shRNA沉默P115基因对胃癌细胞增殖相关蛋白表达的影响及机制  

Effect of P115 gene silence by shRNA on expression of gastric carcinoma cell proliferation-associated proteins and relevant mechanism

作　　者：邓玮[1] 易永芬[1] 屈玉玲[1] 闫田静[1] 文雪[1] 

机构地区：[1]重庆医科大学病理学教研室分子医学与肿瘤研究中心,重庆400016

出　　处：《中国生物制品学杂志》2013年第8期1117-1121,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(30672431);教育部博士点基金(20060631006)

摘　　要：目的探讨高尔基体囊泡转运蛋白(colgi-vesicular transport protein,P115)基因沉默对胃癌细胞株BGC-823中增殖相关因子细胞周期素D1(cyclinD1)、微小染色体维持缺陷蛋白2(mini chromosome maintenance protein 2,Mcm2)和增殖细胞核抗原(proliferating Cell Nuclear Antigen,PCNA)表达的影响及其可能的机制。方法采用脂质体介导法将重组表达质粒P115-shRNA2(P115-shRNA2组)和阴性对照质粒shNC(阴性对照组)转染至高表达P115的胃癌细胞株BGC-823中,并设未转染组,经G418筛选出稳定转染细胞,实时定量PCR(Real-time PCR)法检测细胞中P115、巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)、cyclinD1、Mcm2和PCNA基因mRNA的转录水平;Western blot法检测细胞中P115、MIF、pERK1/2、cyclinD1、Mcm2和PCNA蛋白表达水平;免疫共沉淀法检测BGC-823细胞中P115与MIF间的相互作用;ELISA法检测细胞上清液中MIF的分泌水平。结果与未转染组和阴性对照组相比,P115-shRNA2组细胞中P115、MIF、cyclinD1、Mcm2和PCNA的mRNA和蛋白表达水平均明显降低,ERK1/2的磷酸化水平明显下调,BGC-823细胞培养上清中MIF的含量明显降低;P115蛋白可在抗MIF的免疫沉淀物中检出,P115和MIF在BGC-823细胞中存在特异性的相互作用。结论 P115基因沉默下调胃癌细胞中cyclinD1、Mcm2和PCNA的表达,其分子机制可能与P115通过调控MIF的表达和分泌,进而启动下游的ERK1/2信号通路有关。P115可作为研究胃癌细胞增殖分子机制的新靶点。Objective To investigate the effect of silence of Golgi-vesicular transport protein（P115）gene on expressions of cyclin D1,mini chromosome maintenance protein 2（Mcm2）and proliferating cell nuclear antigen（PCNA）in gastric carcinoma BGC-823 cells as well as the relevant mechanism.Methods Recombinant plasmid P115-shRNA2 and negative control plasmid shNC were transfected to BGC-823 cells in mediation of liposome,using those untransfected as blank control.The stably transfected cells were screened with G418,and determined for transcription levels of P115,macrophage migration inhibitory factor（MIF）,cyclinD1,Mcm2 and PCNA mRNAs by Real-time PCR,for expression levels of P115,MIF,pERK1 / 2,cyclinD1,Mcm2 and PCNA proteins by Western blot,for interaction between P115 and MIF by co-immunoprecipitation,and for secretion level of MIF in culture supernatant by ELISA.Results Compared with those in blank and negative control groups,both the mRNA transcription and protein expression levels of P115,MIF,cyclinD1,Mcm2 and PCNA,the expression level of pERK1 / 2 protein and the MIF content in culture supernatant of BGC-823 cells in p115-shRNA2 group decreased significantly（P 0.01）.P115 was detected in the immunoprecipitate with anti-MIF antibody,which showed specific interaction with MIF in BGC-823 cells.Conclusion P115 gene silence down-regulated the expressions of cyclinD1,Mcm2 and PCNA in gastric cancer cells,of which the mechanism was probably related to the fact that P115 adjusted the expression and secretion levels of MIF and then modulated ERK1 / 2 signaling pathway.P115 gene might be a potential target for study on the molecular mechanism of proliferation of gastric carcinoma cells.

关 键 词：短发夹RNA 胃癌 高尔基体囊泡转运蛋白 巨噬细胞移动抑制因子 细胞周期素D1 微小染色体维持缺陷蛋白2 增殖细胞核抗原 细胞增殖 

分 类 号：R735.2[医药卫生—肿瘤]