Promoter Gene Methylation Regulates Clooxygenase-2 Expression in Androgen-Dependent and Independent Prostate Cancer Cells

Background: Clooxygenase-2 (COX-2) expression is overexpressed in human prostate cancer, and aberrant methylation of the COX-2 promoter has also been elucidated. However, how the methylation of CpG islands at COX-2 regulates its expression in prostate cancer is still unclear. We will determine the methylated 5′ CpG island of the COX-2 gene and its role in the expression of COX-2 in prostate androgen-dependent and androgen-independent cancer cells, LNCaP and DU145. Methods: We used western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to confirm the COX-2 expression in prostate cancer cell lines, including LNCaP (androgendependent) and DU145 (androgen-independent) cells. To investigate whether the COX-2 expression was regulated by the methylation status of the 5′ CpG island, we treated LNCaP and DU145 cells with the DNA methylation inhibitor, 5-aza-2′-deoxycytidine, and determined COX-2 expression in the treated/untreated cells by western blotting and qRT- PCR. Subsequently, bisulfite sequencing was performed to study the methylation sites in the treated/untreated cells. The effects of 5-aza-2′- deoxycytidine to cell proliferation, cell migration and cell cycle process in DU145 and LNCaP cells were determined using Cell Counting Kit-8 (CCK-8) assay, transwell assay and flow cytometry, respectively. Results: The results revealed that the expression of COX-2 in androgen-dependent LNCaP cells was 5.44-fold (in protein level) and 2.46-fold (in mRNA level) higher than that in androgen-independent DU145 cells. After 5-aza-2′-deoxycytidine treatment, COX-2 expression in DU145 cells was elevated significantly, but no change was found in LNCaP cells. The A and C regions of the COX-2 CpG island exhibited reduced methylation along with that an increased expression of COX-2 was noted in DU145 cell after 5-aza-2′-deoxycytidine treatment. Also, the treatment with 5-aza-2′-deoxycytidine inhibited cell proliferation, cell migration and influenced the cell cycle progression in both DU145 and LNCaP cells.