cell. Biochem. 118: 1965-1968, 2017. © 2017 Wiley Periodicals, Inc.
LncRNA COL1A2-AS1 inhibits the scar fibroblasts proliferation via regulating miR-21/Smad7 pathway.
Nong Qingwen,Li Shuntang,Wu Yajun,Liu Daen
Biochemical and biophysical research communications
lncRNA COL1A2-AS1 (COL1A2 antisense RNA 1), a lncRNA overexpressed in hypertrophic scar, has been demonstrated to be involved in the hypertrophic scar formation. However, the mechanisms of lncRNA COL1A2-AS1 inhibiting the scar fibroblasts proliferation remains not well understood. In this study, we demonstrated that lncRNA COL1A2-AS1 was upregulated in hypertrophic scar tissue and fibroblasts, and suppressed fibroblasts proliferation by promoting Smad7 expression. Furthermore, we found that miR-21 was involved in lncRNA COL1A2-AS1-induced expression of Smad7, by which COL1A2-AS1 acted as endogenous sponge to adsorb miR-21 and in turn regulated Smad7 and a cascade of molecular to play a protective role in hypertrophic scar. In addition, overexpression of miR-21 attenuated COL1A2-AS1-mediated proliferation suppression of hypertrophic scar fibroblasts. In conclusion, our study demonstrated that COL1A2-AS1/miR-21/Smad pathway plays an important role in inhibiting hypertrophic scar formation, and suggested this novel pathway may be a new target for hypertrophic scar treatment.
tRNA‑derived small RNAs: A novel class of small RNAs in human hypertrophic scar fibroblasts.
Zhang Yaping,Deng Qin,Tu Longxiang,Lv Dan,Liu Dewu
International journal of molecular medicine
tRNA‑derived small RNAs (tsRNAs) have been shown to play regulatory roles in many physiological and pathological processes. However, their roles in hypertrophic scars remain unclear. The present study investigated differentially expressed tsRNAs in human hypertrophic scar fibroblasts and normal skin fibroblasts via high‑throughput sequencing. Several dysregulated tsRNAs were validated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, target prediction, coexpression networks and competing endogenous RNA (ceRNA) networks were evaluated to discover the principal functions of significantly differentially expressed tsRNAs. In total, 67 differentially expressed tsRNAs were detected, of which 27 were upregulated and 40 downregulated in hypertrophic scar fibroblasts. The GO analysis indicated that the dysregulated tsRNAs are associated with numerous biological functions, including 'nervous system development', 'cell adhesion', 'focal adhesion', 'protein binding', 'angiogenesis' and 'actin binding'. KEGG pathway analysis revealed that the most altered pathways include 'Ras signaling pathway', 'Rap1 signaling pathway' and 'cGMP‑PKG signaling pathway'. The target genes of the differentially expressed tsRNAs participate in several signaling pathways important for scar formation. The results of RT‑qPCR were consistent with those of sequencing. MicroRNA (miR)‑29b‑1‑5p was identified as a target of tsRNA‑23678 and was downregulated in hypertrophic scar fibroblasts, constituting a negative regulatory factor for scar formation. Furthermore, tsRNA‑23761 acted as a ceRNA and bound to miR‑3135b to regulate the expression of miR‑3135b targets, including angiotensin‑converting enzyme. Collectively, these findings reveal that tsRNAs are differentially expressed in human hypertrophic scar fibroblasts, and may contribute to the molecular mechanism and treatment of hypertrophic scars.
miR-155 inhibits the formation of hypertrophic scar fibroblasts by targeting HIF-1α via PI3K/AKT pathway.
