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 myocardi-al infarction by targeting SNX17
郑岚 1赵丹丹 2胡丹丹 1姜秋嘉 1高强 3吕丽芳3
作者信息
- 1. 哈尔滨医科大学基础医学院 生理学教研室;哈尔滨医科大学药学院 药理学教研室,黑龙江 哈尔滨 150081
- 2. 哈尔滨医科大学药学院 药理学教研室,黑龙江 哈尔滨 150081
- 3. 哈尔滨医科大学基础医学院 生理学教研室
- 折叠
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SNX17 3′-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SNX17 3′-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.
Abstract
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 in-farction group was subjected to ligation of the left anterior descending artery to induce myocardi-al infarction.Primary cultures of neonatal rat cardiomyocytes were prepared,and an in vitro hy-poxia 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 lev-els 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 be-tween miR-182 and the 3′-UTR region of SNX17;Intracellular calcium concentrations were ob-served 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,hypoxia-treated 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(P<0.01),and prolonged calcium reuptake time(P<0.01);overex-pression of SNX17 significantly reversed the calcium homeostasis disorders caused by miR-182 overexpression(P<0.05).
关键词
心肌梗死/miR-182/SNX17/心肌细胞内钙稳态Key words
myocardial infarction/miR-182/SNX17/cardiomyocyte intracellular calcium ho-meostasis引用本文复制引用
基金项目
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)
出版年
2024
NETL
NSTL
万方数据