Dual Role of MiR-21-Mediated Signaling in HUVECs and Rat Surgical Flap under Normoxia and Hypoxia Condition.
Chang Chih-Hau,Yen Meng-Chi,Liao Ssu-Hui,Hsu Yu-Ling,Lai Chung-Sheng,Kuo Yur-Ren,Hsu Ya-Ling
International journal of molecular sciences
Restoring sufficient vascularity of the ischemia/hypoxia flap is always the critical issue in flap surgeries. In a previous studies microRNA-21 (miR-21) expression was upregulated after rat skin flap surgery. MiR-21 has been reported to be induced by hypoxia and the function of miR-21 involves in the process of angiogenesis. However, the precise regulatory mechanisms in miR-21-mediated pathways are still unclear. These issues were investigated via in vitro and in vivo experiments in this study. In human umbilical vein endothelial cells (HUVEC), the expression of hsa-miR-21-5p was induced after hypoxic culture and the induction of hsa-miR-21-5p was suppressed after sequential normoxic culture. Moreover, transfection of hsa-miR-21-5p mimic enhanced tube formation capacity in normoxia, but attenuated it in hypoxia. Furthermore, bioinformatic analysis suggested that SMAD7 was a predicted target of hsa-miR-21-5p. Our results demonstrated the effect of hsa-miR-21-5p was different on SMAD7 expression in normoxia and hypoxia. In rat skin flaps, blockage of miR-21-5p significantly increased angiogenesis via analysis of color laser Doppler imaging and repressed SMAD7 expression in ischemic skin tissue. Our study showed the opposite effect of miR-21-5p mediating angiogenesis in normoxia and hypoxia, providing important implications regarding the design of novel miRNA-based therapeutic strategies in flap surgeries.
Serum microRNA-21 and microRNA-221 as potential biomarkers for cerebrovascular disease.
Tsai Pei-Chien,Liao Yi-Chu,Wang Yung-Song,Lin Hsiu-Fen,Lin Ruey-Tay,Juo Suh-Hang Hank
Journal of vascular research
BACKGROUND/AIMS:MicroRNA miR-21, miR-221 and miR-145 have been implicated in the cardiovascular system. We aimed to compare the serum levels of the three microRNAs (miRNAs) in different severities of cerebrovascular diseases and evaluate the feasibility of using these miRNAs as biomarkers for stroke. METHODS:We enrolled 167 subjects with ischemic stroke, 66 atherosclerosis subjects with any carotid plaque score and 157 healthy controls. These three types of subjects represent three levels of severity in cerebrovascular diseases. Analysis of covariance was used to evaluate the relationship between miRNAs and disease severity with adjustment for conventional risk factors. To test the prediction for stroke, we built regression models containing the serum miRNA levels and risk factors. Prediction capabilities were compared by the receiver operating characteristic curves. RESULTS:Stroke patients and atherosclerosis subjects had significantly higher miR-21 and lower miR-221 serum levels than healthy controls, while the miR-145 expression was too low to provide useful information in this regard. The best model showed that miR-21 and miR-221 were independent predictors. There was a 6.2-fold increase for stroke risk when miR-21 levels increase by log₁₀2(-ΔCt) = 1, while a 10.4-fold increase was observed as miR-221 decreases by log₁₀2(-ΔCt) = 1. CONCLUSIONS:Serum miR-145 was not detected in over 50% of the patients and it may not be an ideal marker to predict stroke. MiR-21 and miR-221 are novel biomarkers for atherosclerosis and stroke.
Paeonol protects rat vascular endothelial cells from ox-LDL-induced injury in vitro via downregulating microRNA-21 expression and TNF-α release.
Liu Ya-rong,Chen Jun-jun,Dai Min
Acta pharmacologica Sinica
AIM:Paeonol (2'-hydroxy-4'-methoxyacetophenone) from Cortex moutan root is a potential therapeutic agent for atherosclerosis. This study sought to investigate the mechanisms underlying anti-inflammatory effects of paeonol in rat vascular endothelial cells (VECs) in vitro. METHODS:VECs were isolated from rat thoracic aortas. The cells were pretreated with paeonol for 24 h, and then stimulated with ox-LDL for another 24 h. The expression of microRNA-21 (miR-21) and PTEN in VECs was analyzed using qRT-PCR. The expression of PTEN protein was detected by Western blotting. TNF-α release by VECs was measured by ELISA. RESULTS:Ox-LDL treatment inhibited VEC growth in dose- and time-dependent manners (the value of IC50 was about 20 mg/L at 24 h). Furthermore, ox-LDL (20 mg/L) significantly increased miR-21 expression and inhibited the expression of PTEN, one of downstream target genes of miR-21 in VECs. In addition, ox-LDL (20 mg/L) significantly increased the release of TNF-α from VECs. Pretreatment with paeonol increased the survival rate of ox-LDL-treated VECs in dose- and time-dependent manners. Moreover, paeonol (120 μmol/L) prevented ox-LDL-induced increases in miR-21 expression and TNF-α release, and ox-LDL-induced inhibition in PTEN expression. A dual-luciferase reporter assay showed that miR-21 bound directly to PTEN's 3'-UTR, thus inhibiting PTEN expression. In ox-LDL treated VECs, transfection with a miR-21 mimic significantly increased miR-21 expression and inhibited PTEN expression, and attenuated the protective effects of paeonol pretreatment, whereas transfection with an miR-21 inhibitor significantly decreased miR-21 expression and increased PTEN expression, thus enhanced the protective effects of paeonol pretreatment. CONCLUSION:miR-21 is an important target of paeonol for its protective effects against ox-LDL-induced VEC injury, which may play critical roles in development of atherosclerosis.
microRNA-21 protects against ischemia-reperfusion and hypoxia-reperfusion-induced cardiocyte apoptosis via the phosphatase and tensin homolog/Akt-dependent mechanism.
Yang Qiong,Yang Kan,Li Anying
Molecular medicine reports
Myocardial tissue injury caused by ischemia and hypoxia is a major cause of fatal diseases, including coronary atherosclerosis resulting from myocardial infarction and stroke. A number of microRNAs have been demonstrated to function as protectors against ischemia-reperfusion (I/R) and/or hypoxia-reperfusion (H/R)-induced myocardial injury, including microRNA-21 (miR-21). However, the protective mechanism of miR-21 has not been fully elucidated. The present study demonstrated that miR-21 had an anti-apoptotic role in I/R-induced myocardial damage in vivo and in H/R-induced H9C2 cell death in vitro. Of note, the present study indicates that a common molecular mechanism is likely to exist in I/R- and H/R-induced cardiocyte apoptosis. During I/R and H/R, forced expression of miR-21 upregulated the Akt signaling activity via suppressing the expression of phosphatase and tensin homolog (PTEN) and the increased activity of Akt signaling further inhibited apoptosis partially by increasing the ratio of B-cell lymphoma 2(Bcl-2)/Bcl-2-associated X protein, which further suppressed the expression of caspase-3. In conclusion, to the best of our knowledge, it was shown for the first time that miR-21 had a protective role in I/R- and H/R-induced cardiocyte apoptosis via the PTEN/Akt-dependent mechanism. The present study indicates that miR-21 may be a promising agent for the treatment of I/R and H/R-induced myocardial injury.
MicroRNA-21, induced by high glucose, modulates macrophage apoptosis via programmed cell death 4.
Shang Yuan-Yuan,Fang Ning-Ning,Wang Feng,Wang Hui,Wang Zhi-Hao,Tang Meng-Xiong,Peng Jie,Zhang Yun,Zhang Wei,Zhong Ming
Molecular medicine reports
MicroRNA-21 (miR-21) has been found to promote cell proliferation and survival. It has also been shown to exhibit an increased expression in a number of forms of cardiovascular disease. However, the mechanisms underlying the involvement of miR-21 in atherosclerosis remain to be elucidated. In the present study, it was demonstrated that miR-21 was upregulated in a time-dependent manner in response to high-concentration glucose stimulation in Raw 264.7 macrophages. High concentrations of glucose induce macrophage apoptosis. miR-21-inhibited macrophages treated with a normal concentration of glucose exhibited increased levels of cell apoptosis and augmented levels of activated caspase-3, while cells treated with an miR-21 inhibitor and a high concentration of glucose, revealed significantly increased levels of apoptosis. In addition, inhibition of miR-21 increased mRNA and protein levels of programmed cell death 4 (PDCD4), which, by contrast, were reduced in miR-21-inhibited cells that had been treated with a high concentration of glucose. In conclusion, miR-21 is sensitive to high-concentration glucose treatment in macrophages, and appears to have a protective effect in macrophage apoptosis induced by high concentrations of glucose via PDCD4.
MiR-21 suppresses endothelial progenitor cell proliferation by activating the TGFβ signaling pathway via downregulation of WWP1.
Zuo Keqiang,Li Maoquan,Zhang Xiaoping,Lu Chenghui,Wang Shi,Zhi Kangkang,He Bin
International journal of clinical and experimental pathology
Endothelial damage is strongly associated with cardiovascular diseases such as atherosclerosis. Bone marrow-derived endothelial progenitor cells (EPCs) play an important role in the maintenance of endothelial homeostasis and contribute to re-endothelialization of injured vessels as well as revascularization of ischemic tissues. MicroRNAs (miRNAs) have been reported to regulate EPC biological functions. In this study, we found that EPCs of atherosclerosis patients and EPCs exposed to hypoxia have increased expression of miRNA-21 (miR-21) as well as diminished ability to proliferate. MiR-21 knockdown rescued hypoxia-induced growth arrest in EPCs. Next, we used a luciferase reporter assay to demonstrate that miR-21 downregulates the expression of WW domain-containing protein 1 (WWP1), a negative regulator of TGFβ signaling, by directly targeting the 3'-UTR of WWP1. Finally, miR-21 overexpression or WWP1 knockdown in EPCs significantly activates the TGFβ signaling pathway and inhibits cell proliferation. Taken together, our results indicate that miR-21 suppresses EPC proliferation by activating the TGFβ signaling pathway via downregulation of WWP1. These findings may help the development of strategies to enhance the vitality of EPCs for therapeutic applications.
MicroRNA-21 negatively regulates Treg cells through a TGF-β1/Smad-independent pathway in patients with coronary heart disease.
Li Sihui,Fan Qian,He Shaolin,Tang Tingting,Liao Yuhua,Xie Jiangjiao
Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology
BACKGROUND:CD4+CD25+FoxP3+ regulatory T cells (Treg cells) play a protective role against the development and progression of the inflammatory disease atherosclerosis (AS). MicroRNA-21 (miR-21) is expressed in Treg cells and is up-regulated in the context of AS and other inflammatory diseases. AIMS:This study aimed to determine the role of miR-21 in Treg cell regulation and gene expression during the development of AS in patients with coronary heart disease (CHD). METHODS AND RESULTS:MiR-21 expression in peripheral blood mononuclear cells (PBMCs) was significantly up-regulated in patients with CHD (acute myocardial infarction (AMI) group, n=24; unstable angina (UA) group, n=21; stable angina (SA) group, n=24) compared with patients with chest pain syndrome (CPS, n=27), and miR-21 expression showed an increasing trend from SA to UA to AMI patients. Moreover, flow cytometry analysis indicated that the frequencies of circulating Treg cells decreased in a manner proportionate opposite with the level of miR-21. Quantitative real-time PCR (qRT-PCR) revealed a decrease in mRNA expression of forkhead box P3 (foxp3), transforming cell growth factor beta 1(TGF-β1) and smad7 (a known target gene of miR-21). ELISA analysis revealed a decrease in TGF-β1 secreted into the plasma. In addition, we transfected PBMCs with a miRNA negative control (NS-m), a miR-21 mimic (miR-21-m), a miRNA inhibitor negative control (NS-i), or a miR-21 inhibitor(miR-21-i). Up-regulation of miR-21 decreased the frequency of circulating Treg cells, decreased the expression levels of foxp3, TGF-β1 and smad7, and decreased the amount of TGF-β1 secreted into the plasma. Consistent with these observations, miR-21 down-regulation increased the frequency of circulating Treg cells, increased the expression of foxp3, TGF-β1 and smad7, and increased the amount of TGF-β1 secreted into the plasma. CONCLUSIONS:Because the smad7 expression pattern was similar to that of TGF-β, our study suggests that miR-21 can negatively regulate the frequency of circulating Treg cells through a TGF-β1/smad-independent signaling pathway in PBMCs.
Association of serum microRNA-21 levels with Visfatin, inflammation, and acute coronary syndromes.
Darabi Faramarz,Aghaei Mahmoud,Movahedian Ahmad,Elahifar Armin,Pourmoghadas Ali,Sarrafzadegan Nizal
Heart and vessels
MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression. It seems that microRNA-21 (miR-21) and Visfatin, a novel adipocytokine, play roles in inflammation and atherosclerosis. The aim of this study was to investigate the association of miR-21 with Visfatin, inflammation, atherosclerosis and acute coronary syndrome (ACS). Based on coronary angiography and electrocardiogram (ECG), 53 patients with ACS and 52 patients with stable CAD were enrolled in this study. We assayed serum miR-21, Visfatin, and routine chemistries using quantitative reverse transcriptase polymerase chain reaction (QRT-PCR), enzyme-linked immunosorbent assay (ELISA) and automated analyzer, respectively. We used a regression analysis to describe the relationship between the variables. Serum miR-21 level in 2 value was significantly higher in ACS patients (10.52 ± 1.01-fold) than the stable CAD patients (4.4 ± 0.79-fold) (F = 4.59, p < 0.001). In addition, serum Visfatin was significantly higher in ACS patients (17.5 ± 0.61 ng/ml) than the stable CAD patients (12.7 ± 0.49 ng/ml) (F = 2.62, p < 0.001). Furthermore, the serum miR-21 level correlated positively with serum Visfatin level (r = 0.26, p = 0.008), hs-CRP (r = 0.29, p = 0.003), age (r = 0.21, p = 0.034) and negatively with HDL-cholesterol (r = -0.28, p = 0.004). We concluded that the increased serum miR-21 and Visfatin may be involved in the pathogenesis of ACS through promoting inflammation or may result from inflammatory responses to ACS. Furthermore, the potential role of miR-21 and Visfatin in plaque instability and inflammation warrants further investigations.
Ursolic acid induced anti-proliferation effects in rat primary vascular smooth muscle cells is associated with inhibition of microRNA-21 and subsequent PTEN/PI3K.
Jiang Qixiao,Han Yantao,Gao Hui,Tian Rong,Li Ping,Wang Chunbo
European journal of pharmacology
This study focused on the anti-proliferation effects of ursolic acid (UA) in rat primary vascular smooth muscle cells (VSMCs) and investigated underlying molecular mechanism of action. Rat primary VSMCs were pretreated with UA (10, 20 or 30μM) or amino guanidine (AG, 50μM) for 12h or with PI3K inhibitor LY294002 for 30min or with Akt inhibitor MK2206 for 24h, then 10% fetal bovine serum was used to induce proliferation. CCK-8 was used to assess cell proliferation. To explore the mechanism, cells were treated with UA (10, 20 or 30μM), LY294002 or MK2206, or transient transfected to inhibit miRNA-21 (miRNA-21) or to overexpress PTEN, then quantitative real-time PCR was used to assess the mRNA levels of miRNA-21 and phosphatase and tensin homolog (PTEN) for cells treated with UA or miRNA-21 inhibitor; western blotting was used to measure the protein levels of PTEN and PI3K. UA exerted significant anti-proliferation effects in rat primary VSMCs. Furthermore, UA inhibited the expression of miRNA-21 and subsequently enhanced the expression of PTEN. PTEN was found to inhibit the expression of PI3K. In conclusion, UA exerts anti-proliferation effects in rat primary VSMCs, which is associated with the inhibition of miRNA-21 expression and modulation of PTEN/PI3K signaling pathway.
MicroRNA-21 suppresses ox-LDL-induced human aortic endothelial cells injuries in atherosclerosis through enhancement of autophagic flux: Involvement in promotion of lysosomal function.
Tang Feng,Yang Tian-Lun,Zhang Zhen,Li Xiao-Gang,Zhong Qiao-Qing,Zhao Ting-Ting,Gong Li
Experimental cell research
Atherosclerosis is a common pathological basis of cardiovascular disease and remains the leading cause of mortality. Endothelial cell (EC) injury and autophagy dysfunction have been proved to contribute to the development of atherosclerosis. Recently, accumulating evidence confirms that microRNAs (miRNAs) have emerged as vital regulators and fine-tuners of various pathophysiological cellular impacts and molecular signaling pathways involved in atherosclerosis. Herein, the objective of the present study was to explore the biological function of miR-21 in oxidized low-density lipoprotein (ox-LDL)-induced human aortic endothelial cells (HAECs) injury and the underlying molecular mechanism. The results showed that ox-LDL treatment significantly decreased HAECs viability, increased caspase-3 activity, apoptosis ratio and Bax protein expression, and reduced Bcl-2 protein expression resulting in EC injuries. Simultaneously, ox-LDL treatment obviously reduced miR-21 level in a time-and dose-dependent manner. Notably, ox-LDL-induced EC injuries were abolished by miR-21 mimics transfection. In addition, miR-21 mimics alleviated ox-LDL-induced impaired autophagic flux as illustrated by the increases in LC3-II/LC3-I ratio and Beclin-1 protein expression, and the decrease in p62 protein expression in HAECs. Moreover, ox-LDL suppressed the expressions of lysosomal membrane protein (LAMP1) and cathepsin D proteins, and attenuated cathepsin D activity in HAECs, leading to lysosomal dysfunction, while these effects were also blocked by miR-21 mimics. These findings indicated that miR-21 restored impaired autophagic flux and lysosomal dysfunction, thereby attenuating ox-LDL-induced HAECs injuries.
MicroRNA-21 drives the switch to a synthetic phenotype in human saphenous vein smooth muscle cells.
