Jiang Chang-Ke, Gong Fang
Department of Pediatrics, Yongchuan Hospital Affiliated to Chongqing Medical University, Chongqing 402160, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2017 Jun 8;33(6):514-518. doi: 10.12047/j.cjap.5601.2017.122.
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.
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.
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 (<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 (<0.01). Knockdown of DNMT1 blocked the role of miR-148a in differentiation of hMSCs.
miR-148a was upregulated in cardiomyocyte differentiation of hMSCs, and miR-148a promoted myocardial differentiation of hMSCs targeting DNMT1.
探讨5-氮杂胞苷（5-aza）诱导人骨髓间充质干细胞（hMSCs）向心肌细胞分化过程中miR-148a的表达情况，并研究miR-148a对hMSCs心肌分化的影响。
采用免疫荧光分析检测5-aza诱导hMSCs心肌分化过程中心肌特异性蛋白（α-MHC）相关标志基因的表达。分别用qRT-PCR和蛋白质免疫印迹法分析hMSCs心肌分化后miR-148a和DNA甲基转移酶1（DNMT1）的表达。通过蛋白质免疫印迹法检测转染合成的miR-148模拟物和miR-148a抑制剂后α-MHC的表达。利用生物信息学分析预测miR-148a的潜在靶点，并用双荧光素酶报告基因系统验证该预测。将DNMT1 shRNA和miR-148a抑制剂共转染hMSCs后，探讨miR-148a在hMSCs心肌分化过程中的调控作用及机制。
5-氮杂胞苷诱导后α-MHC显著增加。hMSCs向心肌细胞分化过程中miR-148a显著增加，而此过程中DNMT1的基因和蛋白表达水平显著降低（<0.01）。当miR-148a诱导hMSCs向心肌细胞分化并过表达时，α-MHC的表达显著上调。相反，miR-148a的下调抑制了α-MHC的表达（<0.01）。敲低DNMT1可阻断miR-148a在hMSCs分化中的作用。
hMSCs向心肌细胞分化过程中miR-148a上调，且miR-148a通过靶向DNMT1促进hMSCs的心肌分化。
