Abstract
Cytochrome P450 1A2 (CYP1A2) is one of the most abundant and important drug metabolizing enzymes in human liver. However, little is known about the post-transcriptional regulation of CYP1A2, especially the mechanisms involving microRNAs (miRNAs). This study applied a systematic approach to investigate the post-transcriptional regulation of CYP1A2 by miRNAs. Candidate miRNAs targeting the 3'-untranslated region (3'-UTR) of CYP1A2 were screened in silico, resulting in the selection of sixty-two potential miRNAs for further analysis. The levels of two miRNAs, hsa-miR-132-5p and hsa-miR-221-5p, were inversely correlated with the expression of CYP1A2 mRNA transcripts in normal human liver tissue samples represented in The Cancer Genome Atlas (TCGA) dataset. The interactions between these miRNAs and cognate CYP1A2 mRNA sequences were evaluated using luciferase reporter gene studies and electrophoretic mobility shift assays, by which a direct interaction was confirmed involving hsa-miR-132-5p and a cognate binding site present in the CYP1A2 3'-UTR. Experiments by which hsa-miR-132-5p or random miRNA controls were introduced into HepG2, Huh-7 and HepaRG hepatic cell lines showed that only hsa-miR-132-5p suppressed the endogenous and lansoprazole-induced expression of CYP1A2, at biological activity, protein production, and mRNA transcript levels. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays showed that hsa-miR-132-5p attenuates CYP1A2-mediated, lansoprazole-enhanced, flutamide-induced hepatic cell toxicity. Results from multilayer experiments demonstrate that hsa-miR-132-5p suppresses the expression of CYP1A2 and that this suppression is able to decrease the extent of an adverse drug-drug interaction involving lansoprazole and flutamide.