miR-22-5p and miR-29a-5p Are Reliable Reference Genes for Analyzing Extracellular Vesicle-Associated miRNAs in Adipose-Derived Mesenchymal Stem Cells and Are Stable under Inflammatory Priming Mimicking Osteoarthritis Condition. Ragni Enrico,Perucca Orfei Carlotta,De Luca Paola,Viganò Marco,Colombini Alessandra,Lugano Gaia,Bollati Valentina,de Girolamo Laura Stem cell reviews and reports During the last two decades, mesenchymal stem cells (MSCs) gained a place of privilege in the field of regenerative medicine. Recently, extracellular vesicles (EVs) have been identified as major mediators of MSCs immunosuppressive as well as pro-regenerative activities in many disease models, including inflammatory/degenerative conditions as joint diseases and osteoarthritis. In order to shed light on EVs potential, a rigorous profiling of embedded proteins, lipids and nucleic acids (mRNA/miRNA) is mandatory. Nevertheless, reliable strategies to efficiently score miRNA cargo and modulation under diverse experimental conditions or treatments are missing. The aim of this work was to identify reliable reference genes (RGs) to analyze miRNA content in EVs secreted by adipose-derived MSCs (ASCs) and verify their consistency under inflammatory conditions that were proposed to enhance ASC-EVs immunomodulatory and regenerative potential. RefFinder algorithm, that integrates the currently available major computational programs (geNorm, NormFinder, BestKeeper, and Delta Ct method), allowed to identify miR-22-5p and miR-29a-5p as the most stable RGs. Notably, both miRNAs maintained the highest stability when EVs isolated from IFNg-treated ASCs were included in the analysis. In addition, considerable effects of suboptimal RGs choice on the reliable quantification of miRNAs involved at different levels (tissue homeostasis or macrophage polarization) in the osteoarthritis phenotype, and thus considered as promising therapeutic molecule, have clearly been demonstrated. In conclusion, a proper normalization method is not only needed for research purposes but also mandatory to characterize clinical products and predict their therapeutic potential, especially in the emerging field of MSCs derived-EVs as new tools for regenerative medicine. 10.1007/s12015-019-09899-y
    Upregulation of miR-22 promotes osteogenic differentiation and inhibits adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells by repressing HDAC6 protein expression. Huang Shan,Wang Shihua,Bian Chunjing,Yang Zhuo,Zhou Hong,Zeng Yang,Li Hongling,Han Qin,Zhao Robert Chunhua Stem cells and development Mesenchmal stem cells (MSCs) can be differentiated into either adipocytes or osteoblasts, and a reciprocal relationship exists between adipogenesis and osteogenesis. Multiple transcription factors and signaling pathways have been reported to regulate adipogenic or osteogenic differentiation, respectively, yet the molecular mechanism underlying the cell fate alteration between adipogenesis and osteogenesis still remains to be illustrated. MicroRNAs are important regulators in diverse biological processes by repressing protein expression of their targets. Here, miR-22 was found to regulate adipogenic and osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hADMSCs) in opposite directions. Our data showed that miR-22 decreased during the process of adipogenic differentiation but increased during osteogenic differentiation. On one hand, overexpression of miR-22 in hADMSCs could inhibit lipid droplets accumulation and repress the expression of adipogenic transcription factors and adipogenic-specific genes. On the other hand, enhanced alkaline phosphatase activity and matrix mineralization, as well as increased expression of osteo-specific genes, indicated a positive role of miR-22 in regulating osteogenic differentiation. Target databases prediction and validation by Dual Luciferase Reporter Assay, western blot, and real-time polymerase chain reaction identified histone deacetylase 6 (HDAC6) as a direct downstream target of miR-22 in hADMSCs. Inhibition of endogenous HDAC6 by small-interfering RNAs suppressed adipogenesis and stimulated osteogenesis, consistent with the effect of miR-22 overexpression in hADMSCs. Together, our results suggested that miR-22 acted as a critical regulator of balance between adipogenic and osteogenic differentiation of hADMSCs by repressing its target HDAC6. 10.1089/scd.2012.0014
    Evaluation of miR-22 and miR-20a as diagnostic biomarkers for gastric cancer. Jafarzadeh-Samani Zahra,Sohrabi Sareh,Shirmohammadi Khadije,Effatpanah Hosein,Yadegarazari Reza,Saidijam Massoud Chinese clinical oncology BACKGROUND:Gastric cancer (GC) is the fourth most common cancers and the second reason for cancer-related death around the world, particularly in East Asian countries. Diagnosing GC in its early stages is followed by more successful treatment. Unfortunately, there is no accurate method for GC diagnosis in its early stages. Recently, miRNAs have been investigated in the most cancer researches which have demonstrated that they have been dysregulated in many cancers. METHODS:This case-control study aims to investigate the expression rate of miR-22 and miR-20a in 32 cancerous tissues as well as 32 healthy adjacent tissues. Quantitative reverse transcriptase PCR (q-RT PCR) was used for investigating the expression rate of these miRNAs. RESULTS:The expression rate of miR-20a in cancerous tissues was significantly increased (8.9 times) in comparison with their healthy tissues (P<0.001), while the expression rate of miR-22 in cancerous tissues was significantly decreased (1.9 times) (P<0.05). CONCLUSIONS:The obtained results suggest miR-22 and miR-20a as good diagnostic biomarkers for early detection of GC. However more research is needed to investigate their efficacy. 10.21037/cco.2017.03.01
