Abstract
UGT2B15 is an important androgen-metabolizing UDP-glucuronosyltransferase (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, noncanonical 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, cotransfection of both microRNAs (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 Cancer Genome Atlas (TCGA) hepatocellular carcinoma data set 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-metabolizing UGTs.
Footnotes
- Received October 25, 2017.
- Accepted November 30, 2017.
↵1 D.G.H. and R.M. contributed equally to this work.
R.A.M. is a Cancer Council/SA Health Beat Cancer Professorial Chair. During the preparation period, P.I.M. was a National Health and Medical Research Council Senior Principal Research Fellow; R.M. was an Australian Research Council Future Fellow.
This study was supported by the National Health and Medical Research Council of Australia [Grant ID1020931 (to P.I.M.) and Grant ID1085410 (to P.I.M., R.A.M., and R.M.)]. The project was also supported by funding from the Flinders Medical Centre Foundation.
↵This article has supplemental material available at jpet.aspetjournals.org.
- Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics
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