MiR-214 Supresses Fibrosis-associated Genes Expression Through Targeting EZH1 And EZH2 In Cardiac Fibroblasts

Recently, the prevalence of cardiovascular diseases shows a trend of rapid growth in China. At present, about 290 million people suffer from cardiovascular diseases, which including 4.5 million patients of heart failure. Cardiovascular disease is the leading cause of mortality in China, which results in the death of about. 3.5 millon people per year. As an important aspect of cardiac remodeling, cardiac fibrosis is associated with the initiation and progression of various cardiovascular diseases. Chronic myocardial fibrosis is very likely to cause heart failure, fatal arrhythmia and sudden cardiac arrest. It is reported that mutiple stimulus inculding ischemia-reperfusion injury, pressure load, and diabetes mellitus might induce cardiac fibrosis by regulating the expression of fibrosis associated genes. Cardiac fibrosis can profoundly affect cardiac function even resulting in heart failure by increasing myocardial stiffness, impairing electrical conduction and inducing cardiomyocytes death, which are the common pathlogical basis of various cardiovascular diseases. However, since the mechanism of cardiac fibrosis is very complex, the precise molecule and cell signaling involved in cardiac fibrosis are still not quite clear. Therefore, to further explore the mechanism of cardiac fibrosis can not only help us to better understand the pathophysiologic basis of diseases, but also contribute to the dignosis and treament of cardiac fibrosis associated diseases.Cardiac fibrosis is characterized by proliferation of cardiac fibroblasts and misbalance of degradation and synthesis of extracellular matrix protein which finally result in excessive deposition of ECM (especially collenge Ⅰ and collenge Ⅲ) in myocardium. A critical event of cardiac fibrosis is the transdifferentiation of cardiac fibroblasts to activer myofibroblasts, which markedly express a-SMA and can secrete a mass of ECM, cytokine and growth factor.A growing body of evidence suggests that the renin-angiotensin-aldosterone system(RAAS), inflammatory cytokines and chemokines, reactive oxygen species (ROS), intracullar Ca2+, extracellular matrix and regulatory growth factors (such as TGF-β, PDGF) are implicated in the initiation and progression of cardiac fibrosis. At molecular and cellular level, various stimulus signals transmit to the cell nucleus through receptors and related cellular signaling pathways, which regulate the promotors of fibrosis associated genes (such as Collagen Ⅰ, Collagen Ⅲ,α-SMA, MMP, fibronectin and so on) to control their expression. Recent reports have demonstrated that the turnover of ECM in cardiac fibroblasts is mediated by at least 4 signaling cascades, including TGF- β/Smads, mitogenactivated protein kinase(MAPK)-P38, RhoA-actin dynamics-myocardin-related transcription factor (MRTF-A), and transient receptor potential cation channel C6 (TRPC6)-calcineurin. There is a complex relationship between these pathways and stimulus signals, which consquently affect the development of cardiac fibrosis.MicroRNAs are endogenous, non-coding RNAs of 18-25 nucleotides that participate in cardiovascular physiology and diseases by interacting with the 3’UTR of their target genes to repress translation or enhance mRNA degradation. Recent studies have demonstrated that miRNAs are implicated in myocardial fibrosis. For examples, miR-21,-29,-30,-133 and miR-590 modulate fibrosis-related genes expression in animal models, including ischemia/ reperfusion myocardial infarction, trans-aortic constriction (TAC)and nicotine-induced cardiac fibrosis. some miRNAs might offer new targets as anti-fibrotic agents. According to the results of real time PCR and miRNAs microarray, we choose miR-214 for further study. It is reported that miR-214 protects injuried heart from ischemia reperfusion by improving calcium overload and cell death, while extensive fibrosis and impaired cardiac function were observed in miR-214 KO mice after ischemia reperfusion for 7d versus wild-type mice. However, whether miR-214 participates in cardiac fibrosis is unknown.Bioinformatics analysis indicated that enhancer of zeste homologl and 2 (EZH1, EZH2) might be direct targets of miR-214. As