Alshanwani Aliah R,Riches-Suman Kirsten,O'Regan David J,Wood Ian C,Turner Neil A,Porter Karen E
Cardiovascular disease is a leading cause of morbidity and mortality. Smooth muscle cells (SMC) comprising the vascular wall can switch phenotypes from contractile to synthetic, which can promote the development of aberrant remodelling and intimal hyperplasia (IH). MicroRNA-21 (miR-21) is a short, non-coding RNA that has been implicated in cardiovascular diseases including proliferative vascular disease and ischaemic heart disease. However, its involvement in the complex development of atherosclerosis has yet to be ascertained. Smooth muscle cells (SMC) were isolated from human saphenous veins (SV). miR-21 was over-expressed and the impact of this on morphology, proliferation, gene and protein expression related to synthetic SMC phenotypes monitored. Over-expression of miR-21 increased the spread cell area and proliferative capacity of SV-SMC and expression of MMP-1, whilst reducing RECK protein, indicating a switch to the synthetic phenotype. Furthermore, platelet-derived growth factor BB (PDGF-BB; a growth factor implicated in vasculoproliferative conditions) was able to induce miR-21 expression via the PI3K and ERK signalling pathways. This study has revealed a mechanism whereby PDGF-BB induces expression of miR-21 in SV-SMC, subsequently driving conversion to a synthetic SMC phenotype, propagating the development of IH. Thus, these signaling pathways may be attractive therapeutic targets to minimise progression of the disease. © 2018 IUBMB Life, 70(7):649-657, 2018.
Long noncoding RNA TCONS_00024652 regulates vascular endothelial cell proliferation and angiogenesis via microRNA-21.
Halimulati Muertiza,Duman Bagedati,Nijiati Julaiti,Aizezi Abudoureyimu
Experimental and therapeutic medicine
Acute coronary syndrome caused by the rupture of atherosclerotic plaques is one of the primary causes of major cardiovascular events, and neovascularization within the plaque is closely associated with its stability. Long noncoding RNA (lncRNAs) is a type of noncoding RNA that serves a crucial role in regulating vascular endothelial cells (VECs). The aim of the present study was to investigate the effect of lncRNA TCONS_00024652 on the proliferation and angiogenesis of VECs following stimulation with TNF-α. The expression of lncRNA and miRNA was measured in human umbilical vein endothelial cells (HUVECs) by reverse transcription-quantitative polymerase chain reaction. Cell proliferation was measured using a Cell Counting Kit-8 assay. Wound healing and tube formation assays were performed to determine cell migration and angiogenesis. Interactions between TCONS_00024652 and miR-21 were determined using bioinformatics and a dual-luciferase reporter assay. The results demonstrated that TCONS_00024652 is highly expressed in TNF-α-induced HUVECs. Functional assays demonstrated that the dysregulated expression of TCONS_00024652 promotes endothelial cell proliferation and angiogenesis, whereas TCONS_00024652 knockdown induces the opposite effects. Bioinformatics analysis using starBase predicted putative binding at the 3'-untranslated region of TCONS_00024652 and miR-21 and luciferase reporter assays further verified this interaction. The results of the present study suggest that the targeting of TCONS_00024652 by miR-21 may be a potential method of improving vascular endothelial dysfunction, neovascularization maturation and plaque stabilization.
miRNA-21 inhibition inhibits osteosarcoma cell proliferation by targeting PTEN and regulating the TGF-β1 signaling pathway.
Hu Xiaoming,Li Lihua,Lu Yao,Yu Xiangui,Chen Hai,Yin Qingshui,Zhang Yu
The present study aimed to investigate the role of microRNA (miRNA)-21 in the growth of osteosarcoma. A total of 46 patients with osteosarcoma and 20 healthy controls were included in the study. The expression of miRNA-21 was detected by reverse transcription-quantitative polymerase chain reaction in tumor tissues and adjacent healthy tissues from patients with osteosarcoma, as well as the serum of patients with osteosarcoma and the healthy controls was. Receiver operating characteristic curve analysis was performed to evaluate the diagnostic values of serum miRNA-21 for osteosarcoma at different T stages. Survival curves plotted using the Kaplan-Meier method were used to evaluate the prognostic value. miRNA-21 knockdown osteosarcoma cell lines were established and their effects on cell proliferation were explored using a Cell Counting Kit-8 assay. The effect of miRNA-21 knockdown on the protein expression of phosphatase and tensin homolog (PTEN) and transforming growth factor (TGF)-β1 was detected by western blot analysis. The expression levels of miRNA-21 in tumor tissues were significantly higher compared with the adjacent healthy tissues in the majority of patients with osteosarcoma. The serum miRNA-21 increased as the T-stage of osteosarcoma increased. Serum miRNA-21 may be used to effectively diagnose osteosarcoma and predict the prognosis of the disease. miRNA-21 knockdown inhibited the proliferation of osteosarcoma and promoted the expression of PTEN and TGF-β1 proteins in the osteosarcoma cells. However, TGF-β1 inhibitor treatment reduced the inhibitory effects of miRNA-21 knockdown on osteosarcoma cell proliferation. In conclusion, miRNA-21 inhibition may inhibit osteosarcoma cell proliferation by targeting PTEN and regulating the TGF-β1 signaling pathway.
MicroRNA-21 mediates high phosphate-induced endothelial cell apoptosis.
Li Zhaoyu,Wiernek Szymon,Patterson Cam,Wang Huanchen,Qi Guoxian,Dai Xuming
American journal of physiology. Cell physiology
Hyperphosphatemia, the elevated level of inorganic phosphate (Pi) in serum, is associated with increased cardiovascular morbidities and mortality. The effects of high Pi on endothelial cells are not well studied. This study investigated high Pi-induced endothelial cell apoptosis and the role of microRNA-21. Mouse myocardial endothelial cells (MEC) were cultured in normal (1 mM) and high (5 mM) Pi conditions. Apoptosis was detected by TUNEL staining and flow cytometry. MicroRNA profiles of MEC response to changes in Pi concentration were obtained using gene expression arrays. Expression levels of the microRNA-21 target genes, programmed cell death gene 4 ( PDCD4), poly(ADP-ribose) polymerase ( PARP), and phosphatase and tensin homolog ( PTEN), as well as NF-κB were measured by Western blotting and RT-PCR. MicroRNA-21-specific inhibitors and mimics were used to study effects of microRNA-21 on MEC apoptosis and gene expression regulations. High Pi induced MEC apoptosis and upregulated microRNA-21 expression. MicroRNA-21-specific mimics reproduced high Pi-induced apoptosis in normal Pi medium, and microRNA-21 inhibitors ameliorated the high Pi induction of apoptosis, suggesting that microRNA-21 mediated high Pi-induced MEC apoptosis. The microRNA-21 targets PDCD4, PTEN, PARP, and NF-κB were significantly downregulated in high Pi conditions. High Pi-induced downregulation of PDCD4 was abolished by microRNA-21 inhibitors and selective ERK inhibitor (selumetinib) and was reproduced by microRNA-21 mimics. Inhibitors and mimics of microRNA-21 did not have effects on high Pi-induced NF-κB downregulation. Selumetinib blocked high Pi-induced NF-κB downregulation. MicroRNA-21 mediates high Pi-induced endothelial cell apoptosis, which involves an ERK1/2/microRNA-21/PDCD4 pathway. High Pi-induced downregulation of NF-κB expression is mediated by an ERK1/2 signaling-dependent but microRNA-21-independent mechanism.
MicroRNA-21 attenuates oxygen and glucose deprivation induced apoptotic death in human neural stem cells with inhibition of JNK and p38 MAPK signaling.
Chen Rui,Tai Yingchun,Zhang Yurong,Wang Li,Yang Yang,Yang Nan,Ma Shuyun,Xue Fangfang,Wang Jianjun
Neural stem cells (NSCs) persist in the mammalian brain throughout life and protect against hypoxia-ischemia injury. NSCs are being increasingly recognized as a novel therapeutic target for various neurological disorders. Previous research indicates that miR-21 attenuates hypoxia-ischemia induced apoptotic death in various cell types. However, whether miR-21 plays a role in this protective effect mediated by NSCs is unknown, particularly in human NSCs (hNSCs). The present study investigated whether miR-21 could prevent hNSC injury induced by oxygen and glucose deprivation (OGD). Upon challenge with OGD treatment, loss of cell viability was observed in cultured hNSCs, as shown by CCK-8 assay. Moreover, quantitative real-time PCR (qRT-PCR) analysis indicated that expression of miR-21 increased in a time-dependent manner. TUNEL staining and Western blotting analysis showed that overexpression of miR-21 inhibited excessive hNSCs death induced by OGD treatment. Accordingly, knock down of miR-21 attenuated the neuroprotective effect observed in response to OGD treatment. Furthermore, JNK and p38 MAPKs inhibition was observed after overexpression of miR-21, and knock down of miR-21 had the opposite effect. We suggest that miR-21 prevents OGD-induced hNSCs death and apoptotic-associated protein activities through inhibiting JNK and p38 pathways in cultured hNSCs. Our findings may help to develop strategies for enhancing resident and transplanted NSCs survival after hypoxia-ischemic brain damage.
MicroRNA-21 increases the expression level of occludin through regulating ROCK1 in prevention of intestinal barrier dysfunction.
Liu Zhihua,Li Chao,Chen Shihua,Lin Hongcheng,Zhao Huan,Liu Min,Weng Jinsheng,Liu Ting,Li Xiaomei,Lei Chao,Li Chen,Jiang Yanqiong,Moyer Mary Pat,Yin Chunxia,Zhou Xinke
Journal of cellular biochemistry
OBJECTIVE:The aim of this study is to investigate the role of molecular mechanism of microRNA (miR)-21 on tight junction (TJ)-proteins and its protective effects on the intestinal barrier. METHODS:TJ proteins and target genes expression were analyzed in miR-21 inhibition and overexpression NCM460 cell lines. To further verify the role of miR-21, the mmu-miR-21 intestinal epithelial conditional knockout (IKO) mice model was established. MiR-21 expression was detected in clinical specimens of acute stercoral obstruction patients. RESULTS:Rho-associated protein kinase 1 (ROCK1) were identified as target genes of miR-21. There is a negative correlation between miR-21 expression level and TJ proteins levels. TJ protein and ROCK1 were significantly decreased in miR-21 IKO mice, which presented intestinal inflammation response and intestinal barrier dysfunction (both P < 0.05). Determination of clinical samples showed consistent results with NCM460 cell line and miR-21 IKO mice. CONCLUSIONS:MiR-21 could be a protective factor of intestinal barrier dysfunction, which promoting the expression of TJ protein by targeting ROCK1 in vivo and in vitro.
Total flavonoids from Smilax glabra Roxb blocks epithelial-mesenchymal transition and inhibits renal interstitial fibrosis by targeting miR-21/PTEN signaling.
Luo Qihan,Cai Zhaowei,Tu Jue,Ling Yun,Wang Dejun,Cai Yueqin
Journal of cellular biochemistry
BACKGROUND:Smilax glabra Roxb, a traditional Chinese herb, has been widely used in folk medicine. The current study was performed to investigate the protective effect of S. glabra Roxb extract, pure total flavonoids from Smilax glabra Roxb (PTFS), on renal interstitial fibrosis (RIF) and its underlying mechanism. METHODS:First, a surgical model of unilateral ureteral obstruction was established in rats to induce RIF. Then, rats were grouped and treated with PTFS at different concentration. Second, HK-2 cells underwent an epithelial-mesenchymal transition (EMT) by the addition of transforming growth factor-β1 (TGF-β1). Additionally, HK-2 cells after inducing for EMT were transfected with microRNA-21 (miR-21) mimic or inhibitor. These HK-2 cells were grouped and treated with PTFS at different concentration. Finally, real-time polymerase chain reaction and Western blot analysis were performed to detect the expression of possible signaling factor involved in RIF in renal tissues or HK-2 cells after PTFS treatment. RESULTS:In vivo and in vitro experiments indicated that PTFS treatment could decrease the expression of α-smooth muscle actin (α-SMA; mesenchymal marker) and increase the expression of E-cadherin (epithelial marker) in both messenger RNA and protein level. Moreover, PTFS also attenuated the expression of TGF-β1/Smad signaling in both renal tissues and HK-2 cells that underwent EMT. Overexpression or inhibition of miR-21 in HK-2 cells activated or blocked the PI3K/Akt signaling via targeting phosphatase and tension homolog (PTEN), and then promoted or suppressed the progress of TGF-β1-induced EMT by regulating the expression of α-SMA and E-cadherin. Furthermore, PTFS treatment inhibited TGF-β1-induced EMT progress by blocking miR-21/PTEN/PI3K/Akt signaling. CONCLUSION:PTFS has strong anti-EMT and antifibrosis effects both in vitro and in vivo. The mechanism underlying these effects may be related to inhibition of TGF-β1/Smad, and their downstream miR-21/PTEN signaling, leading to blocks of EMT process during RIF.
Antisense Oligonucleotides against miR-21 Inhibit the Growth and Metastasis of Colorectal Carcinoma via the DUSP8 Pathway.
Ding Tao,Cui Panpan,Zhou Ya,Chen Chao,Zhao Juanjuan,Wang Hairong,Guo Mengmeng,He Zhixu,Xu Lin
Molecular therapy. Nucleic acids
Accumulating research has documented that microRNA-21 (miR-21) plays an important role in the development of human colorectal carcinoma (CRC). Our recent work also showed that antisense oligonucleotides (ASOs) against miR-21 can impair the growth of CRC cells in vitro. However, the potential role of miR-21 in gene therapy against CRC remains to be fully elucidated. Here, we further observed the effect of ASOs against miR-21 on the growth and metastasis of CRC in vivo using a xenograft model of human CRC. We found that ASOs could effectively inhibit the growth and metastasis of CRC in vivo, accompanied by downregulated expression of miR-21 and reduced transduction of the AKT and ERK pathway. Mechanically, global gene expression analysis showed that the expression of DUSP8, a novel target of miR-21, was upregulated in tumor mass. Furthermore, overexpression of DUSP8 could remarkably suppress the proliferation and migration of CRC cells in vitro. Finally, downregulation of DUSP8 could abrogate the effects of ASOs against miR-21 on the proliferation and migration of CRC cells, as well as altered transduction of the AKT and ERK signaling pathway. Together, these data suggest that ASOs against miRNAs are an attractive and potential therapeutic for the treatment of human CRC and warrant further development.
Mutations in GAS5 affect the transformation from benign prostate proliferation to aggressive prostate cancer by affecting the transcription efficiency of GAS5.
Zhu Lizhen,Zhu Qi,Wen Huihuang,Huang Xiang,Zheng Ge
Journal of cellular physiology
BACKGROUND:In this study, we aimed to explore the effects of GAS5 single-nucleotide polymorphisms (SNPs) on GAS5 expression. And the signaling pathways underlying the function of GAS5 during the pathogenesis of prostate cancer (PC) were clarified. MATERIALS AND METHODS:Patients with PC were recruited and grouped according to their specific genotypes of rs55829688 and rs145204276. Kaplan-Meier overall survival curves were calculated and compared among different groups. Real-time polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry (IHC) assays were conducted to examine the expression of different factors involved in PC. And computational analyses and luciferase assays were conducted to clarify the regulatory relationships among the above factors. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide), flow cytometry, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays were used to evaluate cell viability and apoptosis. RESULTS:The expression of GAS5, PDCD4, PTEN, and AKT was decreased gradually in the order of patient Group 1-4, whereas the expression of microRNA-21 (miR-21) and miR-1284 showed an opposite trend. GAS5 was identified to target miR-21 and miR-1284, whereas miR-21 and miR-1284 regulated the expression of PDCD4/PTEN and AKT, respectively. Moreover, the GAS5/miR-21/PDCD4/PTEN and GAS5/miR-1284/AKT signaling pathway was found to be closely related to the tumorigenesis of PC. CONCLUSIONS:GAS5 SNPs affected the survival rate and prognosis in patients with PC via regulating the expression of miR-21/miR-1284, which in turn affected the expression of PDCD4, PTEN, and AKT. GAS5 downregulated the expression of miR-21/miR-1284, thus leading to the elevated expression of key regulators of apoptosis. Therefore, the GAS5 SNPs may be used as key indicators for the diagnosis and prognosis prediction of PC.
Low expression of microRNA-21 inhibits trophoblast cell infiltration through targeting PTEN.
Lou C-X,Zhou X-T,Tian Q-C,Xie H-Q,Zhang J-Y
European review for medical and pharmacological sciences
OBJECTIVE:We investigate whether microRNA-21 could increase the infiltration ability of trophoblast cells via regulating PTEN expression, thus participating in the occurrence and development of preeclampsia. PATIENTS AND METHODS:MicroRNA-21 expression in the placenta tissues of preeclampsia women and normal pregnant women was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The effects of microRNA-21 on cell proliferation and infiltration were examined by cell counting kit-8 (CCK-8) and transwell assay, respectively. The dual-luciferase reporter gene assay was used to determine the binding relationship between microRNA-21 and PTEN. Western blot was performed to detect PTEN and microRNA-21 in trophoblasts. RESULTS:QRT-PCR results showed that the microRNA-21 expression was significantly lower in the placenta of the preeclampsia women than those of normal pregnant women. Overexpression of microRNA-21 in HTR-8/SVneo cells had no effect on cell proliferation, but enhanced cell infiltration ability. Inhibition of microRNA-21 in trophoblasts showed the opposite effects. The results of luciferase activity assay and Western blot showed that microRNA-21 could target PTEN and downregulate its expression. Overexpression of PTEN in HTR-8/SVneo cells partially reversed the enhanced invasive ability induced by microRNA-21 overexpression. CONCLUSIONS:Low expression of microRNA-21 attenuated cell infiltration of trophoblasts via direct regulating PTEN expression.
Growth arrest-specific 5 attenuates cisplatin-induced apoptosis in cervical cancer by regulating STAT3 signaling via miR-21.
