miR-29a-3p通过靶向Bak1参与高糖诱导的心肌细胞凋亡

MiR-29a-3p regulates high glucose-induced cardiomyocyte apoptosis by targeting Bak1

目的探讨miR-29a-3p通过靶向Bak1参与调控高糖诱导的心肌细胞凋亡的作用机制。方法体外培养大鼠H9c2心肌细胞,分为正常葡萄糖组(NG)、高糖组(HG)、高糖+miR-29a-3p模拟物组(HG+miR-29a-3p mimics)、高糖+模拟物阴性对照组(HG+NC mimics)。qRT-PCR检测miR-29a-3p的表达,流式细胞术检测细胞凋亡水平。蛋白印迹法检测凋亡相关因子Bcl-2、Mcl-1、Bax、Caspase-3和Bak1的表达水平。双荧光素酶报告基因试验检测miR-29a-3p和Bak1的靶向关系。结果与NG组比较,HG组miR-29a-3p表达水平降低,心肌细胞凋亡率明显升高(P<0.05);与HG+NC mimics比较,HG+miR-29a-3p mimics组心肌细胞凋亡水平明显降低(P<0.05),心肌细胞凋亡蛋白Bax、Caspase-3、Bak1的表达显著降低(P<0.05),抗凋亡蛋白Bcl-2和Mcl-1的表达明显增加(P<0.05)。双荧光素酶报告基因技术验证了Bak1是miR-29a-3p的直接靶基因。结论高糖能够诱导心肌细胞凋亡,过表达miR-29a-3p可能通过靶向调控Bak1减轻高糖诱导的心肌细胞凋亡。

Objective To investigate the role and mechanism of miR-29a-3p in regulation of high glucose-induced cardiomyocyte apoptosis by targeting Bak1.Methods H9c2 cells were cultured in vitro and divided into normal glucose group(NG),high glucose group(HG),HG+miR-29a-3p mimics group and HG+NC mimics group.The expression of miR-29a-3p was detected by qRT-PCR,and the level of apoptosis was measured by flow cytometry.The expression levels of apoptosis-related factors Bcl-2,Mcl-1,Bax,Caspase-3,and Bak1 were determined by Western blotting.The dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-29a-3p and Bak1.Results Compared with NG group,the expression of miR-29a-3p was decreased and the rate of cardiomyocyte apoptosis was significantly increased in HG group(P<0.05).Compared with HG+NC mimics group,the level of cardiomyocyte apoptosis was significantly reduced in HG+miR-29a-3p mimics group(P<0.05),and the expression of apoptosis-related proteins Bax,Caspase-3,and Bak1 was significantly decreased(P<0.05),while the expression of anti-apoptotic proteins Bcl-2 and Mcl-1 was significantly increased(P<0.05).The dual-luciferase reporter gene assay confirmed that Bak1 was a direct target gene of miR-29a-3p.Conclusion High glucose can induce the cardiomyocyte apoptosis,and the overexpression of miR-29a-3p may alleviate high glucose-induced cardiomyocyte apoptosis by targeting and regulating Bak1.