the enzymatically core subunit of Polycomb repressive complex2 (PRC2), EZH1 and EZH2 tri-melthylate H3K27 in the promoter of target genes and facilitate chromatin compaction and gene silencing. Whether miR-214 regulates EZH1 and EZH2 in cardiac fibroblasts is still unclear.In the present study, we use Ang-Ⅱ infused C57/BL6 mice as cardiac fibrotic animal model. We investigated the expression and role of miR-214 in Ang-Ⅱ infusion induced mouse fibrotic myocardium. Meanwhile, we confirmed whether miR-214 could regulate the expression of its target genes EZH1 and EZH2 at transcriptional level. Moreover, we analyzed the role of EZH1 and EZH2 in miR-214 involved cardiac fibrosis. In this study, we will demostrate the antifibrotic role and associated molecular mechanism of miR-214 in remodeling heart, which may offer a new opportunity as a anti-fibrotic agent.Part1 The expression of miR-214 in myocardium of cardiac remodeling miceObjective:To establish the cardiac fibrotic animal model. To investigate the expression of miR-214 in myocardium of cardiac remodeling mice.Methods:1. We used the C57/BL6 mouse with angiotensin Ⅱ infusion (1.46mg/kg/2w) for 14 days to establish the cardiac fibrotic animal model; 16 male SPF mice (20-25g) were randomly assigned to 2 groups for treatment; 2. Systolic blood pressure was measured by tail cuff method, and the heart weight and tibia length were measured to calculate the ratio; 3. Use echocardiography to assess the cardiac function; 4. Heart sections were stained with Masson trichrome for the collagen volume fraction (CVF) analysis; 5. The mRNA and protein level of fibrosis associated genes (including collal, col3al, a-SMA) in mice myocardium were detected by real-time PCR assay and Western Blot, respectively; 6.Use miRNAs microarray and real-time PCR assay to investigate the miRNAs expression profile in myocardium of Ang-Ⅱ infused mice.Results:1. Systolic blood pressure and the heart weight to tibial length ratio were both increased in Ang Ⅱ infusion group in comparision to saline group.2. Result of Echocardiography showed the increased ventricular wall, decreased ventricular volume, compensatory enhanced cardiac function in Ang Ⅱ infusion mice.3; Results of Masson trichrome staining revealed that the perivascular and interstitial fibrosis were markedly increased in myocardium of Ang-Ⅱ infused mice. 4. Result of qPCR and Western Blot showed the increased expression of fibrosis associated genes (collal, col3al, a-SMA) both in mRNA and protein level.5. A miRNAs-profiling array revealed that miRNAs were dysregulated in the myocardium of Ang-Ⅱ infused mice. Results of qRT-PCR demonstrated that the expression of miR-214 was significantly down-regulated in the myocardium of Ang-Ⅱ infused mice compared to control group.Conclusion:1. Pathological cardiac fibrosis were observed in myocardium of Ang-II infused mice compared to control group, which indicated that our animal model was succesful.2. miR-214 was significantly down-regulated in the myocardium of Ang-II infused mice compared to control group.Part2 the effect of miR-214 on Ang-Ⅱ induced cardiac fibrosisObjective:To investigate the effect of miR-214 on cardiac fibrosis in vivo.Methods:1. We used the C57/BL6 mouse with angiotensin Ⅱ infusion (1.46mg/kg/2w) for 14 days to establish the cardiac fibrotic animal model. Meanwhile, the saline group and Ang-Ⅱ infusion group were injected with agomiR-214 and agomiR-NC, respectively.24 male SPF mice (20-25g) were randomly assigned to 4 groups for treatment; 2. The heart weight and tibia length were measured to calculate the ratio; 3. Use echocardiography to assess the cardiac function; 4. Heart sections were stained with Masson trichrome for the collagen volume fraction (CVF) analysis.5. Use Western Blot to detect the protein level of fibrosis associated genes (including collal, col3al, α-SMA) in mice myocardium.Results:1. The heart weight to tibial length ratio was increased in Ang Ⅱ infusion group in comparision to saline group, while decreased in agomiR-214 group versus agomiR-NC group.2. Result of Echocardiography showed the increased ventricular wall, decreased ventricular volume, compensatory enhanced cardiac function in Ang Ⅱ infusion mice, which were improved in agomiR-214 group versus