Methylation-associated miR193b silencing activates master drivers of aggressive prostate cancer.

Abstract

240 Background: Epigenetic changes in DNA methylation, histone modifications and miRNA expression have major roles in prostate cancer (PC) initiation and progression. Previous screening for epigenetically regulated miRNAs in PC revealed hypermethylation of the miR-193b promoter, but no related functional studies in PC have been reported. Methods: Methylation was measured by methylation-specific PCR. Quantitative RT-PCR was used to validate the regulation of potential miR-193b targets. Luciferase reporter assays were applied to detect the regulation of miR-193b-targets. The promoter methylation status was modified by DNMT and HDAC inhibitors (5-azacytidine and mocetinostat). Results: miR-193b level is inversely correlated with the degree of its promoter’s methylation in the TCGA cohort. Distinct miR-193b promoter methylation patterns were detected in multiple normal and PC cell lines. Overexpression of miR-193b in PC cells with low miR193b level induced cell growth inhibition, apoptosis, or inhibition of invasion. Previously reported top 150-downregulated genes by miR-193b in liposarcoma showed a high correlation with PC progression in multiple PC cohorts. We further identified FOXM1 and RRM2 as the direct targets of miR-193b. Knockdown of FOXM1 and RRM2 in PC cells phenocopied miR-193b with respect to inhibition of invasion and induction of apoptosis. Furthermore, combination treatment with DNMT and HDAC inhibitors released the methylation-regulated silencing of miR-193b in PC cells, resulting the inhibition of FOXM1 and RRM2 expression. Conclusions: We revealed the tumor suppressive function of miR-193b in PC. Overexpression of miR-193b in PC cells with low miR193b level induced cell growth inhibition, apoptosis, or inhibition of invasion. A gene set identified in liposarcoma cells by overexpression of miR-193b showed a high correlation with PC progression in multiple PC cohorts. FOXM1 and RRM2 may be the key targets of miR-193b in PC. Our findings suggest that methylation-silencing miR-193b in PC may release the inhibition of some key oncogenes to contribute PC progression, which could provide a possible therapeutic mechanism for PC therapy.