敲除Dock180抑制大鼠H9C2心肌细胞系增殖和促凋亡

Dock180 knockout inhibits proliferation and promotes apoptosis of rat derived H9C2 cardiomyocytes strain

目的探讨敲除细胞质移动蛋白1(Dock180)对大鼠H9C2心肌细胞系增殖和凋亡的影响及其机制。方法用CRISPR/Cas9系统构建single guide RNA(sgRNA)Dock180质粒,并将其包装成敲除Dock180重组慢病毒再进行筛选,建立敲除Dock180的H9C2心肌细胞稳定株。实验分为A:阴性慢病毒组(Cas9)、B:敲除Dock180组、C:阴性慢病毒低氧组、D:敲除Dock180低氧组。RT-PCR检测各组细胞Dock180 mRNA表达水平,Western blot检测相关蛋白的表达水平,MTT检测细胞增殖率,流式细胞术检测细胞凋亡率。结果 B组和D组Dock180 mRNA和蛋白无表达;分别较A组和C组,B组和D组p-ERK1/2、Bcl-2蛋白和细胞增殖率均降低(P<0.01),Bax蛋白和细胞凋亡率均增加(P<0.05,P<0.01);较A组,C组Dock180 mRNA和蛋白表达降低,p-ERK1/2、Bcl-2蛋白和细胞增殖率均降低,Bax蛋白和细胞凋亡率均增加(P<0.05,P<0.01);较B组、D组p-ERK1/2、Bcl-2蛋白和细胞增殖率均降低,Bax蛋白和细胞凋亡率均增加(P<0.05,P<0.01)。结论敲除Dock180可抑制H9C2心肌细胞增殖,促进细胞凋亡,其作用可能通过p-ERK1/2、Bax和Bcl-2分子介导。

Objective To investigate the effects of dedicator of cytokinesis 1（ Dock180） knockout on proliferation and apoptosis in rat derived H9C2 cardiomyocytes and their mechanisms. Methods A single guide RNA（ sgRNA）targeting rat Dock180 gene was designed and constructed using CRISPR/Cas9 system. A plasmid contained above sgRNA was packaged into lentivirus and selected to knockout Dock180 in the cardiomyocytes. A single clone of cardiomyocyte with Dock180 knockout was established. Cardiomyocytes were divided into negative lentivirus group（ Cas9,A group）,Dock180 knockout group（ B group）,Cas9 lentivirus hypoxia group（ C group）,Dock180 knockout hypoxia group（ D group）. The expression of Dock180 mRNA was examined by RT-PCR,and relevant proteins were detected by Western blot. The cell proliferation rate of the cardiomyocytes was determined by MTT,and the apoptotic rate was measured by flow cytometry. Results Dock180 mRNA and protein were absent in B andD groups. Compared with A and C groups,p-ERK1/2 and Bcl-2 protein expression and cell proliferation rate were lower in B and D groups respectively（ P〈0. 01）,while Bax protein expression and cell apoptosis rate were higher in B and D groups respectively（ P〈0. 05,P〈0. 01）; Compared with A group,Dock180 mRNA and protein,p-ERK1/2 and Bcl-2 proteins and cell proliferation rate were reduced,while Bax protein and cell apoptosis rate were increased in C group（ P〈0. 05,P〈0. 01）. Compared with B group,p-ERK1/2 and Bcl-2 proteins and cell proliferation rate were decreased,while Bax protein and cell apoptosis rate were increased in D group（ P〈0. 05,P〈0. 01）. Conclusions Dock180 knockout with CRISPR/Cas9 can inhibit proliferation and promote apoptosis via p-ERK1/2,Bcl-2 and Bax in H9C2 cardiomyocytes.