Yao Tingting,Lu Rongbiao,Zhang Jun,Fang Xingyu,Fan Li,Huang Chunxian,Lin Rongchun,Lin Zhongqiu
Journal of cellular physiology
Cervical cancer is the most common cause of female cancer-related mortality worldwide. Decreased expression of long noncoding RNA growth arrest-specific 5 (GAS5) is found in human cervical cancer tissues and associated with poor prognosis. However, the studies on associations between GAS5 level and malignant phenotypes, as well as sensitivity to chemotherapeutic drug in cervical cancer cells are limited. In this study, overexpression of GAS5 in cervical cancer cells resulted in prohibited cell proliferation and colony formation, which were promoted by siGAS5. Enhanced GAS5 increased cell percentage in the G0/G1 phase and decreased cells percentage in the S phase, whereas reduced expression did not. The malignant behaviors of cervical cancer cells, manifested by cell migration and invasion, could be weakened by the GAS5 overexpression and enhanced by siGAS5. Furthermore, in cisplatin-induced cell, overexpression of GAS5 reduced cells viability and enhanced apoptosis, whereas in cells transfected with siGAS5, apoptosis eliminated. We have reported the upregulation of microRNA-21 (miR-21) and its oncogenetic roles in cervical cancer previously. In this study, we found the negative relationship between the GAS5 and miR-21. Moreover, the decrease of miR-21 associated proteins phosphorylated STAT3 and E2F3 was seen in GAS5 overexpressed cells, both of which could be increased by siGAS5. The GAS5 deficiency also reduced miR-21 target proteins TIMP3 and PDCD4 expressions. Taken together, the GAS5 expression level is inversely associated with malignancy, but positively associated with sensitivity to cisplatin-induced apoptosis, suggesting that GAS5 could be a biomarker of cisplatin-resistance in clinical therapy of human cervical cancer.
Bone marrow stem cells-derived exosomes extracted from osteoporosis patients inhibit osteogenesis via microRNA-21/SMAD7.
Jiang L-B,Tian L,Zhang C-G
European review for medical and pharmacological sciences
OBJECTIVE:To explore whether bone marrow stem cells (MSCs)-derived exosomes extracted from osteoporosis patients could inhibit osteogenesis via microRNA-21/SMAD7. PATIENTS AND METHODS:MSCs from osteoporosis patients were isolated and cultured. MSCs morphology was observed, and the specific surface antigens were identified by flow cytometry. The osteogenic ability of MSCs was detected by alizarin red staining and oil red staining. Exosomes were collected from MSCs suspension by ultracentrifugation, and microRNA-21 expression in MSCs derived-exosomes was detected. Moreover, protein and mRNA levels of ALP, Bglap, and Runx2 in MSCs treated with different sources of MSCs-derived exosomes were detected by qRT-PCR (quantitative real-time polymerase chain reaction) and Western blot, respectively. ALP activity in MSCs was accessed by a relative commercial kit. Furthermore, binding sites of microRNA-21 and SMAD7 were predicted by Targetscan, miRWalk, and miRDB, and were further verified by luciferase reporter gene assay. SMAD7 expression in MSCs derived-exosomes was also detected. RESULTS:MSCs extracted from healthy adults, and osteoporosis patients were in adherent growth and exhibited elongated morphology, which could differentiate into osteoblasts and lipoblasts after different inductions. MicroRNA-21 expression in MSCs-derived exosomes extracted from osteoporosis patients was remarkably higher than those extracted from healthy adults. Decreased Runx2 expression and ALP activity were found after treatment of MSCs-derived exosomes extracted from osteoporosis patients. SMAD7 was confirmed to bind to microRNA-21 and was downregulated in osteoporosis patients in comparison with healthy adults. Overexpression of SMAD7 resulted in downregulated ALP, Bglap, and Runx2. CONCLUSIONS:MicroRNA-21 inhibits osteogenesis through regulating MSCs-derived exosomes extracted from osteoporosis patients via targeting SMAD7.
Inhibition of profibrotic microRNA-21 affects platelets and their releasate.
Barwari Temo,Eminaga Seda,Mayr Ursula,Lu Ruifang,Armstrong Paul C,Chan Melissa V,Sahraei Mahnaz,Fernández-Fuertes Marta,Moreau Thomas,Barallobre-Barreiro Javier,Lynch Marc,Yin Xiaoke,Schulte Christian,Baig Ferheen,Pechlaner Raimund,Langley Sarah R,Zampetaki Anna,Santer Peter,Weger Martin,Plasenzotti Roberto,Schosserer Markus,Grillari Johannes,Kiechl Stefan,Willeit Johann,Shah Ajay M,Ghevaert Cedric,Warner Timothy D,Fernández-Hernando Carlos,Suárez Yajaira,Mayr Manuel
Fibrosis is a major contributor to organ disease for which no specific therapy is available. MicroRNA-21 (miR-21) has been implicated in the fibrogenetic response, and inhibitors of miR-21 are currently undergoing clinical trials. Here, we explore how miR-21 inhibition may attenuate fibrosis using a proteomics approach. Transfection of miR-21 mimic or inhibitor in murine cardiac fibroblasts revealed limited effects on extracellular matrix (ECM) protein secretion. Similarly, miR-21-null mouse hearts showed an unaltered ECM composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In plasma samples from the community-based Bruneck Study, we found a marked correlation of miR-21 levels with several platelet-derived profibrotic factors, including TGF-β1. Pharmacological miR-21 inhibition with an antagomiR reduced the platelet release of TGF-β1 in mice. Mechanistically, Wiskott-Aldrich syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. miR-21-null mice had lower platelet and leukocyte counts compared with littermate controls but higher megakaryocyte numbers in the bone marrow. Thus, to our knowledge this study reports a previously unrecognized effect of miR-21 inhibition on platelets. The effect of antagomiR-21 treatment on platelet TGF-β1 release, in particular, may contribute to the antifibrotic effects of miR-21 inhibitors.
Compound Astragalus and Salvia miltiorrhiza extract inhibits hepatocellular carcinoma progression via miR-145/miR-21 mediated Smad3 phosphorylation.
Wu Chao,Chen Weiyang,Fang Meng,Boye Alex,Tao Xiangming,Xu Yuanyuan,Hou Shu,Yang Yan
Journal of ethnopharmacology
ETHNOPHARMACOLOGICAL RELEVANCE:Compound Astragalus and Salvia miltiorrhiza extract (CASE), containing astragalosides, astragalus polysaccharide extracted from Astragalus membranaceus (Fisch.) Bge. and salvianolic acids from Salvia miltiorhiza Bge., has been found to inhibit hepatocarcinogenesis via mediating transforming growth factor-β (TGF-β)/Smad signaling, especially Smad3 phosphorylation. The crucial interaction between microRNA-145/microRNA-21 (miR-145/miR-21) and Smad3 phosphorylation is implicated in the pathogenesis and progression of hepatocellular carcinoma (HCC). However, effects of CASE on HCC progression involved in the expression of miR-145/miR-21 and their interaction with Smad3 phosphorylation downstream of TGF-β/MAPK/Smad pathway remain unclear. This study addressed above questions using in vitro (HepG2 cells) and in vivo (Xenografts of nude mice) models of HCC. MATERIALS AND METHODS:In vivo [Diethylnitrosamine (DEN)-induced HCC in rats] and in vitro [TGF-β-stimulated HepG2 cells] models of HCC were established and co-administrated using graded doses/concentrations CASE (60, 120, 240 mg/kg used in rats; 20, 40, 80 µg/ml used in HepG2 cells), miR-145 and miR-21 were measured. HepG2 cells were transfected with miR-145 antagomir, miR-21 agomir and Smad3C/L plasmids (Smad3 EPSM, Smad3 3S-A and Smad3 WT related to up-regulated expression of pSmad3C, pSmad3L and pSmad3C/3L respectively) and then treated by CASE (80 µg/ml). Similarly, HepG2 cell xenografted nude mice were administered with miR-145 antagomir, miR-21 agomir and CASE (310 mg/kg); Smad3 WT, Smad3 EPSM and Smad3 3S-A plasmids stably transfected HepG2 cell lines were constructed respectively and their xenografted nude mice were established, and then treated by CASE (310 mg/kg). Cell proliferation, migration, apoptosis, tumor growth and histopathologic characteristics of xenografts were assessed; also, domain-specific Smad3 phosphorylation isoforms (pSmad3C/pSmad3L), activated MAPKs (pERK1/2, pJNK1/2, pp38) and miR-145, miR-21 were measured. RESULTS:CASE up-regulated miR-145 while down-regulated miR-21 expression in both rats with DEN-induced HCC and TGF-β-stimulated HepG2 cells; CASE inhibited cell migration, proliferation and tumor growth while facilitated cell apoptosis in TGF-β-stimulated HepG2 cells and xenografts of nude mice with miR-145 antagomir/miR-21 agomir treatment via increasing miR-145 and facilitating miR-145 modulated pSmad3L→pSmad3C signaling switch while decreasing miR-21 and inhibiting miR-21 modulated MAPK-dependent Smad3L phosphorylation. Also, up-regulated pSmad3C enhanced inhibited effect of CASE on tumor growth and facilitated effect of CASE on cell apoptosis involved in increased miR-145 while decreased miR-21 expression, however, inverse phenomena were observed when up-regulated pSmad3L. CONCLUSION:Our results suggest that CASE inhibits HCC progression via mediating the interaction of miR-145/miR-21 and Smad3 phosphorylation, especially miR-145/miR-21 mediated Smad3 phosphorylation, which maybe provides an important theoretical foundation for CASE's anti-HCC therapy used for patients in a near future.
Methyl helicterte ameliorates liver fibrosis by regulating miR-21-mediated ERK and TGF-β1/Smads pathways.
Huang Quanfang,Zhang Xiaolin,Bai Facheng,Nie Jinlan,Wen Shujuan,Wei Yuanyuan,Wei Jinbin,Huang Renbin,He Min,Lu Zhongpeng,Lin Xing
Methyl helicterate (MH) has been reported to have protective effects against CCl-induced hepatic injury and fibrosis in rats, but its protective mechanism, especially on hepatic stallete cells (HSCs), remains unclear. Recently, our pilot experiment showed that MH could inhibit miR-21 expression in HSC-T6 cells, suggesting that miR-21 may be one of the targets of MH to intervene liver fibrosis. To verify the hypothesis, the present study would focus on the regulatory effect of MH on the miR-21-mediated ERK and TGF-β1/Smads pathways. Briefly, rats were intraperitoneally injected with 0.5 ml porcine serum (PS) twice a week for 24 weeks to induce liver fibrosis, and meanwhile, the rats were treated with MH from weeks 16 to 24. In vitro experiment, miR-21 expression in HSC-T6 cells was up- or down-regulated using lentiviral transfection assay. Collagen accumulation, inflammatory cytokines, cell apoptosis, miR-21 expression, and activation of the ERK and TGF-β1/smad2/3 pathways were then assessed. The results showed that MH treatment markedly alleviated PS-induced liver injury, as evidenced by the attenuation of histopathological changes and the decrease in serum alanine and aspartate aminotransferases activity. MH significantly decreased the content of inflammatory cytokines and recruited the anti-oxidative defense system. Moreover, MH treatment significantly decreased miR-21 expression and inhibited the activation of the ERK and TGF-β1/smad2/3 pathways in liver tissues. In vitro experiments showed that MH strongly inhibited HSC-T6 cell activation and reduced collagen accumulation. Interestingly, miR-21 overexpression significantly promoted HSC-T6 cell proliferation, reduced HSC apoptosis, and increased collagenation, while these abnormal changes induced by miR-21overexpression were significantly reversed by MH treatment. Furthermore, miR-21 overexpression notably activated the ERK and TGF-β1/Smads pathways via repressing SPRY2 and Smad7 expression respectively, however, these effects were largely abolished by MH treatment. In conclusion, our study demonstrates that MH significantly alleviates PS-induced liver injury and fibrosis by inhibiting miR-21-mediated ERK and TGF-β1/Smads pathways.
SphK1/S1P Mediates PDGF-Induced Pulmonary Arterial Smooth Muscle Cell Proliferation via miR-21/BMPRII/Id1 Signaling Pathway.
Li Fangwei,Wang Jian,Zhu Yanting,Liu Lu,Feng Wei,Shi Wenhua,Wang Qingting,Zhang Qianqian,Chai Limin,Li Manxiang
Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology
BACKGROUND/AIMS:The underlying molecular mechanisms involved in sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate (S1P) mediation of platelet-derived growth factor (PDGF)-induced pulmonary arterial smooth muscle cell (PASMC) proliferation are still unclear, and the present study aims to address this issue. METHODS:Small interfering RNA (siRNA) and microRNA inhibitor transfection was performed to block the expression of SphK1, bone morphogenetic protein receptor II (BMPRII) and microRNA-21 (miR-21). Gene expression levels of SphK1, BMPRII and inhibitor of DNA binding 1 (Id1) were detected by immunoblotting, miR-21 expression level was examined with qRT-PCR, and S1P production was measured by ELISA. Additionally, PASMC proliferation was determined by BrdU incorporation assay. RESULTS:Our results indicated that PDGF increased the expression of SphK1 protein and S1P production, up-regulated miR-21 expression, reduced BMPRII and Id1 expression, and promoted PASMCs proliferation. Pre-silencing of SphK1 with siRNA reversed PDGF-induced S1P production, miR-21 up-regulation, BMPRII and Id1 down-regulation, as well as PASMC proliferation. Pre-inhibition of miR-21 also blocked BMPRII and Id1 down-regulation as well as PASMC proliferation caused by PDGF. Knockdown of BMPRII down-regulated Id1 expression in PASMCs. We further found that inhibition of PI3K/Akt and ERK signaling pathways, particularly ERK cascade, suppressed PDGF-induced above changes. CONCLUSION:Our study indicates that SphK1/S1P pathway plays an important role in PDGF-induced PASMC proliferation via miR-21/BMPRII/Id1 axis and targeting against SphK1/S1P axis might be a novel strategy in the prevention and treatment of pulmonary arterial hypertension (PAH).
Dynamic Single Molecular Rulers: Toward Quantitative Detection of MicroRNA-21 in Living Cells.
Li Mei-Xing,Zhao Wei,Wang Hui,Li Xiang-Ling,Xu Cong-Hui,Chen Hong-Yuan,Xu Jing-Juan
Innovative techniques to measure microRNA (miRNA) in vivo could greatly improve the fundamental understanding of complex cellular processes. Herein, we report a novel method for real-time, quantitative miRNA detection inside living cells based on core-satellite plasmon rulers (PRs). This approach allows for the statistical analysis of single hybridization event caused by target miRNA. We investigated hundreds of satellite leaving events and found that the distribution of the time range for one strand displacement event is miRNA concentration-dependent, which obeyed Poisson statistics. Probing several such PRs under dark-field microscopy would provide precise determination of miRNA in vitro and in living cells, without photobleaching or blinking of the fluorophores. We believe the simple and practical approach on the basis of dynamic PRs with single-molecule sensitivity combined with statistical analysis hold promising potential to visualize native nucleic acids with short sequence and low-abundance.
MicroRNA-21 contributes to high glucose-induced fibrosis in peritoneal mesothelial cells in rat models by activation of the Ras-MAPK signaling pathway via Sprouty-1.
Gao Qing,Xu Lin,Yang Qian,Guan Tian-Jun
Journal of cellular physiology
Peritoneal fibrosis remains to be one of the most severe causes of failure in continuous peritoneal dialysis. The current study cultured peritoneal mesothelial cells in high glucose to stimulate the environment of peritoneal fibrosis model in rats, and investigate whether microRNA-21 (miR-21) targeting Sprouty-1 affects high glucose-induced fibrosis in peritoneal mesothelial cells via the rennin angiotensin system (Ras)-mitogen-activated protein kinase (MAPK) signaling pathway, as well as potential mechanisms. Peritoneal tissues in fibrosis rats were collected to extract peritoneal mesothelial cells, which, after in vitro culture, were transfected with a series of mimic or inhibitor of miR-21, or the small interfering RNA (siRNA) against Sprouty-1. Reverse-transcription quantitative polymerase chain reaction and western blot analyses were performed to determine the levels of related genes or proteins. MTT assay and flow cytometry were conducted to observe the cell viability and cell apoptosis of peritoneal mesothelial cells. Dual-luciferase reporter gene assay revealed that Sprouty-1 is the target gene of miR-21. Peritoneal fibrosis manifested with elevated miR-21, extracellular-signal-regulated kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), RAS and p38MAPK but reduced Sprouty-1. Cells transfected with miR-21 mimic exhibited decreased Sprouty-1 expressions, but increased levels of ERK, JNK, RAS, and p38MAPK. As for cellular process, miR-21 mimic or siRNA against Sprouty-1 exposure reduced cell viability, which resulted in more cells arrested at the G1 stage, and induced apoptosis. In contrast, miR-21 inhibitor exposure was observed to have induced effects on peritoneal mesothelial cells. These key findings provide evidence that miR-21 inhibits Sprouty-1 to promote the progression of fibrosis in peritoneal mesothelial cells by activating the Ras-MAPK signaling pathway.
MicroRNA-21 abrogates palmitate-induced cardiomyocyte apoptosis through caspase-3/NF-κB signal pathways.
