Bruckmueller Henrike, Martin Paul, Kähler Meike, Haenisch Sierk, Ostrowski Marek, Drozdzik Marek, Siegmund Werner, Cascorbi Ingolf, Oswald Stefan
Institute of Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein , Campus Kiel, Kiel 24105, Germany.
Department of General and Transplantation Surgery, Pomeranian Medical University , Szczecin 70-001, Poland.
Mol Pharm. 2017 Jul 3;14(7):2245-2253. doi: 10.1021/acs.molpharmaceut.7b00076. Epub 2017 Jun 2.
Intestinal drug transporters are crucial determinants for absorption and oral bioavailability of drugs. In healthy tissue donors, a recent study revealed profound discrepancies between mRNA expression and protein abundance as well as differences in the protein content between small and large intestine for clinically relevant multidrug transporters as the ATP binding cassette transporter subfamily B member 1 (ABCB1) and subfamily C member 3 (ABCC3) and the solute carrier family 15 member 1 (SLC15A1, PEPT1). As the mechanisms underlying these observations remained unclear, the aim of the present study was to elucidate the intestinal regiospecific microRNA profile under physiological conditions and identify specific microRNAs contributing to the post-transcriptional regulation of major drug transporters. For this purpose, tissue samples were collected from six intestinal sites obtained from six healthy tissue donors. The expression of 754 microRNAs was determined using qRT-PCR based low density arrays, and microRNA expression levels were correlated with transporter protein abundance quantified by targeted proteomics. A total of 241 microRNA-transporter pairs were identified, showing significant negative correlations to protein abundance (p < 0.05). Out of these, for nine pairs, the binding of the microRNA to the respective transporter 3'-UTR was predicted in silico. Besides the already known interactions of miR-27a-3p-ABCB1 and miR-193a-3p-PEPT1, reporter gene assays confirmed binding of miR-192-5p to the ABCC3 3'-UTR (reduction of reporter gene activity by 31%; p = 0.0012), miR-409-3p to the ABCB1 3'-UTR (reduction by 38%; p = 0.0006), and miR-193b-3p as well as miR-27a-3p to PEPT1 3'-UTR (reduction by 49% (p = 0.0012) and 20% (p = 0.0043), respectively). These results suggest that mucosal microRNA expression contributes to the explanation of discrepancies between mRNA expression and protein abundance as well as site-dependent differences in protein content along the human intestine under physiological conditions, as exemplified for ABCB1, ABCC3, and PEPT1.
肠道药物转运体是药物吸收和口服生物利用度的关键决定因素。在健康组织供体中，最近一项研究揭示了临床相关多药转运体（如ATP结合盒转运体B亚家族成员1（ABCB1）、C亚家族成员3（ABCC3）以及溶质载体家族15成员1（SLC15A1，即PEPT1））在mRNA表达与蛋白质丰度之间存在显著差异，并且在小肠和大肠的蛋白质含量也有所不同。由于这些观察结果背后的机制尚不清楚，本研究的目的是阐明生理条件下肠道区域特异性微小RNA谱，并鉴定对主要药物转运体进行转录后调控的特定微小RNA。为此，从六名健康组织供体获取的六个肠道部位采集了组织样本。使用基于qRT-PCR的低密度阵列测定了754种微小RNA的表达，并将微小RNA表达水平与通过靶向蛋白质组学定量的转运体蛋白质丰度进行关联。总共鉴定出241对微小RNA-转运体对，它们与蛋白质丰度呈现显著负相关（p < 0.05）。其中，对于9对，通过计算机模拟预测了微小RNA与各自转运体3'-UTR的结合。除了已知的miR-27a-3p-ABCB1和miR-193a-3p-PEPT1相互作用外，报告基因检测证实了miR-192-5p与ABCC3 3'-UTR的结合（报告基因活性降低31%；p = 0.0012），miR-409-3p与ABCB1 3'-UTR的结合（降低38%；p = 0.0006），以及miR-193b-3p和miR-27a-3p与PEPT1 3'-UTR的结合（分别降低49%（p = 0.0012）和20%（p = 0.0043））。这些结果表明，黏膜微小RNA表达有助于解释生理条件下人类肠道中mRNA表达与蛋白质丰度之间的差异以及蛋白质含量的部位依赖性差异，ABCB1、ABCC3和PEPT1即为例证。
