Department of Clinical Pharmacology and Toxicology, University Hospital Zurich, University of Zurich, Zürich, Switzerland.
Department of Surgery, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
Liver Int. 2019 Dec;39(12):2350-2359. doi: 10.1111/liv.14212. Epub 2019 Sep 3.
BACKGROUND & AIMS: The organic anion-transporting polypeptide 1B1 (OATP1B1) is an anion exchanger expressed at the hepatocyte sinusoidal membrane, which mediates the uptake of several endogenous metabolites and drugs. OATP1B1 expression level and activity are major sources of inter-patient variability of pharmacokinetics and pharmacodynamics of several drugs. Besides the genotype, factors that contribute to the inter-individual variability in OATP1B1 expression level are practically unknown. The aim of this work was to uncover novel epigenetic mechanisms of OATP1B1 regulation.
A functional screening strategy to assess the effect of microRNAs on the uptake of estrone-3-sulphate, an OATP1B1 substrate, into human hepatocellular carcinoma (Huh-7) cells was used. microRNA-206 (miR-206) expression in human liver tissues was measured by real-time RT-PCR. OATP1B1 expression in Huh-7 and in human liver tissues was assessed by real-time RT-PCR, Western blotting and immunostaining. The mRNA-miRNA interaction was assessed by reporter assay.
miR-206 mimic repressed mRNA and protein expression of OATP1B1 in Huh-7 cells. The intracellular accumulation of estrone-3-sulphate was reduced by 30% in cells overexpressing miR-206. The repressive effect of miR-206 on the activity of the firefly luciferase gene 2 under the control of the OATP1B1 3' untranslated region was lost upon deletion of the predicted miR-206 binding site. Hepatic miR-206 level negatively correlated with OATP1B1 mRNA and protein levels extracted from normal human liver tissues.
miR-206 exerts a suppressive effect on OATP1B1 expression by an epigenetic mechanism. Individuals with high hepatic levels of miR-206 appear to display lower level of OATP1B1.
有机阴离子转运多肽 1B1（OATP1B1）是一种表达于肝细胞窦状膜的阴离子交换器，可介导多种内源性代谢物和药物的摄取。OATP1B1 的表达水平和活性是多种药物药代动力学和药效学个体间变异性的主要来源。除了基因型外，导致 OATP1B1 表达水平个体间变异性的因素实际上尚不清楚。本研究旨在揭示 OATP1B1 调控的新的表观遗传机制。
采用功能性筛选策略，评估 microRNA 对人肝癌细胞（Huh-7）摄取雌酮-3-硫酸盐（OATP1B1 的底物）的影响。采用实时 RT-PCR 法测量人肝组织中的 microRNA-206（miR-206）表达。采用实时 RT-PCR、Western blot 和免疫染色法评估 Huh-7 细胞和人肝组织中的 OATP1B1 表达。采用报告基因检测评估 mRNA-miRNA 相互作用。
miR-206 模拟物可抑制 Huh-7 细胞中 OATP1B1 的 mRNA 和蛋白表达。转染 miR-206 的细胞内雌酮-3-硫酸盐的蓄积减少了 30%。在缺失预测的 miR-206 结合位点后，荧光素酶基因 2 在 OATP1B1 3'非翻译区控制下的活性的抑制作用丧失。从正常人肝组织中提取的 miR-206 水平与 OATP1B1 mRNA 和蛋白水平呈负相关。
miR-206 通过表观遗传机制对 OATP1B1 表达发挥抑制作用。肝组织中 miR-206 水平高的个体，OATP1B1 水平可能较低。