Zhou Xiaodi,Chang Bo,Gu Yuzhong
Anatolian journal of cardiology
OBJECTIVE:The aim of the study was to investigate the role of microRNA-21 (miR-21) in cardiomyocyte apoptosis and to determine a possible mechanism. METHODS:H9c2 embryonic rat heart-derived cells were used in the study. Cell viability was determined using the 3-(4.5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide (MTT) assay, and flow cytometry was used to evaluate cell apoptosis. Reverse transcription-polymerase chain reaction and western blot assays were used to detect mRNA and protein expression of the apoptosis-related proteins and miR-21. ELISA was used to detect reactive oxygen species (ROS). RESULTS:Palmitate exposure greatly reduced miR-21 expression in cardiomyocytes. Apoptosis increased when miR-21 was inhibited with or without palmitate exposure. Consistently, reduced apoptosis was observed when miR-21 was overexpressed in cardiomyocytes. Caspase-3 activity was reduced after palmitate exposure. Bcl-2 protein expression was increased in H9c2 cells when transfected with the miR-21 mimic. MiR-21 overexpression alone did not induce ROS or DNA fragmentation; however, in conjunction with palmitate exposure, miR-21 mimic reduced ROS and DNA fragmentation. Moreover, palmitate administration overcame the antioxidant effect of 3 mM N-acetylcysteine to significantly inhibit apoptosis, DNA fragmentation, and caspase-3 activity. The exposure to palmitate greatly reduced p65 and p-p38 expression in the nucleus. A p38 inhibitor had no effect on the expression of Bcl-2 and cleaved caspase-3 in H9c2 cells alone; however, when combined with exposure to palmitate the p38 inhibitor induced Bcl-2 expression and inhibited caspase-3 activity. The p38 inhibitor by itself did not induce apoptosis, ROS production, or DNA fragmentation in H9c2 cells, but when palmitate was included with the p38 inhibitor, apoptosis, ROS production, and DNA fragmentation were reduced. CONCLUSION:miR-21 protects cardiomyocytes from apoptosis that is induced by palmitate through the caspase-3/NF-κB signal pathways.
Long noncoding RNA HAND2-AS1 inhibits cancer cell proliferation, migration, and invasion in esophagus squamous cell carcinoma by regulating microRNA-21.
Yan Yusheng,Li Shaobin,Wang Suihai,Rubegni Pietro,Tognetti Linda,Zhang Jiaqing,Yan Lu
Journal of cellular biochemistry
Long noncoding RNA (lncRNA) HAND2-AS1 is a well-characterized tumor suppressor in several types of malignancies, while its role in esophagus squamous cell carcinoma (ESCC) is unknown. In this study, we found that lncRNA HAND2-AS1 was downregulated, while microRNA-21 ( miRNA-21) was upregulated in tumor tissues than in adjacent healthy tissues of ESCC patients. Expression levels of lncRNA HAND2-AS1 and miRNA-21 were significantly and inversely correlated in tumor tissues but not in healthy tissues. Plasma levels of lncRNA HAND2-AS1 were lower in ESCC patients than in healthy controls, and downregulation of plasma lncRNA HAND2-AS1 distinguished early stage ESCC patients from healthy controls. lncRNA HAND2-AS1 overexpression resulted in downregulation of miRNA-21 in cells of ESCC cell lines and inhibited cell proliferation, migration, and invasion. miRNA-21 overexpression failed to affect lncRNA HAND2-AS1 expression but significantly attenuated the inhibitory effect of lncRNA HAND2-AS1 overexpression on cancer cell proliferation, migration, and invasion. Therefore, lncRNA HAND2-AS1 may inhibit cancer cell proliferation, migration, and invasion in ESCC by regulating miRNA-21.
Collagen biomaterial stimulates the production of extracellular vesicles containing microRNA-21 and enhances the proangiogenic function of CD34 cells.
McNeill Brian,Ostojic Aleksandra,Rayner Katey J,Ruel Marc,Suuronen Erik J
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
CD34 cells are promising for revascularization therapy, but their clinical use is limited by low cell counts, poor engraftment, and reduced function after transplantation. In this study, a collagen type I biomaterial was used to expand and enhance the function of human peripheral blood CD34 cells, and potential underlying mechanisms were examined. Compared to the fibronectin control substrate, biomaterial-cultured CD34 cells from healthy donors had enhanced proliferation, migration toward VEGF, angiogenic potential, and increased secretion of CD63CD81 extracellular vesicles (EVs). In the biomaterial-derived EVs, greater levels of the angiogenic microRNAs (miRs), miR-21 and -210, were detected. Notably, biomaterial-cultured CD34 cells had reduced mRNA and protein levels of Sprouty (Spry)1, which is an miR-21 target and negative regulator of endothelial cell proliferation and angiogenesis. Similar to the results of healthy donor cells, biomaterial culture increased miR-21 and -210 expression in CD34 cells from patients who underwent coronary artery bypass surgery, which also exhibited improved VEGF-mediated migration and angiogenic capacity. Therefore, collagen biomaterial culture may be useful for expanding the number and enhancing the function of CD34 cells in patients, possibly mediated through suppression of Spry1 activity by EV-derived miR-21. These results may provide a strategy to enhance the therapeutic potency of CD34 cells for vascular regeneration.-McNeill, B., Ostojic, A., Rayner, K. J., Ruel, M., Suuronen, E. J. Collagen biomaterial stimulates the production of extracellular vesicles containing microRNA-21 and enhances the proangiogenic function of CD34 cells.
MicroRNA-21 Deficiency Alters the Survival of Ly-6C Monocytes in ApoE Mice and Reduces Early-Stage Atherosclerosis-Brief Report.
Chipont Anna,Esposito Bruno,Challier Inès,Montabord Mélanie,Tedgui Alain,Mallat Ziad,Loyer Xavier,Potteaux Stephane
Arteriosclerosis, thrombosis, and vascular biology
Objective- To determine the role of microRNA-21 (miR-21) on the homeostasis of monocyte subsets and on atherosclerosis development in ApoE (apolipoprotein E) mice. Approach and Results- In ApoE mice, miR-21 expression was increased in circulating Ly-6C nonclassical monocytes in comparison to Ly-6C monocytes. The absence of miR-21 significantly altered the survival and number of circulating Ly-6C nonclassical monocytes in ApoE mice. In the early stages of atherosclerosis, the absence of miR-21 limited lesion development both in the aortic sinus (by almost 30%) and in the aorta (by almost 50%). This was associated with less monocyte availability in circulation and increased apoptosis of local macrophages in plaques. At later stages of atherosclerosis, lesion size in the aortic root was similar in ApoE and ApoE miR-21 mice, but plaques showed a less stable phenotype (larger necrotic cores) in the latter. The loss of protection in advanced stages was most likely because of excessive inflammatory apoptosis related to an impairment of local efficient efferocytosis. Conclusions- Gene deletion of miR-21 in ApoE mice alters Ly-6C nonclassical monocytes homeostasis and contribute to limit early-stage atherosclerosis.
MicroRNA-21 attenuates BDE-209-induced lipid accumulation in THP-1 macrophages by downregulating Toll-like receptor 4 expression.
Zhi Hui,Yuan Na,Wu Jiang-Ping,Lu Lin-Ming,Chen Xiao-Yun,Wu Si-Kang,Mai Bi-Xian
Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association
Growing evidence demonstrates a possible response of specific microRNA (miRNA) to environmental pollutant stimuli in multiple biological processes. We previously reported that a persistent organic pollutant, decabromodiphenyl ether (BDE-209), can enhance Toll-like receptor 4 (TLR4)-dependent lipid uptake in THP-1 macrophages; whether miRNAs are involved in this process remains unclear. In the present study, we investigated the levels of several miRNAs related to TLR4 signaling, including miRs-9, -21, -27b, -125b, -132, -146a, -147, -155, and -let-7e, in THP-1 macrophages after stimulation by BDE-209 and oxidized low-density lipoprotein. The results showed that the levels of miR-21 were significantly suppressed by BDE-209 at concentrations of 6.25, 12.5 and 25 μM, in a dose-dependent manner; whereas there was no significant changes for the other miRNAs investigated. Moreover, the suppression of miR-21 was accompanied by an upregulated TLR4 expression, at both mRNA and protein levels. Further analysis showed that the up-regulated TLR4 induced by BDE-209 was inhibited in macrophages transfected with miR-21 mimic; meanwhile opposite results were exhibited when an anti-miR-21 inhibitor was transfected to the macrophages. Additionally, transfection with miR-21 mimic effectively attenuated BDE-209-induced lipid accumulation in macrophages. Together, these data illustrate that miR-21 inhibits BDE-209-triggered lipid accumulation in macrophages through down-regulating TLR4 expression.
MicroRNA-21-5p are involved in apoptosis and invasion of fibroblast-like synoviocytes through PTEN/PI3K/AKT signal.
Yan Xin,Liu Yake,Kong Xaoli,Ji Juan,Zhu Hai,Zhang Zexu,Fu Ting,Yang Junling,Zhang Zhongyuan,Liu Fan,Gu Zhifeng
The function of microRNA-21-5p (miR-21) in fibroblast-like synoviocytes in RA was still unclear. In our study, we used tumor necrosis factor alpha (TNFα) (10 ng/ml) to mimic RA-FLSs and we found that normal FLS stimulated with TNFα caused the increasing expression of miR-21, a disintegrin and metalloproteinase with thrombospondin motifs 5 and matrix metalloproteinase 3, which were in accord with RA-FLSs changes. Our data showed that miR-21 overexpression significantly increased cell invasion and decreased apoptosis in FLSs. Knockdown of miR-21 in FLSs causes the opposite result. However, miR-21 may not affect the proliferation of FLSs. Meanwhile, we showed that miR-21 activated the PI3K/AKT signaling pathway to participate in RA by inhibiting PTEN expression. Taken together, our results suggested that miR-21 may play a positive role in RA and may be a promising new therapeutic target for RA.
SAV1, regulated by microRNA-21, suppresses tumor growth in colorectal cancer.
Jiang Jianwu,Chang Wei,Fu Yang,Gao Yongshun,Zhao Chunlin,Zhang Xiefu,Zhang Shuijun
Biochemistry and cell biology = Biochimie et biologie cellulaire
This study investigated the role and action of the Salvador 1 protein (SAV1, also called WW45) in colorectal cancer (CRC). For this, CRC SW480 and HCT116 cells were infected with lentiviruses of SAV1 overexpression vector (lenti-SAV1) and SAV1 short hairpin RNA (sh-SAV1) to overexpress and silence SAV1 respectively, or transfected with microRNA-21 (miR-21) mimic to overexpress miR-21. Relative mRNA levels of SAV1 and relative miR-21 levels in CRC tissues or cells were detected. The effects of SAV1 and miR-21 on cell proliferation and apoptosis were evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and annexin V - fluorescein isothiocyanate (FITC) - propidium iodide (PI) flow cytometry, respectively. Our results revealed that SAV1 was downregulated in CRC tissues compared with the adjacent noncancerous tissues. Furthermore, SAV1 overexpression inhibited proliferation and promoted apoptosis in SW480 and HCT116 cells, whereas knockdown of SAV1 exerted the opposite effect. Additionally, the tumorigenesis of SW480 cells in xenografted mice was significantly inhibited by SAV1 overexpression but promoted by SAV1 knockdown. MiR-21 levels significantly and negatively correlated with SAV1 expression in CRC tissues. More importantly, miR-21 overexpression significantly abolished the SAV1-mediated inhibition of proliferation and stimulation of apoptosis of SW480. In conclusion, SAV1 suppresses tumor growth in CRC and is regulated by miR-21.
Targeting MicroRNA-21 Suppresses Gastric Cancer Cell Proliferation and Migration via PTEN/Akt Signaling Axis.
Zhou Hao,Liu Hongyan,Jiang Miao,Zhang Shaoren,Chen Junfeng,Fan Xiaoming
MicroRNA plays a pivotal role in various human cancers, especially in human gastric cancer. In the present study, we evaluated the effect of microRNA-21 (miR-21) on the gastric cancer cell proliferation, migration, apoptosis and the related signaling cascades. Here, we showed that down-regulation of miR-21 markedly reduced gastric cancer cell proliferation (AGS and NCI-N87 cells) in a time dependent manner. Moreover, our findings revealed that silencing miR-21 dramatically blocked gastric cancer cell migration and movement, which might be related to down-regulation of vimentin expression. We also found that down-regulation of miR-21 promoted cell apoptosis and repressed cell cycle progression. Further investigation showed that down-regulation of miR-21 significantly increased phosphatase and tensin homolog (PTEN) protein expression level, but not transcription level (mRNA level), which in turn decreased Akt phosphorylation at Thr308 and Ser473. Collectively, our results uncover that miR-21 targets PTEN/Akt signaling pathway and regulates cell proliferation, migration and apoptosis in human gastric cancer cells. Our findings may provide a therapeutic target for treatment of human gastric cancer.
Curcumin regulates the miR-21/PTEN/Akt pathway and acts in synergy with PD98059 to induce apoptosis of human gastric cancer MGC-803 cells.
Qiang Zhanrong,Meng Lingyu,Yi Caixia,Yu Lianying,Chen Wenxia,Sha Weihong
The Journal of international medical research
OBJECTIVE:PD98059 is a potent and selective inhibitor of mitogen-activated protein kinase. Substantial preclinical evidence has shown an anti-tumor effect of curcumin on various solid tumors. This study aimed to investigate whether curcumin synergistically acts with PD98059 in exerting anti-gastric cancer effects. METHODS:The cell counting kit-8 assay was used to detect cell proliferation of the human gastric cancer MGC-803 cell line. Flow cytometry was performed to detect apoptosis. Western blotting was used to detect phosphatase and tensin homolog (PTEN) and phosphorylated Akt (p-Akt) expression levels. Quantitative reverse transcription-polymerase chain reaction was used to determine microRNA-21 (miR-21). RESULTS:A dose of 5 to 40 µM curcumin inhibited proliferation of MGC-803 cells in a dose- and time-dependent manner. A high dose of curcumin strongly inhibited p-Akt protein expression. With increasing curcumin levels, PTEN expression increased and miR-21 levels decreased. These results suggest that curcumin negatively modulated the miR-21/PTEN/Akt pathway. Moreover, after pretreatment with PD98059, cell apoptosis induced by curcumin was significantly increased. Additionally, the inhibitory effects of curcumin on the miR-21/PTEN/Akt pathway were significantly enhanced. CONCLUSION:PD98059 combined with curcumin may be a potential strategy for managing gastric cancer.
miR-21 reverses impaired decidualization through modulation of KLF12 and NR4A1 expression in human endometrial stromal cells†.
Yan Qiang,Yan Guijun,Zhang Chunxue,Wang Zhilong,Huang Chenyang,Wang Junxia,Zhou Jidong,Liu Yang,Ding Lijun,Zhang Qun,Zhen Xin,Jiang Yue,Sun Haixiang
Biology of reproduction
Impaired decidualization has been considered a major cause of infertility in adenomyosis. However, the mechanism remains poorly understood. Recent studies suggest that microRNAs (miRNA) play a crucial role in embryo implantation. The aim of the present study was to identify the role of miR-21 in human endometrial stromal cell (hESC) decidualization in vitro. To explore the roles of miR-21 in decidualization, we detected the expression of miR-21 in the endometrium of fertile control and adenomyosis patients, and analyzed the effects of miR-21 on the biological behaviors of hESC decidualization. The results demonstrated that miR-21 was downregulated in the endometrium of adenomyosis patients compared with the control endometrium. miR-21 effectively promoted the expression of the 8Br-cAMP plus medroxyprogesterone acetate (MPA)-induced hESC decidualization marker genes PRL and IGFBP-1 and morphological transformation through the modulation of KLF12 and NR4A1 expression; conversely, inhibition of miR-21 expression compromised hESC decidualization in vitro. In addition, Luciferase reporter, western blotting, and quantitative real-time PCR (qRT-PCR) assays confirmed that miR-21 interacted with the 3' untranslated region of the transcription factor KLF12 and downregulated KLF12 at the transcriptional and translational levels. KLF12 overexpression abolished miR-21-enhanced 8Br-cAMP plus MPA-induced decidualization. Taken together, these results illustrate that miR-21 promotes endometrial decidualization by inhibiting KLF12, and miR-21 overexpression reverses the poor decidual response of hESCs in patients with adenomyosis in vitro.
The effects of Radix Angelica Sinensis and Radix Hedysari ultrafiltration extract on X-irradiation-induced myocardial fibrosis in rats.
Ma Chengxu,Fu Zhaoyuan,Guo Huan,Wei Huiping,Zhao Xinke,Li Yingdong
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Radix Angelica Sinensis and Radix Hedysari are traditional Chinese medicines that are used for preventing and treating various diseases. This study aimed to investigate the effect and possible underlying mechanisms of Radix Angelica Sinensis and Radix Hedysari ultrafiltration extract (RAS-RH) on X-irradiation-induced cardiac fibrosis in rats. Our data demonstrated that (a) a single dose of total body irradiation (TBI) at 8 Gy resulted in cardiac fibrosis, whereas the control hearts exhibited less collagen and fibrosis. RAS-RH mitigated these morphological injuries. (b) TBI resulted in an increase in the serum levels of transforming growth factor β1 (TGF-β1) and troponin-I (TnI). RAS-RH inhibited the release of TBI-induced serum TGF-β1 and the TnI levels. (c) TBI inhibited the apoptosis of primary rat cardiac fibroblasts, whereas RAS-RH induced the apoptosis of primary rat cardiac fibroblasts after X- irradiation. (d) TBI resulted in an increase in the expression of osteopontin (OPN), c-fos, c-jun, miRNA-21 and collagen1α (COL1α) in primary rat cardiac fibroblasts, and RAS-RH mitigated the TBI-induced increased expression of OPN, c-jun, miRNA-21 and COL1α. In conclusion, these results demonstrate that RAS-RH exerts antifibrotic effects possibly through inducing the apoptosis of fibroblasts, inhibiting the release of serum TGF-β1, reducing the levels of serum TnI and reducing the expression of OPN, c-jun, miRNA-21 and COL1α. Therefore, RAS-RH may potentially be developed as a medical countermeasure for the mitigation of radiation-induced myocardial fibrosis.
IgE Downregulates PTEN through MicroRNA-21-5p and Stimulates Airway Smooth Muscle Cell Remodeling.
