EBVaGC肿瘤差异表达的基因表达谱芯片筛选

GENE EXPRESSION MICROARRAY STUDY FOR TUMOR ASSOCIATED GENES IN EBVaGC

目的应用全基因组表达谱芯片技术,分析EB病毒(EBV)阳性胃癌细胞系与EBV阴性胃癌细胞系基因表达谱差异,筛选EBV相关胃癌(EBVaGC)相关基因。方法采用TRIzol一步法提取3种EBV阳性胃癌细胞系(GT38、PT、SNU-719)和3种EBV阴性胃癌细胞系(SGC7901、HGC-27、MKN-45)总RNA并纯化,逆转录合成cDNA,利用荧光染料(Cy3)标记aaUTP,转录合成标记的cRNA,与Agilent人类全基因组表达谱芯片杂交,扫描荧光信号图像,对芯片原始数据扣除本底和归一化处理,利用倍数差异和t检验计算筛选差异表达的基因,采用上海博豪生物在线分析系统进行基因的功能注释和关联分析,明确差异基因的生物学功能。将处理前后差异表达倍数>2.0或<0.5,且在不同样本间的具有统计学差异的基因判断为差异表达基因。选取特定的差异表达基因,应用实时荧光PCR技术验证芯片结果的可靠性。结果聚类热图及矩阵图分析显示,EBV阳性胃癌细胞系与阴性胃癌细胞系之间表达谱存在明显差异。EBV阳性胃癌细胞系与阴性胃癌细胞系之间有322条基因的表达有明显差异,其中与肿瘤相关的基因涉及转录翻译、信号转导、黏附、细胞增殖和凋亡以及免疫应答等多种生物学过程。选取6条差异基因进行验证,结果有3条基因表达上调,3条基因表达下调,表达趋势与芯片结果一致。结论 EBV阳性胃癌细胞系与阴性胃癌细胞系基因表达存在明显差异,提示EBV感染可影响胃癌细胞相关基因的表达,分析这些差异表达基因有助于阐明EBV感染在EBVaGC发生发展中的作用机制,为进一步筛选EBVaGC特异性肿瘤标记物以及进行生物治疗提供理论依据。

Objective To detect and compare the gene expression profile between EBV positive and negative gastric carci- noma cell lines using eDNA mieroarray method and screen differentially expressed genes potentially related with EBVaGC. Me- thods Total RNA was extracted and purified from six gastric carcinoma cell lines （GT38, PT and SNU-719 were EBV positive; SGC7901, HGC-27 and MKN-45 were EBV negative）. The first and second eDNA strains were generated and then transcribed into cRNA, fluorescent dye Cy3 was used to label the cRNA. The labeled cRNA was used to hybrid with the Agilent expression profile chip, the fluorescence signal was then scanned by the computer and the expression profile of two groups was compared. Differenti- ally-expressed genes were classified according to their functions. Specific differentially-expressed genes were chosen for further veri- fication using real-time RT-PCR. Results Heat map and matrix plot analysis found distinctly different expression profie in EBV positive and EBC negative gastric carcinoma cell lines,322 genes were detected to be expressed significantly different, genes which related with neoplasm involve many biological process, such as transcription, translation, signal transduction, adhesion, cell proli- feration, apoptosis and immune response. The expression trend of six genes for further verification was consistent with the microar- ray （three up-regulated and three down-regulated）. Conclusion Distinctly different expression profiles exist in EBV positive and EBC negative gastric carcinoma cell lines, suggesting that EBV infection may change the expression of associated genes, analyzing these genes may help clarify the action mechanism of EBV infection in the formation and development of EBVaGC, providing theo- ry basis for specific tumor markers and biological therapy of EBVaGC.