Background: DNA methyltransferase 1 (DNMT1) mutants display altered methylation patterns that may contribute to oncogenesis. We hypothesized that the silencing or inhibition of DNMT1 may affect the malignancy of hepatocellular carcinoma (HCC) cells.
Methods: The HCC cell line KYN2 was used to construct 3 experimental groups: i) a DNMT1-siRNA group transfected with a green fluorescent protein (GFP) lentiviral vector to silence endogenous DNMT1 gene expression, which was confirmed by real-time quantitative polymerase chain reaction, ii) a 5-Aza-CdR group transfected with a null GFP lentiviral vector and treated with the DNMT1 inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR), and iii) a control group transfected with a null GFP lentiviral vector. Cellomics ArrayScan VTI imaging and MTT assays were conducted to assess cell proliferation. Cell cycle phase arrest and apoptosis were assayed by flow cytometry. Colony formation was assessed by fluorescence microscopy.
Results: DNMT1 mRNA expression was significantly inhibited in DNMT1-silenced cells relative to control cells (p < 0.05), indicating successful transfection and gene expression knockdown. Cell proliferation was significantly inhibited in DNMT1-siRNA and 5-Aza-CdR cells relative to control cells (p < 0.05). G1-to-S phase shifts were significantly increased in DNMT1-siRNA and 5-Aza-CdR cells relative to control cells (p < 0.05). Apoptosis was significantly increased in DMNT1-siRNA and 5-Aza-CdR cells relative to control cells (p < 0.05). DMNT1-siRNA and 5-Aza-CdR cells displayed significantly reduced colony formation relative to control cells (p < 0.05). Notably, 5-Aza-CdR had more pronounced effects upon all these parameters than DNMT1 silencing.
Conclusion: DNMT1 activity appears to positively contribute to the malignancy of HCC cells.
© 2016 S. Karger GmbH, Freiburg.