Reversal effect of 5-aza-2-deoxycytidine on the maternally expressed gene 3 promoter hypermethylation and its inhibitory effect on the proliferation of epithelial ovarian cancer cells

Abstract

To investigate the reversal effects of different concentrations of DNA methylation inhibitor, 5-aza-2-deoxycytidine, on the hypermethylation of maternally expressed gene 3 (MEG3) gene promoter, and then the inhibitory effect of restoration of MEG3 expression on the proliferation of ovarian cancer cells. Human ovarian cancer OVCAR3 cells were treated with various concentration of 5-aza-2-deoxycytidine (0, 1, 5, 10, 20 µmol/L, respectively) for 6 days. Then the methylation status of MEG3 promoter was detected by methylation specific PCR (MSP). The alteration of MEG3 gene expression was detected by RT-PCR. Cell proliferation was determined by MTT assay and EdU incorporation assay. After treated with 5-aza-2-deoxycytidine, the methylation status of MEG3 in the 0, 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine groups were 1.00 ± 0.00, 0.79 ± 0.00, 0.67 ± 0.00, 0.65 ± 0.03 and 0.61 ± 0.01 folds, respectively (P < 0.05 for all). The relative expressions of MEG3 mRNA in the 0, 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine groups were 1.00 ± 0.00, 2.04 ± 0.16, 2.44 ± 0.17, 3.19 ± 0.34 and 5.34 ± 0.39, respectively (P < 0.05 for all). In contrast to the negative control, the inhibition rates of the OVCAR3 cell growth were increased significantly when treated with 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine in 2, 4 and 6 days. There were (40.78 ± 0.80)%, (35.65 ± 0.33)%, (31.81 ± 0.66)%, (27.33 ± 1.27)% and (17.75 ± 1.85)% of EdU-positive cells in the 0, 1, 5, 10 and 20 µmol/L 5-aza-2-deoxycytidine groups (P < 0.01 for all). Maternally expressed gene 3 promoter hypermethylation is reversed by 5-aza-2-deoxycytidine in ovarian cancer cells. The downregulation of MEG3 gene might be resulted from the methylation, and the re-expression of MEG3 partly contribute to the growth inhibition of epithelial ovarian cancer cells.