miR-148a 通过DNMT1 调控MSCs 向心肌分化的内在作用机制研究

目的:验证DNA 甲基转移酶1(DNMT1)是miR-148a 直接调控的靶基因,探究miR-148a 通过靶向DNMT1 调控骨髓间充质干细胞(MSCs)向心肌分化的作用机制。方法:MSCs 细胞经5忆鄄氮胞苷(5’-aza)诱导向心肌分化后,使用miRNA芯片筛选出诱导前后表达差异的miRNAs。Targetscan 软件分析异常表达的miR-148a 的靶基因,将野生型或突变型DNMT1 的3’-UTR 区域克隆到荧光素酶报告基因的下游(DNMT1-wt 或DNMT1-mut)并分别与miR-148a mimics(miR-148a 模拟物)或scramble(miR-148a 阴性对照)共转染,荧光素酶报告基因实验验证所预测的靶基DNMT1,qRT-PCR 和Western blot 分别检测转染miR-638 mimics 和scramble 后,对DNMT1 的mRNA 和蛋白表达的影响。构建miR-148a 慢病毒质粒,分别在转染到MSCs 后的第1、7、14 和28 天后检测miR-148a 对GATA 结合因子4(Gata-4)基因上游DNA 调控序列CpG 甲基化水平的影响、qRT-PCR 和Western blot 分别检测转染miR-148a 慢病毒质粒后对心肌特异蛋白:球蛋白重链(MHC) 和心脏肌钙蛋白T(cTnT)、MSCs 分化特征蛋白CD90 和CD29 表达的影响,以及对心肌特异转录因子心脏特异性同源盒基因(Nkx2.5)和Gata-4表达的影响。结果:miRNA 芯片筛选5’-aza 诱导MSCs 向心肌分化后表达异常的miRNAs,包括miR-146a-5p、miR-148a、miR-539 等,其中miR-148a 表达明显上调。Targetscan、荧光素酶报告基因实验和qRT-PCR 验证DNMT1 是miR-148a 直接调控的靶基因。miR-148a 慢病毒感染MSCs 细胞,证实在转染的不同时间,Gata-4 上游基因甲基化水平发生改变,且随着转染时间延长,Gata-4 甲基化水平呈不断降低趋势,MSCs 细胞内MHC 和cTnT 表达提高,CD90 和CD29 表达降低,Nkx2.5 和Gata-4 表达提高,MSCs 细胞出现向心肌分化。结论:miR-148a 可通过靶向调控DNMT1 而调控MSCs 细胞的向心肌分化。

Study on miR-148a regulation function of cardiac differentiation on MSCs by targeting DNMT1

Objective:To verify DNA methyltransferase 1(DNMT1) is the direct target of miR-148a and explore the internal mechanism by which miR-148a regulates the cardiac differentiation of mesenchymal stem cells ( MSCs) by directly targeting DNMT1.Methods:miRNA microarray was used to screen out the abnormally expressed miRNAs in MSCs before and after 5’-azacytidine(5’-aza) treatment.Target scan was uesd to predict the target of miR-148a.miR-148a mimics and DNMT1-wt,scramble andDNMT1-wt,miR-148a and DNMT1-mut,scramble and DNMT1-mut were co-transfected in MSCs cells and luciferase activity were detected by single photon.MSCs cells were transfected with miR-148a lentivirus plasmid.Respectively extract DNA,RNA and protein1,7,14 and 28 days after transfection.The CpG methylation level on DNA regulatory sequences of Gata-4 upstream gene was detected by methylation detection,the mRNA and protein expressions of myosin heavy chain(MHC),cardiac troponin T(cTnT),CD90,CD29,Nkx2.5 and Gata-4 were detected by qRT-PCR and Western blot.Results:miRNA Microarray screened out the abnormally expressed miRNAs in MSCs before and after 5’-aza-induced,including miR-146a-5p,miR-148a,miR-539,etc.Among which miR-148a was remarkably upregulated.Targetscan,Luciferase Reporter Gene and qRT-PCR verified that DNMT1 was the direct target of miR-148a.By infected MSCs cells with miR-148a lentivirus plasmid,we confirmed that the methylation level of Gata-4 gene upstream was changed,and with prolong of transfection,the methylation level of Gata-4 was decreased,the expression of MHC and cTnT were increased,while CD90 and CD2 were continually decreased,the mRNA expression of Nkx2.5 and Gata.4 were increased MSCs cells started myocardial differentiation.Conclusion:miR-148a can regulate the cardiac differentiation of MSCs by directly targeting DNMT1.