Fang Lei,Wang Xinggang,Sun Qingzhu,Papakonstantinou Eleni,S'ng Chongteck,Tamm Michael,Stolz Daiana,Roth Michael
International journal of molecular sciences
The patho-mechanism leading to airway wall remodeling in allergic asthma is not well understood and remodeling is resistant to therapies. This study assessed the effect of immunoglobulin E (IgE) in the absence of allergens on human primary airway smooth muscle cell (ASMC) remodeling in vitro. ASMCs were obtained from five allergic asthma patients and five controls. Proliferation was determined by direct cell counts, mitochondrial activity by expression of cytochrome c, protein expression by immunoblotting and immuno-fluorescence, cell migration by microscopy imaging, and collagen deposition by cell based ELISA and RNA expression by real time PCR. Non-immune IgE activated two signaling pathways: (i) signal transducer and activator of transcription 3 (STAT3)→miR-21-5p→downregulating phosphatase and tensin homolog (PTEN) expression, and (ii) phosphatidylinositol 3-kinases (PI3K)→protein kinase B (Akt)→mammalian target of rapamycin (mTOR)→ribosomal protein S6 kinase beta-1 (p70s6k)→peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1-α)→peroxisome proliferator-activated receptor-γ (PPAR-γ)→cyclooxygenase-2 (COX-2)→mitochondrial activity, proliferation, migration, and extracellular matrix deposition. Reduced PTEN expression correlated with enhanced PI3K signaling, which upregulated ASMC remodeling. The inhibition of microRNA-21-5p increased PTEN and reduced mTOR signaling and remodeling. Mimics of microRNA-21-5p had opposing effects. IgE induced ASMC remodeling was significantly reduced by inhibition of mTOR or STAT3. In conclusion, non-immune IgE alone is sufficient for stimulated ASMC remodeling by upregulating microRNA-21-5p. Our findings suggest that the suppression of micoRNA-21-5p may present a therapeutic target to reduce airway wall remodeling.
miRNA-21 promotes osteogenesis via the PTEN/PI3K/Akt/HIF-1α pathway and enhances bone regeneration in critical size defects.
Yang Chi,Liu Xiaohan,Zhao Kai,Zhu Youming,Hu Bin,Zhou Yong,Wang Mohan,Wu Yiqun,Zhang Chengfei,Xu Jianguang,Ning Yujie,Zou Duohong
Stem cell research & therapy
BACKGROUND:Functional reconstruction of maxillofacial bone defects is a considerable clinical challenge. Many studies have emphasized the osteogenic and angiopoietic abilities of stem cells for tissue regeneration. We previously showed that microRNA-21 (miRNA-21) can promote angiogenesis in human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs). In the present study, the role of miRNA-21 in osteogenic differentiation of bone marrow-derived stem cells (BMSCs) was investigated. METHODS:Western blotting and qPCR were performed to investigate the influences of miRNA-21 on osteogenic differentiation of BMSCs. The effects of miRNA-21 on PTEN/PI3K/Akt/HIF-1α pathway were also assessed using western blotting. To further evaluate the roles of miRNA-21 in osteogenesis in vivo, we conducted animal experiments in rat and canine. New bone formation was assessed using micro-CT and histological methods. RESULTS:In the present study, we found that miRNA-21 promotes the migration and osteogenic differentiation of bone marrow-derived stem cells (BMSCs) in vitro. Using gain- and loss-of-function studies, we found that miRNA-21 promoted the osteogenic ability of BMSCs by increasing P-Akt and HIF-1α activation. Finally, we verified the essential role of miRNA-21 in osteogenesis by implanting a miRNA-21-modified BMSCs/β-tricalcium phosphate (β-TCP) composite into critical size defects. Radiography, micro-CT, and histology revealed significantly greater volume of new bone formation in the miRNA-21 group than in the control group. CONCLUSION:In conclusion, our study demonstrated an essential role of miRNA-21 in promoting maxillofacial bone regeneration via the PTEN/PI3K/Akt/HIF-1α pathway.
Luteolin protects PC-12 cells from HO-induced injury by up-regulation of microRNA-21.
Zhang Zhiti,Xu Peng,Yu Haiyan,Shi Lei
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
BACKGROUND:Ischemic cerebrovascular disease (ICVD) is the third leading cause of death worldwide. Luteolin is a naturally flavonoid widely distributed in many plant leaves. This study aimed to explore the effects of luteolin on HO-induced ICVD cell oxidative injury model, as well as underlying molecular mechanisms. METHODS:Viability and apoptosis of PC-12 cells and rat brain microvascular endothelial cells (rBMECs) were detected using CCK-8 assay and FITC-Annexin V/PI staining, respectively. The levels of ROS and MDA were measured using DCFH-DA staining and MDA assay kit, respectively. Cell transfection was conducted to change the expression level of miR-21. Expression levels of key factors involved in cell proliferation, oxidative stress, apoptosis, PI3K/AKT and PDCD4/p21 pathways were evaluated using western blotting. RESULTS:Low concentration of luteolin had no significant effect on PC-12 cell viability and presented protective effects on HO-induced PC-12 cell viability loss, proliferation inhibition, ROS generation, oxidative stress increase and apoptosis. Moreover, luteolin up-regulated the expression level of miR-21 in HO-treated PC-12 cells. Overexpression of miR-21 strengthened the protective effects of luteolin on HO-induced PC-12 cell oxidative injury. Suppression of miR-21 had opposite effects. Furthermore, luteolin alleviated HO-induced inactivation of PI3K/AKT pathway and activation of PDCD4/p21 pathway in PC-12 cells by up-regulating miR-21. Besides, luteolin also protected rBMECs from HO-induced oxidative injury. CONCLUSION:Our research revealed the protective effects of luteolin on HO-induced ICVD cell oxidative injury. Luteolin protected PC-12 cells from HO-induced oxidative injury by up-regulating miR-21, activating PI3K/AKT pathway and inactivating PDCD4/p21 pathway.
PI3K/Akt Pathway and miR-21 are Involved in N-Ethyl-N-Nitrosourea-Induced F1 Mouse Lung Tumorigenesis: Effect of Inositol Hexaphosphate.
Sahay Satya,Tiwari Prakash,Pandey Manuraj,Gupta Krishna P
Journal of environmental pathology, toxicology and oncology : official organ of the International Society for Environmental Toxicology and Cancer
The risk of cancer development in offspring due to carcinogen exposure during pregnancy is a serious issue. In this study, we explored the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and microRNA-21 (miR-21) in transplacental lung tumorigenesis and its prevention by dietary compound inositol hexaphosphate (IP6) in F1 mice. Balb/c mice were exposed to the N-ethyl-N-nitrosourea (ENU) intraperitoneally on the 17th day of gestation. After weaning, half of the litters were fed with oral 2% IP6. At the end of 30, 120, or 240 days, we did not observe any effect on fetal viability or weight between ENU-exposed and non-exposed litters and the same was true of IP6. Altered expressions of the PI3K/Akt pathway were observed in F1 mice. Further, miR-21 expressions were found to be modulated at the respective time as well, along with the activation of matrix metalloproteinase (MMP-9) and vascular endothelial growth factor expression. Akt activation also enhanced the expression of cyclin D1, cyclooxygenase-2 (COX-2), nuclear factor-κB (NF-κBp50), and mammalian target of rapamycin (mTOR). IP6-fed F1 mice showed reduced tumorigenesis along with reduced expression of the PI3K/Akt pathway miR-21 and downstream targets. The PI3K/Akt pathway and miR-21 are involved in transplacental lung tumorigenesis, whereas IP6 seemed to affect lung tumorigenesis by suppressing the expression of the PI3K/Akt pathway in F1 mice.
The uremic toxin p-cresyl sulfate induces proliferation and migration of clear cell renal cell carcinoma via microRNA-21/ HIF-1α axis signals.
Wu Tsai-Kun,Wei Chyou-Wei,Pan Ying-Ru,Hsu Ren-Jun,Wu Chung-Yi,Yu Yung-Luen
p-Cresyl sulfate (pCS), a uremic toxin, can cause renal damage and dysfunction. Studies suggest that renal dysfunction increases the prevalence of renal cancer. However, the effect of pCS on the proliferation and migration of renal cancer is unclear. Clear cell renal cell carcinoma (ccRCC) expresses mutant von Hippel-Lindau gene and is difficult to treat. Hypoxia-inducible factor-1α and 2-α (HIF-1α and HIF-2α) as well as microRNA-21 (miR-21) can regulate the proliferation and migration of ccRCC cells. However, the association between HIF-α and miR-21 in ccRCC remains unclear. Therefore, the effects of pCS on ccRCC cells were investigated for HIF-α and miR-21 signals. Our results showed that pCS induced overexpression of HIF-1α and promoted the proliferation and regulated epithelial-mesenchymal transition-related proteins, including E-cadherin, fibronectin, twist and vimentin in ccRCC cells. pCS treatment increased miR-21 expression. Specifically, inhibition of miR-21 blocked pCS-induced proliferation and migration. Taken together, the present results demonstrate that pCS directly induced the proliferation and migration of ccRCC cells through mechanisms involving miR-21/HIF-1α signaling pathways.
Ski-related novel protein suppresses the development of diabetic nephropathy by modulating transforming growth factor-β signaling and microRNA-21 expression.
Wang Yuanyuan,Liu Lingling,Peng Wei,Liu Huiming,Liang Luqun,Zhang Xiaohuan,Mao Yanwen,Zhou Xingcheng,Shi Mingjun,Xiao Ying,Zhang Fan,Zhang Yingying,Liu Lirong,Yan Rui,Guo Bing
Journal of cellular physiology
Unveiling the mechanisms that drive the pathological phenotypes of diabetic nephropathy (DN) could help develop new effective therapeutics for this ailment. Transforming growth factor-β1 (TGF-β1)/Smad3 signaling is aberrantly induced in DN, leading to elevated microRNA-21 (miR-21) expression and tissue fibrosis. Ski-related novel protein (SnoN) negatively regulates the TGF-β pathway, but the relationship between SnoN and miR-21 has not been described in the context of DN. In this study, this association was investigated in vivo (streptozotocin-induced rat model of diabetes) and in vitro (NRK-52E model system under high glucose conditions). In both model systems, we observed reduced amounts of the SnoN protein and elevated miR-21 amounts, indicative of an inverse relationship. These changes in SnoN and miR-21 amounts were accompanied by reduced E-cadherin and elevated α-smooth muscle actin and collagen III levels, consistent with epithelial to mesenchymal transition (EMT). In vitro overexpression of SnoN in NRK-52E cells downregulated miR-21 at the transcriptional and posttranscriptional levels and repressed EMT and extracellular matrix (ECM) deposition. In contrast, knockdown of SnoN resulted in miR-21 upregulation, particularly at the transcriptional level. We further demonstrated that overexpression and inhibition of miR-21 promoted and suppressed EMT and ECM deposition, respectively, without affecting SnoN levels. Our results indicated that SnoN suppresses the development of DN as well as renal fibrosis by downregulating miR-21, and therefore represents a novel and promising therapeutic target for DN.
MicroRNA-21 and its impact on signaling pathways in cervical cancer.
Wang Yong,Zhou Shiying,Fan Kefeng,Jiang Chen
Oncogenic microRNA-21 (miR-21/miRNA-21) is a stable inhibitor of gene expression that is often upregulated in cervical cancer, a disease that affects the health of women and tends to transform and spread. Previous studies investigating miR-21 in biopsies and cells from cervical cancer patients have identified that miR-21 binds target mRNAs in signaling pathways or long non-coding RNAs (lncRNA). Furthermore, studies have elucidated the molecular mechanisms of two tumor necrosis factor α (TNF-α) signaling pathways that promote cell proliferation and inhibit cell apoptosis. miR-21 inhibits the TNF receptor 1 (TNFR1) signaling pathway and activates the TNFR2 signaling pathway. Moreover, miR-21 enhances cervical cancer cell proliferation by influencing the protein kinase B/mammalian target of rapamycin and RAS p21 protein activator 1 signaling pathways. The present review discusses the evidence that miR-21 may impact cervical cancer through inhibiting apoptosis and enhancing proliferation, and may therefore be a target for clinical intervention.
Atrasentan alleviates high glucose-induced podocyte injury by the microRNA-21/forkhead box O1 axis.
Wang Jie,Shen Lanyu,Hong Hong,Li Jie,Wang Hongtai,Li Xiuzhen
European journal of pharmacology
Diabetic nephropathy (DN) is the most common complication of diabetes mellitus. Atrasentan (Atr) has potential therapeutic values for DN. MicroRNAs (miRNAs) function as vital regulators in the pathophysiology of kidney diseases including DN. Our present study aimed to further explore whether Atr could alleviate kidney injury by regulating microRNA-21(miR-21)/forkhead box O1 (FOXO1) in DN mouse models and cell models. Blood glucose concentration and ACR ratio were determined by matching commercial kits. MiR-21 and FOXO1 mRNA level was measured by RT-qPCR assay. Protein levels of FOXO1, LC3Ⅰ, LC3Ⅱ and p62 were measured by western blot assay. Cell apoptotic index was examined by flow cytometry. The interaction of miR-21 and FOXO1 was tested by bioinformatics analysis, luciferase assay and RIP assay. We found that Atr alleviated kidney injury by inhibiting miR-21 expression and promoting autophagy in DN mice. Moreover, miR-21 loss suppressed apoptosis and induced autophagy in high glucose (HG)-treated podocytes. And, Atr inhibited cell apoptosis and improved cell autophagic activity by downregulating miR-21 in HG-cultured podocytes. Moreover, FOXO1 was identified as a target of miR-21. MiR-21 exerted its pro-apoptosis and anti-autophagy effects by targeting FOXO1 in HG-cultured podocytes. Atr enhanced FOXO1 expression by downregulating miR-21 in HG-cultured podocytes. We concluded that Atr mitigated kidney injury in DN mice and alleviated HG-mediated apoptosis increase and autophagy inhibition in podocytes by regulating miR-21/FOXO1 axis, further elucidating the molecular basis by which Atr hampered DN progression.
lncRNA mortal obligate RNA transcript was downregulated in ovarian carcinoma and inhibits cancer cell proliferation by downregulating miRNA-21.
Chen Xiao,Wu Wanyin,Cao Xiaolong,Zhao Xiaoshan,Li Weizhan,Deng Caijiu,Huang Zhen
Journal of cellular biochemistry
microRNA-21 (miRNA-21) is a well-characterized oncogenic miRNA in human cancers. In the present study, we found that miRNA-21 was upregulated, while long noncoding RNA Mortal Obligate RNA Transcript (lncRNA MORT), which has been reported to be silenced in 16 types of cancers, was downregulated in tumor tissues than in adjacent healthy tissues of patients with ovarian carcinoma. Expression of lncRNA MORT in tumor tissues was found to be significantly affected by tumor size but not by tumor metastasis. Expression levels of lncRNA MORT and miRNA-21 were significantly and inversely correlated in both tumor tissues and adjacent healthy tissues. Overexpression of lncRNA MORT inhibited miRNA-21, while miRNA-21 overexpression failed to significantly affect lncRNA MORT expression. Overexpression of lncRNA MORT inhibited, while miRNA-21 overexpression promoted the proliferation of cells of ovarian cancer cell lines. In addition, miRNA-21 overexpression partially reversed the inhibitory effects of lncRNA MORT overexpression on cancer cell proliferation. However, overexpression of lncRNA MORT showed no significant effects on cancer cell migration and invasion. Therefore, lncRNA MORT was downregulated in ovarian carcinoma and lncRNA MORT overexpression inhibited cancer cell proliferation, possibly by downregulating miRNA-21.
Effect of microRNA-21 on hypoxia-inducible factor-1α in orthodontic tooth movement and human periodontal ligament cells under hypoxia.
Zhang Xueqin,Chen Dongru,Zheng Jinxuan,Deng Lidi,Chen Zhengyuan,Ling Junqi,Wu Liping
Experimental and therapeutic medicine
Orthodontic tooth movement can lead to temporary hypoxia of periodontal tissues. Periodontal ligament cells (PDLCs) react to hypoxia, releasing various biological factors to promote periodontal tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) is one of the most sensitive factors involved in the response to hypoxia. HIF-1α has been identified to be involved in osteogenic and osteoclast differentiation ; however, few studies have investigated the expression of HIF-1α in the periodontal ligament (PDL) during orthodontic movement . In a previous study, microRNA-21 (miR-21) was demonstrated to be highly expressed in a rat model of orthodontic tooth movement. Additionally, miR-21 can increase the expression of HIF-1α in certain tumor cell types and is involved in tumor bioactivities. In the present study, HIF-1α exhibited expression patterns in a similar way to miR-21 in PDL samples from a rat model of orthodontic tooth movement, with expression initially increased and followed by a decrease over time. Furthermore, human PDLCs were exposed to a hypoxic environment , which induced significant upregulation of HIF-1α and miR-21 expression. Furthermore, miR-21 mimics increased HIF-1α expression and promoted osteogenic differentiation, indicated by upregulated expression of the osteogenic markers osteopontin, runt-related gene-2 and alkaline phosphatase. miR-21 inhibitors suppressed HIF-1α expression and downregulated the osteogenic markers. In conclusion, the results revealed that miR-21 has a positive effect on HIF-1α expression in PDLCs under hypoxia and has important roles in osteogenic differentiation during orthodontic tooth movement. These findings provide a theoretical basis by which to promote tissue reconstruction during orthodontic tooth movement.
Activation of the STAT3/microRNA-21 pathway participates in angiotensin II-induced angiogenesis.
