Regulation of UDP-Glucuronosyltransferase 2B15 by miR-331-5p in prostate cancer cells involves canonical and non-canonical target sites.

UGT2B15 is an important androgen metabolizing UGT and the mechanisms controlling its expression are of considerable interest. Recent studies showed that miR-376c regulates UGT2B15 in prostate cancer cells via a canonical target site in the 3'untranslated region (3'UTR). The UGT2B15 3'UTR also contains a canonical miR-331-5p target site; previous work indicated that deleting this site reduced, but did not abolish, the ability of miR-331-5p to repress a luciferase reporter carrying the UGT2B15 3'UTR. We report here the discovery and characterization of a second, non-canonical miR-331-5p target site in the UGT2B15 3'UTR. miR-331-5p-mediated repression of a UGT2B15 3'UTR-reporter was partly inhibited by mutating either of the two miR-331-5p target sites separately, but completely abolished by mutating the two sites simultaneously, indicating that the two sites act cooperatively. miR-331-5p mimics significantly reduced both UGT2B15 mRNA levels and glucuronidation activity in prostate cancer cells, confirming that the native transcript is a miR-331-5p target. Transfection of either miR-331-5p or miR-376c mimics repressed the activity of the UGT2B15 3'UTR-reporter; however, co-transfection of both miRNAs further reduced activity, indicating cooperative regulation by these two miRNAs. A significant negative correlation between miR-331 and UGT2B15 mRNA levels was observed in a tissue RNA panel, and analysis of the TCGA Hepatocellular Carcinoma dataset provided further evidence that miR-331 may play an important role in regulation of UGT2B15 in vivo. There was no significant correlation between miR-331 and UGT2B15 mRNA levels in the TCGA Prostate Adenocarcinoma cohort, which may reflect the complexity of androgen-mediated regulation in determining UGT2B15 levels in prostate cancer. Finally we show that miR-331-5p does not regulate UGT2B17, providing the first evidence for a post-transcriptional mechanism that differentially regulates these two important androgen-metabolising UGTs.