Abstract 3089: miR-491-3p regulation of the UDP-glucuronosyltransferase (UGT) 1A gene family.

Abstract

Abstract Background: The UDP-Glucuronosyltransferases (UGTs) are phase II metabolic enzymes that prime endogenous and exogenous compounds for excretion from the body. The UGTs are often found to have high inter-individual differences in expression, and this impacts patients’ response to drug therapy. Studies examining the epigenetic regulation of the UGT enzymes have not previously been performed. Recently, it has been shown that the phase I metabolic cytochrome P-450s (CYPs) are negatively regulated by microRNA (miRNA). miRNA are short, endogenous strands of RNA roughly 22-24 nucleotides in length that regulate gene expression. MiRNA specifically bind to the 3’ untranslated region (UTR) of mature messenger RNA (mRNA) and post-transcriptionally repress protein expression. Purpose: The goal of this study is to identify and evaluate miRNA regulation of UGT1A expression and whether this regulation contributes to differences in UGT1A enzymatic activity. Results: miR-491-3p was identified using the miRanda prediction program. Luciferase assays showed a significant reduction in luciferase activity with an attached UGT1A 3’UTR in the presence of miR-491-3p mimic (P<0.03). This reduction was lost when the miR-491-3p binding site in the UGT1A 3’ UTR was mutated. Mature miR-491-3p expression levels were identified and quantified in numerous cancer cell lines and normal human tissues. There was a significant increase in expression of miR-491-3p in four human liver cancer cell lines (HepG2, Hep3B, HuH-7, and SK-HEP-1) compared to normal liver (P<0.001) and a significant decrease in expression between normal human colon compared to Caco-2 colorectal cancer cells (P=0.0041), and between normal lung compared to A-549 lung cancer cells (P=0.0002). In HuH-7 liver cancer cells, we determined that transient transfection of miR-491-3p significantly reduced mRNA levels of UGT1A1 (P<0.0001), UGT1A3 (P<0.05), and UGT1A6 (P<0.001). Inversely, inhibitor knockdown of endogenous miR-491-3p levels in HuH-7 cells showed a significant increase in the expression of UGT1A1 (P<0.003) and UGT1A10 (P<0.05) mRNA. No alteration in expression was observed for UGT2B7. miR-491-3p over-expression affected the enzymatic activity of the UGT1As against the chemotherapeutic compound raloxifene (P<0.01), a drug metabolized by several members of the UGT1A family. Conclusion: miR-491-3p regulates the expression of several members of the UGT1A gene family. This regulation affects UGT1A enzymatic activity against the drug raloxifene in vitro. This is the first evidence implicating the epigenetic regulation of the UGT enzymes by miRNA. Citation Format: Douglas F. Dluzen, Anna Salzberg, Dongxiao Sun, Nathan Jones, Ryan Bushey`, Philip Lazarus. miR-491-3p regulation of the UDP-glucuronosyltransferase (UGT) 1A gene family. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3089. doi:10.1158/1538-7445.AM2013-3089