miR-30e-3p调控的自噬对缺血缺氧致H9c2心肌细胞损伤的保护作用

Protective effect of miR-30e-3p-regulated autophagy on H9c2 cardiomyocyte injury induced by ischemia and hypoxia

目的:研究miR-30e-3p在缺血缺氧(IH)环境中对H9c2心肌细胞自噬的调控,以及对心肌细胞损伤的影响。方法:使用H9c2心肌细胞进行体外研究,将慢病毒作为载体转染H9c2心肌细胞以上调或者抑制miR-30e-3p表达。根据本课题组前期研究取9 h作为干预时间点,将H9c2心肌细胞随机分为6组,分别为正常对照(Con)组、IH组、IH+miR-30e-3p阴性对照(IH+NC)组、IH+miR-30e-3p mimic(IH+MIR)组、IH+miR-30e-3p inhibitor(IH+MIR inhibitor)组和IH+miR-30e-3p mimic+3-MA(IH+MIR+3-MA)组。以实时荧光定量聚合酶链反应(qPCR)检测miR-30e-3p表达水平,MTS法检测心肌细胞存活率,乳酸脱氢酶(LDH)测定心肌细胞损伤,蛋白质印迹(Western blotting)检测自噬关键蛋白LC3与p62表达变化,共聚焦显微镜观察细胞自噬流。结果:与Con组比较,IH组miR-30e-3p表达与心肌细胞存活率下降,心肌细胞损伤增加,自噬蛋白LC3Ⅱ表达减少,p62表达升高(均P<0.05);与IH组比较,过表达miR-30e-3p后心肌细胞存活率升高,心肌损伤减少,LDH、自噬蛋白LC3Ⅱ表达增加,p62表达降低,同时自噬流增多(均P<0.05)。结论:过表达miR-30e-3p能增加IH环境中H9c2心肌细胞自噬,减轻细胞损伤,起到保护心肌细胞的作用。

Objective:To study the regulation of miR-30 e-3 p on autophagy of H9 c2 cardiomyocyte and its effect on cardiomyocyte injury in theenvironmentof ischemia and hypoxia(IH).Methods:H9 c2 cardiomyocytes were used for in vitro study.Lentivirus was used as a vector to transfect H9 c2 cardiomyocytes in order to up-regulate or inhibit the expression of miR-30 e-3 p.According to the previous study,9 h was taken as intervention time point.H9 c2 cardiomyocytes were randomly divided into 6 groups:normal control group(Con),IH group,IH+miR-30 e-3 p negative control group(IH+NC),IH+miR-30 e-3 p mimic(IH+MIR)group,IH+miR-30 e-3 p inhibitor(IH+MIR inhibitor)group and IH+miR-30 e-3 p mimic+3-MA(IH+MIR+3-MA)group.The expression of miR-30 e-3 p was detected by real-time fluorescence quantitative polymerase chain reaction(qPCR).The survival rate of cardiomyocytes was detected by MTS method.The injury of cardiomyocytes was detected by lactate dehydrogenase(LDH).The expression changes of the key proteins of autophagy LC3 and p62 were detected by Western blotting.The autophagy flow was observed by confocal microscope.Results:Compared with Con group,the expression of miR-30 e-3 p and cardiomyocyte survival rate weredecreased,thecardiomyocyte injury was increased,the expressionof autophagy protein LC3 II was decreased,and the expression of p62 was increased in IH group(all P<0.05).Compared with IH group,the cardiomyocyte survival rate was increased,the myocardial injury was decreased,the expression of LDH and autophagy protein LC3 II were increased,the expression of p62 was decreased,and the autophagy flow was increased after overexpression of miR-30 e-3 p(all P<0.05).Conclusion:Overexpression of miR-30 e-3 p can increase autophagy of H9 c2 cardiomyocytes,reduce cellular injury and protect cardiomyocytesin IH environment.