下调miR-320a表达对缺氧/复氧诱导的心肌细胞增殖和凋亡的影响

Effect of down-regulation of miR-320a expression on proliferation and apoptosis of cardiomyocytes induced by hypoxia/reoxygenation

目的:探讨下调miR-320a表达对心肌细胞缺氧/复氧(H/R)损伤模型的影响,并阐明其相关作用机制。方法:采用实时荧光定量PCR(RT-qPCR)法检测急性心肌梗死(AMI)患者血清中和H/R诱导的心肌H9C2细胞中miR-320a表达水平。将miR-320a inhibitor、inhibitor NC、小干扰Janus激酶(si-JAK2)和si-NC质粒分别转染至H9C2细胞中,同时设空白对照组,确定转染成功后进行H/R处理。H9C2细胞分为对照组、H/R组、H/R+inhibitor NC组、H/R+miR-320a inhibitor组、H/R+miR-320a inhibitor+si-NC组和H/R+miR-320a inhibitor+si-JAK2组。双荧光素酶报告基因检测miR-320a与Janus激酶2(JAK2)的靶向关系,CCK-8法检测各组细胞增殖率,生化法检测各组细胞中超氧化物歧化酶(SOD)活性、丙二醛(MDA)水平和细胞培养上清液中乳酸脱氢酶(LDH)活性,流式细胞术检测各组细胞凋亡率,RT-qPCR法检测各组细胞中miR-320a和JAK2 mRNA表达水平,Western blotting法检测各组细胞中B淋巴细胞瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、剪切型含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved-caspase-3)、JAK2、信号转导因子和转录激活因子3(STAT3)及磷酸化STAT3(p-STAT3)蛋白表达水平。结果:AMI患者血清中和H/R组H9C2细胞中miR-320a表达水平明显高于对照组(P<0.05)。双荧光素酶报告基因检测结果提示miR-320a可与JAK2靶向结合。与对照组比较,H/R组细胞增殖率和细胞中SOD活性明显降低(P<0.05),细胞凋亡率、细胞中MDA水平和细胞培养上清液中LDH活性明显升高(P<0.05),细胞中Bcl-2和JAK2蛋白表达水平及p-STAT3/STAT3比值明显降低(P<0.05),Bax和cleaved-caspase-3蛋白表达水平明显升高(P<0.05)。与H/R组比较,H/R+miR-320a inhibitor组细胞增殖率和细胞中SOD活性明显升高(P<0.05),细胞凋亡率、细胞中MDA水平和细胞培养上清液中LDH活性明显降低(P<0.05),细胞中Bcl-2和JAK2蛋白表达水平及p-STAT3/STAT3比值明显升高(P<0.05),Bax和cleaved-caspase-3蛋白表达水平明显降低(P<0.05)。与H/R+miR-320ainhibitor+si-NC组比较,H/R+miR-320a inhibitor+si-JAK2组细胞增殖率和细胞中SOD活性明显降低(P<0.05),细胞凋亡率、细胞中MDA水平和细胞培养上清液中LDH活性明显升高(P<0.05)。结论:下调miR-320a表达可抑制H/R诱导的心肌细胞凋亡,增加细胞增殖活性,其作用机制与靶向调控JAK2/STAT3信号通路有关。

Objective:To discuss the effect of down-regulation of miR-320a expression on the myocardial hypoxia/reoxygenation(H/R)injury model,and to clarify its related mechanism.Methods:Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of miR-320a in serum of the patients with actue myocardial infarction(AMI)and the myocardial H9C2 cells induced by H/R.The miR-320a inhibitor,inhibitor NC,small interference Janus kinase 2(si-JAK2),and si-NC plasmids were transfected into the H9C2 cells respectively,and blank control group was set up.After successful transfection,the H/R treatment was performed.The H9C2 cells were divided into control group,H/R group,H/R+inhibitor NC group,H/R+miR-320a inhibitor group,H/R+miR-320a inhibitor+si-NC group and H/R+miR-320a inhibitor+si-JAK2 group.The targeting relationship between miR-320a and Janus kinase 2(JAK2)was detected by double luciferase reporter gene;the proliferation rate of cells in various groups were detected by CCK-8 assay;the activities of superoxide dismutase(SOD)and levels of malonaldehyde(MDA)in cells and the levels of lactate dehydrogenase(LDH)in cell culture supernanant in various groups were detected by biochemical method;the apoptotic rates of cells in various groups were detected by flow cytometry;the expression levels of miR-320a and JAK2 mRNA in cells in various groups were detected by RT-qPCR method;the expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),cleaved-cysteinyl aspartate specific proteinase-3(cleaved-caspase-3),JAK2,signal transducers and activator of transcription 3(STAT3),and phosphorylated STAT3(p-STAT3)proteins in cells in various groups were detected by Western blotting method.Results:The expression levels of miR-320a in serum of the patients with AMI and the myocardial H9C2 cells in H/R group were significantly higher than those in control group(P<0.05).The results of double Luciferase reporter gene detection suggested that miR-320a could targetedly bind with JAK2.Compared with control group,the proliferation rate of the cells and SOD activity in the cells in H/R group were decreased significantly(P<0.05),the apoptotic rate of the cells,MDA level and LDH activity in the cells were significantly increased(P<0.05),the expression levels of Bcl-2 and JAK2 proteins and ratio of p-STAT3/STAT3 in the cells were significantly decreased(P<0.05),and the expression levels of Bax and cleaved caspase-3 proteins in the cells were significantly increased(P<0.05).Compared with H/R group,the proliferation rate of the cells and SOD activity in the cells in H/R+miR-320a inhibitor group were increased(P<0.05),while the apoptotic rate of the cells,MDA level in the cells,LDH activity in the cell culture supernanant were decreased(P<0.05),the expression levels of Bcl-2 and JAK2 proteins and ratio of p-STAT3/STAT3 in the cells were significantly increased(P<0.05),the expression levels of Bax and cleaved-caspase-3 proteins in the cells were significantly decreased(P<0.05).Compared with H/R+miR-320a inhibitor+si-NC group,the proliferation rate of the cells and SOD activity in the cells in H/R+miR-320a inhibitor+si-JAK2 group were decreased(P<0.05),and the apoptotic rate of the cells,MDA level in the cells,and LDH activity in the cell culture supernanant were increased(P<0.05).Conclusion:Down-regulation of miR-320a expression can inhibit the apoptosis of the cardiomyocytes induced by H/R and increase the proliferation activity of cells,and its mechanism is related to the targeted regulation of JAK2/STAT3 signaling pathway.