Wu Xue,Li Jun,Yang Xuekang,Bai Xiaozhi,Shi Jihong,Gao Jianxin,Li Yan,Han Shichao,Zhang Yijie,Han Fu,Liu Yang,Li Xiaoqiang,Wang Kejia,Zhang Julei,Wang Zheng,Tao Ke,Hu Dahai
Journal of molecular histology
Hypertrophic scar (HS) is a serious skin fibrotic disease characterized by the excessive proliferation of fibroblasts and often considered as a kind of benign skin tumor. microRNA-155 (miR-155) is usually served as a promising marker in antitumor therapy. In view of the similarities of hypertrophic scar and tumor, it is predicted that miR-155 may be a novel therapeutic target in clinical trials. Here we found the expression levels of miR-155 was gradually down regulated and HIF-1α was upregulated in HS tissue and HS derived fibroblasts (HFs). And cell proliferation was inhibited when miR-155 was overexpressed or HIF-1α was silenced. Moreover, overexpression of miR-155 in HFs could reduce the expression of collagens in vitro and inhibit the collagen fibers arrangement in vivo, whereas miR-155 knockdown gave opposite results. Furthermore, we found that miR-155 directly targeted the HIF-1α, which could also independently inhibit the expression of collagens in vitro and obviously improved the appearance and architecture of the rabbit ear scar in vivo when it was silencing. Finally, we found that PI3K/AKT pathway was enrolled in these processes. Together, our results indicated that miR-155 was a critical regulator in the formation and development of hypertrophic scar and might be a potential molecular target for hypertrophic scar therapy.
Regulation of RhoA by STAT3 coordinates glial scar formation.
Renault-Mihara Francois,Mukaino Masahiko,Shinozaki Munehisa,Kumamaru Hiromi,Kawase Satoshi,Baudoux Matthieu,Ishibashi Toshiki,Kawabata Soya,Nishiyama Yuichiro,Sugai Keiko,Yasutake Kaori,Okada Seiji,Nakamura Masaya,Okano Hideyuki
The Journal of cell biology
Understanding how the transcription factor signal transducer and activator of transcription-3 (STAT3) controls glial scar formation may have important clinical implications. We show that astrocytic STAT3 is associated with greater amounts of secreted MMP2, a crucial protease in scar formation. Moreover, we report that STAT3 inhibits the small GTPase RhoA and thereby controls actomyosin tonus, adhesion turnover, and migration of reactive astrocytes, as well as corralling of leukocytes in vitro. The inhibition of RhoA by STAT3 involves ezrin, the phosphorylation of which is reduced in STAT3-CKO astrocytes. Reduction of phosphatase and tensin homologue (PTEN) levels in STAT3-CKO rescues reactive astrocytes dynamics in vitro. By specific targeting of lesion-proximal, reactive astrocytes in - mice, we show that reduction of PTEN rescues glial scar formation in mice. These findings reveal novel intracellular signaling mechanisms underlying the contribution of reactive astrocyte dynamics to glial scar formation.
Umbilical Cord-Derived Mesenchymal Stem Cell-Derived Exosomal MicroRNAs Suppress Myofibroblast Differentiation by Inhibiting the Transforming Growth Factor-β/SMAD2 Pathway During Wound Healing.
Fang Shuo,Xu Chen,Zhang Yuntong,Xue Chunyu,Yang Chao,Bi Hongda,Qian Xijing,Wu Minjuan,Ji Kaihong,Zhao Yunpeng,Wang Yue,Liu Houqi,Xing Xin
Stem cells translational medicine
: Excessive scar formation caused by myofibroblast aggregations is of great clinical importance during skin wound healing. Studies have shown that mesenchymal stem cells (MSCs) can promote skin regeneration, but whether MSCs contribute to scar formation remains undefined. We found that umbilical cord-derived MSCs (uMSCs) reduced scar formation and myofibroblast accumulation in a skin-defect mouse model. We found that these functions were mainly dependent on uMSC-derived exosomes (uMSC-Exos) and especially exosomal microRNAs. Through high-throughput RNA sequencing and functional analysis, we demonstrated that a group of uMSC-Exos enriched in specific microRNAs (miR-21, -23a, -125b, and -145) played key roles in suppressing myofibroblast formation by inhibiting the transforming growth factor-β2/SMAD2 pathway. Finally, using the strategy we established to block miRNAs inside the exosomes, we showed that these specific exosomal miRNAs were essential for the myofibroblast-suppressing and anti-scarring functions of uMSCs both in vitro and in vivo. Our study revealed a novel role of exosomal miRNAs in uMSC-mediated therapy, suggesting that the clinical application of uMSC-derived exosomes might represent a strategy to prevent scar formation during wound healing. SIGNIFICANCE:Exosomes have been identified as a new type of major paracrine factor released by umbilical cord-derived mesenchymal stem cells (uMSCs). They have been reported to be an important mediator of cell-to-cell communication. However, it is still unclear precisely which molecule or group of molecules carried within MSC-derived exosomes can mediate myofibroblast functions, especially in the process of wound repair. The present study explored the functional roles of uMSC-exosomal microRNAs in the process of myofibroblast formation, which can cause excessive scarring. This is an unreported function of uMSC exosomes. Also, for the first time, the uMSC-exosomal microRNAs were examined by high-throughput sequencing, with a group of specific microRNAs (miR-21, miR-23a, miR-125b, and miR-145) found to play key roles in suppressing myofibroblast formation by inhibiting excess α-smooth muscle actin and collagen deposition associated with activity of the transforming growth factor-β/SMAD2 signaling pathway.
