miR1-2 induces the differentiation of bone marrow mesenchymal stem cells into cardiomyocytes via Wnt/beta-catenin signal pathway

doi:10.3969/j.issn.2095-4344.1503

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) have the potential to differentiate into cardiomyocytes, but exhibit a low differentiation rate. Moreover, its differentiation mechanism is still unclear.
OBJECTIVE: To investigate the role of miR1-2 in the differentiation of mouse BMSCs into cardiomyocytes and to reveal the signaling pathways involved in this process.
METHODS: Mouse BMSCs were treated with miR1-2 and 5-azacytosine. The cardiomyocyte marker GATA4 in BMSCs was detected by qPCR. Apoptosis in BMSCs was detected by flow cytometry and the activity of Wnt/β-catenin signaling pathway was evaluated by measuring the upstream protein of this signal pathway.
RESULTS AND CONCLUSION: After 24 hours of transfection with miR1-2 mimics, the expression of miR1-2 in BMSCs was significantly increased. After 48 hours of transfection with miR-2 mimics, there was no significant difference in the apoptotic rate of BMSCs. After 48 hours of transfection with miR1-2 mimics, the expression of GATA4 increased significantly with time. After 72 hours of transfection with miR1-2 mimics, the expression of these genes was significantly higher in the miR1-2 group than in the 5-azacytosine group and the control group. Compared with miR1-2-mimics-NC, treatment with miR1-2 mimics could significantly increase the relative expression of β-catenin and Wnt11 at mRNA and protein levels, while addition of Wnt/β-catenin pathway inhibitor LGK-974 reduced the relative expression of β-catenin and Wnt11. To conclude, miR1-2 in BMSCs has the potential to effectively induce the differentiation of BMSCs into cardiomyocytes via the Wnt/β-catenin pathway.

Key words: Mesenchymal Stem Cells, Myocytes, Cardiac, Cell Differentiation, Tissue Engineering

CLC Number:

Gao Wei, Li Wenwei, Ren Zengfu, Chen Shiyun. miR1-2 induces the differentiation of bone marrow mesenchymal stem cells into cardiomyocytes via Wnt/beta-catenin signal pathway[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(1): 7-12.

Figures/Tables 3

2.3 miR1-2的高度表达诱导骨髓间充质干细胞中心脏特异性基因的表达为了确定骨髓间充质干细胞中miR1-2的过度表达是否能诱导这些细胞分化成心肌细胞，使用qPCR检测心脏特异性基因GATA4的表达水平。数据表明，GATA4的表达在5-氮杂胞嘧啶处理48 h后显著增加，但是72 h后GATA4的表达与对照组相比没有显著增加。在转染miR1-2模拟物48 h后，与对照相比，GATA4的表达随着时间的延长也显著增加，处理72 h后，这些基因在miR1-2模拟物处理组中的表达显著高于5-氮杂胞嘧啶组和对照组，见图3。这些结果表明，过表达miR1-2可以通过增加心脏基因的表达水平诱导骨髓间充质干细胞向心肌细胞分化。 2.4 高表达miR1-2激活Wnt/β-catenin信号通路为了确定miR1-2的过度表达是否通过Wnt/β-catenin信号通路诱导小鼠骨髓间充质干细胞的心脏分化。为此，实验检测了miR1-2模拟物处理后骨髓间充质干细胞中Wnt/β-catenin信号通路上游蛋白β-catenin和Wnt11的转录水平。结果显示，与miR1-2-mimics-NC相比，用miR1-2模拟物处理后，β-catenin和Wnt11的相对表达均显著增加，见图4。这些结果表明Wnt信号通路可能在miR1-2诱导的骨髓间充质干细胞向心肌细胞分化过程中发挥重要作用。 2.5 miR1-2通过Wnt/β-catenin信号传导激活诱导心肌细胞分化为了研究miR1-2的高表达诱导的心脏特异性基因的增加水平是否通过Wnt信号传导，使用Wnt/β-catenin信号通路抑制剂LGK-974来抑制Wnt/β-catenin信号通路的激活。结果显示，miR1-2模拟物处理后，β-catenin和Wnt11的相对表达量均显著增加，而添加LGK-974后，β-catenin和Wnt11的相对表达量均显著降低，见图5。与qPCR分析结果相一致，Western blot分析也证实经72 h处理后，骨髓间充质干细胞增加了GATA4蛋白的表达，Wnt抑制剂处理后，与没有Wnt抑制剂处理的miR1-2模拟物组相比，GATA4的蛋白质表达显著降低，见图6。 "

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