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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.

ABSTRACT: 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 (Rs?=?-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.

SUBMITTER: Papageorgiou I

PROVIDER: S-EPMC6188638 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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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.

Papageorgiou Ioannis I Court Michael H MH

Biochemical pharmacology 20170928

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 re ...[more]

PMID: 28962835

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Project description:Because human prostate-distributed UDP-glucuronosyltransferase (UGT) 2B15 metabolizes 5α-dihydrotestosterone (DHT) and 3α-androstane-5α,17β-diol metabolite, we sought to determine whether 2B15 requires regulated phosphorylation similar to UGTs already analyzed. Reversible down-regulation of 2B15-transfected COS-1 cells following curcumin treatment and irreversible inhibition by calphostin C, bisindolylmaleimide, or röttlerin treatment versus activation by phorbol 12-myristate 13-acetate indicated that 2B15 undergoes PKC phosphorylation. Mutation of three predicted PKC and two tyrosine kinase sites in 2B15 caused 70-100 and 80-90% inactivation, respectively. Anti-UGT-1168 antibody trapped 2B15-His-containing co-immunoprecipitates of PKCα in 130-140- and >150-kDa complexes by gradient SDS-PAGE analysis. Complexes bound to WT 2B15-His remained intact during electrophoresis, whereas 2B15-His mutants at phosphorylation sites differentially dissociated. PKCα siRNA treatment inactivated >50% of COS-1 cell-expressed 2B15. In contrast, treatment of 2B15-transfected COS-1 cells with the Src-specific activator 1,25-dihydroxyvitamin D(3) enhanced activity; treatment with the Src-specific PP2 inhibitor or Src siRNA inhibited >50% of the activity. Solubilized 2B15-His-transfected Src-free fibroblasts subjected to in vitro [γ-(33)P]ATP-dependent phosphorylation by PKCα and/or Src, affinity purification, and SDS gel analysis revealed 2-fold more radiolabeling of 55-58-kDa 2B15-His by PKCα than by Src; labeling was additive for combined kinases. Collectively, the evidence indicates that 2B15 requires regulated phosphorylation by both PKCα and Src, which is consistent with the complexity of synthesis and metabolism of its major substrate, DHT. Whether basal cells import or synthesize testosterone for transport to luminal cells for reduction to DHT by 5α-steroid reductase 2, comparatively low-activity luminal cell 2B15 undergoes a complex pattern of regulated phosphorylation necessary to maintain homeostatic DHT levels to support occupation of the androgen receptor for prostate-specific functions.

Dataset Information

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.

Publications

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.

Similar Datasets

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