Armartmuntree Napat, Jusakul Apinya, Sakonsinsiri Chadamas, Loilome Watcharin, Pinlaor Somchai, Ungarreevittaya Piti, Yong Chern Han, Techasen Anchalee, Imtawil Kanokwan, Kraiklang Ratthaphol, Suwannakul Nattawan, Kaewlert Waleeporn, Chaiprasert Timpika, Thanan Raynoo, Murata Mariko
Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand.
J Cancer. 2021 Mar 5;12(9):2673-2686. doi: 10.7150/jca.52378. eCollection 2021.
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.
启动子区域的DNA高甲基化通过表观遗传变化导致基因沉默。我们之前报道过，早期B细胞因子1（EBF1）在胆管癌（CCA）组织中表达下调，且与肿瘤进展相关。因此，我们推测EBF1启动子的DNA高甲基化会抑制CCA中EBF1的表达并促进其进展。在本研究中，利用公开的全基因组关联数据数据库，分析了CCA和正常胆管（NBD）组织中EBF1的DNA甲基化状态和mRNA表达水平。结果显示，与NBD组织相比，CCA组织中EBF1启动子区域的DNA甲基化显著增加。甲基化程度与EBF1 mRNA表达水平呈负相关。采用甲基化特异性PCR技术，对72例CCA组织中EBF1启动子区域的DNA甲基化率进行了研究。肿瘤组织中EBF1启动子区域甲基化率高的CCA患者（54/72）预后较差。所有CCA细胞系中EBF1启动子区域的甲基化率均高于永生化胆管细胞系（MMNK1）。用DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷（5-Aza-dC）处理后，CCA细胞中EBF1表达水平升高，DNA甲基化率降低。此外，CCA细胞中EBF1表达的恢复导致细胞生长、迁移和侵袭受到抑制。另外，RNA测序分析表明EBF1参与抑制癌症中的多种信号通路。综上所述，EBF1启动子区域的DNA高甲基化抑制EBF1表达并诱导CCA进展，具有侵袭性的临床结局。