    Circulating miR-22-5p and miR-122-5p are promising novel biomarkers for diagnosis of acute myocardial infarction. Wang Yu,Chang Wenguang,Zhang Yuan,Zhang Lei,Ding Han,Qi Hongzhao,Xue Sheng,Yu Hua,Hu Longgang,Liu Dacheng,Zhu Wenjie,Wang Yin,Li Peifeng Journal of cellular physiology BACKGROUND/AIMS:This study sought to evaluate the potential of circulating microRNAs (miRNAs) as novel indicators for acute myocardial infarction (AMI). METHODS:Plasma samples were collected from each participant, and total RNA was extracted. Quantitative real-time polymerase chain reaction were used to investigate the expression of circulating miRNAs. We measured circulating levels of six individual miRNAs, which are known to be relevant to AMI, in the plasma samples from 66 AMI patients and 70 non-AMI healthy comparisons. RESULTS:Five small RNAs were specifically expressed in AMI patients, plasma miR-122-5p levels is significantly elevated (p < 0.0001) in AMI patients, while plasma miR-22-5p ( p < 0.05) levels were significantly decreased. In addition, significant correlations between miR-22-5p and miR-122-5p ( R = 0.773), miR-122-5p and creatine kinase isoenzyme (CK-MB; R = 0.6296) were detected. Further, receiver operating characteristic (ROC) analysis indicated that miR-22-5p showed considerable diagnostic efficiency for predicting AMI (area under the curve [AUC] = 0.975). Combining miR-22-5p and miR-122-5p in a panel increased the sensitivity (98.6%) of distinguishing between patients with AMI and healthy comparisons. CONCLUSION:Circulating miR-22-5p and miR-122-5p could be considered promising novel diagnostic biomarkers for AMI. 10.1002/jcp.27274
    Serum miR-22 Could be a Potential Biomarker for the Prognosis of Breast Cancer. Shao Yuxin,Yao Yi,Xiao Peng,Yang Xiaoxia,Zhang Dianlong Clinical laboratory BACKGROUND:This study aims to evaluate whether miR-22 could be used as a potential biomarker to screen breast cancer patients from healthy controls. This has never been explored. METHODS:Real time PCR analysis was carried out to explore the expression of serum miR-22 in breast cancer patients. Chi-square test was used for counting data. Log rank test was used for comparing survival curves. CoX regression model was used for univariate and multivariate prognosis analysis. In addition, we also evaluated the role of miR-22 on the migration capacity of MCF-7 cells using a wound healing assay. RESULTS:We found that low expression of miR-22 was significantly associated with late TNM stage, lymph node metastasis, local recurrence, and distant metastasis. Meanwhile, low expression of miR-22 was significantly associated with short survival and poor prognosis in all patients and lymph node subgroups. Analysis of CoX univariate and multivariate models demonstrated that miR-22 is an independent prognostic marker of breast cancer. In ad-dition, overexpression of miR-22 significantly decreased the migration of MCF-7 cells, validating the tumor suppressor role of miR-22 in breast cancer cells. CONCLUSIONS:In summary, low miR-22 expression may be a potential biomarker to screen breast cancer patients from healthy control. 10.7754/Clin.Lab.2018.180825
    miR-22 regulates C2C12 myoblast proliferation and differentiation by targeting TGFBR1. Wang Han,Zhang Qian,Wang BinBin,Wu WangJun,Wei Julong,Li Pinghua,Huang Ruihua European journal of cell biology Recently, miR-22 was found to be differentially expressed in different skeletal muscle growth period, indicated that it might have function in skeletal muscle myogenesis. In this study, we found that the expression of miR-22 was the most in skeletal muscle and was gradually up-regulated during mouse myoblast cell (C2C12 myoblast cell line) differentiation. Overexpression of miR-22 repressed C2C12 myoblast proliferation and promoted myoblast differentiation into myotubes, whereas inhibition of miR-22 showed the opposite results. During myogenesis, we predicted and verified transforming growth factor beta receptor 1 (TGFBR1), a key receptor of the TGF-β/Smad signaling pathway, was a target gene of miR-22. Then, we found miR-22 could regulate the expression of TGFBR1 and down-regulate the Smad3 signaling pathway. Knockdown of TGFBR1 by siRNA suppressed the proliferation of C2C12 cells but induced its differentiation. Conversely, overexpression of TGFBR1 significantly promoted proliferation but inhibited differentiation of the myoblast. Additionally, when C2C12 cells were treated with different concentrations of transforming growth factor beta 1 (TGF-β1), the level of miR-22 in C2C12 cells was reduced. The TGFBR1 protein level was significantly elevated in C2C12 cells treated with TGF-β1. Moreover, miR-22 was able to inhibit TGF-β1-induced TGFBR1 expression in C2C12 cells. Altogether, we demonstrated that TGF-β1 inhibited miR-22 expression in C2C12 cells and miR-22 regulated C2C12 cell myogenesis by targeting TGFBR1. 10.1016/j.ejcb.2018.03.006
