Objective: To investigate the expression of miR-148a in the process of myocardial differentiation of human mesenchymal stem cells (hMSCs) induced 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 protein (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 methyltransferase 1 (DNMT1) after myocardial differentiation 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 bioinformatics analysis to predict the potential target of miR-148a, and the dual luciferase report gene system was used to verify the predication. After co-transfected with DNMT1 shRNA and miR-148a inhibitors, hMSCs were used to explore the regulatory role and mechnism of miR-148a in the process of myocardial differentiation of hMSCs.
Results: α-MHC was increased significantly after induced by 5-azacytidine. miR-148a was increased significantly in cardiomyocyte differentiation of hMSCs, while the gene and protein expression levels of DNMT1 were decreased significantly in this progress (P<0.01). The expression of α-MHC was up-regulated significantly in hMSCs when miR-148a was induced into cardiomyocyte differentiation and overexpressed. Instead, downregulation of miR-148a suppressed α-MHC expression (P<0.01). Knockdown of DNMT1 blocked the role of miR-148a in differentiation of hMSCs.
Conclusions: miR-148a was upregulated in cardiomyocyte differentiation of hMSCs, and miR-148a promoted myocardial differentiation of hMSCs via targeting DNMT1.
Keywords: DMNT1; cardiac differentiation; mesenchymal stem cells; miR-148a.