Promoter hypermethylation of early B cell factor 1 (EBF1) is associated with cholangiocarcinoma progression.

ABSTRACT

DNA hypermethylation in a promoter region causes gene silencing via epigenetic changes. We have previously reported that early B cell factor 1 (EBF1) was down-regulated in cholangiocarcinoma (CCA) tissues and related to tumor progression. Thus, we hypothesized that the DNA hypermethylation of EBF1 promoter would suppress EBF1 expression in CCA and induce its progression. In this study, the DNA methylation status of EBF1 and mRNA expression levels were analyzed in CCA and normal bile duct (NBD) tissues using a publicly available database of genome-wide association data. The results showed that the DNA methylation of EBF1 promoter region was significantly increased in CCA tissues compared with those of NBD. The degree of methylation was negatively correlated with EBF1 mRNA expression levels. Using methylation-specific PCR technique, the DNA methylation rates of EBF1 promoter region were investigated in CCA tissues (n=72). CCA patients with high methylation rates of EBF1 promoter region in the tumor tissues (54/72) had a poor prognosis. Higher methylation rates of EBF1 promoter region have shown in all CCA cell lines than that of an immortal cholangiocyte cell line (MMNK1). Upon treatment with the DNA methyltransferase inhibitor 5-Aza-dC, increased EBF1 expression levels and reduced DNA methylation rates were observed in CCA cells. Moreover, restoration of EBF1 expression in CCA cells led to inhibition of cell growth, migration and invasion. In addition, RNA sequencing analysis suggested that EBF1 is involved in suppression of numerous pathways in cancer. Taken together, DNA hypermethylation in the EBF1 promoter region suppresses EBF1 expression and induces CCA progression with aggressive clinical outcomes.