Induction of alpha-methylacyl-CoA racemase by miR-138 via up-regulation of β-catenin in prostate cancer cells.

Abstract

Alpha-methylacyl-CoA racemase (AMACR) is highly overexpressed in prostate cancer (PCa) and its transcriptional regulators include various transcription factors and CTNNB1/β-catenin. Our previous findings suggested a post-transcriptional regulation by the tumor-suppressive microRNA miR-138 in PCa. Thus, the aim of this study was to demonstrate the direct interaction of miR-138 with the 3'-UTR of AMACR. Furthermore, the influence of miR-138 on the expression of AMACR and selected AMACR regulators was investigated in PCa cells. Using DU-145, PC-3, and LNCaP PCa cells, the effect of exogenous miR-138 on AMACR and selected AMACR regulators was determined by quantitative PCR and Western blot. Luciferase reporter assays were used to verify target and promoter interaction. Using a luciferase reporter assay a direct interaction of miR-138 with the AMACR-3'-UTR was confirmed. Surprisingly, AMACR expression was up-regulated by up to 125% by exogenous miR-138 in PCa cells. The lack of any miR-138 binding sites within the AMACR promoter suggested an indirect mechanism of up-regulation. Therefore, the effect of miR-138 on selected AMACR regulators including CTNNB1/β-catenin, RELA, SMAD4, SP1, and TCF4 was evaluated. MiR-138 solely evoked an up-regulation of CTNNB1 mRNA expression and β-catenin protein levels by up to 75%. Further in silico analysis revealed a binding site for miR-138 within the CTNNB1 promoter. MiR-138 could enhance the activity of the CTNNB1 promoter, which in turn could contribute to the observed AMACR up-regulation. The present findings suggest that miR-138 can indirectly up-regulate AMACR via transcriptional induction of CTNNB1, at least in vitro in PCa cells.