ENST00000418539.1调控miR-24对H_(2)O_(2)诱导心肌细胞氧化应激损伤的影响

Effects of ENST00000418539.1 on H_(2)O_(2)-induced Oxidative Stress in Cardiomyocyte by Regulating the Expression of miR-24

目的探讨长链非编码RNA ENST00000418539.1(LncRNA ENST00000418539.1)是否通过调控miR-24的表达影响过氧化氢(H_(2)O_(2))诱导心肌细胞氧化应激损伤。方法体外培养大鼠心肌细胞H9c2,200μmol/L H_(2)O_(2)处理心肌细胞48 h建立模型,分别将乱序无意义阴性序列(si-NC)、ENST00000418539.1小分子干扰RNA(si-ENST00000418539.1)、si-ENST00000418539.1与阴性对照(anti-miR-NC)、si-ENST00000418539.1与miR-24特异性寡核苷酸抑制剂(anti-miR-24)转染至心肌细胞,使用H_(2)O_(2)处理心肌细胞。实时荧光定量聚合酶链反应检测ENST00000418539.1、miR-24的表达量;流式细胞术检测细胞凋亡率;检测丙二醛(MDA)、乳酸脱氢酶(LDH)水平;双荧光素酶报告实验验证ENST00000418539.1、miR-24的靶向关系;蛋白免疫印迹法检测半胱氨酰天冬氨酸特异性蛋白酶3(Caspase-3)、半胱氨酰天冬氨酸特异性蛋白酶9(Caspase-9)的表达。结果与对照组比较,模型组细胞凋亡率明显升高(P<0.05),Caspase-3、Caspase-9蛋白水平明显升高(P<0.05),MDA、LDH水平明显升高(P<0.05);与模型组、si-NC组比较,si-ENST00000418539.1组细胞凋亡率明显降低(P<0.05),Caspase-3、Caspase-9蛋白水平明显降低(P<0.05),MDA、LDH水平明显降低(P<0.05);双荧光素酶报告实验证实ENST00000418539.1可靶向结合miR-24。与si-ENST00000418539.1+anti-miR-NC组比较,si-ENST00000418539.1+anti-miR-24组细胞凋亡率明显升高(P<0.05),Caspase-3、Caspase-9蛋白水平明显升高(P<0.05),MDA、LDH水平明显升高(P<0.05)。结论干扰ENST00000418539.1表达可负向调控miR-24的表达,从而抑制H_(2)O_(2)诱导心肌细胞凋亡及减轻氧化应激损伤。

Objective To investigate the LncRNA ENST00000418539.1 affects H_(2)O_(2) induced oxidative stress injury by regulating the expression of miR-24.Methods Rat cardiomyocytes H9c2 were cultured in vitro,and cardiomyocytes were treated with 200μmol/L H_(2)O_(2) for 48 h to establish the model.si-NC,si-ENST00000418539.1,si-ENST00000418539.1 and anti-miR-NC,si-ENST00000418539.1 were established respectively.Transfected with anti-miR-24 into cardiomyocytes and treated them with H_(2)O_(2).qRT-PCR was used to detect the expression of ENST00000418539.1 and miR-24.Flow cytometry was used to detect the apoptosis rate.The levels of malondialdehyde(MDA)and lactic dehydrogenase(LDH)were detected.The double luciferase report experiment verified the targeting relationship of ENST00000418539.1 and miR-24.Western blot was used to detect the expression of Caspase-3 and Caspase-9.Results Compared with control group,the apoptosis rate in model group significantly increased(P<0.05),the levels of Caspase-3 and Caspase-9 proteins significantly increased(P<0.05),and the levels of MDA and LDH significantly increased(P<0.05).Compared with model group and si-NC group,the apoptosis rate of si-ENST00000418539.1 group significantly reduced(P<0.05),the levels of Caspase-3 and Caspase-9 proteins significantly reduced(P<0.05),and the levels of MDA and LDH significantly reduced(P<0.05).Double luciferase reporting experiments confirm that ENST00000418539.1 could target miR-24.Compared with si-ENST00000418539.1+anti-miR-NC group,the apoptosis rate of si-ENST00000418539.1+anti-miR-24 group significantly increased(P<0.05),and the levels of Caspase-3 and Caspase-9 proteins were significantly higher(P<0.05),and the levels of MDA and LDH increased significantly(P<0.05).Conclusion Interfering with the expression of ENST00000418539.1 cannegatively regulate the expression of miR-24,thereby inhibiting H_(2)O_(2) inducing myocardial cell apoptosis and reducing oxidative stress injury.