收稿日期: 2018-10-26
网络出版日期: 2019-02-26
基金资助
国家自然科学基金面上资助项目(81670362);国家自然科学基金面上资助项目(81470515);嘉定区科委卫生系统科研资助项目(2014-KW-02)
Role of miR-19b in protecting cardiomyocytes from apoptosis by activating Akt signaling
Received date: 2018-10-26
Online published: 2019-02-26
[目的] 基于新生大鼠心肌细胞氧糖剥离再灌注(oxygen glucose deprivation/reperfu-sion, OGD/R)模型, 研究 miR-19b 对心肌细胞凋亡的调控作用, 并初步说明其分子机制.[方法] 采用酶解法提取新生大鼠心肌细胞, 分别转染 miR-19b 模拟物(mimics) 和抑制物 (inhibitor), 然后对细胞进行氧糖剥离再灌注处理, 模拟心肌缺血再灌注损伤过程. 采用末端脱氧核苷酸转移酶介导的三磷酸脱氧尿甘(2'-deoxyuridine 5'-triphosphate, dUTP)缺节末端标记(terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling, TUNEL)法, 检测心肌细胞凋亡. 采用蛋白质免疫印迹法检测凋亡蛋白(Bcl2, Bax 和 Caspase3)以及下游分 子同源磷酸酶-张力蛋白 (phosphatase and tensin homolog, PTEN)和 Akt 的表达.[结果] TUNEL 染色表明, miR-19b mimics 可以明显降低 OGD/R 模型中凋亡心肌细胞比率, 而 miR-19b inhibitor 作用则相反. 蛋白质免疫印迹法检测 Bcl2, Bax 和 Caspase3 的表达变化也与 TUNEL 染色结果一致. miR-19b mimics 可下调 PTEN, 激活 Akt, 而 miR-19b inhibitor 的作用则相反. 同时转染 miR-19b inhibitor 和 PTEN-siRNA 进行功能逆转实验, 发现 PTEN-siRNA 可以逆转 miR-19b inhibitor 对细胞凋亡的促进作用.[结论] miR-19b 可通过下调 PTEN 激活 Akt 信号通路, 保护心肌细胞凋亡.
朱宏文, 喻溥蛟, 许嘉鸿 . miR-19b 通过激活 Akt 信号通路保护心肌细胞凋亡[J]. 上海大学学报(自然科学版), 2019 , 25(1) : 10 -17 . DOI: 10.12066/j.issn.1007-2861.2097
[Objective] The purpose of the research is to investigate the effects of miR-19b on apoptosis and its underlying mechanisms in the oxygen glucose deprivation/reperfusion (OGD/R) model of neonatal rat cardiomyocytes (NRCMs). [Methods] NRCMs are obtained from neonatal rats by enzymatic hydrolysis and then transfected by miR-19b mimics and miR-19b inhibitor for the following study. A model of OGD/R has been built in NRCMs to mimic myocardial ischemia and reperfusion injury (MIRI). Terminal deoxynucleotidyl transferase (TdT)-mediated 2'-deoxyuridine 5'-triphosphate (dUTP)-biotion nick end labeling (TUNEL) assay has been performed to detect cardiomyocyte apoptosis. Western blot has been applied for detecting the expression of apoptotic proteins (Bcl2, Bax and Caspase3) and the downstream proteins of miR-19b. TUNEL staining indicates that miR-19b mimics could reduce the ratio of TUNEL positive nucleus to total nucleus and miR-19b inhibitor has opposite effects. [Results] Results based on western blot support the observations above. Moreover, miR-19b mimics could down-regulate the expression of phosphatase and tensin homolog (PTEN) and activate Akt signaling while miR-19b inhibitor has opposite effects. The rescue experiment has confirmed that PTEN-siRNA could reverse the promotion of miR-19b inhibitor on cardiomyocyte apoptosis. [Conclusion] miR-19b may protect cardiomyocytes from apoptosis through the activation of Akt signaling via regulating PTEN negatively.
Key words： cardiomyocytes apoptosis; miR-19b; Akt signaling
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