Demethylation of the miR-146a promoter by 5-Aza-2'-deoxycytidine correlates with delayed progression of castration-resistant prostate cancer

Abstract

Background: Androgen deprivation therapy is the primary strategy for the treatment of advanced prostate cancer; however, after an initial regression, most patients will inevitably develop a fatal androgen-independent tumor. Therefore, understanding the mechanisms of the transition to androgen independence prostate cancer is critical to identify new ways to treat older patients who are ineligible for conventional chemotherapy.Methods: The effects of 5-Aza-2'-deoxycytidine (5-Aza-CdR) on the viability and the apoptosis of the androgen-dependent (LNCaP) and androgen-independent (PC3) cell lines were examined by MTS assay and western blot analysis for the activation of caspase-3. The subcutaneous LNCaP xenografts were established in a nude mice model. MiR-146a and DNMTs expressions were analyzed by qRT-PCR and DNA methylation rates of LINE-1 were measured by COBRA-IRS to determine the global DNA methylation levels. The methylation levels of miR-146a promoter region in the different groups were quantified by the bisulfite sequencing PCR (BSP) assay.Results: We validated that 5-Aza-CdR induced cell death and increased miR-146a expression in both LNCaP and PC3 cells. Notably, the expression of miR-146a in LNCaP cells was much higher than in PC3 cells. MiR-146a inhibitor was shown to suppress apoptosis in 5-Aza-CdR-treated cells. In a castrate mouse LNCaP xenograft model, 5-Aza-CdR significantly suppressed the tumors growth and also inhibited prostate cancer progression. Meanwhile, miR-146a expression was significantly enhanced in the tumor xenografts of 5-Aza-CdR-treated mice and the androgen-dependent but not the androgen-independent stage of castrated mice. In particular, the expression of miR-146a was significantly augmented in both stages of the combined treatment (castration and 5-Aza-CdR). Additionally, the methylation percentage of the two CpG sites (-444 bp and -433 bp), which were around the NF-κB binding site at miR-146a promoter, showed the lowest methylation levels among all CpG sites in the combined treatment tumors of both stages.Conclusion: Up-regulating miR-146a expression via the hypomethylation of the miR-146a promoter by 5-Aza-CdR was correlated with delayed progression of castration-resistant prostate cancers. Moreover, site-specific DNA methylation may play an important role in miR-146a expression in androgen-dependent prostate cancer progression to androgen-independent prostate cancer and therefore provides a potentially useful biomarker for assessing drug efficacy in prostate cancer. © 2014 Wang et al.; licensee BioMed Central Ltd.