基因RNA干扰载体的构建及其对乳腺癌细胞耐药性的影响

目的:构建针对CIAPIN1基因的慢病毒siRNA表达载体并稳定转染人乳腺癌多柔比星耐药细胞MCF-7/ADM,观察该基因对乳腺癌细胞耐药性的影响。方法:设计合成针对CIAPIN1的siRNA重组质粒表达载体,并筛选出最有效的干扰序列,使用病毒包装系统进行慢病毒颗粒的包装和生产,获取ADM-CIAPIN1 RNAi稳定表达细胞株;MTT法检测CIAPIN1基因干扰前后细胞对于不同化疗药物IC50值的变化。结果:测序验证针对CIAPIN1的siRNA重组质粒构建成功,并筛选CIAP-IN1-siRNA1为最佳干扰序列;以慢病毒为载体将干扰表达质粒稳定转染入乳腺癌细胞MCF-7/ADM后,抑制CIAPIN1表达水平超过88%。RNA干扰后紫杉醇、多柔比星及吉西他滨3种抗肿瘤药物对于MCF-7/ADM细胞的IC50值均显著下降(7.12±0.31)、(11.21±1.79)、(49.72±4.52)vs(1.13±0.06)、(4.51±0.20)、(18.30±1.27)μg/ml,P<0.01],说明该细胞的耐药性明显减弱。结论:针对CIAPIN1基因的慢病毒siRNA表达载体可以有效抑制MCF-7/ADM细胞中该基因的表达,CIAPIN1基因表达下调可使乳腺癌细胞的多药耐药性发生逆转。

Construction of CIAPIN1 -RNAi vector and its effect on drug resistance of breast cancer cells

Objective: To construct lentiviral expressed vector of siRNA targeting CIAPIN1 and establish breast cancer cells with a stable expression of siRNA-CIAPIN1,and to investigate the effect of CIAPIN1 on breast cancer cells multi-drug resistance.Methods: The expressed vectors of recombined plasmid of siRNA targeting CIAPIN1 were designed and synthesized.Select the most efficient interfering sequence,spackage,and produce a lentiviral vector with it.Stably transfect CIAPIN1-RNAi into MCF-7/ADM cells.Detect IC50 value of different drugs in MCF-7/ADM cells before and after CIAPIN1 interference by MTT.Results: The expressed vectors of recombined plasmid of siRNA targeting CIAPIN1 were successfully synthesized and the most efficient interfering sequence was CIAPIN1-siRNA1.Stable transfection of CIAPIN1-RNAi into MCF-7/ADM cells by a lentiviral vector suppressed the expression of CIAPIN1 in MCF-7/ADM cells more than 88%.After RNA interference,IC50 value of MCF-7/ADM cells to anticancer drugs(paclitaxel,doxorubicin and gemcitabine) significantly decreased from(7.12±0.31),(11.21±1.79),(49.72±4.52) to(1.13±0.06),(4.51±0.20),(18.30±1.27) μg/ml respectively,suggesting a significant decrease in the drug resisstance of the cells.Conclusion: Lentiviral expressed vector of CIAPIN1-siRNA can efficiently interfere the expression of CIAPIN1 in MCF-7/ADM cells.The study also confirmed the regulation effect of CIAPIN1 on breast cancer cell multi-drug resistance(MDR).