The involvement of miRNAs in CYP450 enzymes and UDP-glucuronosyltransferases regulation in the human liver.

MicroRNAs play a key role in post-transcriptional regulation of genes. There is substantial evidence that miRNA regulate the transcription of genes involved in drug metabolism and transport. As the liver is the main organ contributing to the biotransformation of exogenous compounds including drugs, this study aimed to identify miRNAs that regulate the function of genes encoding enzymes crucial for drug biotransformation in the human liver. The identification of miRNAs potentially regulating pivotal cytochrome P450 enzymes and UDP-glucuronosyltransferase genes was performed via three different widely available web tools. Among the 479 predicted microRNA-target gene interactions, 22 unique miRNAs were detected at the quantitative level determined using qPCR low density arrays. The expression levels of these selected miRNAs were correlated with the target enzyme protein abundance quantified via mass spectrometry-based targeted proteomics. Significant negative correlations between miRNA expression and enzyme protein abundance were observed for CYP2C8, CYP2C9, CYP3A4, UGT1A1, UGT1A3 and UGT2B15. To verify the microRNA expression results, additional miRNA expression analysis (qPCR) in higher reaction volumes was performed. This analysis confirmed the statistically significant negative correlations between CYP2C8-hsa-miR-655-3p and UGT1A3-hsa-miR-200a-3p, and to validate the predicted interactions, in vitro experiments were conducted for these two miRNA-target pairs. For this purpose, HepG2 cells were co-transfected with pre-miR miRNA precursors (hsa-miR-655-3p and hsa-miR-200a-3p) and the respective enzyme 3'UTR constructs. A significant reduction in reporter activity confirmed the direct targeting of UGT1A3 by miR-200a-3p, while miR-655-3p had no significant effect on CYP2C8. These results provide insights into the miRNA-mediated epigenetic regulation of drug-metabolizing enzymes.