ABCC2 (MRP2) is an important export pump, expressed at tissue barriers. The genetic variants À24C4T, 1249G4A and 3972C4T are leading to inter-individual differences of bioavailability of various endogenous and exogenous compounds. Considering ABCC2 haplotypes, we investigated DNA-protein binding properties, mRNA secondary structure, mRNA stability, protein expression and transport activity in various cell lines and analyzed the bioavailability of talinolol in 24 healthy Caucasian volunteers; À24C4T had no clear influence on DNA-protein binding and the mRNA stability did not differ significantly. In transfected HEK293T/17 cells, haplotypes H9 (CGT), H10 (TGC) and H12 (TGT) had significantly lower protein expression, whereas H2 (CAC) exhibited significantly increased protein expression compared to the wild type (H1, CGC): 32.7 ± 8.8, 73.1 ± 6.3; 44.0 ± 15.5 and 115.2±8.2%, respectively. This corresponded with efflux rates of the fluorescent dye glutathione-methylfluorescein in vitro and by trend with talinolol bioavailability in vivo. In conclusion our results show a haplotypedependent influence on transport capacity of ABCC2, which seems to be mainly based on posttranscriptional modification of protein expression rather than transport rates.
microRNAs (miRNAs), which contribute to the post-transcriptional processing through 3Ј-untranslated region-interference, have been shown to be involved in the regulation of ATPbinding 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 miRNAmediated post-transcriptional gene regulation. We screened the expression of 377 human miRNAs in HepG2 cells after 48 h of treatment with 5 M 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-offunction 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 Ϯ 0.43 after 24 h. In addition, miR-379 increased (maximally 4.10 Ϯ 1.33-fold after 48 h), whereas ABCC2 protein decreased with a maximal fold change of 0.47 Ϯ 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.
Our study suggests an association of imatinib treatment, miRNA downregulation and ABCG2 overexpression, possibly contributing to the mechanisms involved in imatinib distribution and response in CML therapy.
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