Institute for Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein, Bldg. 30, Arnold-Heller-Str. 3, D-24105 Kiel, Germany.
Mol Pharmacol. 2011 Aug;80(2):314-20. doi: 10.1124/mol.110.070714. Epub 2011 May 3.
microRNAs (miRNAs), which contribute to the post-transcriptional processing through 3'-untranslated region-interference, have been shown to be involved in the regulation of ATP-binding cassette (ABC) membrane transporters. The aim of this study was to investigate whether ABCC2, an important efflux transporter for various endogenous and exogenous compounds at several compartment barriers, is subject to miRNA-mediated post-transcriptional gene regulation. We screened the expression of 377 human miRNAs in HepG2 cells after 48 h of treatment with 5 μM rifampicin [a pregnane X receptor (PXR) ligand] or vehicle using reverse transcription-polymerase chain reaction-based low-density arrays. Specific miRNA, ABCC2 mRNA, and protein expression were monitored in HepG2 cells undergoing rifampicin treatment for 72 h. Loss- and gain-of-function experiments and reporter gene assays were performed for further confirmation. Highly deregulated miRNAs compared with in silico data revealed miRNA (miR) 379 as candidate miRNA targeting ABCC2 mRNA. Under rifampicin treatment, ABCC2 mRNA increased significantly, with a maximal fold change of 1.56 ± 0.43 after 24 h. In addition, miR-379 increased (maximally 4.10 ± 1.33-fold after 48 h), whereas ABCC2 protein decreased with a maximal fold change of 0.47 ± 0.08 after 72 h. In contrast, transfection of miR-379 inhibitor led to an elevation of ABCC2 protein expression after rifampicin incubation for 48 h. We identify a miRNA negatively regulating ABCC2 on the post-transcriptional level and provide evidence that this miRNA impedes overexpression of ABCC2 protein after a PXR-mediated external transcriptional stimulus in HepG2 cells.
微小 RNA（miRNAs）通过 3'非翻译区干扰参与转录后加工，已被证明参与 ATP 结合盒（ABC）膜转运体的调节。本研究旨在探讨 ABCC2（多种内源性和外源性化合物在几个隔室屏障中的重要外排转运体）是否受 miRNA 介导的转录后基因调控。我们在 HepG2 细胞中用 5μM 利福平（一种孕烷 X 受体（PXR）配体）或载体处理 48 小时后，用逆转录-聚合酶链反应（RT-PCR）低密阵列筛选 377 个人类 miRNA 的表达。在利福平处理 72 小时的 HepG2 细胞中监测特定 miRNA、ABCC2 mRNA 和蛋白表达。进行了缺失和获得功能实验以及报告基因检测以进一步确认。与计算机数据相比，高度失调的 miRNA 揭示了 miRNA（miR）379 是靶向 ABCC2 mRNA 的候选 miRNA。在利福平处理下，ABCC2 mRNA 显著增加，24 小时后最大 fold change 为 1.56±0.43。此外，miR-379 增加（48 小时后最大增加 4.10±1.33 倍），而 ABCC2 蛋白在 72 小时后最大 fold change 为 0.47±0.08 减少。相比之下，miR-379 抑制剂转染后，利福平孵育 48 小时后 ABCC2 蛋白表达升高。我们鉴定了一种负调控 ABCC2 的 miRNA，并提供了证据表明，这种 miRNA 在外源转录刺激后，通过 PXR 介导的转录后水平抑制 ABCC2 蛋白的过表达。
