| Epstein-Barr virus (EBV) is an important DNA virus of the y-herpesvirus family that is ubiquitous in human population, which has close relationship with many kinds of human malignant tumor. EBV could infect human B lymphocytes and stratified squamous epithelium in vivo and results in a variety of tumors derived from either B cell origin or epithelial origin. The EBV gene expression in EBVaGC and NPC cells is restricted, and that might be related to the epigenetic mechanisms on regulating virus gene promoter usage, in particular DNA methylation.Objective To explore the methylation status of EBV early gene BARF1and BHRF1promoters and the influence of the methylation status on transcriptional expression, and further to research the roles of BARF1and BHRF1played in the occurrence of EBVaGC.Methods The methylation status of BARF1and BHRF1promoters in EBV-positive cell lines (GT38, B95-8, GT39, Raji, PT, C666-1, OB, SNU719) and EBVaGC tissues were detected by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS). The methylation status of the promoters was detected by bisulfite genomic sequencing and BARF1and BHRF1gene expression was detected by real-time quantitative PCR before and after the treatment with5-Aza-CdR in GT38, GT39and B95-8cell lines.Results①MSP results showed that BARF1and BHRF1gene promoters were hypermethylated in different degrees. The methylation staus of BARF1and BHRF1gene promoters by MSP were M type in5EBV positive cell lines (GT38, GT39, PT, C666-1and Raji) and M+U type in3EBV positive cell lines (SNU719, B95-8, and OB).②BGS results showed that the cell lines which MSP result is M-type (Raji, GT38, GT39) are highly methylated and the cell line which MSP result is M+U-type (B95-8) is partly methylated.③MSP results in EBVaGCs tissues showed that31cases (31/43,72.1%) were M-type and12cases (12/43,27.9%) were M+U-type in BARF1.36cases (36/43,83.7%) were M-type and7cases (7/43,16.3%) were M+U-type in BHRF1.④The methylation status of BARF1and BHRF1promoters in B95-8, GT39, AGS-EBV and GT38cell lines were significantly reduced after treated with5-Aza-CdR.⑤The expression of BARF1and BHRF1gene was raised after the treatment of5-Aza-CdR, demonstrating that the methylation status could influence the expression. The expression of BARF1gene raised2.13times in B95-8,6.50times in Raji,73.53times in GT38and2.50times in GT39after the treatment. The expression of BHRF1gene raised3.39times in B95-8,2.46times in Raji,6.45times in GT38and16.67times in GT39after the treatmentConclusion①The expressions of EBV early genes BHRF1was closely related to the methylation status of BHRF1promoter. The expression of BARF1might be influenced by the methylation status of BARF1promoter. Methylation is an important mechanism to regulate the activity of promoter of EBV early genes.②The methylation status of promoter and expression of BARF1and BHRF1are related to each other, manifested by the fact that the methylation status of promoter and expression of BARF1and BHRF1are dictinctive in different kinds of EBV-positive cell lines, but in the same cell line, the methylation status of promoter and expression of BARF1and BHRF1are resemble to each other.③The methylation status of BARF1and BHRF1promoters in EBVaGCs tissues are hypermethylated, it is suggested that hypermethylated status mediated BARF1and BHRF1expression, EBV maintain in latency stage to evade the immunological survelillance of the host, and then maintain the persistent infection and play an important role in the occurrence and development of EBVaGC. |
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