摘要
目的探讨Gemin3基因表达在细胞增殖中的作用及其途径。方法采用免疫共沉淀、谷胱苷肽-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发卡RNA干涉敲减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PCR检测Gemin3敲减对p53及其下游基因在转录水平的影响,流式细胞检测Gemin3敲减对细胞增殖的影响。结果 Gemin3的C端与p53的中部DNA结合部位相互结合,Gemin3在转录水平抑制p53基因的表达,敲减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的表达促进细胞的增殖。
[ Objective ] To investigate the role of Gemin3 on p53-mediated apoptosis. [Methods ] Co-immuno- precipitation and GST pull-down assay were used to determine the combination of Gemin3 with p53 and their com- bining domain. The effect of Gemin3 on p53 was checked by luciferase reporter assay. Gemin3 knock-down stable cell lines were made by lentivirus-delivered small hairpin RNA then puromycin selection. Real-time PCR was used to confirm the effect of Gemin3 on p53 and its downstream gene at transcriptional level, and flow cytometric assay was used to analyze the role of Gemin3 in apoptosis. [ Results ] The C-terminal of Gemin3 interacted with DNA combining domain of p53. Gemin3 repressed p53 expression at transcription level. Knock-down Gemin3 expression led to increased apoptosis. [Conclusion] Gemin3 forms complex with p53 and plays an anti-apoptosis role by repressing the transcription activity of p53.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2013年第18期1-11,共11页
China Journal of Modern Medicine
基金
supported by grants from the National Natural Science Foundation of China(No.81171649)
National Natural Science Foundation of Liaoning Province(No.201102267)
