微小核糖核酸-6216对过氧化氢诱导的心肌细胞氧化应激损伤的保护及机制研究  

Protective effect and mechanism of miR-6216 on hydrogen peroxide induced cardiomyocyte oxidative stress Injury

作　　者：吴留广[1] 金辉[1] 朱草原 尚勇[1] WU Liuguang;JIN Hui;ZHU Caoyuan;SHANG Yong(Department of Cardiacvascular Surgery,Zhoukou City Central Hospital,Zhoukou 466000,China)

机构地区：[1]河南周口市中心医院心脏血管外科

出　　处：《心肺血管病杂志》2019年第12期1280-1286,共7页Journal of Cardiovascular and Pulmonary Diseases

摘　　要：目的:探讨微小核糖核酸-6216(miR-6216)对过氧化氢(H2O2)诱导的心肌细胞氧化应激损伤的保护作用及机制。方法:正常体外培养AC16心肌细胞,建立H2O2氧化应激损伤模型,分为对照组、H2O2组、H2O2+miR-6216组、H2O2+miR-con组、H2O2+miR-6216+pc DNA-control组、H2O2+miR-6216+pc DNA-E2F1组。qRT-PCR检测心肌细胞miR-6216表达量;MTT法测定各组心肌细胞存活率;流式细胞数检测心肌细胞凋亡率;应用试剂盒检测CK-MB、MDA、SOD;双荧光素酶报告基因实验miR-6216与E2F1的靶向作用;Western blot法检测Bax、Bcl2、E2F1蛋白表达。结果:H2O2不同剂量组心肌细胞存活率显著降低(P<0.05),miR-6216表达量显著降低(P<0.05),与H2O2+miR-con组相比,H2O2+miR-6216组心肌细胞存活率显著升高(P<0.05);与control组相比,H2O2组心肌细胞CK-MB、MDA含量及Bax蛋白表达水平显著升高(P<0.05),心肌细胞凋亡率显著升高(P<0.05),而SOD水平、Bcl2蛋白表达水平显著降低(P<0.05),上调miR-6216表达能够显著逆转H2O2对心肌细胞凋亡率及氧化损伤的作用;miR-6216可负向调控靶基因E2F1表达;E2F1过表达可逆转上调miR-6216表达对H2O2诱导的心肌细胞氧化应激损伤的保护作用。结论:上调miR-6216表达可通过下调E2F1表达而减轻H2O2诱导的心肌细胞氧化应激损伤。Objective:To investigate the protective effect and mechanism of microRNA-6216(miR-6216)on oxidative stress injury induced by hydrogen peroxide(H2O2)in cardiomyocytes.Methods:AC16 cardiomyocytes were cultured in vitro to establish a H2O2 oxidative stress injury model,which was divided into control group,H2O2 group,H2O2+miR-6216 group,H2O2+miR-con group,H2O2+miR-6216+pc DNA-control group,H2O2+group.miR-6216+pcDNA-E2F1 group.qRT-PCR was used to detect the expression of miR-6216 in cardiomyocytes.The survival rate of cardiomyocytes in each group was determined by MTT method.Flow cytometry was used to detect the apoptosis rate of cardiomyocytes.The kit was used to detect CK-MB,MDA,and SOD.Dual luciferase reporter gene assay miR-6216 and E2 F1 targeting.Western blot was used to detect the expression of Bax,Bcl2 and E2 F1 proteins.Results:The survival rate of cardiomyocytes in H2O2 group was significantly decreased(P<0.05),and the expression of miR-6216 was significantly decreased(P<0.05).Compared with H2O2+miR-con group,the survival rate of H2O2+miR-6216 group was significant.Elevated(P<0.05).Compared with the control group,the levels of CK-MB,MDA and Bax protein in H2O2 group were significantly increased(P<0.05),and the apoptosis rate of cardiomyocytes was significantly increased(P<0.05),but the levels of SOD and Bcl2 were significantly decreased(P<0.05).Up-regulation of miR-6216 expression significantly reversed the effect of H2O2 on cardiomyocyte apoptosis rate and oxidative damage.miR-6216 could negatively regulate the expression of target gene E2F1;E2F1 overexpression could reverse the protective effect of miR-6216 expression on H2O2-induced cardiomyocyte oxidative stress injury.Conclusions:Upregulation of miR-6216 expression attenuates H2O2-induced cardiomyocyte oxidative stress damage by down-regulating E2F1 expression.

关 键 词：微小核糖核酸-6216 氧化应激 E2F1 心肌细胞 

分 类 号：R54[医药卫生—心血管疾病]