RNAi抑制乳腺癌MCF-7/ADR细胞乳腺癌耐药蛋白基因研究

RNA interference inhibits the expression of breast cancer resistance protein gene in MCF-7/ADR

目的利用RNAi技术干扰耐药乳腺癌细胞MCF-7/ADR的BCRP(乳腺癌耐药蛋白)基因的表达,观察MCF-7/ADR细胞BCRP的mRNA变化,BCRP蛋白的表达以及对阿霉素耐药性的变化。方法设计3条不同(BCRP1,BCRP2,BCRP4)的RNA干扰链,构建出pGenesil-BCRP-EGFP(绿色荧光蛋白)质粒,设立对照组,空白质粒(HK)组及RNAi干扰组进行实验。半定量RT-PCR检测不同组的BCRP的mRNA水平,Western-blot检测不同组的BCRP蛋白的水平,MTT法分析RNAi干扰组药敏性变化。结果RT-PCR和Western-blot检测结果显示:BCRP的mRNA和蛋白的表达受抑制,以BCRP1组干扰效果明显,明显弱于其他组。MTT比色法结果显示:BCRP1组对阿霉素的药物敏感性明显提高,与对照组差异有显著性(P<0.05)。结论RNAi能抑制BCRP基因转录和翻译水平的表达,其中以BCRP1的干扰效果明显,能明显提高MCF-7/ADR对阿霉素的药物敏感性。

Objective To observe the changes of breast cancer resistance protein （BCRP） mRNA, BCRP expression, and its chemosensitivity to mitoxantrone through using interfering the expression of BCRP in MCF-7/ADR cell line by RNA interfering technology. Methods Three different siRNA sequences （BCRP1, BCRF2, BCRP4） were designed according to the BCRP sequence. The sequences were cloned into Pgenesil-1 plasmid to construct pGenesil-BCRP-EGFP plasmid. Five groups （control group, HK group, BCRP1 group, BCRF2 group, and BCRP4 group） were set up and plasmids were transfected into cells by liposome. The BCRP expression of MCF-7/ADR cell was measured by RT-PCR and Western blotting methods. MTT test was applied to measure the change of chemosensitive to mitoxantrone. Results RT-PCR and Western blotting demonstrated that BCRP expression was significantly decreased by transfection with pC, enesil-BCRP1 targeting plasmid; MTr test demonstrated the chemosensitivity to mitoxantrone was improved by transfection with RNAi plasmid, especially for BCRP1 group （ P 〈 0. 05）. Conclusion These results show that by inducing RNAi-targeting plasmid, BCRP expression can be effectively decreased at both transeriptional and translational levels, which reversed the drug resistance but improved the chemosensitivity significantly.