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目的:观察siRNA联合介导Livin和Survivin基因的沉默诱导人肾癌细胞786-O化疗敏感性的作用。方法:构建Livin和Suvivin联合靶向的小干扰RNA(siRNA)重组表达载体shRNA,然后将目的载体转染于人肾癌细胞株786-O。应用实时荧光定量聚合酶链反应(Real-time PCR)及Western blot方法分别检测转染前细胞经顺铂、5-FU和丝裂霉素处理后的Livin和Survivin基因的mRNA与蛋白水平的变化。应用CCK-8试验检测转染前后细胞对顺铂、5-氟尿嘧啶(5-FU)和丝裂霉素的半数致死量(IC50)、细胞增殖的变化。结果:CCK-8结果显示,联合介导Livin和Survivin基因沉默组细胞较分别单独介导细胞组化疗的敏感性明显增强(P<0.05),且转染前后细胞对顺铂、5-FU和丝裂霉素的IC50发生显著变化(P<0.05)。结论:SiRNA联合介导Livin和Survivin基因表达下调,发挥协同增强的siRNA作用。
OBJECTIVE: To observe the effect of siRNA combined with silencing of Livin and Survivin gene inducing 786-O chemosensitivity in human renal cell carcinoma cells. Methods: A shRNA targeting Livin and Suvivin targeting siRNA was constructed and transfected into human renal carcinoma cell line 786-O. Real-time PCR and Western blot were used to detect the mRNA and protein levels of Livin and Survivin after cisplatin, 5-FU and mitomycin treatment respectively . CCK-8 assay was used to detect the change of the median lethal dose (IC50) and cell proliferation of cisplatin, 5-fluorouracil and mitomycin before and after transfection. Results: The results of CCK-8 showed that the combination of Livin and Survivin silenced cells was significantly more sensitive than the cell-mediated chemotherapy alone (P <0.05), and the effect of cisplatin, 5-FU and The IC50 of mitomycin changed significantly (P <0.05). CONCLUSIONS: SiRNA-mediated down-regulation of Livin and Survivin gene expression, play a synergistic effect of siRNA.