Epstein-Barr病毒RPMS1基因在鼻咽癌组织中的表达及其细胞内定位

Expression of Epstein-Barr virus RPMS1 gene in nasopharyngealc arcinoma and its subcellular location in vitro

目的:了解RPMS1基因在鼻咽癌组织中的表达特点,并明确该基因在上皮细胞中表达的亚细胞区域,为进一步研究其生物学功能提供依据.方法:提取鼻咽癌组织及各细胞系的DNA和总RNA,以巢式PCR扩增W片段来检测Ep-ste in-Barr病毒(EB病毒)的感染情况,RT-PCR检测RPMS1基因在鼻咽癌组织、细胞株中的表达,并克隆入pEGFP-C2真核表达载体,通过脂质体转染技术将其介导入人胚肾上皮(HEK293)细胞,以激光共聚焦显微镜观察其蛋白表达的亚细胞区域.结果:除B JAB细胞株外,所有标本中均检测到EB病毒W片段;RPMS1 mRNA在鼻咽癌组织中的表达率为83.3%(45/54),在鼻咽非癌组织中则仅为4%(1/25),二者有显著性差异(P<0.001),鼻咽癌外周血淋巴细胞中未检测到RPMS1表达,RPMS1在SUNE1细胞中的表达高于Raji细胞,但不表达于B95.8细胞;RPMS1可稳定表达于HEK293细胞,其编码蛋白的表达以胞核为主.结论:EB病毒RPMS1基因在高发区鼻咽癌组织中存在高水平转录,在体外可稳定表达于上皮细胞的胞核,提示RPMS1编码蛋白可能作为核转录因子通过调控细胞增殖相关的信息传递而参与上皮癌变过程.

AIM： To investigate the expression of RPMS1 mRNA in biopsies of nasopharyngeal carcinoma （NPC） in the area with high incidence and to observe the subcellular fraction of its encoding protein. METHODS： DNA and total RNA were isolated from tissues and cells. Nested PCR was designed to amplify the W sequence of EBV DNA and RT- PCR was used to analyze the transcription of RPMS1 in all samples. RPMS1 coding sequence （CDS） amplified from NPC tissues was cloned into pEGFP-C2. After transfected with this recombinant plasmid by lipofectin mediation, HEK293 cells were analyzed by RT-PCR and laser confocal microscope to observe the expression of RPMS1 and its subcellular fraction. RESULTS. Except for BJAB cells, EBV W sequence was observed in all the samples. RPMS1 expressed in 33.3% （45/54） NPC biopsies and 4% （1/25） noncarcinoma nasopharynx biopsies with significant difference between them but no RPMS1 mRNA was detected in peripheral lymphocytes of NPC. RPMS1 expressed at lower level in Raji than in SUNE1 and was not expressed in B95.8 cells. The recombinant plasmid was constructed and expressed stably in HEK293 cells, in which the fusion protein was found to be nuclear. CONCLUSION： EBV-RPMS1gene is expressed in NPC tissues in the area with high incidence and its encoding protein can stably express in cytoplasm of epithelial cell in vitro, suggesting that RPMS! may function as a nuclear transcriptional factor by regulating the signal conduction related with cellular proliferation.