Study on the role of Hsa-miR-31-5p in hypertrophic scar formation and the mechanism.
Wang X,Zhang Y,Jiang B H,Zhang Q,Zhou R P,Zhang L,Wang Chen
Experimental cell research
Hypertrophic scar (HS) formation is associated with the fibrosis of fibrocytes caused by excessive extracellular matrix (ECM) synthesis and deposition, the initial event of HS formation. Our high throughput screen of miRNA expression profiles identified hsa-miR31-5p, whose transcription level was most differentially in normal skin fibroblasts (NS) and HS among other miRNAs. The level of hsa-miR31-5p in HS was significantly higher than in NS. In-vitro functional experiments showed hsa-miR31-5p knockdown remarkably suppressed the proliferation of hypertrophic scar fibroblasts (HSFBs) under hypoxia, promoted cell invasion, and inhibited the expression of Collagen I and III and Fibronectin (FN), suggesting that hsa-miR31-5p knockdown effectively reduces HS formation caused by excessive ECM synthesis and deposition in HSFBs under hypoxia. Mechanism study showed that the regulation of HS formation by hsa-miR31-5p was mediated by its target gene, factor-inhibiting HIF-1 (FIH): under hypoxia, hsa-miR31-5p down-regulated FIH and thus increased the level of hypoxia inducible factor-1α (HIF-1α), which subsequently activated the HIF-1α fibrosis regulation pathway in HSFBs, and stimulated the proliferation and ECM synthesis in HSFBs, eventually resulting in fibrosis and scar formation. The data also show that knockdown of hsa-miR31-5p in HSFBs impaired the trend of increased proliferation, reduced invasion and excessive ECM synthesis and deposition caused by HIF-1a activation under hypoxia through upregulating FIH, indicating that knockdown of hsa-miR31-5p effectively inhibits the formation of HS. In conclusion, hsa-miR31 -5p plays an important role in HS formation by inhibiting FIH and regulating the HIF-1α pathway. Therefore, hsa-miR31 -5p may be a novel therapeutic target for HS.
MicroRNA-494 targets PTEN and suppresses PI3K/AKT pathway to alleviate hypertrophic scar formation.
He Ting,Zhang Yue,Liu Yang,Wang Hongtao,Zhang Wanfu,Liu Jiaqi,Li Na,Li Yan,Wang Luxu,Xie Songtao,Hu Dahai
Journal of molecular histology
Hypertrophic scar is a common complication after skin injury. MicroRNAs have been reported related to hypertrophic scar through posttranscriptional control of genes. Hypertrophic scar-derived fibroblast model and mice incision model were used to see the expression of microRNA-494 and whether the level changes of microRNA-494 could affect scar formation. It was found that in hypertrophic scar, the expression of microRNA-494 decreased. However, after over-express microRNA-494 in fibroblasts, the levels of scar related molecules such as Col I, Col III increased. And when suppress the level of microRNA-494 in fibroblasts, the levels of collagen decreased. Moreover, the up-regulation of microRNA-494 led to decreased apoptosis of fibroblasts while the down-regulation of it led to increased apoptosis. Further, it was found that PTEN was one of the downstream targets of microRNA-494. The up-regulation of PTEN led to inactivation of PI3K/AKT pathway and the decreased expression of collagens. In conclusion, we confirmed that microRNA-494 could be a key regulator to suppress hypertrophic scar formation. The suppression of microRNA-494 could eliminate its inhibition effect to PTEN and finally decrease the expression of collagen and inhibit hypertrophic scar formation.
microRNA-203 Modulates Wound Healing and Scar Formation via Suppressing Hes1 Expression in Epidermal Stem Cells.
