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[RNA interference-mediated silencing of cytochrome P450 3A4 gene in CHL-3A4 cells]
Gui-zhong Yang1, Hua-rong Yu, Ye Yuan
1Department of Pharmacology, Chongqing Medical University, Chongqing 400016, China.
Objective:
To investigate the effect of small hairpin interfering RNA (shRNA) in suppressing cytochrome P450 3A4 (CYP3A4) gene expression in CHL-3A4 cells.
Methods:
Three shRNA expression vectors targeting CYP3A4 gene (CYP3A4 I, C YP3A4 II, and CYP3A4 III, respectively) were designed, synthesized and transfected into CHL-3A4 cells via liposomes. The inhibitory effect of shRNA on CYP 3A4 gene expression was detected by Western blotting and RT-PCR, and the effect of shRNA transfection in suppressing cyclophosphamide-induced cytotoxicity was measured using MTT assay.
Results:
The vector carrying CYP3A4 III shRNA significantly reduced the expression of CYP3A4 gene at both the mRNA (75%) and protein levels (80%) in CHL3A4 cells. The cytotoxicity of cyclophosphamide was markedly inhibited by CYP3A4 III-mediated suppression of CYP3A4 gene expression by 75% in CHL-3A4 cells.
Conclusion:
The vector-mediated RNA interference can suppress CYP3A4 gene expression in CHL-3A4 cells, and RNA interference technique provides a new means for studying cytochrome P450 gene function in mammalian cells.
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