microRNA-200b下调DNMT3A的表达对SD大鼠心肌纤维化抑制作用的机制研究

Down-regulation of DNMT3A expression by microRNA-200b inhibits myocardial fibrosis in SD rats

目的研究微小RNA200b(miR-200b)靶向下调DNA甲基化转移酶3A(DNMT3A)对SD大鼠心肌纤维化的影响。方法 SD大鼠构建心肌纤维化模型,HE和Masson法观察病理学改变;qRT-PCR检测miR-200b的表达;Western blot法测定α-SMA、Col1A1、DNMT3A的表达。提取SD乳鼠心肌成纤维细胞(CFs),将miR-200b促进剂和抑制剂分别转染于CFs中,同时设立空白及阴性对照组。qRT-PCR检测miR-200b、DNMT3A、Col1A1和α-SMA水平;Western blot测定DNMT3A、Col1A1和α-SMA的蛋白表达;MTT法观察CFs的增殖情况。结果 HE和Masson染色结果显示,模型组SD大鼠心肌组织胶原纤维明显增多,心肌细胞排列杂乱;qRT-PCR显示miR-200b表达降低;Western blot显示,α-SMA、Col1A1、DNMT3A蛋白水平升高。体外实验qRT-PCR结果显示,α-SMA、Col1A1、DNMT3A在miR-200b促进剂组中的表达降低,miR-200b抑制剂组则相反;Western blot显示,miR-200b促进剂组中DNMT3A、Col1A1和α-SMA的表达降低,miR-200b抑制剂组结果则相反;MTT结果显示,外源性过表达miR-200b 24、48、72、96 h后,CFs增殖水平降低,而miR-200b抑制剂组结果相反。结论 miR-200b可能通过下调DNMT3A来影响CFs的活化增殖,从而改善心肌纤维化。

To investigate the effect of microRNA200b(miR-200b)targeting down-regulation of DNA methyltransferase 3A(DNMT3A)on myocardial fibrosis in SD rats.Methods In vivo:Myocardial fibrosis model was established in SD rats,HE and Masson method were used to observe the pathological changes,qRT-PCR was used to detect the expression of miR-200b,and Western blot was used to determine the expression ofα-SMA,Col1A1 and DNMT3A.In vitro:Cardiomyocyte fibroblasts(CFs)were isolated from SD rats and miR-200b mimics and inhibitors were transfected into CFs.At the same time,blank and negative control groups were established.The levels of miR-200b,DNMT3A,Col1A1 andα-SMA were determined by qRT-PCR.The protein expressions of DNMT3A,Col1A1 andα-SMA were determined by Western blot.The proliferation of CFs was observed by MTT assay.Results In vivo:The results of HE and Masson staining in SD rats in the model group showed that the myocardial tissue showed a significant increase of collagen fibers,and the myocardial cells were arranged disorderly;qRT-PCR showed that the expression of miR-200b decreased;Western blot showedα-SMA,Col1A1,DNMT3A protein The level rises.In vitro:qRT-PCR showed that the expression ofα-SMA,Col1A1,and DNMT3A decreased in the miR-200b mimics group,and in the miR-200b inhibitors group.Western blot showed that the expression of DNMT3A,Col1A1,andα-SMA in the miR-200b mimics group was reduced,but the results in the miR-200b inhibitors group were reversed.The results of MTT showed that after exogenous overexpression of miR-200b 24h,48h,72h,96h,the proliferation of CFs decreased,while the results of miR-200b inhibitors group were opposite.Conclusion miR-200b may affect the activation and proliferation of CFs by down-regulating DNMT3A and improve myocardial fibrosis.