    Evaluation of miR-22 and miR-21 as diagnostic biomarkers in patients with epithelial ovarian cancer. Paliwal Nidhi,Vashist Minakshi,Chauhan Minakshi 3 Biotech Among the 11 most common cancers, ovarian cancer is the fifth leading cause of death after lung, breast, colorectal and pancreatic cancer. Although diagnosis of ovarian cancer in early stages is followed by various successful treatments, no accurate and reliable method is available at present. Currently microRNAs are being explored as signature biomarkers for early detection of various types of cancer. However little information is available on expression and correlation of microRNA in ovarian cancer. In this study, we have chosen two microRNA on the basis of their altered frequency in epithelial ovarian cancer cases. The main objective of this study is to evaluate the expression of microRNA-22 and microRNA-21 along with various clinicopathological parameters. Expression level of microRNA-22 and microRNA-21 in different stages and subtypes of epithelial ovarian carcinoma has been analyzed to find out its role as a potential diagnostic biomarker. Present study has been conducted in the serum of 80 epithelial ovarian cancer patients and 80 age matched healthy women. Quantitative real time PCR was used to compare the expression of miR-22 and miR-21 between the cases and control groups. Statistical analysis showed 7.85-fold increase in miR-21 expression and 2.1-fold reduction in miR-22 expression of ovarian cancer patients. Increased serum level of miR-21 in ovarian cancer patients and decreased level of miR-22 has been correlated with advanced international federation of gynecology and obstetrics (FIGO) stage and histological sub types of epithelial ovarian cancer. Serous ovarian carcinoma was the most common cancer in the present study. Calculated fold change value for miR-21 was 3.98 and  - 2.86 for miR-22 in serous ovarian cancer. Fold change value in the miR-21 expression in advanced stage was 6.29 and 4.25 in early stage. Whereas lower calculated fold change was observed for miR-22 in advanced stage than in early stage (- 2.16). Present study revealed up-regulated expression of miR-21 and down regulated expression of miR-22 in the serum of epithelial ovarian cancer patients. Both of these could be validated as good diagnostic biomarkers for early detection of ovarian cancer. 10.1007/s13205-020-2124-7
    MiR-22-3p regulates the proliferation and invasion of Wilms' tumor cells by targeting AKT3. Luo B,Ma L,Xing X,Wang Z-R,Teng Q,Li S-G European review for medical and pharmacological sciences OBJECTIVE:In this study, the regulatory mechanism of miR-22-3p/AKT3 in the development of Wilms' tumor (WT) was investigated. PATIENTS AND METHODS:Twenty-seven pairs of surgical tumor specimens and adjacent normal tissues were obtained from Jining No. 1 People's Hospital. The expression level of miR-22-3p in WT tissues and cell lines was measured by quantitative RT-PCR. MTT and transwell assays were performed to analyze cell proliferation and invasion in WT. The relationship between miR-22-3p and AKT3 was verified by a Dual-Luciferase assay. The protein expression of AKT3 was evaluated by Western blotting analysis. RESULTS:MiR-22-3p was downregulated and AKT3 was upregulated in WT. Functionally, overexpression of miR-22-3p inhibited cell proliferation and invasion in WT. Moreover, miR-22-3p directly targets AKT3. The knockdown of AKT3 suppressed cell proliferation and invasion in WT. In addition, upregulation of AKT3 restored the tumor suppressive effect of miR-22-3p in WT. CONCLUSIONS:MiR-22-3p inhibits the proliferation and invasion of WT cells by downregulating AKT3, indicating that miR-22-3p may be developed as a new biomarker for the diagnosis of WT. 10.26355/eurrev_202006_21493
    as a metabolic silencer and liver tumor suppressor. Wang Lijun,Wang Yu-Shiuan,Mugiyanto Eko,Chang Wei-Chiao,Yvonne Wan Yu-Jui Liver research With obesity rate consistently increasing, a strong relationship between obesity and fatty liver disease has been discovered. More than 90% of bariatric surgery patients also have non-alcoholic fatty liver diseases (NAFLDs). NAFLD and non-alcoholic steatohepatitis (NASH), which are the hepatic manifestations of metabolic syndrome, can lead to liver carcinogenesis. Unfortunately, there is no effective medicine that can be used to treat NASH or liver cancer. Thus, it is critically important to understand the mechanism underlying the development of these diseases. Extensive evidence suggests that microRNA 22 () can be a diagnostic marker for liver diseases as well as a treatment target. This review paper focuses on the roles of in metabolism, steatosis, and liver carcinogenesis. Literature search is limited based on the publications included in the PubMed database in the recent 10 years. 10.1016/j.livres.2020.06.001
    Resveratrol regulates muscle fiber type conversion via miR-22-3p and AMPK/SIRT1/PGC-1α pathway. Wen Wanxue,Chen Xiaoling,Huang Zhiqing,Chen Daiwen,Chen Hong,Luo Yuheng,He Jun,Zheng Ping,Yu Jie,Yu Bing The Journal of nutritional biochemistry This study investigated the effects of resveratrol and miR-22-3p on muscle fiber type conversion in mouse C2C12 myotubes. Here we showed that resveratrol significantly increased the protein level of slow myosin heavy chain (MyHC) and the activities of succinic dehydrogenase and malate dehydrogenase, as well as markedly decreased the protein level of fast MyHC and the activity of lactate dehydrogenase. Immunofluorescence staining showed that resveratrol remarkably upregulated the number of slow MyHC-positive myotubes and downregulated the number of fast MyHC-positive myotubes, suggesting that resveratrol promoted muscle fiber type conversion from fast-twitch to slow-twitch in C2C12 myotubes. We also showed that miR-22-3p had an opposite function on muscle fiber type conversion and resveratrol was able to repress the expression of miR-22-3p. Furthermore, AMP-activated protein kinase (AMPK) inhibitor Compound C and miR-22-3p mimics could attenuate and eliminate muscle fiber type conversion from fast-twitch to slow-twitch cause by resveratrol, respectively. Together, we provided the first evidence that resveratrol promotes muscle fiber type conversion from fast-twitch to slow-twitch via miR-22-3p and AMPK/SIRT1/PGC-1α pathway in C2C12 myotubes. 10.1016/j.jnutbio.2019.108297
