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.

中文翻译：

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miR-199a-5p 抑制阻塞性胆汁淤积症中 ABCB11 的表达。

ATP 结合盒，亚科 B 成员 11 (ABCB11) 是肝小管膜上胆汁酸的外排转运蛋白。这种转运蛋白的表达在胆汁淤积中减少；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 是否有助于小鼠胆汁淤积中 ABCB11/Abcb11 的消耗。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 显着上调了大约四倍。计算机分析预测 miR-199a-5p 将靶向 ABCB11/Abcb11 mRNA 的 3'-非翻译区 (3'-UTR)。共转染 miRNA-199a-5p 模拟物可显着抑制 Huh-7 细胞中 ABCB11-3'-UTR 荧光素酶构建体的表达，这被 miRNA-199a-5p 模拟抑制剂逆转。我们还显示 CBDL 后用强效核受体 FXR 激动剂奥贝胆酸 (OCA) 治疗小鼠显着增加 Abcb11 mRNA 和蛋白质并降低 miR-199a-5p 表达。计算图谱揭示了编码 miR-199a-5 的基因启动子中的一个保守的 FXR 结合位点 (FXRE)，称为 miR199a-2。电动迁移、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是有功能的。最后，小鼠中的 CBDL 导致 miR199a-2 启动子处的核阻遏物 NcoR1 结合的消耗，这促进了 miR199a-2 的转录。在用 OCA 治疗的 CBDL 小鼠中，miR199a-2 FXRE 的 NcoR1 募集维持在假手术小鼠中发现的水平。综上所述，