miR-148a在5-aza诱导间充质干细胞心肌样分化中的调控作用及机制

Regulation and mechanism of miR-148a on cardiomyocyte differentiation induced by 5-aza in mesenchymal stem cells

目的:探讨miR-148a在5-aza诱导人骨髓间充质(hMSCs)成心肌样分化中的表达及miR-148a对hMSCs体外成心肌样分化的生物学作用。方法:免疫荧光检测5-aza诱导hMSCs分化后心肌细胞特异性标志物α-MHC表达水平;qRT-PCR和Western blot分别检测miR-148a和DNMT1在hMSCs成肌样分化中的表达水平。利用Lipofectamine TM 2000将miR-148a mimics和miR-148a inhibitor分别瞬时转染hMSCs,Western blot检测心肌细胞特异性标志物α-MHC的蛋白表达水平。利用生物信息学技术预测miR-148a的靶基因结合位点利用双荧光素酶报告基因系统鉴定其对靶基因3’UTR的结合序列。通过DNMT1 shRNA和miR-148a inhibitors共转到hMSCs中,研究miR-148a在hMSCs成心肌样分化中的调控作用。结果:hMSCs经5-aza诱导分化后,心肌细胞特性标志物α-MHC蛋白水平明显上调。miR-148a在hBMSCs成肌样分化中显著性增加(P<0.01),DNMT1表达水平显著降低。过表达miR-148a能提高hBMSCs中心肌细胞特异性标志物α-MHC表达水平,而抑制miR-148a则能降低其水平(P<0.01)。DNMT1沉默可以阻断miR-148a对hMSCs的诱导成肌样分化作用。结论:miR-148a在hMCCs成肌样分化中表达上调,通过靶定和调控DNMT1基因的表达,并对hMSCs心肌向分化具有正向调控作用。

Objective： To investigate the expression of miR-148a in the process of myocardial differentiation of human mesenchymal stem cells （hMSCs） in- duced by 5-azacytidine （5-aza） and study the effects of miR-148a on myocardial differentiation of hMSCs. Methods： The immunofluorescence analysis was used to detect the expressions of the associated mark genes of cardiac specific prntein （α-MHC） in the process of myocardial differentiation of hMSCs induced by 5-aza. qRT-PCR and Western blot were used to analysis the expressions of miR-148a and DNA methyhransferase 1 （DNMT1） after myocardial dift;erentiation of hMSCs, respectively. The expression of α-MHC after transfection with synthetic miR-148 mimics and miR-148a inhibitor was examined by Western blot. We used bioinfor- mattes analysis to predict the potential target of miR-148a, and the dual luciferase report gene system was used to verify the predication. After en-transfected with DNMTI shRNA and miR-148a inhibitor, hMSCs were used to explore the regulatory role and mcchnism of miR-148a in the process nf myocardial differentiatiion of hMSCs. Results： α-MHC was increasd significantly after induced by 5-azacylidine. miR-148a was inereased significantly in cardiomyocyte diffcrentiatkm of hMSCs, while the gene and protdn expression levels off DNMT1 were decreased significantly in this progress （ P 〈 0.01 ）. The expression of a-MHC was up-regu- lated significantly in hMSCs when miR-148a was induced intn cardiomyocyte differentiation and overexprcssed. Instead, downregulation of miR-148a suppressed a- MHC expression （ P 〈 0.01 ）. Knockdown of DNMTI blocked the role of miR-148a in differentiation of hMSCs, Conclusion： miR-148a was upregulated in cardiomyocyte differentiation of hMSCs, and miR-148a promoted myoeardial differentiation of hMSCs via targeting DNMT1.