agomiR-NC group.3. Results of Masson trichrome staining revealed that the fibrosis were markedly increased in myocardium of Ang-Ⅱ infused mice, while decreased in agomiR-214 group versus agomiR-NC group.4. Result of Western Blot revealed that the expression of Collal and Col3al were markedly increased in myocardium of Ang-Ⅱ infused mice, while decreased in agomiR-214 group versus agomiR-NC group.Conclusion:miR-214 can improve the collagen deposition, cardiac remodeling and the cardiac function induced by Ang-Ⅱinfusion.Part3 miR-214 regulates fibrosis associated genes by targeting EZH1 and EZH2Objective:To investigate the effect of miR-214 on fibrotic phenotype in cardiac fibroblasts, to confirm whether EZH1 and EZH2 are direct targets of miR-214 in cardiac fibroblasts, and to analyze the role of EZH1 and EZH2 in miR-214 involved cardiac fibrosis.Methods:1. CFs were isolated from 4w C57BL/6 mice. Cultured CFs from the third passage were used for experiments. The CFs of third passage were identified by FIHC analysis of a-SMA; 2. The expression of fibrosis-related proteins in CFs were detected by immunofluorescence staining; 3. HEK293 cell was co-transfected with miR-214 mimic and luciferase repoter recombination plasmid containing the binding site of miR-214 and the 3’UTR of EZH1 or EZH2 for 24h. Dual luciferase assay was performed to identify the target genes of miR-214.4. The expression of fibrosis associated genes (including collal, col3al,α-SMA), EZH1, EZH2 were detected by Western Blot in CFs treated by 10-5M Ang-Ⅱ for 24h.5. The expression of fibrosis associated genes (including collal, col3al, a-SMA), EZH1, EZH2 and PPARy were detected by real-time PCR assay and Western Blot in CFs transfected with miR-214 mimic, EZH1 siRNA, EZH2 siRNA respectively.6. The expression of fibrosis associated genes (including collal, col3al, a-SMA), EZH1, EZH2 and PPARy were detected by Western Blot in CFs infected with rAd-EZHl,-EZH2 respectively.7. The expression of fibrosis associated genes (including col laT, col3al, α-SMA) and PPARy were detected by Western Blot in CFs transfected with PPARy siRNA.Results: 1. The expression of fibrosis associated genes (collal, col3al) were attenuated in CFs transfected with miR-214 mimic. 2.Transfection of miR-214 mimic decreased the luciferase activity of EZH1 or EZH2 3’UTR reporter gene, while mutation of the site in the EZH1 or EZH2 3’UTR completely blocked the effect of miR-214 mimic.3.The mRNA and protein levels of EZH1 and EZH2 were decreased in CFs with transfection of miR-214 mimic.4.The protein levels of EZH1 and EZH2 were increased in CFs treated with Ang-Ⅱ.5. The mRNA and protein levels of fibrosis associated genes (collal, col3al, a-SMA) were attenuated while the expression of PPARy was increased in CFs transfected with miR-214 mimic, EZH1 siRNA, EZH2 siRNA, respectively.6. The expression of fibrosis associated genes (collal, col3al, a-SMA) were increased while the expression of PPAR γ were decreased in CFs infected with rAd-EZH1,-EZH2, respectively.7. The protein levels of fibrosis associated genes (collal, col3al, a-SMA) were increased in CFs with transfection of PPARy siRNA.Conclusion:1. EZH2 and EZH2 are direct targets of miR-214.2. MiR-214 supresses the expression of EZH1 and EZH2 at transcriptional level, with consequent augment of PPARy, which result in the decreased expression of fibrosis associated genes and block the progression of cardiac fibrosis.In summary:As we know that,cardiac fibrosis is characterized by accumulation of ECM proteins in the myofibroblasts. In the present study, we found that miR-214 was significantly decreased in the myocardium of Ang-II infusion induced cardiac fibrotic mice. In vivo, miR-214 could improve the collagen deposition, cardiac remodeling and the cardiac function induced by Ang-Ⅱ infusion, which indicated its anti-fibrotic role in Ang-Ⅱ infusion induced cardiac fibrotic mice.In vitro, overexpression of miR-214 supresses the expression of its target genes EZH1 and EZH2 at transcriptional level, with consequent increased expression of PPARy, which leads to the decresaed expression of fibrosis associated genes and blocks the progession of cardiac fibrosis. In conclusion, miR-214 protects injured heart from cardiac fibrosis by targeting EZH1 and EZH2.