南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (07): 810-.doi: 10.12122/j.issn.1673-4254.2019.07.10

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血管紧张素Ⅱ通过上调富含半胱氨酸蛋白61 表达诱导HEK293T细胞凋亡

王俊杰，姜艳，Soulixay Senouthai，付冬冬，尤燕舞   

  • 出版日期:2019-07-20 发布日期:2019-07-20

Angiotensin II induces apoptosis of HEK293T cells by up-regulating Cyr61 expression

  • Online:2019-07-20 Published:2019-07-20

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摘要： 目的探讨富含半胱氨酸蛋白61（Cyr61）在血管紧张素Ⅱ（AngⅡ）诱导HEK293 细胞功能中的改变及作用。方法 HEK293T细胞于体外培养，并应用CRISPR/Cas9技术敲低HEK293T细胞Cyr61基因。将细胞分为四组：（1）对照组；（2）Cyr61 敲低组；（3）AngⅡ组；（4）AngⅡ+Cyr61敲低组，以10-7 mol/L AngⅡ处理细胞，48 h收集细胞用流式细胞仪检测细胞凋亡，通过 实时荧光定量PCR(qRT-PCR)技术及蛋白质免疫印迹技术检测细胞Cyr61及Bcl-2的mRNA及蛋白表达量。结果Cyr61敲低 组Cyr61蛋白水平较正常对照组明显下降（P<0.05），AngⅡ干预48 h 后Cyr61蛋白和mRNA表达水平显著升高（P<0.05），Bcl-2 蛋白和mRNA表达水平明显下降（P<0.05），敲低Cyr61 后Bcl-2 蛋白和mRNA表达水平明显升高（P<0.05），与对照组凋亡率 （11.88±1.46）%相比，Cyr61敲低组凋亡率为（3.87±0. 83）%，AngⅡ干预组HEK293T细胞凋亡率为（26.94±3.73）%（P<0.05），与 AngⅡ干预组相比，Cyr61 敲低+AngⅡ组凋亡率为（15.76±1.31）%（P<0.05）。结论Cyr61 表达量的上调与AngⅡ诱导的 HEK293T细胞损伤有关，下调Cyr61的表达可以有效地保护AngⅡ诱导的细胞损伤。

Abstract: Objective To investigate the role of Cyr61 in angiotensin II (AngII)-induced functional changes in HEK293 cells and explore the mechanism. Methods Cyr61 knockdown in cultured HEK293T cells was achieved by transfection of the cells with CRISPR/Cas9 KO plasmid. The changes in apoptosis and expression levels of Cyr61 and Bcl-2 in the cells with or without Cyr61 knockdown in response to treatment with 10-7 mol/L AngII for 48 h were analyzed using flow cytometry, qRT-PCR and Western blotting. Results The cells with Cyr61 knockdown showed significantly decreased expression of Cyr61 protein as compared with the control cells (P<0.05). AngII treatment for 48 h significantly increased the expression of Cyr61 and lowers the expression of Bcl-2 at both the protein and mRNA levels in HEK293T cells. In HEK293T cells with Cyr61 knockdown, AngII treatment resulted in significantly increased expression of Bcl-2 in HEK293T cells as compared with that of the control group (P<0.05). AngII treatment caused significantly increased apoptotic rate in HEK293T cells as compared with the cells with Cyr61 knockdown [(26.94 ± 3.73)% vs (3.87 ± 0.83)% , P<0.05), and the apoptosis rate was significantly lowered to (15.76 ± 1.31)% in HEK293T cells with Cyr61 knockdown following AngII treatment (P<0.05). Conclusion The up-regulation of Cyr61 expression is related with AngII-induced injury in HEK293T cells, and down-regulating Cyr61 expression can effectively protect HEK293T cells against AngII-induced injury.