miR-133a介导Bcl-xl表达调节内皮细胞的凋亡  

miR-133a mediates Bcl-xl expression to regulate endothelial cell apoptosis

作　　者：张永杰 林飞[1] 闫志刚 刘慧兵[1] 孙四玉 王立波 赵国安[1] ZHANG Yong-jie;LIN Fei;YAN Zhi-gang;LIU Hui-bing;SUN Si-yu;WANG Li-bo;ZHAO Guo-an(Heart Center of the First Affiliated Hospital of Xinxiang Medical College,Xinxiang 453000,Henan,China)

机构地区：[1]新乡医学院第一附属医院心脏中心,新乡453000

出　　处：《医学研究生学报》2020年第8期814-819,共6页Journal of Medical Postgraduates

基　　金：河南省高等学校重点科研项目(18A320005,19A360032)。

摘　　要：目的microRNA-133a(miR-133a)参与动脉粥样硬化(AS)病理过程的具体机制不详。文中旨在探究miR-133a参与内皮细胞凋亡的调控过程。方法培养人冠状内皮细胞(HCAECs),分别给予不同浓度(5、15、25、50、75μg/mL)的氧化低密度脂蛋白(ox-LDL)处理12、24 h,MTT法检测细胞活力,筛选文献证实靶向调节抗凋亡蛋白B淋巴细胞瘤因子2-特大型(Bcl-xl)的miRNA。转染细胞48 h后分为:无义序列组(无义序列NC)、过表达组(过表达miR-133a mimics)、沉默无义序列+诱导组(沉默无义序列in NC+ox-LDL诱导HCAECs)、miRNA沉默+诱导组(miR-133a敲减inhibitor+ox-LDL诱导HCAECs)。Western blot法检测抗凋亡蛋白Bcl-xl及活化的半胱氨酸天冬氨酸蛋白酶3(cleaved-caspase3)表达,流式细胞仪检测细胞凋亡率。采用瞬时转染技术过表达及沉默miR-133a,观察其对靶基因Bcl-xl蛋白表达及内皮细胞凋亡的影响。结果MTT法检测显示,ox-LDL浓度25、50μg/mL的12、24 h细胞活力较0μg/mL降低(P<0.05);ox-LDL 75μg/mL的24 h细胞活力较0μg/mL明显降低(P<0.001)。过表达组细胞中miR-133a表达量(41.80±8.94)较无义序列组(1±0.31)明显增高(P<0.01),miRNA沉默+诱导组细胞中miR-133a表达量(0.20±0.11)较沉默无义序列+诱导组(1±0.11)显著降低(P<0.001)。流式细胞仪检测显示,ox-LDL 75μg/mL的细胞凋亡率[(28.97±3.20)%]较0μg/mL[(8.03±0.73)%]明显升高(P<0.01),miRNA沉默+诱导组细胞凋亡率(18.92±3.00)%]较沉默无义序列+诱导组[(27.1±2.01)%]明显降低(P<0.05),过表达组细胞凋亡率[(14.81±2.60)%]较无义序列组[(8.02±1.22)%]显著增加(P<0.05)。Western blot结果显示,随着ox-LDL浓度(25、50、75μg/mL)增加,抗凋亡蛋白Bcl-xl的表达量逐渐降低,cleaved-caspase3的表达量增加。无义序列组Bcl-xl表达量(1.265±0.049)较过表达组(0.940±0.071)明显升高(P<0.05),沉默无义序列+诱导组(0.795±0.064)较miRNA沉默+诱导组(1.150±0.071)明显降低(P<0.05)。结论miR-133a靶向�Objective The specific mechanism of microRNA-133a(miR-133a)involved in the pathological process of atherosclerosis(As)remains an open question.This study aims to explore the role of miR-133a in the regulation of endothelial cell apoptosis.Methods Cultured human coronary endothelial cells(HCAECs)were treated with oxidized low density lipoprotein(ox-LDL).The cell viability was detected by MTT assay.The mRNA levels of Bcl-xl and miRNA(miR-133a,etc)were detected by qRT-PCR method.The expression of anti-apoptotic protein Bcl-xl and cleaved-caspase3 was detected by Western blotting,and the apoptosis rate was detected by flow cytometry.The transient transfection technique was used to observe the effect of overexpression and silencing of miR-133a,on the expression of target gene Bcl-xl protein and endothelial cell apoptosis.Results Ox-LDL was observed to decrease the viability of HCAECs cells and induce HCAECs apoptosis;miR-133a increased abnormally in the apoptosis model;after silencing miR-133a,the decrease of Bcl-xl and the increase of apoptosis rate induced by ox-LDL were partially reversed;the overexpression of miR-133a,Bcl-xl decreased and the apoptosis rate increased,and the difference was statistically significant.Conclusion miR-133a might target and regulate the anti-apoptotic protein Bcl-xl,to induce endothelial cell apoptosis and promote the formation of AS.

关 键 词：miR-133a B淋巴细胞瘤因子2-特大型 内皮细胞凋亡 动脉粥样硬化 

分 类 号：R541[医药卫生—心血管疾病] R543[医药卫生—内科学]