    miR-22-3p regulates muscle fiber-type conversion through inhibiting AMPK/SIRT1/PGC-1α pathway. Wen Wanxue,Chen Xiaoling,Huang Zhiqing,Chen Daiwen,Zheng Ping,He Jun,Chen Hong,Yu Jie,Luo Yuheng,Yu Bing Animal biotechnology MicroRNAs (miRNAs) are a class of conserved non-coding RNAs that are widely regarded as important regulators in a variety of biological processes. Increasing evidence has revealed that skeletal muscle fiber-type conversion is regulated by miRNAs, but the molecular mechanism is still not fully understood. In this study, we confirmed the role of miR-22-3p on skeletal muscle fiber-type conversion and investigated its potential mechanism in C2C12 myotubes. Here, we found that the miR-22-3p mimics inhibited the expressions of myosin heavy chain I (MyHC I), MyHC IIa and promoted the expression of MyHC IIb, while miR-22-3p inhibitor got inverse results. miR-22-3p mimics also downregulated phosphorylated AMPK, SIRT1 and PGC-1ɑ protein levels, which control the expression of oxidative fiber-related genes. Furthermore, Compound C (AMPK inhibitor) eliminated the effect of miR-22-3p inhibitor on MyHC I, MyHC IIa and MyHC IIb expressions. However, AICAR (AMPK activator) also abolished the effect of miR-22-3p mimics on MyHC I, MyHC IIa and MyHC IIb expressions. Collectively, our results suggest that miR-22-3p regulates skeletal muscle fiber-type conversion through inhibiting AMPK/SIRT1/PGC-1ɑ signaling pathway. 10.1080/10495398.2020.1763375
    MiR-22-3p suppresses sepsis-induced acute kidney injury by targeting PTEN. Wang Xudong,Wang Yali,Kong Mingjian,Yang Jianping Bioscience reports BACKGROUND:Septic acute kidney injury is considered as a severe and frequent complication that occurs during sepsis. The present study was performed to understand the role of miR-22-3p and its underlying mechanism in sepsis-induced acute kidney injury. METHODS:Rats were injected with adenovirus carrying miR-22-3p or miR-NC in the caudal vein before cecal ligation. Meanwhile, HK-2 cells were transfected with the above adenovirus following LPS stimulation. We measured the markers of renal injury (blood urea nitrogen (BUN), serum creatinine (SCR)). Histological changes in kidney tissues were examined by hematoxylin and eosin (H&E), Masson staining, periodic acid Schiff staining and TUNEL staining. The levels of IL-1β, IL-6, TNF-α and NO were determined by ELISA assay. Using TargetScan prediction and luciferase reporter assay, we predicted and validated the association between PTEN and miR-22-3p. RESULTS:Our data showed that miR-22-3p was significantly down-regulated in a rat model of sepsis-induced acute kidney injury, in vivo and LPS-induced sepsis model in HK-2 cells, in vitro. Overexpression of miR-22-3p remarkably suppressed the inflammatory response and apoptosis via down-regulating HMGB1, p-p65, TLR4 and pro-inflammatory factors (IL-1β, IL-6, TNF-α and NO), both in vivo and in vitro. Moreover, PTEN was identified as a target of miR-22-3p. Furthermore, PTEN knockdown augmented, while overexpression reversed the suppressive role of miR-22-3p in LPS-induced inflammatory response. CONCLUSIONS:Our results showed that miR-22-3p induced protective role in sepsis-induced acute kidney injury may rely on the repression of PTEN. 10.1042/BSR20200527
    miR-22-3p Negatively Affects Tumor Progression in T-Cell Acute Lymphoblastic Leukemia. Saccomani Valentina,Grassi Angela,Piovan Erich,Bongiovanni Deborah,Di Martino Ludovica,Minuzzo Sonia,Tosello Valeria,Zanovello Paola Cells T-cell acute lymphoblastic leukemia (T-ALL) is a rare, aggressive disease arising from T-cell precursors. NOTCH1 plays an important role both in T-cell development and leukemia progression, and more than 60% of human T-ALLs harbor mutations in components of the NOTCH1 signaling pathway, leading to deregulated cell growth and contributing to cell transformation. Besides multiple NOTCH1 target genes, microRNAs have also been shown to regulate T-ALL initiation and progression. Using an established mouse model of T-ALL induced by NOTCH1 activation, we identified several microRNAs downstream of NOTCH1 activation. In particular, we found that NOTCH1 inhibition can induce miR-22-3p in NOTCH1-dependent tumors and that this regulation is also conserved in human samples. Importantly, miR-22-3p overexpression in T-ALL cells can inhibit colony formation in vitro and leukemia progression in vivo. In addition, miR-22-3p was found to be downregulated in T-ALL specimens, both T-ALL cell lines and primary samples, relative to immature T-cells. Our results suggest that miR-22-3p is a functionally relevant microRNA in T-ALL whose modulation can be exploited for therapeutic purposes to inhibit T-ALL progression. 10.3390/cells9071726
    Rutin treats myocardial damage caused by pirarubicin via regulating miR-22-5p-regulated RAP1/ERK signaling pathway. Qin Meng,Li Qi,Wang Yadi,Li Tengteng,Gu Zehui,Huang Peng,Ren Liqun Journal of biochemical and molecular toxicology Our experiments have previously demonstrated that rutin (RUT) can improve myocardial damage caused by pirarubicin (THP). However, the underlying molecular mechanisms remain uncertain. In this study, we developed an microRNA (miRNA) chip by replicating the rat model of THP-induced myocardial injury and identified miR-22-5p and the RAP1-member of RAS oncogene family/extracellular regulated protein kinases (RAP1/ERK) signaling pathway as an object of study. Also, in vivo experiments demonstrated that THP caused abnormal changes in the electrocardiogram, cardiac function, and histomorphology in rats (P < .01). THP also reduces the expression of miR-22-5p (P < .01) and increases the levels of RAP1/ERK signaling pathway-related proteins (P < .01, P < .05). RUT significantly improved THP-induced myocardial damage (P < .01), increased the expression of miR-22-5p (P < .01), and decreased the levels of RAP1/ERK signaling pathway-related proteins (P < .01, P < .05). In vitro studies confirmed that Rap1a is one of the target genes of miR-22-5p. miR-22-5p overexpression in cardiomyocytes can affect the RAP1/ERK pathway and reduce reactive oxygen species production and cardiomyocyte apoptosis caused by THP (P < .01), which is consistent with the effect of RUT. Our results indicate that RUT treats THP-induced myocardial damage, which may be achieved by upregulating miR-22-5p, causing changes in its target gene Rap1a and the RAP1/ERK pathway. 10.1002/jbt.22615
