RNAi抑制CYP3A4基因表达的实验研究

RNA interference-mediated silencing of cytochrome P450 3A4 gene in CHL-3A4 cells

目的探讨质粒表达型短发夹RNA(shRNA)对CHL-3A4转基因细胞的细胞色素P4503A4(CYP3A4)基因表达的影响。方法构建3条shRNA真核表达载体(CYP3A4Ⅰ、CYP3A4Ⅱ、CYP3A4Ⅲ),脂质体转染CHL-3A4转基因细胞。转染48h后,RT-PCR和Westernblotting分别观察3条shRNA对CYP3A4mRNA和蛋白表达的影响;噻唑蓝观察shRNA抑制环磷酰胺对CHL-3A4转基因细胞毒性作用。结果CYP3A4Ⅲ表达载体的shRNA分别能抑制CHL-3A4细胞的CYP3A4mRNA表达(75%)及蛋白表达(80%),抑制环磷酰胺对CHL-3A4细胞(75%)细胞毒作用。结论RNA干扰能显著抑制CYP3A4基因的表达,RNA干扰技术为肝细胞色素P450的研究提供了新的实验室方法。

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 m-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 ceils, and RNA interference technique provides a new means for studying cytochrome P450 gene function in mammalian cells.