Identification and validation of microRNAs directly regulating the UDP-glucuronosyltransferase 1A subfamily enzymes by a functional genomics approach

Abstract

Posttranscriptional repression of UDP-glucuronosyltransferase (UGT) 1A expression by microRNAs (miRNAs) may be an important mechanism underlying interindividual variability in drug glucuronidation. Furthermore, the UGT1A 3’-UTR shared by all UGT1A enzymes is polymorphic, containing three linked SNPs (rs10929303, rs1042640, and rs8330) that could influence miRNA binding. The aim of this study was to identify the complete complement of miRNAs that could regulate UGT1A expression through binding to the reference and/or common variant UGT1A 3’-UTR. Luciferase reporter plasmids containing either the reference or variant UGT1A 3’-UTR 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. Four novel miRNAs (miR-103b, miR-141–3p, miR-200a-3p, and miR-376b-3p) were identified that repressed both reference and variant UGT1A 3’-UTR, while two other miRNAs selectively repressed the reference (miR-1286) or variant (miR-21–3p) 3’-UTR. Deletion and mutagenesis studies confirmed the binding site location for each miRNA. rs8330 disrupted miR-1286 binding to the reference UGT1A 3’-UTR, while rs10929303 enhanced miR-21–3p binding to the variant 3’-UTR. Transfection of miR-21–3p, miR-103b, miR-141–3p, miR-200a-3p, and miR-376b-3p mimics into LS180 human intestinal cells showed repression of UGT1A1 and UGT1A6 mediated glucuronidation and mRNA without affecting UGT2B7 activity or mRNA. Furthermore, transfection of miR-21–3p, miR-141–3p, and miR-200a-3p into primary human hepatocytes, repressed UGT1A1 activity and mRNA without affecting CYP3A activity. Finally, miR-21–3p and miR-200a-3p expression were negatively correlated with UGT1A6 activity and mRNA in human liver samples. Thus, UGT1A is regulated by multiple miRNAs with some showing allele-dependent effects.

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

“…Previous results from our laboratory demonstrated that miR-103b and miR-376b-3p have functional MREs in the common UGT1A 3’-UTR [17]. In the current work we showed that miR-103b and miR-376b-3p were also among the miRNAs with functional MREs in the UGT2B15 3’-UTR.…”

“…In recent work we used the same in vitro system to perform a genome-wide screen of the miRNA mimic library with the pMIR-REPORT luciferase vector carrying the reference UGT1A 3’-UTR or the variant 3’-UTR [17]. This way we demonstrated that SNP rs10929303 C>T located in the common UGT1A 3’-UTR created a novel MRE for miR-21–3p.…”

“…For all luciferase assays HEK293 cells were co-transfected with a firefly luciferase construct, the pRL-CMV Renilla luciferase control vector and the miRNA mimic or one of the mimic controls (negative or positive) as described previously [17]. …”

“…Accumulating data demonstrate that miRNAs modulate the expression of drug disposition genes, including cytochrome P450 enzymes, UGTs, and transporters [16]. Recently we showed using an unbiased genome-wide screen that five novel miRNA response elements (MREs) are located in the common UGT1A 3’-UTR [17]. In addition two recent studies identified and validated the functional MREs of miR-376c and miR-331–5p to the 3’-UTRs of UGT2B15 and UGT2B17 in prostate cancer cells [18, 19].…”

MiRNA-20A Upregulates TAK1 and Increases Proliferation in Osteosarcoma Cells

“…A plasmid constitutively expressing renilla luciferase was cotransfected as an internal control to correct for differences in both transfection and harvesting efficiencies [12]. The transfections were performed in duplicate, and at least three independent experiments were performed [13].…”