    NCK1-AS1 enhances glioma cell proliferation, radioresistance and chemoresistance via miR-22-3p/IGF1R ceRNA pathway. Wang Bo,Wang Kai,Jin Tenglong,Xu Qiling,He Yanyang,Cui Bingzhou,Wang Yazhou Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie Glioma is the deadliest disease in human central nerve system. Abnormal expression of long noncoding RNA (lncRNA) expression has been demontrated to be implicated in various cancers. The oncogenic role of lncRNA NCK1-AS1 has been validated in cervical cancer, wheras its role in glioma remians obscure. Our research findings suggested that NCK1-AS1 was upregulated in glioma tissues and cells. NCK1-AS1 deficiency hindered cell proliferation and enhanced cell apoptosis. Additionally, the chemoresistance and radioresistance of glioma cells were impaired by NCK1-AS1 depletion. Moreover, miR-22-3p, a downstream gene of NCK1-AS1, could weaken glioma cell chemoresistance and radioresistance. Similarly, IGF1R was the downstream target gene of miR-22-3p. Further mechanism and function assays demonstrated that NCK1-AS1 promoted glioma cell growth, chemoresistance and radioresistance via sponging miR-22-3p to upregulate IGF1R. Finally, the tumor facilitator function of NCK1-AS1 was also verified by in vivo experiments. Taken together, NCK1-AS1 contributes to glioma cell proliferation, radioresistance and chemoresistance via miR-22-3p/IGF1R ceRNA pathway, which might provide a new insight for improving the radiotherapy and chemotherapy treatments of glioma. 10.1016/j.biopha.2020.110395
    Deficiency of DGCR8 increases bone formation through downregulation of miR-22 expression. Choi Young-Jin,Jeong Sujin,Yoon Kyung-Ae,Sung Hye-Jin,Cho Hye-Sim,Kim Dong Wook,Cho Je-Yoel Bone MicroRNAs (miRNA) significantly contribute to bone formation by post-transcriptional regulation of gene expression. Mature miRNAs are generated following sequential cleavage by DROSHA/DGCR8 and DICER. However, recent studies have identified that some miRNAs require only one of these enzymes. Most studies seeking to clarify the role of miRNA during bone formation have been performed using DICER deletion strategies, but little is known regarding the role of DGCR8. To study the function of DGCR8 in osteogenesis, we generated mice in which Dgcr8 is conditionally deleted in osteoprogenitor cells by Col1a1-Cre. Dgcr8-cKO mice showed increased bone volume (BV/TV), trabecular number (Tb/N), and trabecular thickness (Tb.Th), but decreased trabecular separation (Tb.Sp) in the femur. Von Kossa, tartrate-resistant acid phosphatase staining, and calcein double labeling identified that osteoblast activity is increased in Dgcr8-cKO mice. In an effort to elucidate a detailed cellular mechanism, we found that miR-22 was downregulated in Dgcr8-cKO mice, leading to upregulation of the osteocalcin transcript, a key marker of osteoblasts. Interestingly, the mRNA expression level of Dgcr8 was decreased during osteoblast differentiation. Taken together, these results strongly indicate that DGCR8-dependent generation of miR-22 is essential for bone formation and that miR-22 could be a therapeutic target for individuals with bone disease. 10.1016/j.bone.2017.07.021
    miR-22 inhibits tumor growth and metastasis by targeting ATP citrate lyase: evidence in osteosarcoma, prostate cancer, cervical cancer and lung cancer. Xin Mei,Qiao Zhiguang,Li Jing,Liu Jianjun,Song Shaoli,Zhao Xiaoping,Miao Ping,Tang Tingting,Wang Lei,Liu Weichun,Yang Xiaodi,Dai Kerong,Huang Gang Oncotarget MicroRNAs (miRNAs) are non-coding small RNAs that function as negative regulators of gene expression involving in the tumor biology. ATP citrate lyase (ACLY), a key enzyme initiating de novo lipid synthesis, has been found to be upregulated in cancer cells, and its inhibition causes suppressive effects in a variety of tumors. At present, although several ACLY inhibitors have been reported, the potential role of miRNAs in interfering ACLY still needs further clarification. Herein, four different types of tumor cells including osteosarcoma, prostate, cervical and lung cancers were adopted in our study, and we have demonstrated that miR-22 directly downregulated ACLY. Moreover, miR-22 was proved to attenuate cancer cell proliferation and invasion, as well as promote cell apoptosis via inhibiting ACLY. Additionally, we confirmed the higher ACLY protein levels and the lower miR-22 expressions in hundreds of clinical samples of the four primary tumors, and a negative correlation relationship between ACLY and miR-22 was clarified. Finally, in the four animal models, we found that along with the loss of the ACLY expression, the miR-22-treated mice developed rather smaller tumors, less probabilities of distant metastasis, and fairly longer survivals. De novo lipogenesis suppression triggered by miR-22-ACLY axis may contribute to the inhibition of tumor growth and metastasis. These findings provide unequivocal proofs that miR-22 is responsible for the posttranscriptional regulation of ACLY, which yields promising therapeutic effects in osteosarcoma, prostate, cervical and lung cancers. 10.18632/oncotarget.10020
