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

陈旭翔　侯婧瑛　龙会宝　吴浩　伍权华　杨欢　符佳颖　王彤

[摘要] 目的 觀察血管紧张素受体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。

[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

[中图分类号] R331.31          [文献标识码] A          [文章编号] 1673-7210（2019）05（c）-0012-06

[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.