Identification and validation of the microRNA response elements in the 3′-untranslated region of the UDP glucuronosyltransferase (UGT)2B7 and 2B15 genes by a functional genomics approach

Posttranscriptional repression of UDP-glucuronosyltransferase (UGT) 2B7 and 2B15 expression by microRNAs (miRNAs) may be an important mechanism underlying inter-individual variability in drug glucuronidation. Furthermore, the UGT2B15 3'-UTR contains a common SNP (rs3100) that could influence miRNA binding. The aim of this study was to identify the complete complement of miRNAs that could regulate UGT2B7 and UGT2B15 expression through binding to the reference and/or variant 3'-UTRs. Luciferase reporter plasmids containing either the reference or variant 3'-UTRs were screened against a 2,048 human miRNA library to identify those miRNAs that decrease luciferase activity by at least 30% when co-transfected into HEK293 cells. Six novel miRNAs (miR-1293, miR-3664-3p, miR-4317, miR-513c-3p, miR-4483, and miR-142-3p) were identified that repressed the reference UGT2B7 3'-UTR, while twelve novel miRNAs (miR-770-5p, miR-103b, miR-3924, miR-376b-3p, miR-455-5p, miR-605, miR-624-3p, miR-4712-5p, miR-3675-3p, miR-6500-5p, miR-548as-3p, and miR-4292) repressed both the reference and rs3100 variant UGT2B15 3'-UTR. Deletion and mutagenesis studies confirmed the binding site location of each miRNA. Although the UGT2B15 rs3100 SNP was located within the miR-376c-3p response element, there was no effect on miRNA binding. miR-142-3p, miR-3664-3p, miR-4317, miR-455-5p, miR-376c-3p, miR-770-5p, miR-3675-3p, miR-331-5p, miR-605, and miR-376b-3p transcript levels were measured by quantitative PCR and correlated with UGT2B7 and UGT2B15 enzyme activities in 27 human liver samples. A significant negative correlation (R-S = -0.53; p = 0.005) was demonstrated between hepatic miR-455-5p transcript levels and UGT2B15-mediated S-oxazepam glucuronidation activities. Thus, the UGT2B7 and UGT2B15 3'-UTRs contain miRNA response elements for multiple miRNAs that may contribute to variable drug glucuronidation. (C) 2017 Elsevier Inc. All rights reserved.