miR-451a对慢性心衰细胞模型心肌细胞H9c2凋亡与自噬的影响

Effects of miR-451a on apoptosis and autophagy of cardiomyocytes H9c2 in chronic heart failure

目的探究miR-451a对慢性心衰细胞模型心肌细胞H9c2凋亡与自噬的作用机制。方法采用1μmol/L阿霉素(DOX)建立慢性心衰细胞模型。将细胞实验分为Control组(正常心肌细胞H9c2)、DOX组(DOX培养)、DOX+miR-NC组(DOX培养转染对照miR-NC)、DOX+miR-451a组(DOX培养转染miR-451a模拟物miR-451a)、DOX+miR-451a+CCI-779组(DOX和CCI-779培养转染miR-451a)。qRT-PCR检测各组细胞miR-451a相对表达水平;CCK-8和流式细胞术分别检测细胞增殖活性与凋亡;Western blotting检测剪切的Caspase-3(cleaved Caspase-3)、Bcl-2相关X蛋白(Bax)、Beclin-1、微管相关蛋白1轻链3I(LC3I)、LC3II、哺乳动物雷帕霉素靶蛋白(mTOR)、p70s6k、磷酸化mTOR(p-mTOR)、p-p70s6k蛋白表达。结果成功建立慢性心衰细胞模型。与Control组比较,DOX组miR-451a相对表达水平、细胞活性、p-mTOR、p-p70s6k蛋白表达降低,而凋亡率、cleaved-Caspase-3、Bax、Beclin-1、LC3II/I蛋白表达升高。与DOX+miR-NC组比较,DOX+miR-451a组miR-451a相对表达水平、细胞活性、p-mTOR、p-p70s6k蛋白表达升高,而凋亡率、cleaved-Caspase-3、Bax、Beclin-1、LC3II/I蛋白表达降低。与DOX+miR-451a组比较,DOX+miR-451a+CCI-779组细胞活性降低,凋亡率、cleaved-Caspase-3、Bax、Beclin-1、LC3II/I蛋白表达升高。结论miR-451a通过抑制mTOR信号通路促进慢性心衰细胞模型心肌细胞H9c2凋亡和自噬。

Aim To explore the effects of miR-451a on apoptosis and autophagy of cardiomyocytes H9c2 in chronic heart failure.Methods Chronic heart failure cell model was constructed with 1μmol/L doxorubicin(DOX).The cell experiments were divided into control group(normal myocardial cells H9c2),DOX group(DOX culture),DOX+miR-NC group(DOX culture transfected with miR-NC),DOX+miR-451a group(DOX culture transfected with miR-451a mimic miR-451a)and DOX+miR-451a+CCI-779 group(DOX and CCI-779 culture transfected with miR-451a).The relative expression level of miR-451a was detected by qRT-PCR.The activity of cell proliferation and apoptosis were detected by CCK-8 and flow cytometry,respectively.The expressions of cleaved Caspase-3(cleaved-Caspase-3),Bcl-2-associated X protein(Bax),Beclin-1,microtubule-associated protein 1 light chain 3I(LC3I),LC3II,phosphorylated mammalian target of rapamycin(p-mTOR),mammalian target of rapamycin(mTOR),p-p70s6k and p70s6k proteins were detected by Western blotting.Results The model of chronic heart failure was successfully constructed.Compared with Control group,relative expression level of miR-451a,cell activity,expressions of p-mTOR and p-p70s6k proteins were significantly decreased,while apoptosis rate,expressions of cleaved-Caspase-3,Bax,Beclin-1 and LC3II/I proteins were significantly increased in DOX group.Compared with DOX+miR-NC group,relative expression level of miR-451a,cell activity,expressions of p-mTOR and p-p70s6k proteins were significantly increased,while apoptosis rate,expressions of cleaved-Caspase-3,Bax,Beclin-1 and LC3II/I proteins were significantly decreased in DOX+miR-451a group.Compared with DOX+miR-451a group,cell activity was significantly decreased,while apoptosis rate,expressions of cleaved-Caspase-3,Bax,Beclin-1 and LC3II/I proteins were significantly increased in DOX+miR-451a+CCI-779 group.Conclusion miR-451a can promote apoptosis and autophagy of cardiomyocytes in chronic heart failure by inhibiting mTOR signaling pathway.