Abstract
Extensive DNA methylation is observed in gastric cancer with Epstein-Barr virus (EBV) infection, and EBV infection is the cause to induce this extensive hypermethylaton phenotype in gastric epithelial cells. However, some 5′ regions of genes do not undergo de novo methylation, despite the induction of methylation in surrounding regions, suggesting the existence of a resistance factor against DNA methylation acquisition. We conducted an RNA-seq analysis of gastric epithelial cells with and without EBV infection and found that TET family genes, especially TET2, were repressed by EBV infection at both mRNA and protein levels. TET2 was found to be downregulated by EBV transcripts, e.g. BARF0 and LMP2A, and also by seven human miRNAs targeting TET2, e.g., miR-93 and miR-29a, which were upregulated by EBV infection, and transfection of which into gastric cells repressed TET2. Hydroxymethylation target genes by TET2 were detected by hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq) with and without TET2 overexpression, and overlapped significantly with methylation target genes in EBV-infected cells. When TET2 was knocked down by shRNA, EBV infection induced de novo methylation more severely, including even higher methylation in methylation-acquired promoters or de novo methylation acquisition in methylation-protected promoters, leading to gene repression. TET2 knockdown alone without EBV infection did not induce de novo DNA methylation. These data suggested that TET2 functions as a resistance factor against DNA methylation in gastric epithelial cells and repression of TET2 contributes to DNA methylation acquisition during EBV infection.
Highlights
Aberrant DNA methylation is one of the major epigenomic alterations, and DNA hypermethylation of gene promoter regions inactivates tumor suppressor genes and strongly affects cancer development [1,2,3]
TET2 was markedly downregulated after Epstein-Barr virus (EBV) infection, and TET1 was expressed at low levels in both cells (Figure 1B)
Since TET2 expression was markedly decreased after EBV infection in both MKN7 and GES1 cells among the three TET family genes, and TET2 is involved in cytosine hydroxymethylation, we hypothesized that TET2 downregulation contributes to methylation, at least partially
Summary
Aberrant DNA methylation is one of the major epigenomic alterations, and DNA hypermethylation of gene promoter regions inactivates tumor suppressor genes and strongly affects cancer development [1,2,3]. EpsteinBarr virus (EBV)-positive gastric cancer shows a specific hypermethylation phenotype [4,5,6,7,8], which is reportedly the most extensive hypermethylation phenotype among all human malignancies [9]. By infecting low-methylation gastric cancer cells with EBV in vitro, previous studies have demonstrated that EBV infection itself causes the induction of extensive hypermethylation [7, 10, 11]. The molecular mechanism of methylation induction during EBV infection is largely unknown. Latent EBV infection upregulates DNA methyltransferases (DNMTs), resulting in extensive methylation in the EBV genome and, subsequently, in the host genome. EBV infection downregulates the expression of the host miR-200 family, which targets ZEB1 and ZEB2; the upregulation of ZEB1 and ZEB2 results in CDH1 repression [13]
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