微小RNA-30a对缺氧条件下心肌细胞凋亡的影响及其机制研究

Influence of micro RNA-30a on cardiomyocyte apoptosis under hypoxia condition and mechanism study

目的探讨微小RNA-30a(miR-30a)对缺氧条件下心肌细胞凋亡的影响及机制。方法将H9C2细胞随机分为对照组(未做处理)、缺氧组(缺氧处理24 h)、缺氧+阴性对照(NC)组(转染miR-30a阴性对照后,缺氧处理24 h)和缺氧+miR-30a组(转染miR-30a mimics后,缺氧处理24 h)。采用实时定量PCR检测各组细胞中miR-30a的表达水平,比色法检测细胞上清液中超氧化物歧化酶(SOD)、丙二醛(MDA)和乳酸脱氢酶(LDH)含量,MTT法检测细胞的存活率,流式细胞仪检测细胞的凋亡率,Western blot检测STAT3信号通路相关蛋白信号转导与转录因子3(STAT3)、p-STAT3和切割型活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved caspase-3)的表达。结果缺氧处理后H9C2细胞中miR-30a、cleaved caspase-3和p-STAT3表达明显升高,细胞上清液中SOD含量降低,MDA和LDH含量升高,细胞存活率降低,凋亡率升高,与对照组相比,差异具有统计学意义(P<0.05)。转染miR-30a mimics上调miR-30a表达后,能够明显增强缺氧处理下的上述作用。结论 MiR-30a可通过激活STAT3信号通路增强氧化应激损伤,促进缺氧诱导的细胞凋亡。

Objective To discuss the influence of microRNA-30a(miR-30a) on cardiomyocyte apoptosis under hypoxia condition and mechanism. Methods H9C2 cells were randomly divided into control group(without treatment), hypoxia group(hypoxia treatment for 24 h), hypoxia+negative control group(hypoxia+NC group, transfected with negative control of miR-30a followed by hypoxia treatment for 24 h), and hypoxia+miR-30a group(transfected with miR-30a mimics followed by hypoxia treatment for 24 h). The expression of miR-30a was detected by using real-time fluorescence quantitative polymerase chain reaction(RT-PCR) in all groups, and content of superoxide dismutase(SOD), malondialdehyde(MDA) and lactic dehydrogenase(LDH) in supernatant were detected by using chromatoptometry. The survival rate was detected by using methyl thiazolyl tetrazolium test(MTT), and apoptosis was detected by using flow cytometer. The expressions of STAT3, p-STAT3 and cleaved caspase-3 were detected by using Western blotting assay. Results Compared with control group, the expressions of miR-30a, cleaved caspase-3 and p-STAT3 increased significantly, SOD content decreased, content of MDA and LDH increased, survival rate decreased and apoptosis increased in hypoxia group, hypoxia+NC group and hypoxia+miR-30a group(P<0.05). After transfected with miR-30a mimics followed by miR-30a up-regulation, all above indexes were improved under hypoxia condition. Conclusion MiR-30 a can enhance oxidative stress injury and promote hypoxia-induced apoptosis through activating STAT3 signaling pathway.