    MicroRNA-206 is highly expressed in newly formed muscle fibers: implications regarding potential for muscle regeneration and maturation in muscular dystrophy. Yuasa Katsutoshi,Hagiwara Yasuko,Ando Masanori,Nakamura Akinori,Takeda Shin'ichi,Hijikata Takao Cell structure and function miR-1, miR-133a, and miR-206 are muscle-specific microRNAs expressed in skeletal muscles and have been shown to contribute to muscle development. To gain insight into the pathophysiological roles of these three microRNAs in dystrophin-deficient muscular dystrophy, their expression in the tibialis anterior (TA) muscles of mdx mice and CXMD(J) dogs were evaluated by semiquantitative RT-PCR and in situ hybridization. Their temporal and spatial expression patterns were also analyzed in C2C12 cells during muscle differentiation and in cardiotoxin (CTX)-injured TA muscles to examine how muscle degeneration and regeneration affect their expression. In dystrophic TA muscles of mdx mice, miR-206 expression was significantly elevated as compared to that in control TA muscles of age-matched B10 mice, whereas there were no differences in miR-1 or miR-133a expression between B10 and mdx TA muscles. On in situ hybridization analysis, intense signals for miR-206 probes were localized in newly formed myotubes with centralized nuclei, or regenerating muscle fibers, but not in intact pre-degenerated fibers or numerous small mononucleated cells, possibly proliferating myoblasts and inflammatory infiltrates. Similar increased expression of miR-206 was also found in C2C12 differentiation and CTX-induced regeneration, in which differentiated myotubes or regenerating fibers showed abundant expression of miR-206. However, CXMD(J) TA muscles contained smaller amounts of miR-206, miR-1, and miR-133a than controls. They exhibited more severe and more progressive degenerative alterations than mdx TA muscles. Taken together, these observations indicated that newly formed myotubes showed markedly increased expression of miR-206, which might reflect active regeneration and efficient maturation of skeletal muscle fibers.
    MicroRNA-323-3p promotes myogenesis by targeting Smad2. Qin Jin,Sun Yunmei,Liu Shuge,Zhao Rui,Zhang Qiyue,Pang Weijun Journal of cellular biochemistry Skeletal muscle is an important and complex organ with multiple biological functions in humans and animals. Proliferation and differentiation of myoblasts are the key steps during the development of skeletal muscle. MicroRNA (miRNA) is a class of 21-nucleotide noncoding RNAs regulating gene expression by combining with the 3'-untranslated region of target messenger RNA. Many studies in recent years have suggested that miRNAs play a critical role in myogenesis. Through high-throughput sequencing, we found that miR-323-3p showed significant changes in the longissimus dorsi muscle of Rongchang pigs in different age groups. In this study, we discovered that overexpression of miR-323-3p repressed myoblast proliferation and promoted differentiation, whereas the inhibitor of miR-323-3p displayed the opposite results. Furthermore, we predicted Smad2 as the target gene of miR-323-3p and found that miR-323-3p directly modulated the expression level of Smad2. Then luciferase reporter assays verified that Smad2 was a target gene of miR-323-3p during the differentiation of myoblasts. These findings reveal that miR-323-3p is a positive regulator of myogenesis by targeting Smad2. This provides a novel mechanism of miRNAs in myogenesis. 10.1002/jcb.29187
    miRNA-34c inhibits myoblasts proliferation by targeting YY1. Wang Meng,Liu Chuncheng,Su Yang,Zhang Kuo,Zhang Yuying,Chen Min,Ge Mengxu,Gu Lijie,Lu Tianyu,Li Ning,Yu Zhengquan,Meng Qingyong Cell cycle (Georgetown, Tex.) miRNAs are increasingly being implicated as key regulators of cell proliferation, apoptosis, and differentiation. miRNA-34c appears to play a crucial role in cancer pathogenesis wherein it exerts its effect as a tumor suppressor. However, the role of miR-34c in myoblast proliferation remains poorly understood. Here, we found that overexpression miR-34c inhibited myoblasts proliferation by reducing the protein and mRNA expression of cell cycle genes. In contrast, blocking the function of miR-34c promoted myoblasts proliferation and increased the protein and mRNA expression of cell cycle genes. Moreover, miR-34c directly targeted YY1 and inhibited its expression. Similar to overexpression miR-34c, knockdown of YY1 by siRNA suppressed myoblasts proliferation. Our study provides novel evidence for a role of miR-34c in inhibiting myoblasts proliferation by repressing YY1. Thus, miR-34c has the potential to be used to enhance skeletal muscle development and regeneration. 10.1080/15384101.2017.1281479
    Transforming growth factor-beta-regulated miR-24 promotes skeletal muscle differentiation. Sun Qiang,Zhang Yan,Yang Guang,Chen Xiaoping,Zhang Yingai,Cao Guojun,Wang Jian,Sun Yanxun,Zhang Peng,Fan Ming,Shao Ningsheng,Yang Xiao Nucleic acids research MicroRNAs (miRNAs) have recently been proposed as a versatile class of molecules involved in regulation of a variety of biological processes. However, the role of miRNAs in TGF-beta-regulated biological processes is poorly addressed. In this study, we found that miR-24 was upregulated during myoblast differentiation and could be inhibited by TGF-beta1. Using both a reporter assay and Northern blot analysis, we showed that TGF-beta1 repressed miR-24 transcription which was dependent on the presence of Smad3 and a Smads binding site in the promoter region of miR-24. TGF-beta1 was unable to inhibit miR-24 expression in Smad3-deficient myoblasts, which exhibited accelerated myogenesis. Knockdown of miR-24 led to reduced expression of myogenic differentiation markers in C2C12 cells, while ectopic expression of miR-24 enhanced differentiation, and partially rescued inhibited myogenesis by TGF-beta1. This is the first study demonstrating a critical role for miRNAs in modulating TGF-beta-dependent inhibition of myogenesis, and provides a novel mechanism of the genetic regulation of TGF-beta signaling during skeletal muscle differentiation. 10.1093/nar/gkn032
