摘要
目的通过敲除与过表达肺腺癌A549细胞的Rac家族小GTP酶3(rac family small GTPase 3,Rac 3)基因,探讨了Rac 3对A549细胞自噬的影响及机制。方法设计靶向人Rac 3的sgRNA,构建簇状规则间隔的短回文重复序列/簇状规则间隔的短回文重复序列蛋白9(clustered regulatory interspaced short palindromic repeat/cas9,CRISPR/cas9)敲除质粒并导入A549细胞,通过T7E1酶切、碱基测序及Western Blot验证基因编辑。EBSS诱导自噬,检测LC3和SQSTM-1/p62的表达评估自噬水平,caspase3活化水平评估Rac 3对凋亡影响,黑色素瘤抗原A6(melanoma antigen A6,MAGEA6)、AMPKα、ERK1/2、AKT的表达探讨Rac 3影响A549细胞自噬的机制。结果通过CRISPR/cas9技术成功敲除A549细胞的Rac 3基因。Rac 3敲除后诱导自噬,LC3 II/I比值上升、SQSTM-1/p62水平下降,caspase3活化水平升高,MAGEA6表达下降,AMPKα及磷酸化水平上升,Rac 3过表达上述蛋白呈相反方向变化。结论Rac 3通过MAGEA6抑制AMPKα的表达及磷酸化水平,进而抑制肺腺癌A549细胞的自噬和凋亡。
Objective To explore the effect and mechanism of Rac 3 on autophagy of A549 cells by knocking out and overexpressing the Rac 3 gene in lung adenocarcinoma A549 cells.Methods SgRNA of targeting human Rac 3 was designed,CRISPR/cas9 knockout plasmids was constructed and A549 cells were imported.Gene editing was confirmed by T7 endonuclease,base sequencing technology and Western Blot.Autophagy was induced by EBSS;the expressions of LC3 and SQSTM-1/p62 were detected to evaluate the level of autophagy;the caspase3 activation levels was detected to evaluate the effect of Rac 3 on apoptosis;and the expressions of MAGEA6,AMPKα,ERK1/2 and AKT were detected to explore the mechanism of Rac 3 affecting autophagy in A549 cells.Results Rac 3 gene in A549 cells was successfully knocked out by CRISPR/Cas9 technique.Rac 3 autophagy was induced after knockout.LC3 II/I ratio increased,SQSTM-1/p62 level decreased,activation level of caspase3 increased,MAGEA6 expression decreased,AMPKαand its phosphorylation levels increased,and the above-mentioned proteins changed in the opposite direction when Rac 3 was overexpressed.Conclusion Rac 3 inhibits the expression and phosphorylation of AMPKαby MAGEA6,which in turn leads to the inhibition of autophagy and apoptosis of lung adenocarcinoma A549 cells.
作者
杜天宇
肖旭阳
Du Tianyu;Xiao Xuyang(The First Affiliated Hospital of Jinzhou Medical University,Jinzhou 121000 China)
出处
《锦州医科大学学报》
2021年第3期5-10,共6页
Journal of Jinzhou Medical University
基金
辽宁省教育厅自然科学类基础项目,项目编号:JYTJCZR201908。