miR-133b靶向YES1抑制心肌缺血再灌注引起的心肌细胞凋亡和活性氧簇的积累  

miR-133b inhibits myocardial ischemia-reperfusion-induced cardiomyocyte apoptosis and accumulation of reactive oxygen species in rats by targeting YES1

作　　者：彭兴 林玲 周祥群 杨大英 曹阳 尹涛源 刘媛媛[3] PENG Xing;LIN Ling;ZHOU Xiangqun;YANG Daying;CAO Yang;YIN Taoyuan;LIU Yuanyuan(Department of Cardiovascular Medicine,Sanya Central Hospital,Sanya 572000,China;Department of Cardiovascular Medicine,First Affiliated Hospital of Harbin Medical University,Harbin 150000,China;Department of Cardiology,Heilongjiang Provincial Hospital,Harbin 150000,China)

机构地区：[1]三亚中心医院心血管内科,海南三亚572000 [2]哈尔滨医科大学第一附属医院心血管内科,黑龙江哈尔滨150000 [3]黑龙江省医院心内科,黑龙江哈尔滨150000

出　　处：《南方医科大学学报》2020年第10期1390-1398,共9页Journal of Southern Medical University

基　　金：黑龙江省卫生计生委科研课题(2016-540)。

摘　　要：目的探究miR-133b对心肌缺血再灌注(I/R)诱导心肌细胞凋亡的作用及机制。方法体内实验,将大鼠随机分为4组:假手术组、模型组、转染腺病毒对照组、转染miR-133b腺病毒组,每组9只。在左心室心肌的6个随机点注射转染复合物后,利用左侧冠状动脉前降支打活结方法诱导心肌I/R。通过RT-qPCR检测miR-133b的表达水平,FDP-1 HRV和BRS分析系统测定心功能,酶联免疫吸附法测定血清肌酸激酶同工酶(CK-MB)和心肌肌钙蛋白I(CTnI)水平,HE染色观察心肌损伤,流式细胞术检测细胞凋亡,2,7-二氯荧光素双乙酸酯(DCFH-DA)探针测定ROS含量。体外实验,心肌H9C2细胞经缺氧/复氧(H/R)处理后,转染miR-NC或miR-133b mimic,RT-qPCR检测miR-133b的表达水平,流式细胞术检测细胞凋亡,DCFH-DA探针测定ROS含量;双荧光素酶报告实验验证靶向关系;转染pc-YES1或miR-133b mimic后,Western blot检测YES1的表达水平,流式细胞术检测细胞凋亡,DCFH-DA探针测定ROS含量。结果与I/R组相比,AdmiR-133b组大鼠心肌中miR-133b的表达显著上调,LVEDP、cTnI和CK-MB水平显著降低,LVSP、+dp/dt、-dp/dt、HR和CF水平显著升高,病理损伤显著减轻,心肌细胞凋亡和ROS积累显著减少(P<0.01)。与H/R组相比,miR-133b mimic组H9C2细胞的miR-133b表达显著上调,细胞凋亡和ROS积累显著减少(P<0.01)。miR-133b与YES1在3’UTR区存在靶向结合位点,共转染YES1 WT和miR-133b mimic可以显著降低荧光素酶活性(P<0.01)。miR-133b mimic组与H/R组相比H9C2细胞中YES1蛋白的表达显著下调(P<0.01)。共转染pc-YES1逆转miR-133b过表达对心肌细胞凋亡和ROS积累的作用。结论无论是在体内还是体外,miR-133b靶向YES1抑制I/R引起的心肌细胞凋亡和ROS的积累,从而减轻心肌I/R损伤。Objective To investigate the effect of miR-133b on cardiomyocyte apoptosis induced by myocardial ischemia-reperfusion(I/R)and explore the mechanism.Methods Thirty-six adult SD rats were randomized into sham-operated group,I/R group,AdmiR-NC group and AdmiR-133b group,and rat models of myocardial I/R were established in the latter 3 groups with myocardial injections of saline or recombinant adenoviruses in the left ventricle.The expression of MiR-133b was detected using RT-qPCR,and cardiac function of the rats was determined using FDP 1 HRV and BRS analysis system.Serum CK-MB and cTnI levels were determined by ELISA,myocardial injury was evaluated with HE staining,cardiomocyte apoptosis was detected by flow cytometry,and ROS content was determined using a DCFH-DA probe.In the in vitro experiment,H9C2 myocardial cells with hypoxia/reoxygenation(H/R)treatment were transfected with Mir-NC or MiR-133b mimic,and the cellular expression of MiR-133b,cell apoptosis,and ROS content were determined.Dual luciferase reporter assay was performed to verify the targeting relationship between miR-133b and YES1.The effects of pc-YES1 or miR-133b mimic transfection on YES1 expression,apoptosis,and ROS content in H9C2 cells were evaluated.Results Compared with those in I/R group,miR-133b expression was obviously up-regulated,LVEDP,cTnI and CK-MB levels were significantly decreased,and LVSP,+dp/dt,-dp/dt,HR and CF levels were increased in admiR-133b group(P<0.01).The rats in admiR-133b group showed obviously reduced pathological damage,cell apoptosis and ROS content compared with those in I/R group(P<0.01).In H9C2 cells exposed to H/R,transfection with miR-133b mimic significantly up-regulated miR-133b expression and decreased cell apoptosis and ROS content(P<0.01).The results of dual luciferase reporter assay suggested a direct targeting relationship between miR-133b and YES1,and MiR-133b mimic transfection significantly down-regulated YES1 protein expression in cells with H/R exposure(P<0.01).Co-transfection with pc-YES1 reversed t

关 键 词：心肌缺血再灌注 miR-133b YES1 细胞凋亡 活性氧 

分 类 号：R542.2[医药卫生—心血管疾病]