微小RNA-509-3p促进氧化型低密度脂蛋白诱导的小鼠主动脉内皮细胞凋亡  

miR-509-3p promotes oxidized low-density lipoprotein-induced apoptosis in mouse aortic endothelial cells

作　　者：张蕊[1] 宋衍秋[2,3] 赵福梅 刘婷[2] 丛洪良[1,2,3] 赵辉[4] Zhang Rui;Song Yanqiu;Zhao Fumei;Liu Ting;Cong Hongliang;Zhao Hui(Department of Cardiology,Tianjin Chest Hospital,Tianjin 300222,China;Tianjin Cardiovascular Diseases Institute,Tianjin Chest Hospital,Tianjin 300222,China;Tianjin Key Laboratory of Cardiovascular Emergency and Critical Care,Tianjin Municipal Science and Technology Bureau,Tianjin 300222,China;Department of Cardiology,Tianjin Hospital,Tianjin 300211,China)

机构地区：[1]天津市胸科医院心内科,天津300222 [2]天津市胸科医院心血管病研究所,天津300222 [3]天津市心血管急危重症重点实验室,天津300222 [4]天津市天津医院心内科,天津300211

出　　处：《中华危重病急救医学》2023年第12期1291-1297,共7页Chinese Critical Care Medicine

基　　金：天津市卫生健康委员会科技项目(ZC20137);天津市胸科医院科研基金项目(2018XKZ09);天津市医学重点学科(专科)建设项目(TJYXZDXK-055B)。

摘　　要：目的探讨微小RNA-509-3p(miR-509-3p)对动脉粥样硬化血管内皮细胞凋亡的影响。方法将小鼠主动脉内皮细胞(MAEC)分为正常对照组、氧化型低密度脂蛋白(ox-LDL)组、miR-509-3p过表达组、miR-509-3p过表达对照组、miR-509-3p抑制剂+ox-LDL组、miR-509-3p抑制剂对照+ox-LDL组。在MAEC中加入100 mg/L ox-LDL诱导24 h后,加入miR-509-3p过表达/抑制剂及相应对照转染48 h。采用实时荧光定量聚合酶链反应(RT-qPCR)检测ox-LDL诱导的MAEC miR-509-3p表达,流式细胞仪检测细胞凋亡水平,细胞计数试剂盒(CCK-8)检测细胞增殖活性。应用生物信息学分析预测miR-509-3p的直接基因靶点,并使用双荧光素酶报告基因检测确认。分别用RT-qPCR和蛋白质免疫印迹试验(Western blotting)检测Bcl-2的mRNA和蛋白表达。结果ox-LDL刺激后,MAEC miR-509-3p表达较正常对照组显著上调(1.68±0.85比1.00±0.30,t=2.398,P<0.05)。miR-509-3p过表达转染MAEC后,BCL23'非翻译区(3'UTR)野生型(WT)报告基因的荧光素酶活性较miR-509-3p过表达对照组显著降低(0.83±0.06比1.00±0.07,t=4.531,P=0.001);而BCL23'UTR突变型(MUT)报告基因的荧光素酶活性与miR-509-3p过表达对照组差异无统计学意义(0.94±0.05比1.00±0.08,t=1.414,P=0.188)。与正常对照组和miR-509-3p过表达对照组比较,miR-509-3p过表达组转染MAEC后细胞增殖活性降低〔(0.60±0.06)%比(1.00±0.09)%、(0.89±0.04)%,均P<0.01〕,细胞凋亡率增加〔(23.46±2.02)%比(7.66±1.52)%、(10.40±0.78)%,均P<0.05〕,Bcl-2 mRNA及蛋白表达降低(Bcl-2 mRNA相对表达:0.52±0.13比1.00±0.36、1.10±0.19,Bcl-2蛋白相对表达:0.42±0.07比1.00±0.11、0.93±0.10,均P<0.01)。与ox-LDL组比较,抑制miR-509-3p表达可增加ox-LDL诱导的MAEC增殖活性〔(0.64±0.35)%比(0.34±0.20%)%,P<0.05〕,降低细胞凋亡率〔(13.59±2.22)%比(29.84±5.19)%,P<0.01〕,同时上调ox-LDL诱导的MAEC Bcl-2 mRNA和蛋白表达(Bcl-2 mRNA相对表达:0.82±0.09比0.52±0.10,Bcl-2蛋白相对�Objective To investigate the effect of microRNA-509-3p(miR-509-3p)on the apoptosis of atherosclerotic vascular endothelial cells.Methods Mouse aortic endothelial cells(MAECs)were divided into normal control group,oxidized low-density lipoprotein(ox-LDL)group,miR-509-3p overexpression group,miR-509-3p overexpression control group,miR-509-3p inhibitor+ox-LDL group,and miR-509-3p inhibitor control+ox-LDL group.MAEC were induced with 100 mg/L ox-LDL for 24 hours,and then transfected with miR-509-3p overexpression/inhibitor and corresponding control for 48 hours.The miR-509-3p expression in MAECs exposed to ox-LDL was detected using real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).Flow cytometry was used to detect the level of apoptosis,and cell counting kit(CCK-8)was used to detect the proliferation activity of MAECs.The direct gene targets of miR-509-3p were predicted using bioinformatics analyses and confirmed using a dual luciferase reporter assay.The expression of Bcl-2 mRNA and protein was detected by RT-qPCR and Western blotting,respectively Results Compared with the normal control group,miR-509-3p was significantly upregulated in ox-LDL-stimulated MAECs(1.68±0.85 vs.1.00±0.30,t=2.398,P<0.05).After transfection of MAECs with miR-509-3p overexpression,the luciferase activity of the BCL23'UTR WT reporter gene was significantly lower than that of miR-509-3p overexpression control group(0.83±0.06 vs.1.00±0.07,t=4.531,P=0.001).The luciferase activity of the BCL23'-UTR mutant(MUT)reporter gene was not significantly different from that of miR-509-3p overexpression control group(0.94±0.05 vs.1.00±0.08,t=1.414,P=0.188).Compared with the normal control group and miR-509-3p mimics control group,the cell proliferation activity was decreased[(0.60±0.06)%vs.(1.00±0.09)%,(0.89±0.04)%,both P<0.01],the percentage of apoptotic cells were increased[(23.46±2.02)%vs.(7.66±1.52)%,(10.40±0.78)%,both P<0.05],and the mRNA and protein expression of Bcl-2 were significantly downregulated(Bcl-2 mRNA:0.52�

关 键 词：动脉粥样硬化 内皮细胞 凋亡 脂蛋白 BCL-2 微小RNA-509-3p 

分 类 号：R543.5[医药卫生—心血管疾病]