Institute for Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein, Kiel, Germany.
Pharmacogenet Genomics. 2014 Jun;24(6):283-91. doi: 10.1097/FPC.0000000000000046.
The functional influence of single-nucleotide polymorphisms (SNPs) of the ATP-binding cassette (ABC) transporter ABCC2 (MRP2) has been characterized in numerous studies. The aim of this study was to address the question of whether distinct ABCC2 haplotypes, which differ in their mRNA secondary structures, show an influence on the degree of mRNA and protein downregulation through miRNA interaction.
A model using human peripheral blood monocytic cells (PBMCs) isolated from healthy Caucasian volunteers, with three defined ABCC2 haplotypes comprising the 5'-UTR SNP -24C>T, the 1249G>A SNP (V417I), and the silent 3972C>T SNP, was outlined. Cells were transiently transfected with miRNA-379, already known to target ABCC2 in HepG2 cells.
ABCC2 was downregulated through miR-379 in a haplotype-dependent manner: the wild-type CGC/CGC was modestly affected (mRNA: -12.7±4.2%, protein: -9.9±0.1%), whereas variant haplotypes were more strongly suppressed: CGT/CGT (mRNA: -36.7±2.4%, protein: -21.6±0.4%) and TGT/TGT (mRNA: -55.7±1.2%, protein: -46.3±4.0%). In addition, glutathione-methylfluorescein efflux was significantly reduced in miR-379-transfected peripheral blood monocytic cells corresponding to ABCC2 protein expression.
This observation may suggest a differential suppression of ABCC2 by miR-379 caused by haplotype-dependent differences in mRNA secondary structures, resulting in changes in mRNA target accessibility or mRNA stability.
已有大量研究对三磷酸腺苷结合盒（ABC）转运体 ABCC2（MRP2）的单核苷酸多态性（SNP）的功能影响进行了描述。本研究旨在探讨不同 ABCC2 单倍型（在其 mRNA 二级结构上存在差异）是否通过 miRNA 相互作用对 mRNA 和蛋白下调程度产生影响。
本研究构建了一个模型，使用分离自健康白种人志愿者的外周血单核细胞（PBMC），其中包含三个定义明确的 ABCC2 单倍型，分别是 5'-UTR 中的 -24C>T 单核苷酸多态性、1249G>A 单核苷酸多态性（V417I）和沉默的 3972C>T 单核苷酸多态性。细胞瞬时转染已在 HepG2 细胞中靶向 ABCC2 的 miRNA-379。
ABCC2 的下调呈 miRNA-379 依赖性的单倍型依赖方式：野生型 CGC/CGC 受到适度影响（mRNA：-12.7±4.2%，蛋白：-9.9±0.1%），而变异单倍型受到更强的抑制：CGT/CGT（mRNA：-36.7±2.4%，蛋白：-21.6±0.4%）和 TGT/TGT（mRNA：-55.7±1.2%，蛋白：-46.3±4.0%）。此外，转染 miRNA-379 的外周血单核细胞中谷胱甘肽-甲基荧光素外排明显减少，与 ABCC2 蛋白表达相对应。
这种观察结果可能表明，miR-379 对 ABCC2 的抑制作用存在单倍型依赖性差异，这种差异是由 mRNA 二级结构的差异引起的，导致 mRNA 靶标可及性或 mRNA 稳定性发生变化。
