Objective: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
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