Moreover, CCA cell lines and MMNK1 cell line were treated with the DNMT inhibitor 5-Aza-dC and subjected to MSP and EBF1 expression analyses using real-time quantitative polymerase chain reaction (RT-qPCR) and immunocytochemistry (ICC) techniques to confirm that the EBF1 expression level of CCA is regulated by its promoter hypermethylation. The gene discussed is EBF1; the disease is cholangiocarcinoma.