下调miR-31抑制心肌细胞凋亡并防止心肌缺血再灌注损伤的机制研究

Effect of miR-31 on Cardiomyocytes Apoptosis and Myocardial Ischemia-Reperfusion Injury

目的:探究microRNA-31(miR-31)在心肌细胞缺氧/复氧(H/R)损伤及大鼠心肌缺血再灌注损伤(MIRI)中的作用。方法:通过inhibitor-miR-31及空病毒载体构建miR-31低表达心肌细胞,采用H/R建立心肌细胞损伤模型,缺氧4 h再复氧2 h,采用定量逆转录聚合酶链式反应(qRT-PCR)检测细胞miR-31 mRNA相对水平,流式细胞术检测细胞凋亡率,蛋白免疫印迹法(Western Blot)检测细胞半胱氨酸蛋白酶3(Caspase-3)、活化半胱氨酸蛋白酶3(Cleaved Caspase-3)、活化半胱氨酸蛋白酶9(Cleaved Caspase-9)、半胱氨酸蛋白酶9(Caspase-9)、Bcl相关X蛋白(Bax)、B细胞淋巴瘤/白血病-2(Bcl-2)蛋白表达。将Wistar大鼠分为对照组、MIRI组、MIRI+inhibitor-NC组和MIRI+inhibitor-miR-31组,每组24只,MIRI+inhibitor-NC组和MIRI+inhibitor-miR-31组大鼠心肌内注射携带inhibitor-NC、inhibitor-miR-31的慢病毒载体,7 d后,采用左冠状动脉结扎法构建MIRI模型。慢病毒载体注射后14 d,超声仪检查大鼠心率(HR)、左心室壁厚度(LVWT)、左心室收缩末期容积(LVESV)和左室射血分数(LVEF),qRT-PCR检测心肌组织miR-31 mRNA相对水平,氯化三苯基四氮唑(TTC)法检测心脏梗死面积百分比,苏木精-伊红(HE)染色观察大鼠心肌组织病理损伤,缺口末端标记法(TUNEL)染色观察心肌组织凋亡率,酶联免疫吸附法检测血清心肌肌钙蛋白-I(cTnI)、肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)水平,Western Blot检测心肌组织凋亡相关蛋白表达。结果:与对照组比较,H/R组心肌细胞miR-31 mRNA相对水平、细胞凋亡率和Cleaved Caspase-3、Cleaved Caspase-9、Bax蛋白表达升高(P<0.05),Bcl-2蛋白表达降低(P<0.05);与H/R组比较,H/R+inhibitor-miR-31组心肌细胞miR-31mRNA相对水平、细胞凋亡率和Cleaved Caspase-3、Cleaved Caspase-9、Bax蛋白表达降低(P<0.05),Bcl-2蛋白表达升高(P<0.05)。与对照组比较,MIRI组大鼠心肌组织miR-31 mRNA相对水平、梗死面积百分比、细胞凋亡率、LVESV和血清cTnI、CK、CK-MB水平升高(P<0.05),Cleaved Caspase-3、Cleaved Caspase-9、Bax蛋白表达升高(P<0.05),HR、LVWT、LVEF、Bcl-2蛋白表达降低(P<0.05);与MIRI组比较,MIRI+inhibitor-miR-31组大鼠心肌组织miR-31 mRNA相对水平、梗死面积百分比、细胞凋亡率、LVESV和血清cTnI、CK、CK-MB降低(P<0.05),Cleaved Caspase-3、Cleaved Caspase-9、Bax蛋白表达降低(P<0.05),HR、LVWT、LVEF和Bcl-2蛋白表达升高(P<0.05)。结论:抑制miR-31表达能通过增强心肌细胞抗凋亡作用,改善心肌缺氧再灌注损伤。

Objective:To explore the effects of microRNA-31(miR-31)on cardiomyocytes hypoxia-reoxygenation(H/R)injury and myocardial ischemia-reperfusion injury(MIRI)in rats.Methods:The cardiomyocytes with low-expression miR-31 were constructed by inhibitor-miR-31 and empty virus vector.The models of cardiomyocyte injury were constructed by H/R.After 4 h of hypoxia and 2 h of reoxygenation,relative level of miR-31 mRNA was detected by quantitative reverse transcriptase polymerase chain reaction(qRT-PCR).The cells apoptosis was detected by flow cytometry.The expressions of cysteine protease-3(Caspase-3),cleaved cysteine protease-3(Cleaved Caspase-3),cleaved cysteine protease-9(Cleaved Caspase-9),cysteine protease-9(Caspase-9),Bcl-associated X protein(Bax),and B-cell lymphoma/leukemia-2(Bcl-2)proteins were detected by Western Blot.Wistar rats were divided into control group,MIRI group,MIRI+inhibitor-NC group,and MIRI+inhibitor-miR-31 group,with 24 cases in each group.The rats in MIRI+inhibitor-NC group and MIRI+inhibitor-miR-31 group were given myocardial injection of lentiviral vectors carrying inhibitor-NC and inhibitor-miR-31.After 7 days,the MIRI models were constructed by left coronary ligation.At 14 d after injection of lentiviral vector,heart rate(HR),left ventricular wall thickness(LVWT),left ventricular end-systolic volume(LVESV),and left ventricular ejection fraction(LVEF)were detected by ultrasonic apparatus.The relative level of miR-31 mRNA in myocardial tissues was detected by qRT-PCR.The percentage of cardiac infarction area was detected by triphenyltetrazolium chloride(TTC).The pathological damage of myocardial tissues was observed by hemotoxylin-eosin(HE)staining.The apoptosis rate of myocardial tissues was observed by terminal deoxyribonucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL)staining.The levels of cardiac troponin-I(cTnI),creatine kinase(CK)and creatine kinase MB(CK-MB)were detected by enzyme-linked immunosorbent assay.The expressions of apoptosis-related proteins in myocardial tissues were detected by Western blot.Results:Compared with the control group,relative level of miR-31 mRNA in cardiomyocytes,apoptosis rate,expression levels of Cleaved Caspase-3,Cleaved Caspase-9 and Bax proteins increased in H/R group(P<0.05),while expression of Bcl-2 protein decreased(P<0.05).Compared with H/R group,relative level of miR-31 mRNA in cardiomyocytes,apoptosis rate,expression levels of Cleaved Caspase-3,Cleaved Caspase-9 and Bax proteins decreased in H/R+inhibitor-miR-31 group(P<0.05),while expression of Bcl-2 protein increased(P<0.05).Compared with control group,relative level of miR-31 mRNA in myocardial tissues,percentage of infarction area,apoptosis rate,levels of LVESV,serum cTnI,CK and CK-MB increased in MIRI group(P<0.05),expressions of Cleaved Caspase-3,Cleaved Caspase-9 and Bax proteins increased(P<0.05),HR,LVWT,LVEF,and Bcl-2 decreased(P<0.05).Compared with MIRI group,relative level of miR-31 mRNA in myocardial tissues,percentage of infarction area,apoptosis rate,levels of LVESV,serum cTnI,CK and CK-MB decreased in MIRI+inhibitor-miR-31 group(P<0.05),expressions of Cleaved Caspase-3,Cleaved Caspase-9 and Bax proteins decreased(P<0.05),HR,LVWT,LVEF,and Bcl-2 increased(P<0.05).Conclusion:Inhibition of miR-31 expression can relive MIRI by enhancing anti-apoptotic effect of cardiomyocytes.