Abstract
The UDP-glucuronosyltransferase (UGT) family of enzymes are important in the metabolic elimination of a variety of endogenous compounds such as bile acids, steroids, and fat-soluble vitamins, as well as exogenous compounds including many pharmaceuticals. The UGT2B subfamily are the predominant isoforms expressed in human liver. The identification of novel mechanisms including post-transcriptional regulation by microRNA (miRNA) contribute to inter-individual variability in UGT2B expression and are crucial components in predicting patient drug response. In the present study, a high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was employed to measure UGT2B protein levels in a panel of human liver microsomal samples. Concurrent in silico analysis identified eight candidate miRNA as potential regulators of UGT2B enzymes. Comparison of UGT2B protein expression and candidate miRNA levels from human liver samples demonstrated a significant (P<0.05) inverse correlation between UGT2B10 and one of these candidate miRNAs, miR-485-5p. In vitro analysis using luciferase-containing vectors suggested an interaction of miR-485-5p within the 3′ untranslated region (UTR) of UGT2B10 (P=0.0073). A significant reduction in luciferase activity (P=0.022) was also observed for luciferase vectors containing the UGT2B7 3' UTR, an effect manifested by one of two potential miR-485-5p-binding sites. UGT2B10 and UGT2B7 activities were probed using nicotine and AZT, respectively, and significant decreases in glucuronidation activity were observed for both substrates in HuH-7 and Hep3B cells (P<0.0003 and P=0.016, and P=0.017 and P=0.030, respectively) upon over-expression of miR-485-5p mimic. This is the first study demonstrating a regulatory role for miR-485-5p for multiple UGT2B enzymes.
SIGNIFICANCE STATEMENT The purpose of this study was to identify novel epigenetic microRNA regulators of the UDP-glucuronosyltransferase (UGT) 2B drug-metabolizing enzymes in healthy human liver samples. Our results indicate that microRNA 485-5p is a novel regulator of UGTs 2B7 and 2B10, which play an important role in the metabolism of many commonly prescribed medications, carcinogens and endogenous compounds. This study identified potential miRNA-UGT2B mRNA interactions using a novel proteomic approach, with in vitro experiments undertaken to validate these interactions.
- The American Society for Pharmacology and Experimental Therapeutics