摘要
目的:探索miR-182基因对心肌细胞增殖凋亡、活性氧簇(ROS)水平及磷脂酰肌醇-3激酶/丝氨酸苏氨酸激酶(PI3K/AKT)信号通路的影响。方法:将H9C2心肌细胞分为对照组、NC组、H_2O_2组、miR-182mimics组和miR-182inhibitor组,各组细胞均培养24h,逆转录-聚合酶链反应(RT-PCR)检测miR-182的mRNA表达;CCK8法检测各组细胞增殖;流式细胞仪检测细胞凋亡及ROS含量;Western bloting检测增殖相关蛋白ki67和细胞增殖核抗原(PCNA),凋亡相关蛋白含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)和含半胱氨酸的天冬氨酸蛋白水解酶9(Caspase-9)及PI3K/AKT信号通路磷脂酰肌醇3-激酶(PI3K)、磷酸化的丝氨酸苏氨酸激酶(p-AKT)的蛋白表达。结果:与对照组比较,H_2O_2组细胞miR-182、ki67、PCNA、PI3K、p-AKT表达及细胞增殖均显著降低,细胞凋亡率、ROS含量及Caspase-3和Caspase-9的表达均显著升高(均P<0.05);与H_2O_2组比较,miR-182mimics组miR-182、ki67、PCNA、PI3K、p-AKT表达及细胞增殖均显著升高,细胞凋亡率、ROS含量及Caspase-3和Caspase-9表达均显著降低(均P<0.05),miR-182inhibitor组miR-182、ki67、PCNA、PI3K、p-AKT表达及细胞增殖显著降低,细胞凋亡率、ROS含量及Caspase-3和Caspase-9表达均显著升高(均P<0.05)。结论:H_2O_2刺激可降低H9C2心肌细胞miR-182基因表达,过表达miR-182可促进细胞增殖,降低细胞凋亡,激活PI3K/AKT信号通路,其中对细胞增殖凋亡的影响方式是降低ROS含量,上调ki67和PCNA表达,下调Caspase-3和Caspase-9表达;抑制miR-182表达的结果则相反。
Objective:To explore the effect of miR-182 gene on proliferation and apoptosis,the level of ROS and PI3 K/AKT signaling pathway in myocardial cells.Method:H9 C2 cells were divided into control group,NC group,H_2O_2group,miR-182 mimics group and miR-182 inhibitor group,cells were cultured for 24 h,RT-PCR was used to detect the mRNA expression of miR-182;cell proliferation was detected by CCK8 assay;cell apoptosis and ROS content were detected by flow cytometry;Western bloting were used to detect Ki67,PCNA,Caspase-3,Caspase-9,PI3 K,and p-AKT protein expression.Result:Compared with the control group,Ki67,miR-182,PCNA,PI3 K,pAKT expression and cell proliferation in H_2O_2group were significantly decreased,cell apoptosis rate,the content of ROS and Caspase-3 and Caspase-9 expression were significantly increased(P<0.05);compared with H_2O_2group,Ki67,miR-182,PCNA,PI3 K,p-AKT expression and cell proliferation in miR-182 mimics group were significantly increased,cell apoptosis rate,the content of ROS and Caspase-3 and Caspase-9 expression were significantly decreased(P<0.05),miR-182,Ki67,PCNA,PI3 K,p-AKT expression and cell proliferation in miR-182 inhibitor group were significantly decreased,cell apoptosis rate,the content of ROS and Caspase-3 and Caspase-9 expression were significantly increased(P<0.05).Conclusion:H_2O_2stimulation can reduce miR-182 gene expression in H9 C2 Cardiomyocytes,overexpression of miR-182 can promote cell proliferation,reduce cell apoptosis,activated the PI3 K/AKT signaling pathway,the effect of cell proliferation and apoptosis was to reduce the content of ROS,up regulate the expression of Ki67 and PCNA,and down regulate the expression of Caspase-3 and Caspase-9.Inhibition of miR-182 expression is the opposite.
引文
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