Upregulation Of NDRG1 Expression By Silencing DNA Methyltransferases Influences The Biological Behaviors Of Prostate Cancer DU145 Cells

Objective To investigate the influence of upregulation of NDRG1 through silencing DNA methyltransferases(DNMTs) by si RNAs on the biological behaviors of DU145 cells.Methods1.12 cases of prostate cancer(PCa) tissue samples and 12 cases of benign prostatic hyperplasia(BPH) tissue samples were collected from Tianjin Institute of Urology. The expression levels of DNMT1, DNMT3 b and NDRG1 in PCa and BPH tissues were detected by immunohistochemistry and RT-q PCR.2.Human PCa cell lines(DU145, PC-3) and normal prostate epithelial cell line(RWPE-1) were cultured in vitro. Western blot and RT-q PCR were performed to analyze the expression levels of DNMT1, DNMT3 b and NDRG1 in these cell lines.3.DNMT1-si RNA, DNMT3b-si RNA, DNMT1-si RNA+DNMT3b-si RNA and their negative control(NC), which were designed by us referring to the relevant literatures, were transferred into DU145 cells respectively. Western blot and RT-q PCR were performed to analyze the expression levels of DNMT1, DNMT3 b and NDRG1 in these cells. CCK8 assay, Muse cell analyzer, wound healing assay and Transwell migration assay were used to investigate the changes of cell biological behaviors. The cell group which had the most significant changes in biological cell behaviors and the highest expression level of NDRG1 were chosen for the subsequent experiments.4.NDRG1-si RNA and its negative control were transferred into the DU145 cells which had the most significant upregulation of NDRG1. Western blot and RT-q PCR were performed to analyze the expression level of NDRG1 in these cells. And the proliferation, apoptosis, migration and invasion of DU145 cells were also detected.Finally, the results were statistically analyzed.Results1.Immunohistochemistry and RT-q PCR results showed that the expression levels of DNMT1, DNMT3 b in prostate cancer tissues were higher than these in normal prostate tissues(P<0.05), and NDRG1 expression in normal prostate tissues was higher than that in prostate cancer tissues(P<0.05).2.Western blot and RT-q PCR showed that the expression levels of DNMT1,DNMT3 b in DU145 and PC-3 cells were higher than these in RWPE-1 cells(P<0.05),and NDRG1 expression in RWPE-1 cells was higher than that in DU145 and PC-3cells(P<0.05). And the expression of DNMT1, DNMT3 b in DU145 cells was higher than these in PC-3 cells(P<0.05).3.Western blot and RT-q PCR showed that DNMT1-si RNA and DNMT3b-si RNA could significantly inhibit the expression of DNMT1, DNMT3 b and improve the expression of NDRG1(P<0.05). CCK8 assay, wound healing assay and Transwell migration assay showed that the proliferation, migration and invasion of DU145 cells were significantly inhibited(P<0.05). Muse cell analyzer showed that the apoptosis of DU145 cells were significantly enhanced(P<0.05). Among the five cell groups,DNMT1-si RNA group had the most significant effect, but the co-transfection had no significant synergistic effect(P>0.05).4.Western blot and RT-q PCR showed that NDRG1-si RNA could significantly inhibit the expression of NDRG1(P<0.05). CCK8 assay, Muse cell analyzer, wound healing assay and Transwell migration assay showed that the biological behaviors of DU145 cells were significantly reversed(P<0.05).Conclusions1.The expression of DNMT1, DNMT3 b in prostate cancer cells is higher these in normal prostate cells; The expression of NDRG1 in normal prostate cells is higher that in prostate cancer cells.2.NDRG1 implicated as a tumor suppressor gene could inhibit the proliferation and invasion of prostate cancer cells.3.DNMT1, DNMT3 b could increase the methylation of NDRG1, which could downregulate the expression of NDRG1, promoting the proliferation and invasion of prostate cancer cells. And DNMT1 plays a major role in this process.4.Inhibiting the expression of DNMT1, DNMT3 b could restore the expression of NDRG1 partially, inhibiting the proliferation and invasion of prostate cancer cells.