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

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

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10-7mol/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。

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.