RNA干扰CYP1B1基因沉默抑制多不饱和脂肪酸诱导的人乳腺癌MDA-MB-231细胞增殖

Small interference RNA-mediated CYP1B1 gene silencing inhibits the proliferation of human breast cancer MDA-MB-231 cells induced by polyunsaturated fatty acids

目的:探讨小干扰RNA(small interference RNA,siRNA)介导的细胞色素P4501B1(cytochrome P-4501B1,CYP1B1)基因沉默对二十碳五烯酸(eicosapentaenoic acid,EPA)和花生四烯酸(arachidonic acid,AA)作用下乳腺癌MDA-MB-231细胞增殖的影响。方法:应用细胞计数试剂盒(cell counting kit-8,CCK-8)检测经EPA和AA处理后MDA-MB-231细胞的增殖情况。采用RNA干扰(RNA interference,RNAi)技术干扰CYP1B1的表达,随后应用荧光实时定量PCR(real-time-uorescence quantitative PCR,RFQ-PCR)和蛋白免疫印迹法检测转染效率,以及siRNA干扰后EPA和AA作用下MDA-MB-231细胞中CYP1B1和儿茶酚氧位甲基转移酶(catechol-O-methyltransferase,COMT)mRNA和蛋白的表达情况。采用CCK-8法检测siRNA干扰后EPA和AA作用下MDA-MB-231细胞的增殖情况。结果:EPA处理组的MDA-MB-231细胞数明显少于对照组,而AA处理组的MDA-MB-231细胞则明显多于对照组(P<0.05)。转染CYP1B1siRNA的MDA-MB-231细胞中,CYP1B1mRNA和蛋白的表达均有所下降,而COMT mRNA和蛋白的表达水平则有所上升。CYP1B1siRNA转染的MDA-MB-231细胞的增殖能力下降,且EPA处理组的MDA-MB-231细胞数明显多于阴性对照组(P<0.05)。结论:CYP1B1基因沉默能够抑制MDA-MB-231细胞的增殖,逆转EPA对细胞增殖的抑制作用。EPA可能通过调节CYP1B1的表达来抑制乳腺癌细胞的增殖。

Objective： To investigate the effect of CYP1B1 （cytochrome P-450 1B1） gene silencing induced by small interference RNA （siRNA） on the proliferation of MDA-MB-231 cells treated with EPA （eicosapentaenoic acid） and AA （arachidonic acid）. Methods： The proliferation of MDA-MB-231 cells treated with EPA or AA was detected by CCK-8 （cell counting kit-8） assay. The expression of CYP1B1 was interfered by RNAi （RNA interference） technique. The transfection efficiency was examined by real- time fluorescence quantitative PCR （RFQ-PCR） and Western blotting, respectively. The expression levels of CYP1B1 and COMT （catechol-O-methyltransferase） mRNAs and proteins in MDA-MB-231 cells interfered with siRNA and treated with EPA or AA were determined by RFQ-PCR and Western blotting, respectively. The viability of breast cancer MDA-MB-231 cells interfered with siRNA and treated with EPA or AA was detected by CCK-8 assay. Results：The cell number of EPA-treated group was lower while the cell number of AA-treated group was higher than that of the control group （P〈0.05）. The expression levels of CYP1B1 mRNA and protein were decreased in MDA-MB-231 cells transfected with CYP1B1 siRNA, while the expression levels of COMT mRNA and protein were increased. The proliferation of MDA-MB-231 cells transfected with CYP1B1 siRNAwas inhibited, and the number of MDA-MB-231 cells treated with EPA was significantly higher than that of the negative control group （P〈0.05）. Conclusion： CYP1B1 gene silencing inhibits the proliferation of MDA-MB-231 cells and reverses the inhibitory effect of EPA on the cell proliferation. EPA probably inhibits the cell proliferation through regulating the expression of CYP1 B1 in breast cancer.