Department of Pharmaceutical Sciences, Washington State University College of Pharmacy and Pharmaceutical Sciences, Spokane, Washington (A.K.S., C.J.W.W., M.H., G.C., P.L.); and Department of Clinical Pharmacy, University of Michigan College of Pharmacy, Ann Arbor, Michigan (J.S., H.-J.Z.).
Department of Pharmaceutical Sciences, Washington State University College of Pharmacy and Pharmaceutical Sciences, Spokane, Washington (A.K.S., C.J.W.W., M.H., G.C., P.L.); and Department of Clinical Pharmacy, University of Michigan College of Pharmacy, Ann Arbor, Michigan (J.S., H.-J.Z.)
Mol Pharmacol. 2019 Dec;96(6):674-682. doi: 10.1124/mol.119.115881. Epub 2019 Sep 25.
The UDP-glucuronosyltransferase (UGT) family of enzymes is 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 is a major family of UGT enzymes expressed in human liver. The identification of novel mechanisms including post-transcriptional regulation by microRNA (miRNA) contributes to interindividual variability in UGT2B expression and is a crucial component in predicting patient drug response. In the present study, a high-resolution liquid chromatography-tandem mass spectrometry method was employed to measure UGT2B protein levels in a panel of human liver microsomal samples ( = 62). Concurrent in silico analysis identified eight candidate miRNAs as potential regulators of UGT2B enzymes. Comparison of UGT2B protein expression and candidate miRNA levels from human liver samples demonstrated a significant inverse correlation between UGT2B10 and UGT2B15 and one of these candidate miRNAs, miR-485-5p. A near-significant correlation was also observed between UGT2B7 and miR-485-5p expression. In vitro analysis using luciferase-containing vectors suggested an interaction of miR-485-5p within the UGT2B10 3'-untranslated region (UTR), and significant reduction in luciferase activity was also observed for a luciferase vector containing the UGT2B7 3'-UTR; however, none was observed for the UBT2B15 3'-UTR. UGT2B10 and UGT2B7 activities were probed using nicotine and 3'-azido-3'-deoxythymidine, respectively, and significant decreases in glucuronidation activity were observed for both substrates in HuH-7 and Hep3B cells upon overexpression of miR-485-5p mimic. This is the first study demonstrating a regulatory role of miR-485-5p for multiple UGT2B enzymes. SIGNIFICANCE STATEMENT: The purpose of this study was to identify novel epigenetic miRNA regulators of the UGT2B drug-metabolizing enzymes in healthy human liver samples. Our results indicate that miRNA 485-5p is a novel regulator of UGT2B7 and UGT2B10, 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.
UDP-葡糖醛酸基转移酶（UGT）家族的酶在多种内源性化合物（如胆汁酸、类固醇和脂溶性维生素）以及外源性化合物（包括许多药物）的代谢消除中起着重要作用。UGT2B 亚家族是人类肝脏中表达的主要 UGT 酶家族。新机制的鉴定，包括 miRNA（miRNA）的转录后调控，有助于 UGT2B 表达的个体间变异性，是预测患者药物反应的关键组成部分。在本研究中，采用高分辨率液相色谱-串联质谱法测定了一组人肝微粒体样本中的 UGT2B 蛋白水平（= 62）。同时进行的计算机分析鉴定了 8 种候选 miRNA 作为 UGT2B 酶的潜在调节剂。人肝样本中 UGT2B 蛋白表达和候选 miRNA 水平的比较表明，UGT2B10 和 UGT2B15 与其中一种候选 miRNA（miR-485-5p）之间存在显著的负相关。UGT2B7 和 miR-485-5p 表达之间也观察到近乎显著的相关性。使用包含荧光素酶的载体进行的体外分析表明，miR-485-5p 与 UGT2B10 的 3'非翻译区（UTR）相互作用，并且还观察到包含 UGT2B7 3'UTR 的荧光素酶载体的荧光素酶活性显著降低；然而，UGT2B15 的 3'UTR 则没有。使用尼古丁和 3'-叠氮-3'-脱氧胸苷分别探测 UGT2B10 和 UGT2B7 的活性，并且在 HuH-7 和 Hep3B 细胞中转染 miR-485-5p 模拟物后，两种底物的葡萄糖醛酸化活性均显著降低。这是第一项证明 miR-485-5p 对多种 UGT2B 酶具有调节作用的研究。意义陈述：本研究的目的是在健康人肝样本中鉴定 UGT2B 药物代谢酶的新型表观遗传 miRNA 调节剂。我们的结果表明，miRNA-485-5p 是 UGT2B7 和 UGT2B10 的新型调节剂，它们在许多常用药物、致癌物和内源性化合物的代谢中起着重要作用。本研究使用新型蛋白质组学方法鉴定了潜在的 miRNA-UGT2B mRNA 相互作用，并进行了体外实验验证这些相互作用。
