miR-26a-3p靶向调控Survivin表达对H9c2心肌细胞缺氧/复氧损伤的影响  

Effect of miR-26a-3p targeting Survivin on hypoxia/reoxygenation injury of H9c2 cardiomyocyte

作　　者：黄建成[1] 李红英[1] 刘佳[1] 李青泉 张会军[1] 李小兵[1] Huang Jiancheng;Li Hongying;Liu Jia;Li Qingquan;Zhang Huijun;Li Xiaobing(Dept of Cardiovascular Surgery,The First Hospital of Hebei Medical University,Shijiazhuang 050030;Cardiac Intensive Care Unit,The First Hospital of Hebei Medical University,Shijiazhuang 050030)

机构地区：[1]河北医科大学第一医院心脏外科,石家庄050030 [2]河北医科大学第一医院心脏重症监护室,石家庄050030

出　　处：《安徽医科大学学报》2023年第11期1934-1941,共8页Acta Universitatis Medicinalis Anhui

基　　金：河北省医学科学研究课题计划项目(编号:20210594)。

摘　　要：目的探讨miR-26a-3p对缺氧/复氧(H/R)诱导的大鼠心肌细胞(H9c2)损伤的影响及其机制。方法取对数生长期H9c2心肌细胞进行缺氧(1%O 2)6 h,复氧不同时间(2、4、8、12 h)建立细胞H/R模型,另设常氧组,细胞计数试剂盒(CCK-8)检测细胞增殖活性;比色法检测细胞上清液中乳酸脱氢酶(LDH)水平;实时荧光定量PCR(qRT-PCR)检测细胞中miR-26a-3p和生存素(Survivin)mRNA表达水平;Western blot检测细胞中Survivin蛋白表达水平。将miR-26a-3p inhibitor及其阴性对照inhibitor NC、Survivin基因siRNA干扰质粒(si-Survivin)及其阴性对照si-NC共转染至H9c2细胞中,随后进行H/R干预,CCK-8检测各组细胞增殖水平;比色法检测各组细胞中超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量及上清液中LDH水平;流式细胞术检测各组细胞凋亡水平;Western blot检测各组细胞中Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、活化型半胱天冬酶-3(cleaved caspase-3)和Survivin蛋白表达水平;双荧光素酶测定miR-26a-3p和Survivin基因的靶向关系。结果与常氧组细胞比较,随着复氧时间的延长,H9c2细胞增殖活性、Survivin mRNA和蛋白表达水平逐渐降低(P<0.05),而LDH及miR-26a-3p表达水平逐渐升高(P<0.05)。下调miR-26a-3p表达可提高H/R暴露下H9c2细胞增殖活性、SOD活性、Bcl-2蛋白表达水平(P<0.05),同时降低MDA含量、LDH释放量、凋亡率、Bax和cleaved caspase-3蛋白表达水平(P<0.05);而Survivin缺失可明显逆转miR-26a-3p inhibitor对H/R诱导下H9c2细胞的保护作用。双荧光素酶报告基因实验表明Survivin是miR-93-5p的靶基因。结论miR-26a-3p在H/R诱导的心肌细胞损伤中高表达,抑制miR-26a-3p表达可通过靶向上调Survivin表达抑制H/R诱导的心肌细胞凋亡和氧化应激。Objective To investigate the effects of miR-26a-3p on rat myocardial cell(H9c2)injury induced by hypoxia/reoxygenation(H/R)and its mechanism.Methods H9c2 cardiomyocytes in logarithmic growth phase were subjected to hypoxia(1%O 2)for 6 h,and reoxygenated at different times(2,4,8,12 h)to establish H/R model cell.Normoxia group was also set up,and cell proliferation activity was detected by cell counting kit-8(CCK-8).The level of lactic dehydrogenase(LDH)in cell supernatant was determined by colorimetry.The expression levels of miR-26a-3p and Survivin mRNA were detected by real-time fluorescence quantitative PCR(qRT-PCR).The expression level of Survivin protein in the cells was detected by Western blot.H9c2 cells were transfected with miR-26a-3p inhibitor and negative control inhibitor NC,Survivin gene siRNA interference plasmid(si-Survivin)and negative control si-NC,followed by H/R intervention.CCK-8 was used to detect cell proliferation in each group.The activity of superoxide dismutase(SOD)and the content of malonaldehyde(MDA)in cell and the level of LDH in supernatant were determined by colorimetry.The apoptosis level of each group was detected by flow cytometry.The protein expression levels of Bcl-2 associated X protein(Bax),B-cell lymphoma-2(Bcl-2),cleaved caspase-3 and Survivin were detected by Western blot.Targeting relationship between miR-26a-3p and Survivin gene was determined by dual luciferase.Results Compared with the normoxia group,proliferative activity,mRNA and protein expression levels of Survivin in H9c2 cells gradually decreased with the extension of reoxygenation time(P<0.05),while LDH and expression level of miR-26a-3p gradually increased(P<0.05).Down-regulating the expression of miR-26a-3p increased proliferative activity,SOD activity,and expression level of Bcl-2 protein in H9c2 cells exposed to H/R(P<0.05),while MDA content,LDH release amount,apoptosis rate,expression levels of Bax and cleaved caspase-3 protein decreased(P<0.05).Survivin deficiency reversed the protective effect of miR-26a-3

关 键 词：miR-26a-3p 缺氧/复氧 心肌细胞 生存素 细胞凋亡 氧化应激 

分 类 号：R542.2[医药卫生—心血管疾病]