摘要
背景与目的:潜伏膜蛋白LMPl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。本研字己旨在探讨LMP1沉默对AP-1信号转导通路及其下游与细胞转化、增殖和凋亡等相关因子转录表达的影响。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蛋白表达在作用第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。
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 chemically synthetic siRNA targeting LMP1 was transfected into EBV positive gastric carcinoma epithelial cell line by lipofectamine 2000 at 50 nmol/L final concentration. The protein expression of c-Jun, JunB and CDK4 was tested by Western blotting. The mRNA of c-Jun, surviving, CDK4 and MMP9 mRNA were tested by RT-PCR. The expression of survivin were tested by immunohistochemistry. Results： Compared with the cell control, CDK4 mRNA was up regulated（P〈0.05）, c-Jun mRNA and survivin mRNA were downregulated siRNA 24,48,72,96 and 120 hrs post-transfection（P〈0.05）. The level of c-Jun and survivin mRNA were most downregulated by siRNA at 24 hrs and 48 hrs post-transfection, respectively. MMP9 mRNA was also down regulated during the first 3 days（P〈0.05）, and then degraded obviously siRNA 48 hrs post-transfection（P〈0.05）. CDK4 protein expression was also up regulatede at each time point, c-Jun and JunB protein expression was down regulated by siRNA 24, 48, 72 and 96 hrs post-transfection, compared with cell control. [mmunohistochemmistry results showed that the survivin expression was down 24, 48 and 72 hrs posttransfection（P〈0.05）. Conclusion： The specific silence of LMP1 could inhibit cell proliferation and promote cell apoptosis through regulation ofAP-1 signal transduction pathway and its related factors.
出处
《中国癌症杂志》
CAS
CSCD
2008年第11期816-821,共6页
China Oncology
基金
国家自然科学基金资助项目(项目编号:30471623)
山东省科技厅资助项目(项目编号:2004GG3202016)
关键词
EB病毒
潜伏膜蛋白1
活化蛋白-1
胃癌上皮细胞
Epstein-Barr virus
latent membrane protein（LMP 1）
activated protein- 1
gastriccarcinoma epithelial cell
