Department of Pediatrics, Digestive Health Institute, Children's Hospital Colorado, Aurora, Colorado, USA.
Department of Pathology, University of Colorado School of Medicine, Aurora, Colorado, USA.
J Biol Chem. 2021 Dec;297(6):101400. doi: 10.1016/j.jbc.2021.101400. Epub 2021 Nov 12.
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.
三磷酸腺苷结合盒，亚家族 B 成员 11（ABCB11）是肝脏胆小管膜上胆汁酸的外排转运体。这种转运体在胆汁淤积时表达减少；然而，导致这种减少的机制尚不清楚。在这项研究中，我们试图确定 miR-199a-5p 是否有助于在小鼠胆汁淤积中耗尽 ABCB11/Abcb11。在胆总管结扎（CBDL）后小鼠肝脏的 microRNA（miRNA）筛选中，我们发现 miR-199a-5p 显著上调约四倍。计算机分析预测 miR-199a-5p 将靶向 ABCB11/Abcb11 mRNA 的 3'-非翻译区（3'-UTR）。miR-199a-5p 模拟物共转染显著抑制 Huh-7 细胞中 ABCB11-3'-UTR 荧光素酶构建体的表达，而 miR-199a-5p 模拟物抑制剂则逆转了这种抑制。我们还表明，在 CBDL 后用强效核受体 FXR 激动剂奥贝胆酸（OCA）治疗小鼠可显著增加 Abcb11 mRNA 和蛋白的表达，并降低 miR-199a-5p 的表达。计算映射显示在编码 miR-199a-5 的基因启动子中存在一个保守的 FXR 结合位点（FXRE），称为 miR199a-2。电泳迁移率变动分析、染色质免疫沉淀和 miR199a-2 启动子荧光素酶测定证实该结合位点具有功能。最后，在 CBDL 小鼠中，导致 miR199a-2 启动子上核阻遏物 NcoR1 结合的耗尽，从而促进 miR199a-2 的转录。在 CBDL 小鼠中用 OCA 治疗，NcoR1 募集到 miR199a-2 FXRE 的水平保持在 sham 手术小鼠中的水平。总之，我们证明 miR-199a-5p 参与调节 ABCB11/Abcb11 的表达，在阻塞性胆汁淤积中异常上调，并被 FXR 激动剂 OCA 下调。