    MIR-206 regulates connexin43 expression during skeletal muscle development. Anderson Curtis,Catoe Heath,Werner Rudolf Nucleic acids research Skeletal myoblast fusion in vitro requires the expression of connexin43 (Cx43) gap junction channels. However, gap junctions are rapidly downregulated after the initiation of myoblast fusion in vitro and in vivo. In this study we show that this downregulation is accomplished by two related microRNAs, miR-206 and miR-1, that inhibit the expression of Cx43 protein during myoblast differentiation without altering Cx43 mRNA levels. Cx43 mRNA contains two binding sites for miR-206/miR-1 in its 3'-untranslated region, both of which are required for efficient downregulation. While it has been demonstrated before that miR-1 is involved in myogenesis, in this work we show that miR-206 is also upregulated during perinatal skeletal muscle development in mice in vivo and that both miR-1 and miR-206 downregulate Cx43 expression during myoblast fusion in vitro. Proper development of singly innervated muscle fibers requires muscle contraction and NMJ terminal selection and it is hypothesized that prolonged electrical coupling via gap junctions may be detrimental to this process. This work details the mechanism by which initial downregulation of Cx43 occurs during myogenesis and highlights the tight control mechanisms that are utilized for the regulation of gap junctions during differentiation and development. 10.1093/nar/gkl743
    miR-99a-5p Regulates the Proliferation and Differentiation of Skeletal Muscle Satellite Cells by Targeting MTMR3 in Chicken. Cao Xinao,Tang Shuyue,Du Fei,Li Hao,Shen Xiaoxu,Li Diyan,Wang Yan,Zhang Zhichao,Xia Lu,Zhu Qing,Yin Huadong Genes Noncoding RNAs, especially microRNAs (miRNAs), have been reported to play important roles during skeletal muscle development and regeneration. Our previous sequencing data revealed that miR-99a-5p is one of the most abundant miRNAs in chicken breast muscle. The purpose of this study was to reveal the regulatory mechanism of miR-99a-5p in the proliferation and differentiation of chicken skeletal muscle satellite cells (SMSCs). Through the investigation of cell proliferation activity, cell cycle progression, and 5-ethynyl-29-deoxyuridine (EdU) assay, we found that miR-99a-5p can significantly promote the proliferation of SMSCs. Moreover, we found that miR-99a-5p can inhibit myotube formation by decreasing the expression of muscle cell differentiation marker genes. After miR-99a-5p target gene scanning, we confirmed that miR-99a-5p directly targets the 3' untranslated region (UTR) of myotubularin-related protein 3 (MTMR3) and regulates its expression level during chicken SMSC proliferation and differentiation. We also explored the role of MTMR3 in muscle development and found that its knockdown significantly facilitates the proliferation but represses the differentiation of SMSCs, which is opposite to the effects of miR-99a-5p. Overall, we demonstrated that miR-99a-5p regulates the proliferation and differentiation of SMSCs by targeting MTMR3. 10.3390/genes11040369
    MicroRNAs 1, 133, and 206: critical factors of skeletal and cardiac muscle development, function, and disease. Townley-Tilson W H Davin,Callis Thomas E,Wang DaZhi The international journal of biochemistry & cell biology microRNAs (miRNAs) are a class of highly conserved small non-coding RNAs that negatively regulate gene expression post-transcriptionally. miRNAs are known to mediate myriad cell processes, including proliferation, differentiation, and apoptosis. With more than 600 miRNAs identified in humans, it is generally believed that many miRNAs function through simultaneously inhibiting multiple regulatory mRNA targets, suggesting that miRNAs participate in regulating the expression of many, if not all, genes. While many miRNAs are expressed ubiquitously, some are expressed in a tissue specific manner. The muscle specific miR-1, miR-133 and miR-206 are perhaps the most studied and best-characterized miRNAs to date. Many studies demonstrate that these miRNAs are necessary for proper skeletal and cardiac muscle development and function, and have a profound influence on multiple myopathies, such as hypertrophy, dystrophy, and conduction defects. 10.1016/j.biocel.2009.03.002
    miR-221 modulates skeletal muscle satellite cells proliferation and differentiation. Liu Buwei,Shi Yu,He Hongbing,Cai Mingcheng,Xiao Wudian,Yang Xue,Chen Shiyi,Jia Xianbo,Wang Jie,Lai Songjia In vitro cellular & developmental biology. Animal MicroRNAs (miRNAs) are a class of small non-coding RNA molecules, which play important roles in animals by targeting mRNA transcripts for translational repression. Many recent studies have shown that miRNAs are involved in the control of muscle development. In this study, the expression levels of miR-221 in different tissues and during rabbit skeletal muscle satellite cells (SMSCs) differentiation were detected. Gene ontology term enrichment was used to predict the potential biological roles of miR-221. A synthetic miR-221 mimic and a miR-221 inhibitor were used to investigate the functions of miR-221 during SMSCs proliferation and differentiation to further verify the functions of miR-221 in muscle development. In this report, we compared the expression levels of miR-221 in different tissues. The expression levels of miR-221 were upregulated after the induction of differentiation, and then were gradually downregulated during SMSCs differentiation. Overexpression of miR-221 promoted SMSCs proliferation, whereas inhibiting expression restrained proliferation in the EdU and CCK-8 assays. In addition, overexpression of miR-221 led to a decline in the expression levels of the differentiation marker genes MyoG and MHC. miR-221 overexpression suppressed SMSCs myotube formation. On the contrary, inhibition of miR-221 promoted myotube formation. Our data showed that miR-221 increased SMSCs proliferation and decreased differentiation. 10.1007/s11626-017-0210-x
