靶向LMP1基因siRNA作用对AP-1及其相关因子表达的影响

背景与目的: 潜伏膜蛋白LMP1是EBV基因组BamH I Nhet片段上BNLF1编码的一种跨膜蛋白,它是发现最早的能在体外恶性转化细胞系的EBV潜伏期蛋白.本研究旨在探讨LMP1沉默对AP-1信号转导通路及其下游与细胞转化、增殖和凋亡等相关冈子转录表达的影响.方法: 应用50 nmol/L靶向LMP1功能区编码序列的siRNA649转染EBV阳性的胃癌上皮细胞GT38,分别作用24、48、72、96、120 h,采用RT-PCR检测c-Jun,生存素,CDK4和MMP9 mRNA转录水平:免疫组化法检测细胞生存素蛋白表达;Western印迹法检测c-Jun,JunB和CDK4蛋白表达.结果: 靶向LMP1特异性siRNA作用后,各时间点c-Jun和生存素mRNA水平均明显低于细胞对照组(P＜0.05),c-Jun以24 h时降低最为明显,而生存素以48 h时降低最为明显;CDK4 mRNA水平均明显高于细胞对照组(P＜0.05);转染后24、48、72 h时MMP9 mRNA表达水平均明显低于细胞对照组(P＜0.05),96 h和120 h时MMP9则无明显改变(p＞0.05).与细胞对照比较,c-Jun和JunB蛋白表达均下调,JunB蛋白表达往作用第5天时有所恢复,CDK4蛋白表达均上调.免疫组化检测结果显示转染后细胞质中代表生存素阳性的棕色颗粒明显减少.结论: 靶向LMP1功能区编码序列的siRNA转染可影响EBV阳性胃上皮细胞AP-1及其下游相关因子的表达,进而抑制细胞增殖,促进细胞凋亡.

The influence of siRNA targeting LMPI gene on expression of AP-1 and its related factors

Background and purpose:LMP1 was one of the protein encoded by EBV latent gene,which was found to be able to transform cell lines and alter the phenotype of cells due to its oncogenic potential.In human epithelial cells,LMP1 alters many functional properties that are involved in tumor progression and invasions.In this study we investigated the influence of LMP1 silence on AP-1 signal transduction pathway and its downstream factors involved with cell transformation,proliferation and apoptosis.Methods:The chem...