Chen Li-Yuan,Wang Xue,Qu Xiao-Long,Pan Li-Na,Wang Ze-Yang,Lu Yong-Hui,Hu Hou-Yuan
Journal of cellular physiology
Angiotensin II (AngII) facilitates angiogenesis that is associated with the continuous progression of atherosclerotic plaques, but the underlying mechanisms are still not fully understood. Several microRNAs (miRNAs) have been shown to promote angiogenesis; however, whether miRNAs play a crucial role in AngII-induced angiogenesis remains unclear. This study evaluated the functional involvement of miRNA-21 (miR-21) in the AngII-mediated proangiogenic response in human microvascular endothelial cells (HMECs). We found that AngII exerted a proangiogenic role, indicated by the promotion of proliferation, migration, and tube formation in HMECs. Next, miR-21 was found to be upregulated in AngII-treated HMECs, and its specific inhibitor potently blocked the proangiogenic effects of AngII. Subsequently, we focused on the constitutive activation of STAT3 in the AngII-mediated proangiogenic process. Bioinformatic analysis indicated that STAT3 acted as a transcription factor initiating miR-21 expression, which was verified by ChIP-PCR. A reporter assay further identified three functional binding sites of STAT3 in the miR-21 promoter region. Moreover, phosphatase and tensin homolog (PTEN) was recognized as a target of miR-21, and STAT3 inhibition restored AngII-induced reduction in PTEN. Similarly, the STAT3/miR-21 axis was shown to mediate AngII-provoked angiogenesis in vivo, which was demonstrated by using the appropriate inhibitors. Our data suggest that AngII was involved in proangiogenic responses through miR-21 upregulation and reduced PTEN expression, which was, at least in part, linked to STAT3 signaling. The present study provides novel insights into AngII-induced angiogenesis and suggests potential treatment strategies for attenuating the progression of atherosclerotic lesions and preventing atherosclerosis complications.
Ginsenoside Rb1 protects cardiomyocytes from oxygen-glucose deprivation injuries by targeting microRNA-21.
Yang Chuang,Li Bo,Liu Yongsheng,Xing Yue
Experimental and therapeutic medicine
Ginsenoside Rb1 (GS-Rb1) is one of the most important active pharmacological extracts of the traditional Chinese medicine, ginseng, and there is extensive evidence of its cardioprotective properties. However, the microRNA (miR) targets of GS-Rb1 and the underlying mechanisms of GS-Rb1 and miR-21 in the progression of cardiomyocyte apoptosis have not been clearly elucidated. The aim of the current study was to investigate the impact of miR-21 and its target gene, programmed cell death protein 4 (PDCD4), on the protective effect of GS-Rb1 in cardiomyocytes injured by oxygen-glucose deprivation (OGD). The miR-21 expression levels were downregulated, and the percentage of the apoptotic cells and reactive oxygen species (ROS) was increased in OGD-cultured neonatal rat cardiomyocytes; however, the effects were reversed by GS-Rb1 treatment. It was demonstrated that GS-Rb1 could reduce intracellular ROS content, and the expression of cytochrome C and the pro-apoptosis protein, apoptosis regulator B-cell lymphoma associated X (Bax) protein while increasing the expression of the anti-apoptosis protein, apoptosis regulator Bcl-2. The target gene, PDCD4, was significantly upregulated in the OGD group; however, the expression of PDCD4 was inhibited by GS-Rb1 treatment. Furthermore, miR-21 inhibitor transfection reduced GS-Rb1-induced miR-21 upregulation compared with the OGD+GS-Rb1 group, indicating that the miR-21 was involved in the anti-apoptotic effect of GS-Rb1 in cardiomyocytes. The results of the current study highlighted that GS-Rb1 could target miR-21 and its target gene, PDCD4, to protect OGD-injured cardiomyocytes. The results of the current study may provide a novel insight for the treatment of myocardial infarction with Traditional Chinese Medicines, involving miRs as targets.
Celastrol inhibits growth and metastasis of human gastric cancer cell MKN45 by down-regulating microRNA-21.
Yao Shan-Shan,Han Lei,Tian Zi-Bin,Yu Ya-Nan,Zhang Qi,Li Xiao-Yu,Mao Tao,Yang Lin
Phytotherapy research : PTR
Celastrol could inhibit cancer cell growth in vitro. However, effect(s) of celastrol on gastric cancer is not well studied. Therefore, we investigated the effects of celastrol on human gastric cancer cell line MKN45 and the underlying mechanisms. We found that celastrol inhibited cell proliferation, migration, and invasion and induced cell apoptosis and G2/M cell cycle arrest (p < .05, p < .01, or p < .001). Under celastrol treatment, overexpression of microRNA-21 (miR-21) increased cell viability, migration, and invasion and inhibited cell apoptosis compared with negative control (p < .05, p < .01, or p < .001). In addition, the phosphorylation of PTEN was significantly up-regulated, whereas PI3K, AKT, p65, and IκBα phosphorylation was statistically decreased by celastrol (p < .05 or p < .01) and then further reversed by miR-21 overexpression (p < .05 or p < .01). On the other side, miR-21 silence showed contrary results (p < .05) as relative to miR-21 overexpression. In conclusion, celastrol inhibits proliferation, migration, and invasion and inactivates PTEN/PI3K/AKT and nuclear factor κB signaling pathways in MKN45 cells by down-regulating miR-21.
Upregulation of MicroRNA-21 promotes tumorigenesis of prostate cancer cells by targeting KLF5.
Guan Chen,Zhang Lingling,Wang Sixuan,Long Luye,Zhou Huaibin,Qian Shihan,Ma Mengni,Bai Fumao,Meng Qing H,Lyu Jianxin
Cancer biology & therapy
Prostate cancer (PCa) is the second frequently newly diagnosed cancer in men. Androgen deprivation therapy has been widely used to inhibit PCa growth but eventually fails in many patients. Androgen receptor and its downstream molecules like microRNAs could be promising therapeutic targets. We aimed to investigate the involvement of miR-21 in PCa tumorigenesis. We found that miR-21 was an unfavorable factor and correlated positively with tumor grade in PCa patients from TCGA database. MiR-21 was more highly expressed in androgen-independent PCa cells than in androgen-dependent PCa cells. Overexpression of miR-21 promoted androgen-dependent and -independent PCa cell proliferation, migration, invasion, and resistance to apoptosis. Furthermore, increased miR-21 expression promoted mouse xenograft growth. We identified nine genes differentially expressed in PCa tumors and normal tissue which could be potential targets of miR-21 by bioinformatic analyses. We demonstrate that miR-21 directly targeted KLF5 and inhibited KLF5 mRNA and protein levels in PCa. STRING and functional enrichment analysis results suggest that GSK3B might be regulated by KLF5. Our findings demonstrate that miR-21 promotes the tumorigenesis of PCa cells by directly targeting KLF5. These biological effects are mediated through upregulation of GSK3B and activation of the AKT signaling pathway.
LncRNA AWPPH and miRNA-21 regulates cancer cell proliferation and chemosensitivity in triple-negative breast cancer by interacting with each other.
Liu Ai-Na,Qu Hua-Jun,Gong Wen-Jing,Xiang Jin-Yu,Yang Miao-Miao,Zhang Wei
Journal of cellular biochemistry
Long-non-coding RNAs (lncRNA) AWPPH promotes the progression of liver and bladder cancer, indicating its oncogenic role. The current study aimed to explore the involvement of AWPPH in triple-negative breast cancer (TNBC). In the current study, we found that plasma levels of lncRNA AWPPH and microRNA-21 (miRNA-21) were upregulated in patients with TNBC than in healthy controls, and the upregulation of plasma lncRNA AWPPH and miRNA-21 distinguished early-stage patients with TNBC from healthy controls. Plasma levels of lncRNA AWPPH and miRNA-21 were significantly and positively correlated in both patients with TNBC and healthy controls. LncRNA AWPPH and miRNA-21 overexpression led to promoted cancer cells proliferation and improved cancer cell viability under carboplatin treatment, while lncRNA AWPPH small interfering RNA (siRNA) silencing played an opposite role. In addition, miRNA-21 overexpression attenuated the effects of lncRNA AWPPH siRNA silencing on of cancer cell behaviors. LncRNA AWPPH overexpression led to upregulated miRNA-21 in TNBC cells, while miRNA-21 overexpression also led to significantly upregulated lncRNA AWPPH expression. Therefore, lncRNA AWPPH and miRNA-21 may regulate cancer cell proliferation and chemosensitivity in TNBC by interacting with each other.
microRNA-21-5p dysregulation in exosomes derived from heart failure patients impairs regenerative potential.
Qiao Li,Hu Shiqi,Liu Suyun,Zhang Hui,Ma Hong,Huang Ke,Li Zhenhua,Su Teng,Vandergriff Adam,Tang Junnan,Allen Tyler,Dinh Phuong-Uyen,Cores Jhon,Yin Qi,Li Yongjun,Cheng Ke
The Journal of clinical investigation
Exosomes, as functional paracrine units of therapeutic cells, can partially reproduce the reparative properties of their parental cells. The constitution of exosomes, as well as their biological activity, largely depends on the cells that secrete them. We isolated exosomes from explant-derived cardiac stromal cells from patients with heart failure (FEXO) or from normal donor hearts (NEXO) and compared their regenerative activities in vitro and in vivo. Patients in the FEXO group exhibited an impaired ability to promote endothelial tube formation and cardiomyocyte proliferation in vitro. Intramyocardial injection of NEXO resulted in structural and functional improvements in a murine model of acute myocardial infarction. In contrast, FEXO therapy exacerbated cardiac function and left ventricular remodeling. microRNA array and PCR analysis revealed dysregulation of miR-21-5p in FEXO. Restoring miR-21-5p expression rescued FEXO's reparative function, whereas blunting miR-21-5p expression in NEXO diminished its therapeutic benefits. Further mechanistic studies revealed that miR-21-5p augmented Akt kinase activity through the inhibition of phosphatase and tensin homolog. Taken together, the heart failure pathological condition altered the miR cargos of cardiac-derived exosomes and impaired their regenerative activities. miR-21-5p contributes to exosome-mediated heart repair by enhancing angiogenesis and cardiomyocyte survival through the phosphatase and tensin homolog/Akt pathway.
Effect of Celastrus orbiculatus in inhibiting Helicobacter pylori induced inflammatory response by regulating epithelial mesenchymal transition and targeting miR-21/PDCD4 signaling pathway in gastric epithelial cells.
Zhu Yaodong,Liu Lei,Hu Lei,Dong Wenqing,Zhang Mei,Liu Yanqing,Li Ping
BMC complementary and alternative medicine
BACKGROUND:The extract of Celastrus orbiculatus (COE) have been studied for anti-Helicobacter pylori (H. pylori) activity and anti-cancer effects in vitro and in vivo. However, the molecular mechanism by which COE inhibits H. pylori-induced inflammatory response has not been fully elucidated so far. METHODS:The effects of COE on viability, morphological changes, inflammatory cytokine secretion, protein and mRNA expression were analyzed by MTT assay, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, western blot and real-time PCR (RT-PCR), respectively. The methylation level of programmed cell death 4 (PDCD4) promoter was investigated by methylation-specific PCR. (MSP) . RESULTS:COE effectively inhibited the H.pylori-induced inflammatory response by regulating epithelial-mesenchymal transition (EMT). The methylation level of PDCD4 promoter was suppressed by COE, which increased the expression ofPDCD4. Moreover, COE could inhibit microRNA-21 (miR-21) expression, as shown by an enhancement of its target gene PDCD4. Furthermore, both miR-21 over-expression and PDCD4 silencing attenuated the anti-inflammatory effect. of COE. CONCLUSIONS:COE inhibits H. pylori induced inflammatory response through regulating EMT, correlating with inhibition of miR-21/PDCD4 signal pathways in gastric epithelial cells.
lncRNA GAS5 acts as a ceRNA for miR-21 in suppressing PDGF-bb-induced proliferation and migration in vascular smooth muscle cells.
Liu Kefeng,Liu Chen,Zhang Zhengyu
Journal of cellular biochemistry
Aberrant proliferation and migration of vascular smooth muscle cells (VSMCs) contribute significantly to the development of many human cardiovascular diseases, including hypertension. The present study aimed to evaluate the in vitro functional roles of long noncoding RNA growth arrest-specific transcript 5 (GAS5) in VSMCs and explored the underlying molecular mechanisms. We found that GAS5 was significantly downregulated in PDGF-bb-treated VSMCs, and overexpression of GAS5 remarkably attenuated PDGF-bb-induced VSMC proliferation and migration. In addition, miR-21 was observed to potentially bind to GAS5, and we also identified that PDCD4 might be a direct target of miR-21 in VSMCs. Cotransfection of miR-21 mimics remarkably reduced the PDCD4 protein expression in GAS5 overexpressing VSMCs. Further, rescue experiments showed that enforced expression of miR-21 attenuated the inhibitory effects of GAS5 on VSMC proliferation and migration. Collectively, our results demonstrated that GAS5 inhibits PDGF-bb-induced VSMC proliferation and migration, partly through acting as a competing endogenous RNA of miR-21, and provided new evidence that GAS5 may serve as a potential therapeutic target for hypertension.
MiR-21-5p regulates mycobacterial survival and inflammatory responses by targeting Bcl-2 and TLR4 in Mycobacterium tuberculosis-infected macrophages.
Zhao Zhonghua,Hao Jinzhu,Li Xia,Chen Yanfang,Qi Xiaoyan
To date, very little is known about the role of microRNA-21-5p (miR-21-5p) in Mycobacterium tuberculosis (M.tb)-infected macrophages. Here, we show that M.tb infection of RAW264.7 and THP-1 cells increases the expression of miR-21-5p. MiR-21-5p enhances M.tb survival and apoptosis, and attenuates the secretion of inflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α in M.tb-infected macrophages. Dual-luciferase reporter assay revealed that the 3'-UTR of B-cell lymphoma 2 (Bcl-2) or toll-like receptor 4 (TLR4) is a direct target of miR-21-5p. Enforced expressions of Bcl-2 or TLR4 partially attenuate the suppressive effects of miR-21-5p on cell viability and inflammatory cytokines, and effectively decrease bacterial burden. Therefore, the present study highlights a novel role for miR-21-5p in regulation of mycobacterial survival and inflammatory responses by targeting Bcl-2 and TLR4 in M.tb-infected macrophages.
Angiotensin II type i receptor agonistic autoantibodies induces apoptosis of cardiomyocytes by downregulating miR21 in preeclampsia: a mechanism study.
Wang Jin,Yue Jiping,Xia Qin,Jiao Xiangying,Zhi Jianming
American journal of translational research
Angiotensin II type I receptor agonistic autoantibodies (AT-AA) in the plasma of preeclampsia patients can induce apoptosis of cardiomyocytes, and microRNA-21 (miR-21) can exert a protective effect on cardiomyocytes. But whether the pro-apoptotic effect of AT-AA is associated with miR-21 is unclear. The objective of the present study was to explore whether AT-AA induced cardiomyocyte apoptosis was related to its inhibitory of miR-21 expression. In vivo studies, the pregnant rats were divided into two groups: Sham group, Model group. The pathology, cell apoptosis, and relative protein expressions were evaluated by hematoxylin and eosin staining, and Western blot assay. The expression of microRNA was detected by gene microarray. In the cell experiment, the neonatal rat cardiomyocytes were divided into four groups: NC group, AT-AA group, and miR-21 group and AT-AA+miR-21 group. The cell apoptosis and relative proteins' expressions were measured by flow cytometry and Western blot assay. Results: Compared with the Sham group, miR-21 in the cardiac tissue of the model group was downregulated significantly; the expression of p-JNK, Bax and caspases-3 was increased, the expression of Bcl-2 was decreased, and the Bcl-2/Bax ratio became smaller. The expression of miR-21 in AT-AA treated cardiomyocytes was only 52% of the control group, with an apoptosis rate of 32.6%. In addition, the expression of pPTEN, pAKT and pFOXO3a in the model group was significantly higher than that in the NC group. The cardiomyocyte apoptosis rate in miR-21 overexpression group was only 23.7%, which was higher than that in the NC group, but significantly lower than that in AT-AA group. PTEN, AKT and FOXO3a phosporylation in miR-21 overexpression group was also lower than that in AT-AA group. AT-AA induced cardiomyocyte apoptosis by downregulating miR-21, and the PTEN/AKT/FOXO3a signal transduction pathway participated in this process. The result of the present study suggests that miR-21 may prove to be a new target for the diagnosis and treatment of preeclampsia and other cardiovascular diseases.
Long non-coding RNA CASC2 serves as a ceRNA of microRNA-21 to promote PDCD4 expression in oral squamous cell carcinoma.
Pan Lina,Chen Hui,Bai Yang,Wang Qibao,Chen Liang
OncoTargets and therapy
Oral squamous cell carcinoma (OSCC) is a common oral disease with high morbidity and mortality. Recently, long non-coding RNAs (lncRNAs) were identified as critical regulators in OSCC tumorigenesis. The present study aimed to work out the functions and the possible molecular mechanisms of lncRNA CASC2 in human OSCC. The expression levels of CASC2 in clinical OSCC tissue samples and cultured OSCC cell lines were detected by RT-qPCR analysis. MTT assay was performed to detect the proliferation ability of OSCC cells, whereas the apoptosis rate and cell cycle distribution of OSCC cells were determined by flow cytometric analysis. The expression levels of relevant proteins were detected by Western blot assay. Dual-luciferase reporter assay was performed to validate the predicted relationship between CASC2, miR-21 amd PDCD4. The role of CASC2 in OSCC tumorigenesis in vivo was evaluated using a nude mouse tumor model. The results demonstrated that CASC2 was significantly downregulated in clinical OSCC tissue samples and cultured OSCC cell lines. Low CASC2 expression was closely correlated with adverse clinicopathological characteristics of OSCC patients. Functionally, overexpression of CASC2 remarkably inhibited cell proliferation partly through inducing cell cycle arrest and cell apoptosis. Furthermore, bioinformatics analysis and dual-luciferase reporter assay showed that CASC2 might act as a competing endogenous RNA of miR-21 to promote the expression of PDCD4. Rescue experiments also showed that miR-21 blocked the tumor-suppressive role that CASC2 exerted in OSCC cells. Finally, in vivo study indicated that overexpression of CASC2 restrained OSCC tumor growth in volume and weight. In conclusion, these findings indicate that CASC2/miR-21/PDCD4 axis might be a potential regulator of OSCC tumorigenesis, and shed new light on lncRNA-directed diagnostics and therapeutics in OSCC.
Tanshindiol-C suppresses in vitro and in vivo hepatocellular cancer cell growth by inducing mitochondrial-mediated apoptosis, cell cycle arrest, inhibition of angiogenesis and modulation of key tumor-suppressive miRNAs.
