Upregulation of N - myc downstram regulated gene 1 expression by silencing DNA methyltransferases influences the biological behaviors of prostate cancer DU145 cells

Abstract

Objective To investigate the influence of the upregulation of N-myc downstram regulated gene 1 (NDRG1) expression by silencing DNA methyltransferases (DNMTs) using small interfering RNAs (siRNAs) on the biological behaviors of prostate cancer DU145 cells. Methods SiRNAs were used to inhibit the expression of DNMT1, DNMT3b of DU145 cells; Western blotting and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) were used to detect the expression levels of DNMT1, DNMT3b and NDRG1 of DU145 cells; cell counting kit-8 (CCK-8) assay, muse cell analyzer, wound healing assay and Transwell migration assay were used to detect the proliferation, apoptosis, migration and invasion of DU145 cells; then we inhibited the expression of NDRG1 of DU145 cells which had the most significant changes of cell biological behaviors; and we detected the expression level of NDRG1 and the changes of cell biological behaviors. Results Western blotting and RT-qPCR showed that the mRNA (1.59±0.03, 1.23±0.01, 1.36±0.11 vs.0.90±0.15, 1.00±0.03) and protein (1.07±0.16, 0.49±0.01, 0.92±0.09 vs.0.23±0.04, 0.30±0.07) expression levels of NDRG1 of DNMT1 inhibition group, DNMT3b inhibition group and double inhibition group were significantly higher than these in control groups (P<0.05). CCK-8 assay showed that, at 24, 48 and 72 h, the optical density (A) values of DNMT1 inhibition group (0.189±0.030, 0.266±0.048, 0.419±0.058), DNMT3b inhibition group (0.221±0.023, 0.318±0.072, 0.498±0.064) and double inhibition group (0.199±0.021, 0.284±0.062, 0.488±0.091) were significantly lower than control groups (P<0.05). Apoptosis results showed that the apoptosis rates of DNMT1 inhibition group (22.40±1.36)%, DNMT3b inhibition group (14.20±0.80)% and double inhibition group (17.60±0.74)% were significantly higher than control groups (P<0.05). Wound healing assay showed that the healing rates of DNMT1 inhibition group (53.01±3.22)%, DNMT3b inhibition group (70.97±2.12)% and double inhibition group (63.93±4.42)% were significantly lower than control groups (P<0.05). Transwell migration assay showed that the amount of invasion cells in DNMT1 inhibition group [(16±3) cells], in DNMT3b inhibition group [(32±5) cells] and in double inhibition group [(20±4) cells] were significantly less than control groups (P<0.05). Among these groups, DNMT1 inhibition group had the most significant effect (P<0.05), but the co-transfection had no significant synergistic effect. Compared with control groups, NDRG1-siRNA could significantly reverse the changes of cell biological behaviors of DU145 cells (P<0.05). Conclusion Inhibition of DNMT1, DNMT3b of DU145 cells could partially restore NDRG1 expression, which could inhibit the proliferation, apoptosis, migration and invasion of cancer cells. Moreover, DNMT1 inhibition group had the most significant effect, but the co-transfection had no significant synergistic effect, suggesting that DNMT1 could become an effective target for demethylation treatment of prostate cancer. Key words: N-myc downstram regulated gene 1; DNA methylation; DNA methyltransferase; RNA interference; Prostate cancer