27nt-miRNA对过氧化氢诱导血管平滑肌细胞凋亡的影响及分子机制

Effect of 27nt-miRNA on hydrogen peroxide-induced apoptosis in vascular smooth muscle cells and its molecular mechanism

目的:探讨27nt-miRNA对经过氧化氢(H_(2)O_(2))处理的血管平滑肌细胞(VSMCs)凋亡、活力和迁移的影响及作用机制。方法:将大鼠主动脉VSMCs分为5组:空白对照组、H_(2)O_(2)组、anti-27nt-miRNA+H_(2)O_(2)组、阴性对照+H_(2)O_(2)组和27nt-miRNA+H_(2)O_(2)组。空白对照组:不做任何处理;H_(2)O_(2)组:用500μmol/L H_(2)O_(2)处理6 h诱导细胞凋亡;anti-27nt-miRNA+H_(2)O_(2)组:转染27nt-miRNA反义序列慢病毒载体后,用500μmol/L H_(2)O_(2)处理6 h诱导细胞凋亡;阴性对照+H_(2)O_(2)组:转染随机阴性序列慢病毒载体后,用500μmol/L H_(2)O_(2)处理6 h诱导细胞凋亡;27nt-miRNA+H_(2)O_(2)组:转染27nt-miRNA高表达慢病毒载体后,再用500μmol/L H_(2)O_(2)处理6 h诱导凋亡。使用细胞计数试剂盒(CCK-8)法和流式细胞术检测VSMCs存活率、凋亡率,划痕实验检测细胞迁移率,蛋白质免疫印迹法(Western blotting)检测Bcl-2、Bax蛋白表达水平。结果:与空白对照组比较,H_(2)O_(2)组细胞活力和迁移率降低(P<0.05)、细胞凋亡率上升(P<0.05),提示成功构建VSMCs凋亡模型。对比阴性对照+H_(2)O_(2)组,27nt-miRNA+H_(2)O_(2)组可抑制细胞的活力和迁移能力(P<0.05),且细胞凋亡率上升(P<0.05),凋亡相关蛋白Bax表达水平上升,Bcl-2表达水平下降(P<0.05),而anti-27nt-miRNA+H_(2)O_(2)组细胞凋亡率降低、细胞活力和迁移率增加,Bax蛋白表达水平降低,Bcl-2蛋白表达水平升高(P<0.05)。结论:过表达27nt-miRNA可促进H_(2)O_(2)所诱导的VSMCs凋亡和抑制其活力和迁移能力,其机制可能与凋亡相关蛋白Bax、Bcl-2的表达改变密切相关。

Objective:To investigate the effect of 27nt-miRNA on the apoptosis,viability and migration of vascular smooth muscle cells(VSMCs)treated with hydrogen peroxide(H_(2)O_(2))and the mechanism of action.Methods:Rat aortic VSMCs were divided into five groups:blank control group,H_(2)O_(2) group,anti-27nt-miRNA+H_(2)O_(2) group,negative control+H_(2)O_(2) group and 27nt-miRNA+H_(2)O_(2) group.Blank control group:no treatment;H_(2)O_(2) group:apoptosis was induced by treatment with 500μmol/L H_(2)O_(2) for 6 h;anti-27nt-miRNA+H_(2)O_(2) group:apoptosis was induced by treatment with 500μmol/L H_(2)O_(2) for 6 h after transfection with 27nt-miRNA inhibitor lentiviral vector;negative control+H_(2)O_(2) group:VSMCs were transfected with random negative lentiviral vector,then treated with 500μmol/L H_(2)O_(2) for 6 h to induce apoptosis;27nt-miRNA+H_(2)O_(2) group:transfected with 27nt-miRNA high expression lentiviral vector,then treated with 500μmol/L H_(2)O_(2) for 6 h to induce apoptosis.The survival rate and apoptosis rate of VSMCs were detected by using CCK-8 and flow cytometry,respectively.The cell migration rate was detected by scratch assay,and the protein expression levels of Bcl-2 and Bax were detected by western blotting.Results:Compared with the blank control group,the cell viability and migration rate decreased and apoptosis rate increased in the H_(2)O_(2) group(P<0.05),suggesting that the apoptosis model of VSMCs was successfully constructed.Compared with the negative control+H_(2)O_(2) group,the 27nt-miRNA+H_(2)O_(2) group showed inhibited cell viability and migration ability,and the apoptosis rate increased,the expression level of apoptosis-related protein Bax increased and the expression level of Bcl-2 decreased(P<0.05),while the anti-27nt-miRNA+H_(2)O_(2) group showed decreased apoptosis,increased cell viability and migration rate,decreased Bax protein expression level and increased Bcl-2 protein expression level(P<0.05).Conclusion:Overexpression of 27nt-miRNA can promote apoptosis of VSMCs induced by H_(2)O_(2) and inhibit viability and migration ability,and the mechanism may be closely related to the altered expression of apoptosis-related proteins Bax and Bcl-2.