Zhou Ziheng,Shu Bin,Xu Yingbin,Liu Jian,Wang Peng,Chen Lei,Zhao Jingling,Liu Xusheng,Qi Shaohai,Xiong Kun,Wu Jun,Xie Julin
Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology
BACKGROUND/AIMS:Little is known how miR-203 is involved in epidermal stem cells (ESCs) differentiation and scar formation. METHODS:We first used luciferase assay to determine the interaction of miR-203 with the 3'-UTR in regulation of Hes1 expression. We then used flow cytometry to analyze the effects of miR-203 expression on the differentiation of ESCs to MFB by determination of CK15 ratio and α-SMA. To confirm the results of flow cytometry analysis, we used Western blot to examine the expression of α-SMA, Collagen I (Col I), and Collagen III (Col III), as well as the expression of Notch1, Jagged1, and Hes1 in ESCs after the treatment of pre-miR-203 or anti-miR-203. Finally, we examined the effects local anti-miR-203 treatment on would closure and scar formation using a mouse skin wound model. RESULTS:Pre-miR-203 treatment increased ESCs differentiation to MFB cells, as indicated by decreased CK15 ratio and increased MFB biomarkers. This phenomenon was reversed by overexpression of Hes1 in ESCs. In addition, skin incision increased expression of miR-203 in wound tissue. Local treatment of anti-miR-203 could accelerate wound closure and reduce scar formation in vivo, which was associated with increased re-epithelialization, skin attachment regeneration, and collagen reassignment. Finally, we confirmed that anti-miR-203 treatment could inhibit ESCs differentiation in vivo via increasing Hesl expression. CONCLUSION:Taken together, our results suggested that overexpression of miR-203 in ESCs after skin wound may be a critical mechanism underlying the scar formation.
MiR-1908 promotes scar formation post-burn wound healing by suppressing Ski-mediated inflammation and fibroblast proliferation.
Xie Chunhui,Shi Kai,Zhang Xi,Zhao Jingchun,Yu Jiaao
Cell and tissue research
The cell biological basis for scar formation is mainly via excessive fibroblast proliferation accompanied by hypernomic Col I accumulation and inflammation. The role of miR-1908 in scar formation has not been investigated. In this study, we found that miR-1908 expression was inversely associated with the scar suppressor Ski in normal, burn-wounded, healing and scar dermal tissues in humans. Bioinformatics and luciferase reporter gene assays confirmed that miR-1908 targeted the 3'UTR region of Ski mRNA and suppressed Ski expression. Next, human scar epidermal fibroblasts were isolated and the miR-1908 oligonucleotide mimic and inhibitor were respectively transfected into the cells. Western blot analysis proved that Ski expression was sharply reduced by the miR-1908 mimic. MTT and Cell Counting Kit-8 analyses showed that miR-1908 mimic transfection promoted cell proliferation. Simultaneously, data on real-time qPCR analysis indicated that expression of the fibrotic master gene TGF-β1, Ski-suppressing gene Meox2, Col I and proinflammatory markers IL-1α and TNF-α, were all significantly upregulated. In contrast, the miR-1908 inhibitor had a completely opposite effect on cell proliferation and gene expression. The mimic and inhibitor were locally injected into rats with abdominal burn-wounded scars during a 180-day, post-healing experiment. The miR-1908 mimic injection significantly reduced Ski expression, as well as the area, volume and fibrosis of scars in vivo. And, in contrast, the miR-1908 inhibitor injection had an opposite effect to that of the miR-1908 mimic injection. In conclusion, miR-1908 had a positive role in scar formation by suppressing Ski-mediated inflammation and fibroblast proliferation in vitro and in vivo.
Tetrandrine induces microRNA differential expression in human hypertrophic scar fibroblasts in vitro.
