Abstract
ATP-binding cassette, subfamily B member 11 (ABCB11) is an efflux transporter for bile acids on the liver canalicular membrane. The expression of this transporter is reduced in cholestasis; however, the mechanisms contributing to this reduction are unclear. In this study, we sought to determine whether miR-199a-5p contributes to the depletion of ABCB11/Abcb11 in cholestasis in mice. In a microRNA (miRNA) screen of mouse liver after common bile duct ligation (CBDL), we found that miR-199a-5p was significantly upregulated by approximately fourfold. In silico analysis predicted that miR-199a-5p would target the 3′-untranslated region (3′-UTR) of ABCB11/Abcb11 mRNA. The expression of ABCB11-3′-UTR luciferase construct in Huh-7 cells was markedly inhibited by cotransfection of a miRNA-199a-5p mimic, which was reversed by an miRNA-199a-5p mimic inhibitor. We also show treatment of mice after CBDL with the potent nuclear receptor FXR agonist obeticholic acid (OCA) significantly increased Abcb11 mRNA and protein and decreased miR-199a-5p expression. Computational mapping revealed a well-conserved FXR-binding site (FXRE) in the promoter of the gene encoding miR-199a-5, termed miR199a-2. Electromobility shift, chromatin immunoprecipitation, and miR199a-2 promoter-luciferase assays confirmed that this binding site was functional. Finally, CBDL in mice led to depletion of nuclear repressor NcoR1 binding at the miR199a-2 promoter, which facilitates transcription of miR199a-2. In CBDL mice treated with OCA, NcoR1 recruitment to the miR199a-2 FXRE was maintained at levels found in sham-operated mice. In conclusion, we demonstrate that miR-199a-5p is involved in regulating ABCB11/Abcb11 expression, is aberrantly upregulated in obstructive cholestasis, and is downregulated by the FXR agonist OCA.
Highlights
Abcb11 mRNA and protein were significantly depleted after 4 days of common bile duct obstruction (CBDL) in mice, which is confirmed in Figure 1, A–C [1]
After 4 days of Common bile duct ligation (CBDL) in mice treatment with obeticholic acid (OCA) preserved Abcb11 mRNA and protein levels and suppressed miR199a-5a expression at levels found in sham operated mice (Fig. 1, A, B, and D)
We have previously shown that microRNA Let7a-5p was upregulated approximately fourfold after CBDL and targets the 30UTR of ATP-binding cassette sub-family C member 2 (ABCC2) (MRP2) [21], but overexpression of a mimic for this miRNA had no effect on expression of the ABCB11 -30 untranslated region (UTR)-luciferase construct, which lacks a binding site for this miRNA (Fig. 4C)
Summary
The impairmentation of ABCB11 in humans with cholestasis and various animal models of intrahepatic and obstructive cholestasis lead to retention of bile acids in the hepatocyte and exacerbation of liver injury [6, 7]. Normal cellular processes are subject in part to regulation by miRNAs, and alterations in miRNA expression or function can contribute to the pathogenesis of disease in animal models and humans [15,16,17]. In the current study we confirmed that miR-199a-5p was significantly upregulated in this model, and bioinformatic analysis predicted that miR-199a-5p would target the 30 UTR of ABCB11. The goal of this study was to determine the functional importance of miR199a-5p in regulating ABCB11 and how FXR agonism alters miR-199a-5p expression in a liver cell line and cholestatic mice
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