Gemin3基因通过抑制p53表达阻碍细胞凋亡

Gemin3 inhibits cell apoptosis through suppression of p53 expression

目的探讨Gemin3基因表达在细胞凋亡中的作用及途径。方法采用免疫共沉淀、谷胱甘肽-S转移酶共沉淀实验确定Gemin3与p53两种蛋白在体内、体外相互结合及相互作用的结构域。荧光素酶报告基因检测转染Gemin3基因对p53基因的影响，依靠慢病毒载体介导小干扰RNA敲低Gemin3基因的表达，并经嘌呤霉素筛选获得稳定的Gemin3低表达细胞系，实时定量PCR检测Gemin3敲低对p53及其下游基因在转录水平的影响，流式细胞术检测Gemin3敲低对细胞凋亡的影响。结果Gemin3的C端与p53的中部DNA部位相互结合。将p53报告基因、p53蛋白基因以及逐渐增量的Gemin3基因共转染Saos-2细胞，随着Gemin3质粒转染浓度的增高，p53介导的报告基因表达水平逐渐降低，Gemin3在转录水平抑制p53基因的表达。实时定量PCR结果显示，与对照细胞比较，Gemin3敲低细胞中，p53、p21和BaxmRNA的表达水平明显增高。Western blot检测结果显示，Gemin3敲低细胞中，p53的表达明显升高。流式细胞术检测结果显示，敲低Gemin3基因的表达导致细胞凋亡的增加。结论Gemin3与p53相互结合并通过抑制p53的表达阻碍细胞凋亡。

Objective To investigate the role of Gemin3 in cell proliferation and its regulation pathway. Methods Using co-immunoprecipitation and GST pull-down assay to determine the domain of Gemin3 and p53 binding and interaction in vitro and in vivo. To check the effect of Gemin3 on p53 by luciferase reporter assay. Stable Gemin3 knock-down cell lines were generated by lentivirus-delivered small hairpin RNA then puromycin selection. Real-time PCR was used to confirm the effect of Gemin3 level on p53 and its downstream genes, and flow cytometry was used to analyze the effect of Gemin3 on apoptosis. Results The C-terminal of Gemin3 interacted with the DNA binding domain of p53. The p53 reporter gene, PA3M- p53 and increasing amount of GFP-Gemin3 were co-transfected into Saos-2 cells. Gemin3 repressed p53 expression at transcription level. Real-time PCR indicated that the expression of p53, p21 and Bax in Gemin3 knock-down cells was higher than that in the control cells. Western blot showed Gemin3 knock-clown cells had a higher p53 espression. Flow eytometric assay showed that knock-down Gemin3 expression led to an increased cell apoptosis. Conclusion Gemin3 binds with p53 forming a complex and plays an antiapoptotic role by repressing the p53 expression.