Ning P,Peng Y,Liu D W,Hu Y H,Liu Y,Liu D M
Genetics and molecular research : GMR
MicroRNAs (miRNAs) have recently been shown to play a role in normal wound healing process. miRNAs may be linked to pathologic wound healing and closely related to the formation of hypertrophic scars. This study aimed to explore the effects of tetrandrine on the miRNA expression profile in human hypertrophic scar fibroblasts (HSFs) in vitro. HSFs were randomly divided into two groups: the tetrandrine treatment group and the control group. The experimental and control groups were collected and analyzed by miRNA array after a 48-h culture. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to confirm the array results. The targets of differentially expressed miRNA were functionally annotated using bioinformatic approaches. miRNA microarray analysis identified 193 differentially expressed miRNAs and the expression of 186 miRNAs in the experimental group decreased while that of 7 miRNAs increased compared to the control group. The most significantly downregulated miRNA was hsa-miR-1246, and hsa-miR-27b had the highest expression level. Significant differentially expressed miRNAs were predicted to be related to several important signaling pathways related to scar wound healing. The differential miRNA expression identified in this study provides the experimental basis for further understanding the anti-fibrosis effect of tetrandrine.
Effects of photoelectric therapy on proliferation and apoptosis of scar cells by regulating the expression of microRNA-206 and its related mechanisms.
Zhang Song,Zhao Zhen-Min,Xue Hong-Yu,Nie Fang-Fei
International wound journal
Human skin fibroblast (HSF) cells were irradiated with different energy lasers to detect cell proliferation, apoptosis, and expression of microRNA-206 and protein, and to further summarise the therapeutic effect of laser on scar cells. Human scar cell line HSF cells were cultured in three groups. The control group was not irradiated by laser, the low-energy group was irradiated by 10 J/cm laser, and the high-energy group was irradiated by 20 J/cm laser. After irradiation, HSF cells were cultured for 20 hours. Cell proliferation was detected by MTT assay. Cell cycle and apoptosis were detected by flow cytometry. Transwell migration assay was used to detect cell migratory ability. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect miR-206 and mTOR gene levels. The levels of MMP-9, Bax, Bcl-2, cyclin D1, and mTOR signalling pathway proteins were detected by Western blotting assays. The results showed that after laser irradiation, the proliferation of cells decreased, and the difference between the control group and the experimental group was significant (P < .05). The higher the energy was, the greater the upregulation of apoptosis was. Apoptosis and cell migration increased (P < .05). The expressions of microRNA-206, MMP-9, and Bax were upregulated, while the expressions of mTOR, Bcl-2, and cyclin D1 were downregulated. To sum up, laser irradiation can significantly inhibit the proliferation of HSF cells, affect cell cycle, and increase cell apoptosis and migratory ability.
Collagen-based materials combined with microRNA for repairing cornea wounds and inhibiting scar formation.
Zhao Xuan,Song Wenjing,Chen Yawei,Liu Sa,Ren Li
The cornea repair materials amniotic membrane, acellular corneal stroma, and natural polymer-based materials have excellent biocompatibility and support epithelization. However, few researchers have focused on corneal stromal wound healing repair with regard to scar formation and transparency improvements. Herein, we introduced nanocomplexes of gold nanoparticles (AuNPs) and microRNA-133b (miR-133b) into collagen-based material to achieve corneal repair and scar inhibition. We evaluated the cytocompatibility of AuNPs and the ability of miRNA-133b to inhibit myofibroblast transformation in vitro. The AuNPs had no cytotoxicity at working concentrations, and AuNP/miR-133b complexes down-regulated the expression of myofibroblast transformation genes (alpha-smooth muscle actin and type I collagen) in corneal stromal cells. We then loaded AuNP/miR-133b complexes into collagen using two methods: loading on the surface (Col-AMS) and loading inside (Col-AMI) the collagen membrane. The results showed that AuNP/miR-133b introduction did not affect water content, light transmittance, or the mechanical properties of the collagen membrane. MiR-133b can maintain its integrity during the preparation of materials. In vivo lamellar keratoplasty results showed that the cornea epithelized completely and rapidly. Corneas without transplantation and with the transplantation of a non-modified collagen membrane became cloudy after trauma to different degrees after approximately 14 days. Interestingly, cornea transparency was retained after transplantation with Col-AMS and Col-AMI. Hematoxylin-eosin-stained histologic sections revealed that the corneas transplanted with Col-AMS and Col-AMI were similar to healthy corneas. Immunohistochemical staining revealed lower a-SM and Col-1 expression in corneal stroma after transplantation with collagen combined with AuNP/miR-133b. Our results thus suggest that collagen membranes combined with AuNP/miR-133b complexes can rapidly repair corneas and effectively inhibit scar formation.