下调lncRNA KCNQ1OT1抑制H_(2)O_(2)诱导的心肌细胞凋亡和氧化损伤

Down-regulation of lncRNA KCNQ1OT1 Inhibits H_(2)O_(2)-induced Cardiomyocyte Apoptosis and Oxidative Damage

目的探讨下调长链非编码RNA(lncRNA)KCNQ1重叠转录物1(KCNQ1OT1)对过氧化氢(H_(2)O_(2))诱导的心肌细胞凋亡和氧化损伤的影响。方法心肌细胞H9c2分成Control组、H_(2)O_(2)组(H_(2)O_(2)处理)、si-NC组(转染siRNA control,H_(2)O_(2)处理)、si-KCNQ1OT1组(转染KCNQ1OT1 siRNA,H_(2)O_(2)处理)、si-KCNQ1OT1+LiCl组(转染KCNQ1OT1 siRNA,Wnt信号激活剂LiCl和H_(2)O_(2)处理)。CCK-8实验检测细胞增殖,流式细胞术检测细胞凋亡,硫代巴比妥酸法检测丙二醛(MDA)水平、黄嘌呤氧化法检测超氧化物歧化酶(SOD)水平、微量法检测过氧化氢酶(CAT)水平、比色法检测谷胱甘肽过氧化物酶(GSH-Px)水平,Western blot法检测活化的含半胱氨酸的天冬氨酸蛋白水解酶3(C-Caspase-3)、活化的含半胱氨酸的天冬氨酸蛋白水解酶9(C-Caspase-9)、β-连环蛋白(β-catenin)、原癌基因(c-Myc)蛋白表达水平。结果与Control组比较,H_(2)O_(2)组心肌细胞存活率降低,细胞凋亡率升高,细胞中C-Caspase-3、C-Caspase-9、β-catenin、c-Myc蛋白表达水平升高,MDA水平升高,SOD、CAT、GSH-Px水平降低。与si-NC组比较,si-KCNQ1OT1组心肌细胞存活率升高,细胞凋亡率降低,细胞中C-Caspase-3、C-Caspase-9、β-catenin、c-Myc蛋白表达水平降低,MDA水平降低,SOD、CAT、GSH-Px水平升高。与si-KCNQ1OT1组比较,si-KCNQ1OT1+LiCl组心肌细胞存活率降低,细胞凋亡率升高,细胞中C-Caspase-3、C-Caspase-9、β-catenin、c-Myc蛋白表达水平升高,MDA水平升高,SOD、CAT、GSH-Px水平降低。结论下调KCNQ1OT1可以抑制H_(2)O_(2)诱导的心肌细胞凋亡和氧化损伤,其机制与抑制Wnt信号通路有关。

Objective To investigate the effect of down-regulation of long non-coding RNA(lncRNA)KCNQ1 overlapping transcript 1(KCNQ1 OT1)on the apoptosis and oxidative damage of cardiomyocytes induced by hydrogen peroxide(H_(2)O_(2)).Methods Cardiomyocytes H9 c2 were divided into Control group,H_(2)O_(2)group(H_(2)O_(2)treatment),si-NC group(transfection with siRNA control,H_(2)O_(2)treatment),si-KCNQ1 OT1 group(transfection with KCNQ1 OT1 siRNA,H_(2)O_(2)treatment),si-KCNQ1 OT1+LiCl group(transfection with KCNQ1 OT1 siRNA,Wnt signal activator LiCl and H_(2)O_(2)treatment).CCK-8 test was used to detect cell proliferation,flow cytometry to detect cell apoptosis,thiobarbituric acid method to detect malondialdehyde(MDA)level,xanthine oxidation method to detect superoxide dismutase(SOD)level,micro method to detect catalase(CAT)level,colorimetric method to detect glutathione peroxidase(GSH-Px)level,Western blotting method to detect activated cysteine-containing aspartate proteolytic enzyme 3(C-Caspase-3),activated cysteine-containing aspartate proteolytic enzyme 9(C-Caspase-9),β-catenin,and proto-oncogene(c-Myc)protein levels.Results Compared with the Control group,the survival rate of cardiomyocytes in the H_(2)O_(2)group was decreased,the apoptosis rate was increased,the expression levels of C-Caspase-3,C-Caspase-9,β-catenin,and c-Myc protein in the cells increased,and the MDA level increased,and SOD,CAT,GSH-Px levels decreased.Compared with the si-NC group,the survival rate of cardiomyocytes in the si-KCNQ1 OT1 group was increased,the apoptosis rate was decreased,and the expression levels of C-Caspase-3,C-Caspase-9,β-catenin,c-Myc in the cells were decreased,the level of MDA decreased,and the level of SOD,CAT,and GSH-Px increased.Compared with the si-KCNQ1 OT1 group,the survival rate of cardiomyocytes in the si-KCNQ1 OT1+LiCl group was decreased,the apoptosis rate was increased,and the expression levels of C-Caspase-3,C-Caspase-9,β-catenin,and c-Myc increased,MDA level increased,and SOD,CAT,GSH-Px levels decreased.Conclusion Down-regulation of KCNQ1 OT1 inhibits H_(2)O_(2)-induced cardiomyocyte apoptosis and oxidative damage,and the mechanism is related to inhibition of Wnt signaling pathway.