慢病毒介导的敲低UBC9表达抑制ERβ类泛素化修饰水平

Lentivirus-mediated knockdown of human UBC9 inhibits ERβ sumoylation

目的建立稳定敲低UBC9蛋白表达的293T细胞株,检测其对雌激素受体β(ERβ)类泛素化修饰水平的调节作用。方法构建4条针对人UBC9基因的慢病毒短发夹RNA(shRNA)的干扰载体,将其与慢病毒包装辅助质粒共转染293T细胞后,收集病毒上清,感染293T细胞,经嘌呤霉素(puromycin)筛选后获得稳定表达慢病毒介导UBC9 shRNA的混合细胞集落,分别用实时(real-time)PCR和Western印迹方法检测UBC9的表达情况,瞬时转染方法检测UBC9表达水平对ERβ类泛素化修饰水平的影响。结果建立了稳定敲低UBC9的293T细胞株,UBC9降低能够抑制ERβ类泛素化修饰水平。结论 UBC9在ERβ类泛素化修饰反应中发挥重要作用。

Objective To establish 293T cell lines in which UBC9 expression is stably knocked down and to detect the. effect of U BC9 on estrogen receptor β（ERβ） sumoylation. Methods Four short hairpin RNA （shRNA） primers targeting different U BC9 regions were designed and cloned into lentivirus vector pSI H-H1-Puro, which was then packaged with acees- -ory,plasmids into lentvirns in 293T cells. After being infected with lentivirus and selected by puromycin, mixed 293T cohmies stably expressing UBC9 shRNA were obtained and used to detect UBC9 expression with Real-time PCR and West-ern hlotting. Finally, the effect of UBC9 on ERβ sumoylation was determined by transient transfection assay. Result 293T cell lines stably knocking-down UBC9 expression were established and inhibilion of UBC9 expression in this cell line sup-pressed ERβ sunoylation. Conclusion UBC9 plays a key role in ERβ sumoylation.