Si-DNMT1 restore tumor suppressor genes expression through the reversal of DNA hypermethylation in cholangiocarcinoma

Abstract

The aim of our study was to evaluate the effect of shorthairpin RNA plasmid vector knockdown of human DNA methyltransferase 1 on proliferation and the methylation status and expression of tumor suppressor genes in hilar cholangiocarcinoma. The hilar cholangiocarcinoma cell line QBC939 was utilized for this study. QBC939 cells were transfected with a shorthairpin RNA plasmid vector targeting human DNA methyltransferase 1. Control and human DNA methyltransferase 1 shorthairpin RNA plasmid vector-transfected cells were collected at different time points, and the expression levels of human DNA methyltransferase 1 and tumor suppressor genes (cyclin-dependent kinase inhibitor 2B, cyclin-dependent kinase inhibitor 2A, RAS association domain family 1, and cadherin-1) were detected by reverse transcription-polymerase chain reaction. Furthermore, interfering efficiency was confirmed by Western blotting. The methylation status of tumor suppressor genes was detected using methylation-specific polymerase chain reaction. Furthermore, the effect of human DNA methyltransferase 1 knockdown on proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Targeted gene knockout of human DNA methyltransferase 1 restored the expression levels of tumor suppressor genes cyclin-dependent kinase inhibitor 2B, cyclin-dependent kinase inhibitor 2A, RAS association domain family 1, and cadherin-1, indicating that the silencing of these tumor suppressor genes is associated with promoter hypermethylation. In addition, knockdown of human DNA methyltransferase 1 expression significantly inhibited the proliferation of QBC939 cells. Targeted knockdown of human DNA methyltransferase 1 expression restores the expression levels of tumor suppressor genes, thus inhibiting the proliferation of QBC939 cells. These results may provide insight for the development of novel therapies for cholangiocarcinoma.