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miR-92a对H2O2诱导的H9C2心肌细胞凋亡以及MAPK-ERK通路的影响

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摘要:

目的 探讨下调miR-92a表达对过氧化氢(H2O2)诱导H9C2心肌细胞凋亡的影响以及可能的分 子机制。方法 将体外培养的H9C2心肌细胞分为空白对照组(Blank)、H2O2模型组、阴性对照组(NC)、 NC+H2O2组、miR-92a inhibitor组、miR-92a inhibitor+H2O2组。Blank组不经任何特殊处理。NC组、NC+H2O2 组、miR-92a inhibitor组、miR-92a inhibitor+H2O2组细胞进行转染,qRT-PCR法验证转染效率。另H2O2模型 组、NC+H2O2组、miR-92a inhibitor+H2O2组细胞经H2O2诱导6 h,流式细胞术法检测细胞凋亡率。采用试剂盒 检测活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)释放量。Western blot法检测凋亡相关蛋白B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、Caspase-3和丝裂 原活化蛋白激酶(MAPK)-细胞外调节蛋白激酶(ERK)通路蛋白磷酸化情况。结果 成功建立miR-92a inhibitor转染细胞模型。经H2O2处理6 h后,H2O2+miR-92a inhibitor组H9C2细胞的凋亡率、ROS产生量、MDA 和LDH释放量均低于H2O2模型组和H2O2+NC组,而细胞培养上清中SOD水平高于H2O2模型组和H2O2+NC组(P <0.05)。Western blot法检测,与H2O2模型组相比,H2O2+miR-92a inhibitor组细胞Bcl-2蛋白表达量上调, Bax、Caspase-3、磷酸化蛋白激酶B(p-AKT)、p-ERK蛋白表达量降低(P<0.05)。H2O2对H9C2细胞AKT 和ERK蛋白表达量无影响(P>0.05)。结论 下调miR-92a表达可抑制H2O2诱导的H9C2心肌细胞凋亡,其可 能的作用机制与降低ROS释放量、调控Bcl-2、Bax、Caspase-3蛋白以及MAPK-ERK信号通路有关。

Abstract:

Objective To investigate the influence of down-regulated expression of miR-92a on H2O2-induced apoptosis of H9C2 cardiomyocytes and possible molecular mechanism. Methods H9C2 cardiomyocytes cultured in vitro were divided into Blank group, H2O2 model group, negative control group (NC group), NC+H2O2 group, miR-92a inhibitor group and miR-92a inhibitor+H2O2 group. Blank group was not given any treatment. The transfection was carried out in NC group, NC+H2O2 group, miR-92a inhibitor group and miR-92a inhibitor+H2O2 group, and the transfection efficacy was verified by using quantitative real-time PCR (qRT-PCR). The apoptosis rate of H9C2 cardiomyocytes was detected by using flow cytometry after H2O2 induction for 6 h in H2O2 model group, NC+H2O2 group and miR-92a inhibitor+H2O2 group. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and lactic dehydrogenase (LDH) were detected by using kit method. The expressions of B cell lymphoma/leukemia-2 (Bcl-2), Bcl-2- related X protein (Bax), Caspase-3 and protein phosphorylation of MAPK-ERK pathway were detected by using Western blotting assay. Results The transfection model of miR-92a inhibitor was established successfully. After H2O2 treatment for 6 h, the apoptosis rate of H9C2 cardiomyocytes, and levels of ROS, MDA and LDH were lower and SOD level was higher in H2O2+miR-92a inhibitor group than those in H2O2 model group and H2O2+NC group (P<0.05). The results of Western blotting assay showed that the protein expression of Bcl-2 increased, protein expressions of Bax, Caspase-3, p-AKT and p-ERK decreased in H2O2+miR-92a inhibitor group compared with H2O2 model group (P<0.05). The protein expressions of AKT and ERK were not affected by H2O2 (P>0.05). Conclusion The down-regulation of miR-92a expression can inhibit H2O2-induced apoptosis of H9C2 cardiomyocytes, and the mechanism may be related to reducing ROS level, controlling proteins of Bcl-2, Bax and Caspase-3 and MAPK-ERK signaling pathway.

基金项目:

2016年河南省医学科技攻关项目(201602334)

参考文献:

  • 2008

  • 1

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