微小RNA-139-3p靶向调控性别决定区相关高迁移率族盒蛋白4减轻心肌细胞氧化应激损伤  

MicroRNA-139-3p reduces oxidative stress damage in cardiomyocyte via targeting sex-determining region Y-related related high-mobility-group box 4

作　　者：冯超[1] 张伟[1] 蒙国鑫 周涛[1] Zhou Tao;Feng Chao;Zhang Wei;Meng Guoxin(Department of Cardiology,Guizhou Provincial People’s Hospital,Guiyang 550002,China)

机构地区：[1]贵州省人民医院心外科,贵阳550002

出　　处：《中华实验外科杂志》2023年第10期2047-2050,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81960050、82160056);贵州省科技计划项目(科学技术基金)(黔科合基础[2020]1Y293号);贵州省卫生健康委员会科学技术基金项目(gzwkj 2021-193);贵州省人民医院国家自然科学基金后补助基金(GPPH-NSFC-2019-24,GPPH-NSFC-2021-16)。

摘　　要：目的研究微小RNA(miR)-139-3p对H_(2)O_(2)诱导的大鼠心肌细胞系H9c2细胞氧化应激的影响及其可能机制。方法分别将miR-139-3p模拟物(mimics)及其阴性对照(miR-NC)、性别决定区相关高迁移率族盒蛋白4(Sox4)过表达载体(pc-Sox4)及其对照(pcDNA3.1)转染H9c2细胞,细胞共分为6组:对照组、H_(2)O_(2)组、mimic组(转染mimics)、miR-NC组(转染miR-NC)、pcDNA3.1组(转染mimics和pcDNA3.1)和pc-Sox4组(转染mimics和pc-Sox4),除对照组外的其他细胞均在转染后建立H_(2)O_(2)诱导的H9c2细胞氧化应激损伤模型。细胞计数试剂盒(CCK-8)法检测H9c2细胞活力;比色法检测细胞培养上清中乳酸脱氢酶(LDH)的含量;原位缺口末端标记法(TUNEL)染色检测H9c2细胞凋亡;蛋白质印迹法(Western blot)检测Sox4表达;荧光定量聚合酶链反应(PCR)检测miR-139-3p表达水平;生物信息学网站预测miR-139-3p与Sox4的互补结合位点,双荧光素酶报告基因实验验证两者之间的靶向关系。各组间差异比较采用方差分析。结果H_(2)O_(2)组H9c2细胞miR-139-3p水平低于对照组(0.28±0.03比1.00±0.01,t=11.484,P<0.05),miR-139-3p过表达后mimics组细胞活力高于miR-NC组(0.71±0.07比0.44±0.04,t=7.348,P<0.05)、LDH水平低于miR-NC组[(48.21±6.38)U/L比(69.76±7.32)U/L,t=4.759,P<0.05]、细胞凋亡率低于miR-NC组[(21.31±4.02)%比(47.28±5.69)%,t=8.851,P<0.05]。使用TargetScan生物预测网站预测和双荧光素酶报告基因实验证实Sox4是miR-139-3p的靶基因,同时miR-139-3p上调使mimic组Sox4蛋白低于miR-NC组(2.54±0.41比5.38±0.73,t=4.212,P<0.05)。Sox4过表达逆转了miR-139-3p对H_(2)O_(2)诱导的细胞损伤的作用,pc-Sox4组Sox4蛋白表达高于pc-DNA3.1组(5.25±0.71比2.61±0.47,t=4.212,P<0.05)、细胞活力低于pc-DNA3.1组(0.45±0.07比0.69±0.08,t=7.788,P<0.05)、LDH水平高于pc-DNA3.1组[(68.62±7.55)U/L比(44.76±5.21)U/L,t=9.368,P<0.05],细胞凋亡率高于pc-DNA3.1组[(41.24±7.36)%比(24.76±4.28)%,t=9.578,P<0.05]。结�Objective To investigate the effect of microRNA(miR)-139-3p on H_(2)O_(2) induced oxidative stress in H9c2 cells.Methods H9c2 cells were transfected with miR-139-3p mimics and negative control(miR-NC),sex-determining region Y-related related high-mobility-group box 4(Sox4)overexpression vector(pc-Sox4)and its control(pcDNA3.1).The cells were divided into 6 groups:control group,H_(2)O_(2) group,mimic group(transfected mimics),miR-NC group(transfected miR-NC),pcDNA3.1 group(transfected mimics and pcDNA3.1),and pc-Sox4 group(transfected mimics and pc-Sox4).Except for control group,all other cells established H_(2)O_(2) induced oxidative stress injury models.Cell counting kit-8(CCK-8)was used to detect the activity of cells.Colorimetric assay was used to detect the content of lactate dehydrogenase(LDH)in the supernatant of cell culture.Terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)staining was used to detect apoptosis.Western blotting was used to detect the expression of Sox4.Fluorescence quantitative polymerase chain reaction(PCR)was used to detect the expression of miR-139-3p.The bioinformatics website predicts the complementary binding sites between miR-139-3p and Sox4,and verifies the targeting relationship using dual luciferase reporter gene experiments.Differences between groups were compared using analysis of variance.Results The expression of miR-139-3p after H_(2)O_(2) stimulation was lower than control group(0.28±0.03 vs.1.00±0.02,t=11.484,P<0.05).After overexpression of miR-139-3p,the mimics group showed an increase in cell viability compared to miR-NC group(0.69±0.08 vs.0.37±0.04,t=7.348,P<0.05),lower LDH levels[(39.45±4.27)U/L vs.(71.37±8.59)U/L,t=4.759,P<0.05],reduced cell apoptosis rate[(21.31±4.02)%vs.(47.28±5.69)%,t=8.851,P<0.05].TargetScan Bioprediction website prediction and dual luciferase reporter gene experiments confirmed that Sox4 is a downstream target of miR-139-3p,and upregulation of miR-139-3p can inhibit Sox4 expression(2.54±0.41 vs.5.38±0.73,t=4.212,P<0

关 键 词：心肌细胞 氧化应激 微小RNA 性别决定区相关高迁移率族盒蛋白4 

分 类 号：R542.2[医药卫生—心血管疾病]