    MicroRNAs involved in skeletal muscle differentiation. Luo Wen,Nie Qinghua,Zhang Xiquan Journal of genetics and genomics = Yi chuan xue bao MicroRNAs (miRNAs) negatively regulate gene expression by promoting degradation of target mRNAs or inhibiting their translation. Previous studies have expanded our understanding that miRNAs play an important role in myogenesis and have a big impact on muscle mass, muscle fiber type and muscle-related diseases. The muscle-specific miRNAs, miR-206, miR-1 and miR-133, are among the most studied and best characterized miRNAs in skeletal muscle differentiation. They have a profound influence on multiple muscle differentiation processes, such as alternative splicing, DNA synthesis, and cell apoptosis. Many non-muscle-specific miRNAs are also required for the differentiation of muscle through interaction with myogenic factors. Studying the regulatory mechanisms of these miRNAs in muscle differentiation will extend our knowledge of miRNAs in muscle biology and will improve our understanding of the myogenesis regulation. 10.1016/j.jgg.2013.02.002
    Identification and characterization of microRNA from chicken adipose tissue and skeletal muscle. Wang X G,Yu J F,Zhang Y,Gong D Q,Gu Z L Poultry science MicroRNA (miRNA) is small noncoding RNA that is extensively expressed in organisms. Different types play important roles in various biological processes, such as growth and development. In this study, we identified 47 miRNA in chicken adipose tissue and skeletal muscle, of which 38 were known chicken miRNA, 4 were known miRNA homologous to other species, and 5 were potentially novel miRNA. The target genes from adipose tissue and skeletal muscle were predicted. The expression assay indicated that the 10 selected miRNA were differentially expressed in different developmental stages. Both miRNA-133a and miR-1a were muscle-related, whereas miR-122 was adipose-related miRNA. Certain identified miRNA may be essential to growth and development of chicken adipose tissue and skeletal muscle. Further studies of these miRNA will help to understand their functions in growth and development of adipose tissue and skeletal muscle of poultry. 10.3382/ps.2011-01656
    MiR-208b regulates cell cycle and promotes skeletal muscle cell proliferation by targeting CDKN1A. Wang Jian,Song Chengchuang,Cao Xiukai,Li Hui,Cai Hanfang,Ma Yilei,Huang Yongzhen,Lan Xianyong,Lei Chuzhao,Ma Yun,Bai Yueyu,Lin Fengpeng,Chen Hong Journal of cellular physiology Skeletal muscle is the most abundant tissue in the body. The development of skeletal muscle cell is complex and affected by many factors. A sea of microRNAs (miRNAs) have been identified as critical regulators of myogenesis. MiR-208b, a muscle-specific miRNA, was reported to have a connection with fiber type determination. However, whether miR-208b has effect on proliferation of muscle cell was under ascertained. In our study, cyclin-dependent kinase inhibitor 1A (CDKN1A), which participates in cell cycle regulation, was predicted and then validated as one target gene of miR-208b. We found that overexpression of miR-208b increased the expression of cyclin D1, cyclin E1, and cyclin-dependent kinase 2 at the levels of messenger RNA and protein in cattle primary myoblasts in vivo and in vitro. Flow cytometry showed that forced expression of miR-208b increased the percentage of cells at the S phase and decreased the percentage of cells at the G0/G1 phase. These results indicated that miR-208b participates in the cell cycle regulation of cattle primary myoblast cells. 5-Ethynyl-20-deoxyuridine and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed that overexpression of miR-208b promoted the proliferation of cattle primary myoblasts. Therefore, we conclude that miR-208b participates in the cell cycle and proliferation regulation of cattle primary skeletal muscle cell through the posttranscriptional downregulation of CDKN1A. 10.1002/jcp.27146
    Inhibition of the JNK/MAPK signaling pathway by myogenesis-associated miRNAs is required for skeletal muscle development. Xie Shu-Juan,Li Jun-Hao,Chen Hua-Feng,Tan Ye-Ya,Liu Shu-Rong,Zhang Yin,Xu Hui,Yang Jian-Hua,Liu Shun,Zheng Ling-Ling,Huang Mian-Bo,Guo Yan-Hua,Zhang Qi,Zhou Hui,Qu Liang-Hu Cell death and differentiation Skeletal muscle differentiation is controlled by multiple cell signaling pathways, however, the JNK/MAPK signaling pathway dominating this process has not been fully elucidated. Here, we report that the JNK/MAPK pathway was significantly downregulated in the late stages of myogenesis, and in contrast to P38/MAPK pathway, it negatively regulated skeletal muscle differentiation. Based on the PAR-CLIP-seq analysis, we identified six elevated miRNAs (miR-1a-3p, miR-133a-3p, miR-133b-3p, miR-206-3p, miR-128-3p, miR-351-5p), namely myogenesis-associated miRNAs (mamiRs), negatively controlled the JNK/MAPK pathway by repressing multiple factors for the phosphorylation of the JNK/MAPK pathway, including MEKK1, MEKK2, MKK7, and c-Jun but not JNK protein itself, and as a result, expression of transcriptional factor MyoD and mamiRs were further promoted. Our study revealed a novel double-negative feedback regulatory pattern of cell-specific miRNAs by targeting phosphorylation kinase signaling cascade responsible for skeletal muscle development. 10.1038/s41418-018-0063-1