The Methylation Status and Expression of Epstein-Barr Virus Early Genes BARF1 and BHRF1 in Epstein-Barr Virus-Associated Gastric Carcinomas

Abstract

Epstein-Barr virus (EBV) is an important DNA virus which establishes latent infection in human malignancies. Expression of EBV-encoded genes in the associated tumors is strongly modulated by promoter CpG methylation of EBV genome. This study aimed to explore the methylation status of the promoters of EBV BamHI-A rightward frame 1 (BARF1) and BamHI-H rightward open reading frame 1 (BHRF1) and their influence on transcriptional expression, to further understand the roles of BARF1 and BHRF1 in the occurrence of EBV-associated cancer. We evaluated the methylation status of BARF1 and BHRF1 promoters in 43 EBV-associated gastric carcinoma (EBVaGC) tissues and EBV-positive cell lines. Their expressions were evaluated by real-time quantitative PCR. We found that the promoters of BARF1 and BHRF1 were methylated by varying degrees in different EBV-positive cell lines and were almost hypermethylated in all EBVaGC tissues. The methylation status of BARF1 and BHRF1 promoters were significantly reduced by 5-Aza-CdR along with the increasing gene expressions. Hypermethylation of Ap and Hp mediates the frequent silencing of BARF1 and BHRF1 in EBV-associated tumors, which could be reactivated by a demethylation agent, suggesting that promoter demethylation and activation is important for BARF1 and BHRF1 transcription and their further action.

Figure 1

The methylation status of BARF1p and BHRF1p analyzed by MSP in EBV-positive cell lines. (a, c) MSP analysis of the methylation status of BARF1 and BHRF1 gene promoters before 5-Aza-CdR treatment in EBV-positive cell lines: PT, GT38, C666-1, Raji, GT39, SNU719, B95-8, and OB. (b, d) MSP analysis of the methylation status of BARF1 and BHRF1 in EBV-positive cell lines after 5-Aza-CdR treatment. (a, b) BARF 1p. (b, d) BHRF1p. M, methylated; U, unmethylated.

Figure 2

The methylation status of CpG about BARF1and BHRF1 gene promoters (BSP). Each circle is one CpG site and filled circles are methylated CpG sites. (a, b) The methylation status of CpG about BARF1 and BHRF1 gene promoters before 5-aza treatment. There are 50 and 26 CpG sites in BARF1 and BHRF1 promoters, respectively. The methylation ratio of BARF1 before 5-aza in Raji, B95-8, GT39, and GT38 was 98.0%, 46.7%, 48.3%, and 95.3%, respectively, and that of BHRF1 was 83.3%, 32.7%, 16.0%, and 97.4%, respectively. (c, d) The methylation status of CpG about BARF1and BHRF1 gene promoters before and after 5-aza treatment. After 10 μmol/L 5-aza treatment, the methylation ratio of BARF1 in B95-8 and GT38 turned to 0 and 75.0% and that of BHRF1 turned to 0 and 52.6%, respectively.

Figure 3

The methylation status of BARF1 and BHRF1 promoters in gastric carcinoma tissue by MSP and BSP. (a, c) The methylation status of BARF1 and BHRF1 promoters in gastric carcinoma tissue by MSP. M, methylated; U, unmethylated. (b) The methylation status of the 2 early gene promoters in gastric carcinoma tissue by BS. “Black circle” indicates methylated CpG site; “white circle” indicates unmethylated CpG site. The CpG sites in BSP region of BARF1and BHRF1 were 50 and 26. (d) Sequencing result of BARF1 gene promoter in EBV-positive gastric carcinoma cell line named GT38. The 8 CpG sites in the sequence correspond to 32–40 CpG sites in Figure 2(a).

Figure 4

The relative expression of BARF1 and BHRF1. (a) Expression level of BARF1 and BHRF1 in the cell lines before and after treatment with demethylation reagent. Analysis used was the 2^−ΔΔCt method, and the expression level of untreated cells was set as 1. The expression of BARF1 in all cell lines was significantly upregulated after 5-aza treatment (P < 0.05); the expression difference of BHRF1 in SNU719 before and after treatment was not significant. While the difference of BHRF1 in other cell lines was significant (p < 0.05). (b) Expression level of BARF1 and BHRF1 in EBVaGC samples. The level showed in the figure is mean ± SD. The expression of BARF1 and BHRF1 in samples with form M + U (n = 3) was higher than that in the samples with form M (n = 7), but the difference is not significant. EBVaGC, EBV-associated gastric carcinoma. M, methylated; U, unmethylated.