Gene Expression Profile Of EBV Associated Gastric Carcinoma

Objective:①To detect and compare the gene expression profile between EBV positive gastric carcinoma cell lines and negative gastric carcinoma cell lines using the method of cDNA microarray, and screen differencially expressed genes potentially related with EBV-associated gastric carcinoma (EBVaGC).②To detect the expression level of microRNA-155(miR-155) in EBVaGC and EBV negative gastric carcinoma (EBVnGC) and analyze the relationship between EBV infections, microRNA-155 expression and cell differencially-expressed genes.Method:①Total RNA was extracted and purified from 4 gastric carcinoma cell lines (GT38, PT and SNU-719 were EBV positive; SGC7901 was EBV negative), then the first and second cDNA strain was generated, which was further 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, then the fluorescence signal was scanned by the computer and the expression profiles of two groups were compared. Specific differentially-expressed genes were chosen for further verification.②Total RNA was extracted from EBVaGC and EBVnGC tissues using the method of TRIzol or RNeasy FFPE Kit, which was purified afterwards. The expression level of miR-155 was detected by relative quantitative analysis using real-time RT-PCR, and then the relationship between microRNA-155 expression and clinical pathologic features of gastric carcinoma patients was analyzed.③Combining the study of gene expression profile, the potential target genes of miR-155 were calssified using the SAS system of national engeeering center for biochip at Shanghai. The relationship between microRNA-155 expression, EBV infection and cell differencially-expressed genes was analyzed.Result:①By comparing the expression profie between EBV positive gastric carcinoma cell lines and negative gastric carcinoma cell lines,993 genes were detected to be expressed significant differently, genes which may related with neoplasm involve many biological process, such as transcription, translation, signal transduction, adhesion, cell proliferation, apotosis and immune response. The expression trend of 6 genes for further verification was consistent with the microarray.②The delta Ct of miR-155 in EBVaGC and EBVnGC tissues was 6.71±1.94,6.15±1.31, respectively. The expression level of miR-155 in EBVaGC and EBVnGC tissues was not significantly different (t=0.207, P=0.837). The expression level of miR-155 in patients'gender, pathogenic sites, pathological classification or lymph node metastasis has no significant difference, whose P values were all greter than 0.05, however, significantly different in different age groups (F=2.98, P=0.026), namely, the expression level of miR-155 in 50～group was distinctly higher than that in 40～or 60～group (P value was 0.006,0.005, respectively), while no difference between other age groups.③Among the differencially-expressed genes induced by EBV,36 genes may be targeted by miR-155,11 of which were unknown for function. In the 25 function-known genes, most (12/25,48%) were associated with DNA-bingding function and were all up-regulated,7 genes (6 were up-regulated,1 was down-regulated) (7/25,28%) associated with protein-binding function, fewer associated with molecular transportation across the membrane, receptor activity, transcription and cell apotosis.Conclusion:①The expression profiles between EBV positive and negative gastric carcinoma cell lines have distinct difference, suggesting that EBV infection can change the expression of associated genes in gastric carcinoma cells, which may play roles in the formation, development and prognosis of EBVaGC.②The differecially-expressed genes involve many biological processes, suggesting that many genes and their mutations play roles in the formation of EBVaGC, analyzing these genes may help clarify the relationship between the complicated biological features of EBVaGC and cell gene expression, providing theory basis for specific tumor markers and biological therapy for EBVaGC.③In gastric carcinomas, EBV infection does not significantly change the expression level of miR-155, which may be related with the special latent state of EBV in gastric carcinomas; miR-155 was expressed higher in particular age groups, while its expression have no correlation with patients'gender, pathogenic sites, pathological classification or lymph node metastasis.④Among the differentially expressed genes related with miR-155, most of which are associated with transcription, suggesting that EBV and miR-155 may mutually act in the cell gene transcription process, providing theory basis and directions for further study.