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Small Interfering RNA-Mediated Silencing of Cytochrome P450 3A4 Gene

Institute of Clinical Pharmacology, School of Pharmaceutical Sciences (J.C., M.H.) and Department of Clinical Pharmacy, the First Affiliated University Hospital (X.C.), Sun Yat-sen University, Guangzhou, China; Departments of Pharmacy (X.-X.Y., Z.-P.H., E.C., S.-F.Z.) and Biological Sciences (F.-S.S.), Faculty of Science, National University of Singapore, Singapore; Department of Biochemistry, Faculty of Medicine (W.D.), National University of Singapore, Singapore; and Department of Pharmacology, Medical College, Nanchang University, Nanchang, China (M.H.)

RNA interference (RNAi) is a specific and powerful tool used to manipulate gene expression and study gene function. The cytochrome P450 3A4 (CYP3A4) can metabolize more than 50% of drugs. In the present study, we investigated whether vector-expressed small interfering RNAs (siRNAs) altered the CYP3A4 expression and function using the Chinese hamster cell line (V79) overexpressing CYP3A4 (CHL-3A4). Three different siRNA oligonucleotides (3A4I, 3A4II, and 3A4III) were designed and tested for their ability to interfere with CYP3A4 gene expression. Our study demonstrated that transient transfection of CHL-3A4 cells with the 3A4III siRNAs, but not 3A4I and II, significantly reduced CYP3A4 mRNA levels by 65% and protein expression levels by 75%. All these siRNAs did not affect the expression of CYP3A5 at both mRNA and protein levels in V79 cells overexpressing CYP3A5. Transfection of CHL-3A4 cells with 3A4III siRNAs significantly diminished the cytotoxicity of two CYP3A4 substrate drugs, cyclophosphamide and ifosfamide, in CHL-3A4 cells, with the IC₅₀ increased from 55 to 210 µM to >1000 µM. Nifedipine at 5.78, 14.44, and 28.88 µM was significantly (P < 0.01) depleted by approximately 100, 40, and 22%, respectively, in S9 fractions from CHL-3A4 cells compared with parental CHL-pIC19h cells. In addition, transfection of the CHL-3A4 cells with vectors expressing the 3A4III siRNAs almost completely inhibited CYP3A4-mediated nifedipine metabolism. This study demonstrated, for the first time, the specific suppression of CYP3A4 expression and function using vector-based RNAi technique. The use of RNAi is a promising tool for the study of cytochrome P450 family function.

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				Y.-Z. Pan, W. Gao, and A.-M. Yu MicroRNAs Regulate CYP3A4 Expression via Direct and Indirect Targeting Drug Metab. Dispos., October 1, 2009; 37(10): 2112 - 2117. [Abstract] [Full Text] [PDF]