microRNA-182靶向SNX17调控心肌梗死时细胞内钙稳态  

MicroRNA-182 regulates intracellular calcium homeostasis during myocardial infarction by targeting SNX17

作　　者：郑岚 赵丹丹 胡丹丹 姜秋嘉 高强 吕丽芳 ZHENG Lan;ZHAO Dan-dan;HU Dan-dan;JIANG Qiu-jia;GAO Qiang;LU Lifang(Department of Physiology,College of Basic Medicine,Harbin Medical University;Department of Pharmacology,College of Pharmacy,Harbin Medical University,Harbin 150081,China)

机构地区：[1]哈尔滨医科大学基础医学院生理学教研室 [2]哈尔滨医科大学药学院药理学教研室,黑龙江哈尔滨150081

出　　处：《哈尔滨医科大学学报》2024年第4期343-349,354,共8页Journal of Harbin Medical University

基　　金：国家自然科学基金资助项目(82370279);黑龙江省博士启动金(LBH-Q21121);2023年度黑龙江省省属高等学校基本科研业务费项目(2023-KYYWF-0251)。

摘　　要：目的探究心肌梗死(myocardial infarction,MI)时microRNA-182(miR-182)的表达变化并揭示其通过靶向分选连接蛋白17(sorting nexin 17,SNX17)参与调控心肌细胞内钙稳态的机制。方法将12只雄性Wistar大鼠随机分为假手术组和心肌梗死组(n=6),假手术组仅进行胸腔切开操作但不结扎冠状动脉,心肌梗死组通过结扎左前降支构建心肌梗死模型。原代培养乳鼠心肌细胞,构建离体缺氧模型。分别转染miR-182 mimics和AMO-182,过表达和抑制miR-182;转染SNX17质粒过表达SNX17;采用细胞计数试剂盒(cell counting kit-8,CCK-8)检测细胞活性;实时定量PCR检测miR-182和SNX17 mRNA水平;Western blot检测SNX17蛋白水平;荧光素酶报告基因实验验证miR-182与SNX173'-UTR区域的直接结合位点;Fluo-3-AM荧光染料和激光共聚焦显微镜观察细胞内钙浓度。结果与假手术组比较,心肌梗死组大鼠心肌组织中miR-182的表达显著上调(P<0.05)。与对照组比较,缺氧处理的乳鼠心肌细胞活力明显降低,miR-182表达显著上调,SNX17表达显著下调(P<0.05)。荧光素酶报告实验表明,miR-182与SNX173'-UTR区域直接结合并抑制其翻译(P<0.01)。转染miR-182 mimics后,SNX17的蛋白表达进一步下调(P<0.05);转染AMO-182则显著提高了SNX17的蛋白水平(P<0.05)。与对照组比较,过表达miR-182组心肌细胞静息钙水平显著升高(P<0.01),钙瞬变幅度显著降低(P<0.01),钙回落时间显著延长(P<0.01);过表达SNX17显著逆转过表达miR-182引起的钙稳态紊乱现象(P<0.05)。结论miR-182可通过靶向SNX17调控心肌梗死时心肌细胞内钙稳态,有望成为治疗心肌梗死的新靶点。Objective To explore the changes in the expression of microRNA-182(miR-182)during myocardial infarction(MI)and reveals its mechanism in regulating calcium homeostasis in cardiomyocytes by targeting sorting nexin 17(SNX17).Methods Twelve male Wistar rats were randomly divided into sham operation group and myocardial infarction group(n=6).The sham group underwent thoracotomy without coronary artery ligation,while the myocardial infarction group was subjected to ligation of the left anterior descending artery to induce myocardial infarction.Primary cultures of neonatal rat cardiomyocytes were prepared,and an in vitro hypoxia model was constructed.miR-182 mimics and AMO-182 were transfected to overexpress and inhibit miR-182,respectively;SNX17 plasmid was transfected to overexpress SNX17.Cell viability was measured using the cell counting kit-8(CCK-8);miR-182 and SNX17 mRNA levels were determined by real-time quantitative PCR;SNX17 protein levels were detected by Western blotting;a luciferase reporter assay was conducted to verify the direct binding site between miR-182 and the 3'-UTR region of SNX17;Intracellular calcium concentrations were observed using Fluo-3-AM fluorescent dye and laser confocal microscopy.Results Compared with the sham group,miR-182 expression in myocardial tissue of the myocardial infarction group was significantly upregulated(P<0.05).Compared with the control group,hypoxiatreated neonatal rat cardiomyocytes showed significantly reduced viability,upregulated miR-182 expression,and downregulated SNX17 expression(P<0.05).The luciferase reporter assay showed that miR-182 directly binds to the 3'-UTR region of SNX17 and inhibits its translation(P<0.01).After transfection with miR-182 mimics,SNX17 protein expression was further downregulated(P<0.05),while transfection with AMO-182 significantly increased SNX17 protein levels(P<0.05).Compared with the control group,cardiomyocytes overexpressing miR-182 showed significantly increased resting calcium levels(P<0.01),reduced calcium transient amplitude(

关 键 词：心肌梗死 miR-182 SNX17 心肌细胞内钙稳态 

分 类 号：R542.22[医药卫生—心血管疾病]