Zhou Ping,Cheng Yunzhou,Liu Fangli,Wu Kai,Qiu Weilong,Wang Shouqi
Journal of B.U.ON. : official journal of the Balkan Union of Oncology
PURPOSE:Hepatocellular carcinoma causes considerable mortality and no efficient chemotherapy is available. Novel molecules of plant origin may prove beneficial in the development of therapy of hepatocellular carcinoma. In this study we examined the anticancer effects of Tanshindiol-C (TC) against the hepatocellular carcinoma SNU-4235 cell line. METHODS:Proliferation rate of the SNU-2435 cells was determined by MTT assay. Apoptosis was confirmed by DAPI and annexin V/PI staining. Cell cycle analysis was performed by flow cytometry. MicroRNA expression was checked by qRT-PCR and protein expression by western blotting. The in vivo evaluation of TC was performed in xenografted mice models. RESULTS:TC inhibited the growth of the SNU-4235 cells and exhibited an IC50 of 20 µM. Investigation of the underlying mechanism revealed that TC triggered apoptotic death of the SNU-4235 cells which was also associated with enhancement of the expression of Bax and decrease in the expression of Bcl-2. TC also caused arrest of the cells in the G2/M phase of the cell cycle and also exerted angiogenitic effects. TC also enhanced the expression of the tumor suppressor microRNA-21, 222 and 31. In vivo evaluation of TC revealed that it could inhibit the tumor weight volume, suggestive of the anticancer potential of TC. CONCLUSIONS:In brief, tanshindiol-C exerts anticancer effects on hepatocellular carcinoma by induction of apoptosis and cell cycle arrest, along with inhibiting the angiogenesis and the expression of tumor suppressive microRNAs. TC could also inhibit the growth of the xenografted tumors and hence could prove to be a potential anticancer agent.
Ionizing Radiation-inducible microRNA-21 Induces Angiogenesis by Directly Targeting PTEN
Zhang Yongchun,Chen Zhiying,Feng Lingxin,Jiang Peng,Li Xiumei,Wang Xiang
Asian Pacific journal of cancer prevention : APJCP
Background: Previous experimental studies have established that MicroRNAs (miRNAs) can function as oncogenesor tumor suppressors in the regulation of tumor biology or pathology. However, the effects of ionizing radiation (IR)on the expression levels of cellular miRNAs and their further effects on the biological behavior of tumor cells requirefurther investigation. Methods: We determined the proliferation, migration and tube formation of HUVEC cells afterionizing radiation (control, 0h and 24h), and the changes of miR-21, VEFG and HIF-1α levels after ionizing radiation weremeasured by Western blot (WB). The effects of miR-21 mimics and inhibitors on the protein and mRNA expression ofPTEN were determined by RT-PCT and WB. Two independent luciferase reporter plasmids were constructed to detectchanges in PTEN protein expression. Results: We found that both IR and miR-21 increase proliferation, migration andtube formation of HUVEC cells. Ionizing radiation directly targets the inhibition of PTEN expression by causing anincrease in miR-21 expression, and induces the accumulation of VEGF and HIF-1α expression in cells by the PI3K / AKTsignaling pathway. Simultaneous induction of ectopic expression of PTEN can rescue radiation-induced proliferation,migration and tube formation in tumor cells. Conclusion: miR-21 promotes tumor cell proliferation and migration bytargeting inhibition of PTEN expression, which may become a potential target for tumor therapy in the future.
Eupatilin Inhibits Renal Cancer Growth by Downregulating MicroRNA-21 through the Activation of YAP1.
Zhong Weifeng,Wu Zhiming,Chen Nanhui,Zhong Kaihua,Lin Yifeng,Jiang Huiming,Wan Pei,Lu Shanming,Yang Lawei,Liu Siping
BioMed research international
Renal cell carcinoma (RCC) is the second most common human urinary tumor. Eupatilin is the main active ingredient of the traditional Chinese medicine . The effect of Eupatilin on RCC and the underlying mechanism remain unknown. Here, we investigated the anticancer effects and mechanisms of Eupatilin in RCC and , laying an experimental foundation for the clinical application of Eupatilin in the treatment of RCC. The results showed that Eupatilin significantly inhibited 786-O cell viability and migration and promoted apoptosis. Eupatilin inhibited the expression of miR-21 in 786-O cells, and overexpression of miR-21 suppressed the effect of Eupatilin on viability, apoptosis, and migration in 786-O cells. Eupatilin inhibited the growth of renal tumors in nude mice by downregulating miR-21. YAP1, which was identified as a target of miR-21, showed significantly lower expression in RCC tissues than in healthy tissues. miR-21 significantly inhibited YAP1 protein expression in 786-O cells and tumor tissues isolated from nude mice, and YAP1 attenuated the effect of miR-21 on the viability, apoptosis, and migration of 786-O cells. In conclusion, Eupatilin inhibited the expression of miR-21, which mediated the proapoptotic and antimigratory effects of Eupatilin by suppressing YAP1 in renal cancer cells. These results suggested that Eupatilin could be a potent agent for the treatment of RCC.
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.
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.
Skullcapflavone I suppresses proliferation of human lung cancer cells via down-regulating microRNA-21.
Yang Yadong,An Ran,Feng Tao,Qin Xiaofeng,Zhang Jian,Bo Youling,Niu Ben
Experimental and molecular pathology
BACKGROUND:Lung cancer is the most leading cause of cancer-related deaths worldwide. Skullcapflavone I is a flavone compound extracted from Scutellaria baicalensis Georgi (Wogon). The present study investigated the effects of skullcapflavone I on human lung cancer cell proliferation, as well as underlying possible mechanism. METHODS:Cell proliferation was detected using Trypan blue assay. Cell viability was measured using cell counting kit 8 (CCK-8) assay. Quantitative reverse transcription PCR (qRT-PCR) was performed to assess microRNA-21 (miR-21) expression. Cell transfection was conducted to enhance the levels of miR-21 and protein phosphatase 2A (PP2A). Western blotting was used to evaluate the expressions of proliferation cell nuclear antigen (PCNA), Cyclin D1, PP2A, phosphatidylinositol 3-kinase (PI3K), protein kinase 3 (AKT), mechanistic target of rapamycin (mTOR) and p70S6K. RESULTS:Skullcapflavone I significantly suppressed the viability and proliferation of A549 and H1975 cells. The expressions of miR-21 in A549 and H1975 cells were drastically decreased after skullcapflavone I treatment. Overexpression of miR-21 remarkably reversed the skullcapflavone I-induced A549 cell viability inhibition. Moreover, skullcapflavone I enhanced the expression of PP2A in A549 cells. Skullcapflavone I inactivated PI3K/AKT/mTOR signaling pathway in A549 cells via up-regulating PP2A. Besides, skullcapflavone I treatment had no significant influence on human normal bronchial epithelial 16HBE cell viability, proliferation and apoptosis. CONCLUSION:Skullcapflavone I exerted anti-cancer effect on lung cancer cells by down-regulating miR-21 expression, up-regulating PP2A expression and then inactivating PI3K/AKT/mTOR signaling pathway.
[Research progress on miR-21 in heart diseases].
Yang Kun,Hu Xiaosheng
Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences
Pathological processes such as myocardial apoptosis, cardiac hypertrophy, myocardial fibrosis, and cardiac electrical remodeling are involved in the development and progression of most cardiac diseases. MicroRNA-21 (miR-21) has been found to play an important role in heart diseases as a novel type of endogenous regulators, which can inhibit cardiomyocyte apoptosis, improve hypertension and cardiac hypertrophy, promote myocardial fibrosis and atrial electrical remodeling. In this review, we summarize the research progress on the function of miR-21 in heart diseases and its mechanism, and discuss its potential application in diagnosis and treatment of heart diseases.
The Role of MicroRNA-21 in Venous Neointimal Hyperplasia: Implications for Targeting miR-21 for VNH Treatment.
Kilari Sreenivasulu,Cai Chuanqi,Zhao Chenglei,Sharma Amit,Chernogubova Ekaterina,Simeon Michael,Wu Chin-Cheng,Song Hsiang-Lin,Maegdefessel Lars,Misra Sanjay
Molecular therapy : the journal of the American Society of Gene Therapy
The molecular mechanism of hemodialysis access arteriovenous fistula (AVF) failure due to venous neointimal hyperplasia (VNH) is not known. The role of microRNA-21 (miR-21) in VNH associated with AVF failure was investigated by performing in vivo and in vitro experiments. In situ hybridization results revealed that miR-21 expression increased and was associated with fibroblasts in failed AVFs from patients. In a murine AVF model, qRT-PCR gene expression results showed a significant increase in miR-21 and a decrease in miR-21 target genes in graft veins (GVs) compared to contralateral veins in mouse AVF. miR-21 knockdown in GVs was performed using a lentivirus-mediated small hairpin RNA (shRNA), and this improved AVF patency with a decrease in neointima compared to control GVs. Moreover, loss of miR-21 in GVs significantly decreased the Tgfβ1, Col-Ia, and Col-Iva genes. Immunohistochemistry demonstrated a significant decrease in myofibroblasts and proliferation with an increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in miR-21-knockdown vessels, along with a decrease in hypoxia-inducible factor-1 alpha (HIF-1α) and phospho-SMAD2 (pSMAD-2) and phospho-SMAD3 (pSMAD-3) and an increase in phosphatase and tensin homolog (PTEN) staining. Hypoxic fibroblast knockdown for miR-21 showed a significant decrease in Tgfβ-1 expression and pSMAD-2 and -3 levels and a decrease in myofibroblasts. These results indicate that miR-21 upregulation causes VNH formation by fibroblast-to-myofibroblast differentiation.
microRNA-21 promotes breast cancer proliferation and metastasis by targeting LZTFL1.
Wang Hui,Tan Zheqiong,Hu Hui,Liu Hongzhou,Wu Tangwei,Zheng Chao,Wang Xiuling,Luo Zhenzhao,Wang Jing,Liu Shuiyi,Lu Zhongxin,Tu Jiancheng
BACKGROUND:Breast cancer is the most common cancer type in female. As microRNAs play vital role in breast cancer, this study aimed to explore the molecular mechanism and clinical value of miR-21 in breast cancer. METHODS:qRT-PCR was performed to detect miR-21 levels in plasma of 127 healthy controls, 82 benign breast tumor, 252 breast cancer patients, as well as in breast cancer cell lines. Transwell and wound healing assay were used to analyze breast cancer metastasis in response to miR-21 inhibitor. Colony formation and eFluor™ 670 based flow cytometric analysis were used to test breast cancer proliferation following miR-21 inhibitor treatment. Leucine zipper transcription factor-like 1 (LZTFL1), the target gene of miR-21 was predicted by MIRDB, TargetScan 5.1, PicTar and miRanda. Survival analysis of LZTFL1 levels in breast cancer prognosis was estimated with the Kaplan-Meier method by log-rank test according to data from the Cancer Genome Atlas. Luciferase activity assay was performed to confirm the regulation of miR-21 on LZTFL1. LZTFL1 siRNA and miR-21 inhibitor were co-transfected to breast cancer cells, then cell proliferation, migration and epithelial-mesenchymal transition (EMT) makers were tested. BALB/c nude mice were injected in situ with Hs578T cells stably overexpressing miR-21. Breast tumor growth, metastasis and the expression of EMT markers or LZTFL1 were detected in vivo. RESULTS:Plasma miR-21 levels were elevated in breast cancer patients compared with healthy controls and benign breast tumor patients, and the miR-21 levels were significantly decreased after surgery comparing with pre operation in 44 patients. Inhibition of miR-21 suppressed cell proliferation and metastasis in breast cancer cells. LZTFL1 was identified as a novel target gene of miR-21. Knockdown of LZTFL1 overcame the suppression of miR-21 inhibitor on cell proliferation, metastasis and the expression of EMT markers in breast cancer cells. miR-21 overexpression promoted breast cancer cell proliferation and metastasis in vivo. CONCLUSIONS:These results indicate that plasma miR-21 level is a crucial biomarker for breast cancer diagnosis and targeting miR-21-LZTFL1-EMT axis might be a promising strategy in breast cancer therapy. TRIAL REGISTRATION:Retrospectively registered.
Relation between microRNA-21, transforming growth factor β and response to treatment among chronic hepatitis C patients.
Ghazy Amany A,Osman Eman M,Rashwan Eman A,Gaballah Ahmed H,Mostafa Hanan,Tawfik Salwa
Journal of medical virology
BACKGROUND:Persistence of hepatitis C virus (HCV) infection and response to antiviral therapy has been shown to be associated with inappropriate levels of cytokines and microRNAs (miRNAs). miRNA levels have been reported to fluctuate during treatment. Thus they could be useful predictors for responses to treatment among HCV infected patients, thereby reducing ineffective treatments. AIM:The current study aimed to investigate the relation between miRNA-21 expression profiles, transforming growth factor β (TGF-β) serum levels and response to treatment with the new direct antiviral drugs (sofosbuvir + daclatasvir ± ribavirin), among HCV infected Egyptian patients. SUBJECTS AND METHODS:This prospective study was conducted on 50 HCV infected patients (before and after treatment) and 20 healthy volunteers. miRNA expression profiles were determined by real-time polymerase chain reaction and TGF-β1 serum levels were measured by using enzyme-linked immunosorbent assay. RESULTS:There was a significant increase in serum albumin, platelets count and a significant decrease in liver enzymes, serum bilirubin, and prothrombin time after treatment. Significant reduction of viral load among HCV patients after receiving the treatment was reported. Concomitantly, there was an increase in the relative quantity of miRNA-21 (P = .001*) and serum levels of TGF-β1 ( P = .337) among HCV patients after receiving treatment. CONCLUSION:Nearly all responders to direct antiviral drugs showed increased levels of both miRNA-21 and TGF-β1. This may indicate an interplay between TGF-β1 and miRNA-21 during remission or progression of viral infection. Thus miRNA-21 could be used as promising serum biomarker, for assessment of antiviral treatment efficacy and improvement of fibrosis among chronically infected HCV patients.
MicroRNA-21 promotes progression of breast cancer via inhibition of mitogen-activated protein kinase10 (MAPK10).
Xie Yilin,Liu Yaqing,Fan Xiaoyue,Zhang Lan,Li Qing,Li Shenglei,Wang Honglei,Xiao Yi
OBJECTIVE:The present study aims to investigate microRNAs (miRNAs) and messenger RNAs (mRNAs) associated with breast cancer, and to have a better understanding of the mechanism of miRNAs in breast cancer using bioinformatics tools. METHODS:Microarray analysis was performed to predict differentially expressed miRNAs related to breast cancer, followed by prediction and verification of target genes. Human breast cancer cells were transfected and divided into Blank group, NC group, miR-21-5p mimic group, miR-21-5p inhibitor group and siRNA-MAPK10 group. RT-qPCR and Western blot analysis were used to detect mRNA and protein expressions of MAPK10 in tissues and transfected cells, MTT assay, scratch test and Transwell assay for detection of the proliferation, migration and invasion, and Annexin-V-R-PE assay for apoptosis of different cell lines. RESULTS:Highly expressed miR-21-5p and lowly expressed MAPK10 were selected for subsequent experiments, according to the microarray analysis. RT-qPCR showed that the expression of MAPK10 in breast cancer tissues was significantly lower than that in adjacent tissues, while it was reciprocal in expression of miR-21. miR-21-5p negatively regulated MAPK10. The expression of MAPK10 reduced in response to miR-21-5p mimic treatment. Compared with the Blank and NC groups, the proliferation, migration, invasion and metastasis of breast cancer cells decreased, and the apoptosis of breast cancer cells increased in the miR-21-5p inhibitor group and siRNA-MAPK10 group, while it was reciprocal in the miR-21-5p mimic group. CONCLUSION:MiR-21 promotes the proliferation, migration and invasion and inhibits the apoptosis of breast cancer cells by inhibiting MAPK10.
[MicroRNA-21 correlates TGF-β1 pathway of pancreatic ductal adenocarcinoma].
Wu Xiang,Zhuo Shuwei,Zheng Cailing,Gao Ge
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences
OBJECTIVE:To conduct genetic analysis of pancreatic ductal adenocarcinoma tissues and analyze the correlation between targeted microRNA (miRNA) and pathways in pancreatic ductal adenocarcinoma.
Methods: We collected 19 samples of peripheral venous blood serum from patients with pancreatic ductal adenocarcinoma in Hainan Provincial Hospital of Chinese Medicine, and also collected 21 blood serum samples as a control group of non-pancreatic ductal adenocarcinoma. We used the bioinformatics analysis of literature GCBI data platform for screening and analyzing the genetics of pancreatic ductal adenocarcinoma samples. Through GCBI data platform of hierarchy clustering analysis and the enrichment of gene function analysis, the relevant miRNA was screened as a research object in patients with pancreatic ductal adenocarcinoma. The miRNA was screened by literature analysis and pancreatic cancer gene analysis. Real-time PCR and Western blotting were carried out to study the relationship between the selected miRNA and TGF-β1 by overexpression and suppression of the gene in pancreatic ductal adenocarcinoma cells.
Results: MiRNA-21 was screened as a gene associated with pancreatic ductal carcinoma via hierarchy clustering analysis and gene function analysis. MiRNA-21 was highly expressed in the pancreatic ductal carcinoma patients. Expressions of TGF-β1 were inhibired in miRNA-21 overexpressed PANC-1. While the expression of miRNA-21 was inhibited, TGF-β1 expression increased obviously.
Conclusion: MiRNA-21 is highly expressed in patients with pancreatic ductal adenocarcinoma, can regulate the expression of TGF-β1, which may be a mechanism of miRNA-21 in pancreatic ductal adenocarcinoma.
MicroRNA-21 Mediates the Protective Effect of Cardiomyocyte-Derived Conditioned Medium on Ameliorating Myocardial Infarction in Rats.
