Identification of miRNAs that regulate human CYP2B6 expression.

Human hepatic cytochrome P450 2B6 (CYP2B6) expressed is responsible for the metabolism of many drugs, such as cyclophosphamide, ifosfamid, and efavirenz. In the present study, the correlation between CYP2B6 mRNA and protein levels in human liver samples was found to be moderate, indicating a contribution of posttranscriptional regulation of CYP2B6. Thus, we examined the role of microRNAs (miRNAs) in the regulation of CYP2B6. We established two kinds of HEK293 cell lines stably expressing CYP2B6, including or excluding the full-length 3'-untranslated region (3'-UTR) (HEK/2B6+UTR and HEK/2B6 cells, respectively). We tested 14 miRNAs that were predicted to bind to the 3'-UTR of CYP2B6 and found that the overexpression of miR-145, miR-194, miR-222, and miR-378 decreased the CYP2B6 protein level and activity in HEK/2B6+UTR but not in HEK/2B6 cells. These results suggested that miR-145, miR-194, miR-222, and miR-378 negatively regulate CYP2B6 expression by binding to the 3'-UTR. A negative correlation was not observed between the translational efficiency of CYP2B6 and the expression level of miR-145, miR-194, miR-222, or miR-378. This is due to the contribution of multiple miRNAs to CYP2B6 regulation. In conclusion, this study demonstrated that human CYP2B6 is posttranscriptionally regulated by miR-145, miR-194, miR-222, and miR-378.

RNA Editing Enzymes Modulate the Expression of Hepatic CYP2B6, CYP2C8, and Other Cytochrome P450 Isoforms.

A-to-I RNA editing, the most frequent type of RNA editing in mammals, is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes. Recently, we found that there is a large interindividual variation in the expression of ADAR1 protein in the human livers. In this study, we investigated the possibility that A-to-I RNA editing may modulate the expression of cytochrome P450 (P450), causing interindividual variations in drug metabolism potencies. We found that knockdown of ADAR1 or ADAR2 in HepaRG cells resulted in the decreased expression of CYP2B6 and CYP2C8 mRNA and protein. Knockdown of ADARs significantly decreased the stability of CYP2B6 mRNA but not CYP2C8 mRNA. Luciferase assays revealed that the 3'-untranslated region of CYP2B6 and the promoter region of CYP2C8 would be involved in the decrease in their expression by the knockdown of ADARs. We found that the decreased expression of the hepatocyte nuclear factor 4α (HNF4α) protein by the knockdown of ADARs was one of the reasons for the decreased transactivity of CYP2C8. The mRNA levels of other P450 isoforms, such as CYP2A6, 2C9, 2C19, 2D6, and 2E1, which are known to be regulated by HNF4α, were also decreased by ADAR1 or ADAR2 knockdown. Exceptionally, the CYP3A4 mRNA level was significantly increased by ADAR1 knockdown, suggesting the possibility that the change could be due to the change in the expression or function of other regulatory factors. In conclusion, this study revealed that the RNA editing enzymes ADAR1 and ADAR2 are novel regulatory factors of P450-mediated drug metabolism in the human liver.