【摘要】 目的探讨微小RNA-1224-5p(miR-1224-5p)靶向调控血红素加氧酶1(HMOX1)基因对大鼠心肌细胞H9C2缺血再灌注(I/R)损伤的影响。方法体外培养大鼠心肌细胞H9C2,模拟心肌I/R损伤。按照处理方法将大鼠心肌H9C2细胞分为对照组(Control组)、I/R组、I/R+anti-miR-con组、I/R+anti-miR-1224-5p组、I/R+anti-miR-1224-5p+si-con组、I/R+anti-miR-1224-5p+si-HMOX1组。qRT-PCR检测miR-1224-5p表达水平,四甲基噻唑蓝染色法(MTT)检测细胞存活率,流式细胞仪检测细胞凋亡率,酶联免疫吸附法检测细胞培养上清液中超氧化物歧化酶(SOD)、丙二醛(MDA)、乳酸脱氢酶(LDH)和天门冬氨酸氨基转移酶(AST)水平,Western Blot检测HMOX1蛋白表达水平。StarBase在线网站预测显示,HMOX1的3’-非翻译区(3’UTR)存在miR-1224-5p的结合位点,双荧光素酶报告基因实验验证miR-1224-5p与HMOX1靶向关系。结果与Control组比较,I/R组miR-1224-5p、凋亡率、MDA、LDH和AST水平升高(P<0.05),H9C2细胞存活率和SOD水平降低(P<0.05)。与I/R+anti-miR-con组比较,I/R+anti-miR-1224-5p组miR-1224-5p、凋亡率、MDA、LDH和AST水平降低(P<0.05),H9C2细胞存活率和SOD水平升高(P<0.05)。miR-1224-5p在心肌细胞H9C2中抑制HMOX1的表达。与I/R+anti-miR-1224-5p+si-con组比较,I/R+anti-miR-1224-5p+si-HMOX1组H9C2细胞凋亡率、MDA、LDH和AST水平升高(P<0.05),H9C2细胞存活率、HMOX1蛋白和SOD水平降低(P<0.05)。结论过表达miR-1224-5p通过靶向负调控HMOX1表达保护心肌细胞缺血再灌注损伤。更多还原

【Abstract】 Objective To investigate the effects of microRNA-1224-5 p(miR-1224-5 p) on ischemia-reperfusion(I/R) injury in rat myocardial cells-H9 C2 by regulating heme oxygenase 1(HMOX1) gene.Methods The rat myocardial cells-H9 C2 were cultured in vitro to simulate myocardial ischemia-reperfusion injury.H9 C2 cells were divided into control group, I/R group, I/R+anti-miR-con group, I/R+anti-miR-1224-5 p group, I/R+anti-miR-1224-5 p+si-con group, I/R+anti-miR-1224-5 p+si-HMOX1 group.The qRT-PCR was used to detect miR-1224-5 p expression level, and methylthiazoletrazolium(MTT) was used to detect the cell viability, and flow cytometry was used to detect the apoptosis rate, as well as enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of superoxide dismutase(SOD), malondialdehyde(MDA),lactate dehydrogenase(LDH) and aspartate aminotransferase(AST),and Western Blot was used to detect the expression levels of HMOX1 protein.The starBase online website predicted that the 3’-untranslated area(3’UTR) of HMOX1 had binding sites for miR-1224-5 p, and the dual luciferase reporter gene assay verified the targeting relationship between miR-1224-5 p and HMOX1.Results As compared with those in control group, the levels of miR-1224-5 p, apoptotic rate, and the levels of MDA,LDH and AST in I/R group were significantly increased(P<0.05),and the survival rate and SOD levels in H9 C2 cells were significantly decreased(P<0.05).As compared with those in I/R+anti-miR-con group, the miR-1224-5 p levels, apoptotic rate, and the levels of MDA,LDH and AST in I/R+anti-miR-1224-5 p group were significantly decreased(P<0.05),and the survival rate and SOD levels in H9 C2 cells were significantly increased(P<0.05).The miR-1224-5 p could inhibit the expression levels of HMOX1 in cardiomyocyte H9 C2.As compared with those in I/R+anti-miR-1224-5 p+si-con group, the apoptosis rate and the levels of MDA,LDH and AST in I/R+anti-miR-1224-5 p+si-HMOX1 group were significantly increased(P<0.05),however, the H9 C2 cell survival rate, and the levels of HMOX1 protein and SOD were significantly decreased(P<0.05).Conclusion The overexpression of miR-1224-5 p protects cardiomyocytes can protect ischemia-reperfusion injury by targetedly negatively regulating HMOX1 expression.更多还原