ELA/APJ通過上調miR-133a抑制心肌細胞在缺血缺氧條件下凋亡的機制研究

陳旭翔　侯婧瑛　龍會寶　吳浩　伍權華　楊歡　符佳穎　王彤

[摘要] 目的 觀察血管緊張素受體AT1相關的受體蛋白（APJ）的內源性配體ELABELA（ELA）對心肌細胞在缺血缺氧條件下凋亡的影響，并探討miR-133a在其中的調控機制。 方法 將體外培養的心肌細胞分為Control組、ELA治療組、ELA+siAPJ組、ELA+siAPJ NC組、ELA+miR-133a inhibitor組、ELA+ miR-133a inhibitor NC組，在缺血缺氧條件（無血清，1%體積分數O2）下培養24 h。免疫熒光鑒定心肌細胞標志物心肌α肌動蛋白（α-SA）和心肌肌鈣蛋白T（cTnT）表達情況。流式細胞儀檢測各組細胞凋亡情況。Western blot、qRT-PCR檢測各組細胞APJ、miR-133a的表達情況。 結果 ①與Control組比較，ELA治療組細胞早期凋亡率與晚期凋亡率顯著減少（P < 0.01）;②與對應的NC組細胞比較，ELA+siAPJ組、ELA+miR-133a inhibitor組細胞早期凋亡率與晚期凋亡率明顯增加（P < 0.01）;③與Control組比較，ELA治療組細胞APJ的蛋白與mRNA表達量、miR-133a的mRNA表達量均明顯升高（P < 0.01）;④采用siRNAs阻斷APJ的表達后，與ELA+siAPJ NC組比較，ELA+siAPJ組細胞APJ的蛋白與mRNA表達量、miR-133a的mRNA表達量均顯著減少（P < 0.01）。采用siRNAs阻斷miR-133a的表達后，ELA+miR-133a inhibitor組細胞APJ的蛋白與mRNA表達量與ELA+miR-133a inhibitor NC組比較，差異無統計學意義（P > 0.05），miR-133a的mRNA表達量顯著減少（P < 0.01）。 結論 ELA能夠抑制心肌細胞在缺血缺氧環境下的凋亡，此效應可能與其激活APJ之后上調miR-133a有關。

[關鍵詞] ELABELA;血管緊張素受體AT1相關的受體蛋白;缺血缺氧;心肌細胞;細胞凋亡

[中圖分類號] R331.31 ? ? ? ? ?[文獻標識碼] A ? ? ? ? ?[文章編號] 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.