Chen Chih-Hung,Hsu Shu-Yuan,Chiu Chien-Chih,Leu Steve
Conditioned medium derived from ischemic myocardium improves rodent cardiac function after myocardial infarction. Exosomal miRNA-mediated intercellular communication is considered to mediate the protective effect of conditioned medium against ischemic injury. Oxygen-glucose-deprivation (OGD)-treated cardiac cells and a rat model with acute myocardial infarction (AMI) were applied. The expression profiles of myocardial-disease-associated miRNAs in cardiomyocytes, cardiac fibroblasts, ventricular myocardium, and conditioned medium derived from cardiomyocytes under ischemic stresses were analyzed. Primary cultured cell model and a rat model with myocardial infarction were applied to examine the role of miRNA in regulating cardiomyocyte apoptosis, fibroblast activation, immune cell infiltration, and myocardial infarction. Results showed that expression levels of miR-21 in cardiomyocytes, cardiac fibroblasts, and conditioned medium (CM) derived from cardiomyocytes were up-regulated with OGD treatment. With the depletion of miR-21, the protective effect of CM on cardiomyocytes against oxidative stress, enhanced fibroblast activation, and promotion of angiogenesis in endothelial cells were reduced. Administration of CM reduced the infarcted size and immune cell infiltration in myocardium of rats with AMI, while depletion of miR-21 reduced the effect of CM. In conclusion, miR-21 plays a role in intercellular communication among ischemic cardiac cells. The expression of miR-21 is important for the protective effect of conditioned medium against myocardial infarction.
lncRNA DGCR5 inhibits the proliferation of colorectal cancer cells by downregulating miR-21.
Huang Hansheng,Yang Xiaomei,Chen Jun,Fu Junmin,Chen Chaoting,Wen Jianmei,Mo Qiao
Long non-coding RNA (lncRNA) DiGeorge syndrome critical region gene 5 (DGCR5) serves roles as a tumor suppressor or oncogene in different types of cancer. The current study aimed to explore the role of DGCR5 in colorectal cancer (CRC). It was revealed that the expression of DGCR5 was downregulated, while microRNA (miR)-21 was upregulated in CRC. The expression level of DGCR5 in tumor tissue decreased, while expression levels of miR-21 increased, with advancing stages of the disease. The expression levels of DGCR5 and miR-21 were inversely associated in tumor tissues. In CRC cells , miR-21 overexpression failed to significantly affect DGCR5, while DGCR5 overexpression resulted in reduced expression levels of miR-21. DGCR5 overexpression showed no significant effects on cancer cell migration and invasion, but suppressed cancer cell proliferation . miR-21 overexpression increased cancer cell proliferation and attenuated the effects of DGCR5 overexpression. Therefore, lncRNA DGCR5 may inhibit the proliferation of CRC cells by downregulating miR-21.
MicroRNA-21 promotes proliferation in acute myeloid leukemia by targeting Krüppel-like factor 5.
Li Chan,Yan Hua,Yin Jie,Ma Jian,Liao Ang,Yang Shengyou,Wang Lijuan,Huang Yongxiang,Lin Chon,Dong Zhiqiang,Yang Bo,Cao Ting,Liu Guo,Wang Lv
Abnormal expression of microRNA (miR)-21 has been reported in various types of cancers. However, the role and mechanism of miR-21 remain to be elucidated in acute myeloid leukemia (AML). In the present study, it was observed that miR-21 was upregulated and Krüppel-like factor 5 (KLF5) was downregulated in AML cells compared with normal bone marrow cells. Dual luciferase reporter assays revealed that KLF5 was a direct target of miR-21. Indeed, miR-21 overexpression resulted in a downregulation of KLF5 expression, while miR-21 inhibition had the opposite effect in AML cells. In addition, miR-21 overexpression promoted the proliferation of AML cells . Notably, using a mouse xenograft model, miR-21 overexpression was demonstrated to result in enhanced tumor growth and suppressed KLF5 expression in the xenograft tumors . In conclusion, the present results indicated that miR-21 promoted proliferation through directly regulating KLF5 expression in AML cells. miR-21 may thus serve as an oncogene in AML, providing a potential target for AML therapy.
A Novel Chinese Medicine, Xinfeng Capsule, Modulates Proinflammatory Cytokines via Regulating the Toll-Like Receptor 4 (TLR4)/Mitogen-Activated Protein Kinase (MAPK)/Nuclear Kappa B (NF-κB) Signaling Pathway in an Adjuvant Arthritis Rat Model.
Cao Yun-Xiang,Huang Dan,Liu Jian,Zong Rui-Kai,Wan Lei,Huang Chuan-Bing,Zhang Wan-Dong,Wang Yuan
Medical science monitor : international medical journal of experimental and clinical research
BACKGROUND Rheumatoid arthritis (RA) is a chronic autoimmune disease targeting joints. This research aimed to explore the effects of Xinfeng capsules (XFC) on cardiac injury in adjuvant arthritis (AA) model rats and assessed the associated mechanism. MATERIAL AND METHODS An adjuvant arthritis (AA) rat model was established by intracutaneously injection with Freund's complete adjuvant (FCA). Model rats were divided into 4 groups: an AA model group, an astragalus polysaccharides (APS) group, a methotrexate (MTX) group, and an XFC and triptolide (TPT) group. Hematoxylin-eosin (HE) staining was used to observe histopathologic changes. TUNEL assay was utilized to evaluate the apoptosis of cardiomyocytes. ELISA was utilized to evaluate levels of tumor necrosis factor alpha (TNF-alpha), interleukin 17 (IL-17), and interleukin 6 (IL-6) in myocardial tissues. Quantitative RT-PCR (qRT-PCR) was used to detect microRNA-21 (miRNA21) levels. Mitogen-activated protein kinase (MAPK)/p38, Toll-like receptor 4 (TLR4), and nuclear kappa B (NF-kappaB)/p65 levels were evaluated using Western blot. RESULTS XFC significantly improved proinflammatory response compared to the AA model group (p<0.05). XFC treatment significantly decreased the number of cells staining TUNEL-positive compared with the model group (p<0.05). XFC treatment significantly reduced TNF-alpha, IL-17, and IL-6 levels in myocardial tissues compared to the model group (p<0.05). Levels of miRNA21 were significantly lower in the XFC group compared to the AA model group (p<0.05). TLR4, MAPK/p38, and NF-kappaB/p65 expression levels were significantly lower in the XFC group than in the model group (p<0.05). CONCLUSIONS Xinfeng capsule, a traditional Chinese medicine preparation, protects against cardiac injury in AA rats by modulating proinflammatory cytokines expression via the TLR4/MAPK/NF-kappaB signaling pathway.
Long noncoding RNA GAS5 induces abdominal aortic aneurysm formation by promoting smooth muscle apoptosis.
He Xiang,Wang Shifei,Li Mengsha,Zhong Lintao,Zheng Hao,Sun Yili,Lai Yanxian,Chen Xiaoqiang,Wei Guoquan,Si Xiaoyun,Han Yuan,Huang Senlin,Li Xinzhong,Liao Wangjun,Liao Yulin,Bin Jianping
Long noncoding RNAs (lncRNAs) may serve as specific targets for the treatment of abdominal aortic aneurysms (AAAs). LncRNA GAS5, functionally associated with smooth muscle cell (SMC) apoptosis and proliferation, is likely involved in AAA formation, but the exact role of GAS5 in AAA is unknown. We thus explored the contribution of GAS5 to SMC-regulated AAA formation and its underlying mechanisms. : Human specimens were used to verify the diverse expression of GAS5 in normal and AAA tissues. The angiotensin II (Ang II)-induced AAA model in ApoE-/- mice and the CaCl-induced AAA model in wild-type C57BL/6 mice were used. RNA pull-down and luciferase reporter gene assays were performed in human aortic SMCs to detect the interaction between GAS5 and its downstream targets of protein or microRNA (miR). GAS5 expression was significantly upregulated in human AAA specimens and two murine AAA models compared to human normal aortas and murine sham-operated controls. GAS5 overexpression induced SMC apoptosis and repressed its proliferation, thereby promoting AAA formation in two murine AAA models. Y-box-binding protein 1 (YBX1) was identified as a direct target of GAS5 while it also formed a positive feedback loop with GAS5 to regulate the downstream target p21. Furthermore, GAS5 acted as a miR-21 sponge to release phosphatase and tensin homolog from repression, which blocked the activation and phosphorylation of Akt to inhibit proliferation and promote apoptosis in SMCs. The LncRNA GAS5 contributes to SMC survival during AAA formation. Thus, GAS5 might serve as a novel target against AAA.
Overexpression of miRNA-21 Promotes the Proliferation and Invasion in Hepatocellular Carcinoma Cells via Suppressing SMAD7.
Wang Yan,Zhang Ping,Yuan Mei,Li Xiaojie
Technology in cancer research & treatment
PURPOSE:This study aimed to explore the molecular mechanism of microRNA-21 and smad family member 7 in hepatocellular carcinoma. METHOD:A total of 57 participants were divided into control group (healthy participants, n = 10) and hepatocellular carcinoma group (hepatocellular carcinoma patients, n = 37). The expression of microRNA-21 levels were first detected in these two groups. Cell transfection was performed on hepatoma cell lines, followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Transwell assay to reveal proliferation and invasion ability. Furthermore, the relation between microRNA-21 and smad family member 7 was revealed by luciferase reporter gene and RNA immunoprecipitation assay. Finally, a transplantation tumor model of breast cancer in mice was constructed. RESULTS:The serum indicators including α-alanine aminotransferase, aspartate aminotransferase, and albumin were differentially expressed between hepatocellular carcinoma group and control group. Compared to the control group, there was a high expression of microRNA-21 in hepatocellular carcinoma group. Low expression of microRNA-21 inhibited the proliferation and invasion of HepG2.2.15 and Huh7-1.3 cells. Luciferase reporter gene and RNA innumoprecipitation assay showed that smad family member 7 was the target gene of microRNA-21. Moreover, mice model analysis showed that microRNA-21 might regulate the growth of the transplanted tumors in mice by targeting smad family member 7. CONCLUSION:The upregulated microRNA-21 might participate in the proliferation and migration in cells of hepatocellular carcinoma via suppression of smad family member 7. Furthermore, serum indicators such as alanine aminotransferase, aspartate aminotransferase, and albumin might be used as serum diagnostic markers for hepatocellular carcinoma.
Downregulated miR-21 mediates matrine-induced apoptosis via the PTEN/Akt signaling pathway in FTC-133 human follicular thyroid cancer cells.
Li Quanliang,Zhang Shuya,Wang Mingyu,Dong Shouguang,Wang Yan,Liu Song,Lu Tiancheng,Fu Yuqin,Wang Xiuran,Chen Guihong
Matrine is an alkaloid extracted from the leguminous plant . Matrine has clinical effects in the treatment of tumors, including those in lung cancer, nasopharyngeal cancer and liver cancer. However, the effect of matrine on follicular thyroid cancer has not been reported. The aim of the present study was to investigate the effect of matrine on follicular thyroid cancer and its potential mechanism. FTC-133 follicular thyroid cancer cells were treated with different concentrations of matrine, and an MTT assay showed that matrine inhibited the growth of FTC-133 cells in a dose- and time-dependent manner with an IC value of 154.8 µM. Cell apoptosis was analyzed by flow cytometry and the results showed that matrine effectively induced the apoptosis of FTC-133 cells. The expression level of microRNA (miR)-21 was analyzed by reverse transcription-quantitative PCR (RT-qPCR) analysis, and the mRNA and protein expression levels of PTEN, Akt and phosphorylated (p)-Akt were detected by RT-qPCR analysis and western blotting, respectively. The expression of miR-21 was significantly downregulated, PTEN was upregulated at the mRNA and protein expression levels, and p-Akt was downregulated in the FTC-133 cells. The effects of miR-21 mimics and miR-21 inhibitor on the expression of miR-21, PTEN and Akt in FTC-133 cells, and the effect of miR-21 mimics/matrine on the expression of PTEN were also investigated. The results of the present study suggested that matrine inhibited the growth and induced apoptosis of FTC-133 cells via the miR-21/PTEN/Akt signaling pathway.
The protective role of microRNA-21 against coxsackievirus B3 infection through targeting the MAP2K3/P38 MAPK signaling pathway.
He Feng,Xiao Zonghui,Yao Hailan,Li Sen,Feng Miao,Wang Wei,Liu Zhewei,Liu Zhuo,Wu Jianxin
Journal of translational medicine
BACKGROUND:The P38 mitogen-activated protein kinase (MAPK) pathway plays an essential role in CVB3-induced diseases. We previously demonstrated microRNA-21 has potential inhibitory effect on the MAP2K3 which locates upstream of P38 MAPK and was upregulated in mouse hearts upon CVB3 infection. However, the effect and underlying mechanism of miRNA-21 on CVB3 infection remain unclear. METHODS:We detected continuous changes of cellular miRNA-21 and P38 MAPK proteins expression profiling post CVB3 infection in vitro within 12 h. P38 MAPK signaling was inhibited by the specific inhibitor, small interfering RNA and miRNA-21 mimic in vitro, CVB3 replication, cell apoptosis rate and proliferation were detected. Viral load in the mice heart, cardiomyocyte apoptosis rate and histological of the heart were also detected in the mice model of viral myocarditis pretreated with miRNA-21-lentivirus. RESULTS:We observed significant upregulation of miRNA-21 expression followed by suppression of the MAP2K3/P38 MAPK signaling in CVB3-infected Hela cells. The inactivation of the MAP2K3/P38 MAPK signaling by P38 MAPK specific inhibitor, small interfering RNA against MAP2K3, or miRNA-21 overexpression significantly inhibited viral progeny release from CVB3-infected cells. Mechanistically, when compared with control miRNA, miRNA-21 showed no effect on capsid protein VP1 expression and viral load within host cells, while significantly reversing CVB3-induced caspase-3 activation and cell apoptosis rate, further promoting proliferation of infected cells, which indicates the inhibitory effect of miRNA-21 on CVB3 progeny release. In the in vivo study, when compared with control miRNA, miRNA-21 pretreatment remarkably inactivated the MAP2K3/P38 MAPK signaling in mice and protected them against CVB3 infection as evidenced by significantly alleviated cell apoptosis rate, reduced viral titers, necrosis in the heart as well as by remarkably prolonged survival time. CONCLUSIONS:miRNA-21 were reverse correlated with P38 MAPK activation post CVB3 infection, miRNA-21 overexpression significantly inhibited viral progeny release and decreased myocytes apoptosis rate in vitro and in vivo, suggesting that miRNA-21 may serve as a potential therapeutic agent against CVB3 infection through targeting the MAP2K3/P38 MAPK signaling.
MicroRNA-21-5p targeting PDCD4 suppresses apoptosis via regulating the PI3K/AKT/FOXO1 signaling pathway in tongue squamous cell carcinoma.
Liu Changfu,Tong Zhou,Tan Jingyu,Xin Zengxi,Wang Zhiying,Tian Luming
Experimental and therapeutic medicine
The aim of the present study was to analyze the role of microRNA (miRNA)-21-5p in tongue squamous cell carcinoma (TSCC), predict the target gene of miR-21-5p and provide novel strategies for gene therapy in TSCC treatment. The expression levels of miRNA-21-5p in TSCC tissues were analyzed using reverse transcription quantitative polymerase chain reaction, and the effects of miRNA-21-5p on cell proliferation, invasion and apoptosis and the expression levels of target protein PDCD4 in the Cal 27 and SCC9 cell lines were determined. PI3K/AKT/Forkhead Box O1 (FOXO1) pathway-associated protein expression levels were evaluated by western blot analysis. miRNA-21-5p was consistently upregulated in TSCC tissues compared with normal tissues. Inhibition of miR-21-5p inhibited cell proliferation and invasion, and promoted cell apoptosis. A luciferase reporter assay confirmed that PDCD4 was the target of miR-21-5p. Inhibition of miRNA21-5p suppressed the PI3K/Akt/FOXO1 signaling pathway. The results from the present study indicated that miR-21-5p-targeting PDCD4 suppresses apoptosis in human TSCC cell lines. This anti-apoptotic effect was achieved by regulating the PI3K/Akt/FOXO1 signaling pathway. These data represent the basis for a promising novel strategy for the treatment of TSCC.
Overexpression of microRNA-21 mediates Ang II-induced renal fibrosis by activating the TGF-β1/Smad3 pathway via suppressing PPARα.
Lyu Huiyan,Li Xin,Wu Qi,Hao Lirong
Journal of pharmacological sciences
Angiotensin II (Ang II) is an important profibrotic factor, and the tumor-promoting microRNA miR-21 was recently linked to fibrotic disorders. We aimed to investigate whether and how miR-21 mediates Ang II-induced renal fibrosis. In renal tubular epithelial cells, Ang II upregulated miR-21 and fibrosis-related indicators but decreased PPARα expression. miR-21 overexpression promoted PPARα downregulation, activated the TGF-β1/Smad3 pathway and induced fibrogenesis, while miR-21 suppression exerted opposite effects. In Ang II-treated cells, reduced PPARα expression, TGF-β1/Smad3 pathway activation and fibrogenesis were all exacerbated by miR-21 upregulation but alleviated by miR-21 inhibition. The dual-luciferase assay confirmed PPARα as the target of miR-21. PPARα silencing alone could overactivate the TGF-β1/Smad3 pathway in the presence or absence of Ang II. Importantly, the regulatory effects of miR-21 knockdown and the angiotensin type 1 receptor blocker losartan alone or in combination on the PPARα/TGF-β1/Smad3 pathway in Ang II-treated cells were almost the same. More crucially, PPARα restoration abolished the profibrotic effect of miR-21 overexpression. In addition, inhibiting miR-21 in Ang II-treated mice effectively ameliorated the abnormally activated PPARα/TGF-β1/Smad3 pathway, albuminuria, and renal fibrosis without lowering blood pressure. These results demonstrated that miR-21 extensively mediates Ang II-induced kidney fibrosis via amplifying the TGF-β1/Smad3 pathway by targeting PPARα.