MicroRNA-30a调控Beclin-1对缺氧复氧乳鼠心肌细胞的保护效应

目的: 观察microRNA-30a(miR-30a)在原代心肌细胞缺氧复氧中的作用,探讨miR-30a保护缺血再灌注心肌的分子机制。方法: 重组构建慢病毒miR-30a表达载体(LV-GFP-miR-30a)感染原代乳鼠心肌细胞,构建缺氧复氧损伤模型。实验分为正常培养组、单纯缺氧复氧组、LV-GFP加缺氧复氧组、LV-miR-30a-GFP加缺氧复氧组和3-甲基腺嘌呤(3-MA)加缺氧复氧组。Real-time PCR检测缺氧复氧和慢病毒感染对miR-30a的表达影响,Western blotting检测LC3和Beclin-1蛋白表达变化,TUNEL和PI染色检测缺氧复氧后心肌细胞死亡情况。结果: (1)缺氧复氧后心肌miR-30a表达水平下调(P<0.05);(2)慢病毒miR-30a表达载体高效感染后心肌细胞miR-30a表达水平上调(P<0.05),心肌过表达miR-30a下调Beclin-1蛋白表达(P<0.05);(3)心肌过表达miR-30a抑制缺氧复氧后Beclin-1表达(P<0.05);3-MA处理减少缺氧复氧后心肌Beclin-1表达,减少缺氧复氧后LC3-Ⅰ转化为LC3-Ⅱ(P<0.05);(4)过表达miR-30a和3-MA处理减少缺氧复氧后心肌细胞凋亡(P<0.05)。结论: 心肌细胞过表达miR-30a显著下调Beclin-1;抑制自噬可以减少缺氧复氧后心肌细胞死亡。

Proctective effect of microRNA-30a on regulating beclin-1 expression in hypoxia-reoxygenated neonatal rat cardiomyocytes

AIM: To explore the potential mechanism of microRNA-30a(miR-30a) overexpression in neonatal rat cardiomyocytes during hypoxia/reoxygenation(H/R).METHODS: The miR-30a overexpression was induced in primary neonatal rat cardiomyocytes by lentivirus transfection.The cardiomyocytes were divided into 5 groups: normal group,H/R group,LV-GFP+H/R group,LV-GFP-miR-30a+H/R group and 3-methyladenine(3-MA)+H/R group.The expression level of miR-30a after lentivirus transfection and H/R was determined by real-time PCR,while the protein levels of LC3 and Beclin-1 after H/R and lentivirus transfection were detected by Western blotting.The cardiomyocyte death after H/R were measured by TUNEL and PI staining.RESULTS: Compared with LV-GFP group,significant down-regulation of Beclin-1 protein level was observed in cardiomyocytes with miR-30a overexpression,while the protein levels of Beclin-1 and LC3 in the cardiomyocytes with miR-30a overexpression were down-regulated after H/R,and apoptosis of these cells were significantly decreased after H/R.CONCLUSION: The protein level of Beclin-1 is down-regulated in cardiomyocytes with miR-30a overexpression.Inhibition of autophagy decreases the cardiomyocyte death after H/R.