ELA/APJ通过上调miR-133a抑制心肌细胞在缺血缺氧条件下凋亡的机制研究

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摘要目的观察血管紧张素受体AT1相关的受体蛋白（APJ）的内源性配体ELABELA（ELA）对心肌细胞在缺血缺氧条件下凋亡的影响，并探讨miR-133a在其中的调控机制。方法将体外培养的心肌细胞分为Control组、ELA治疗组、ELA+siAPJ组、ELA+siAPJ NC组、ELA+miR-133a inhibitor组、ELA+ miR-133a inhibitor NC组，在缺血缺氧条件（无血清，1%体积分数O2）下培养24 h。免疫荧光鉴定心肌细胞标志物心肌α肌动蛋白（α-SA）和心肌肌钙蛋白T（cTnT）表达情况。流式细胞仪检测各组细胞凋亡情况。Western blot、qRT-PCR检测各组细胞APJ、miR-133a的表达情况。结果 ①与Control组比较，ELA治疗组细胞早期凋亡率与晚期凋亡率显著减少（P ＜ 0.01）；②与对应的NC组细胞比较，ELA+siAPJ组、ELA+miR-133a inhibitor组细胞早期凋亡率与晚期凋亡率明显增加（P ＜ 0.01）；③与Control组比较，ELA治疗组细胞APJ的蛋白与mRNA表达量、miR-133a的mRNA表达量均明显升高（P ＜ 0.01）；④采用siRNAs阻断APJ的表达后，与ELA+siAPJ NC组比较，ELA+siAPJ组细胞APJ的蛋白与mRNA表达量、miR-133a的mRNA表达量均显著减少（P ＜ 0.01）。采用siRNAs阻断miR-133a的表达后，ELA+miR-133a inhibitor组细胞APJ的蛋白与mRNA表达量与ELA+miR-133a inhibitor NC组比较，差异无统计学意义（P ＞ 0.05），miR-133a的mRNA表达量显著减少（P ＜ 0.01）。结论 ELA能够抑制心肌细胞在缺血缺氧环境下的凋亡，此效应可能与其激活APJ之后上调miR-133a有关。

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	陈旭翔1 侯婧瑛1 龙会宝1 吴浩1 伍权华1 杨欢1 符佳颖1 王彤2

关键词 ： ELABELA, 血管紧张素受体AT1相关的受体蛋白, 缺血缺氧, 心肌细胞, 细胞凋亡

Abstract：Objective To investigate the effect of ELABELA (ELA), which is a ligand of angiotensin receptor AT1 associated endogenous protein (APJ), on cardiomyocytes apoptosis under ischemia-hypoxia condition and to explore the regulatory mechanism of miR-133a. Methods The cardiomyocytes were cultured for 24 h under ischemia-hypoxia conditions (without serum, 1% O2) and divided into control group, ELA treatment group, ELA+siAPJ group, ELA+siAPJ NC group, ELA+miR-133a inhibitor group and ELA+miR-133a inhibitor NC group. The expression of myocardial α-actin (α-SA) and cardiac troponin T (cTnT) were observed by immunofluorescence. Flow cytometry was used to detect apoptosis. Western blot and qRT-PCR were used to detect the expression of APJ and miR-133a. Results ①Compared with the control group, the rate of early and late apoptosis in ELA treatment group was significantly decreased (P ＜ 0.01); ②compared with corresponding NC group, the early and late apoptosis rate of ELA+siAPJ group and ELA+miR-133a inhibitor group were significantly increased (P ＜ 0.01); ③the expression of APJ and miR-133a in ELA treatment group was significantly higher than that in control group after 24 h culture (P ＜ 0.01); ④after blocking the expression of APJ and miR-133a by siRNA, compared with that in the corresponding NC group, the protein and mRNA expression level of APJ and mRNA expression level of miR-133a were significantly decreased in ELA+siAPJ group (P ＜ 0.01). The protein and mRNA expression level of APJ did not change significantly in ELA+miR-133a inhibitor group (P ＞ 0.05), but the mRNA expression level of miR-133a was significantly decreased (P ＜ 0.01). Conclusion ELA could inhibit the apoptosis of cardiomyocytes in ischemic and hypoxic environment. This effect might be related to up-regulation of miR-133a after activation of APJ.

Key words： ELABELA Angiotensin receptor AT1 related receptor protein Ischemia-hypoxia Cardiomyocyte Cell apoptosis

基金资助:广东省科技发展专项资金（基础与应用基础研究方向）项目（2017A030313503）；
高校基本科研业务费中山大学资助计划项目（17ykjc18）。

通讯作者: 王彤（1969-），男，博士，主任医师，教授，博士研究生导师，中山大学附属第八医院副院长；研究方向：干细胞与心脑血管疾病。

作者简介: 陈旭翔（1991-），男，中山大学附属孙逸仙纪念医院2016级急诊医学专业在读硕士研究生；研究方向：干细胞与心肺脑复苏。

引用本文:

陈旭翔1 侯婧瑛1 龙会宝1 吴浩1 伍权华1 杨欢1 符佳颖1 王彤2. ELA/APJ通过上调miR-133a抑制心肌细胞在缺血缺氧条件下凋亡的机制研究[J]. 中国医药导报, 2019, 16(15): 12-17.
CHEN Xuxiang1 HOU Jingying1 LONG Huibao1 WU Hao1 WU Quanhua1 YANG Huan1 FU Jiaying1 WANG Tong2. Research on the mechanism of ELA/APJ inhibiting the apoptosis of cardiomyocyte under the condition of ischemia and hypoxia by up-regulating miR-133a. 中国医药导报, 2019, 16(15): 12-17.

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