RNA干扰下调拓扑异构酶Ⅰ在小细胞肺癌细胞株的表达及其与拓扑替康敏感性的关系

Down-regulation of TopⅠ by RNA interference reduces the drug-sensitivity of small cell lung cancercell lines to topotecan

目的检测拓扑异构酶Ⅰ（TopⅠ）在小细胞肺癌（SCLC）细胞株中的表达，并探讨其表达水平对拓扑替康（TPT）敏感性的影响。方法采用Western blot检测TopⅠ在SCLC细胞株H446蛋自水平的表达；应用人工合成并荧光标记的小干扰RNA（siRNA），通过脂质体瞬时转染H446细胞，流式细胞仪检测转染效率；应用定量逆转录聚合酶链反应（RT—PCR）和Western blot检测各干扰序列对TopⅠ在mRNA及蛋白水平的干扰效果，筛选最佳干扰序列；对转染后的细胞株进行药物敏感性实验，观察TopⅠ的表达对TPT敏感性的影响。结果TopⅠ基因在H446中有较高表达；siRNA干扰效果满意，转染效率可达86．7％左右；siRNA oligo TopⅠ-892、siRNA oligo TopⅠ-1152、siRNA oligo TopⅠ-2084和siRNA oligo TopⅠ-2397等4个于扰序列均抑制H446细胞TopⅠ mRNA的表达，抑制率分别为（95．7±1．6）％、（90．8±1．6）％、（96．1±2．7）％和（96．3±1．8）％。序列siRNA oligo TopⅠ-2084和siRNA oligo TopⅠ-2397干扰后，H446细胞TopⅠ蛋白表达明显降低。药物敏感性实验结果显示，相同浓度的TPT对实验组H446细胞的抑制率明显低于转染前及阴性对照的H446细胞（P〈0．01）。结论脂质体介导的人工合成siRNA瞬时转染可有效抑制H446细胞的TopⅠ表达，明显降低细胞对TPT的敏感性。

Objective To investigate the expressions of Top Ⅰ gene in small cell lung cancer cell line H446 ,and explore the influence of Top Ⅰ on the chemosensitivity of the cell line to topotecan （TPT）. Methods Western blot was performed to detect the Top Ⅰ expression in H446 cells. Lipofectamine 2000 was used for the transient transfection of H446 cells by siRNA, and the transfection efficacy was detected. TopⅠ mRNA was analyzed by quantitative RT-PCR and TopI protein was detected by Western blot to selected effective siRNA. The drug-sensitivity to topotecan （TPT） was evaluated by MTT assay. Results TopI gene was expressed in H446 cells. Lipofectamine 2000 mediated the siRNA effectively （ 88.67% ）. Compared with its parental cells, RT-PCR results showed that TopⅠ mRNAs in transfected cells were reduced by （95.7 ± 1.6）%, （90. 8 ± 1. 6）%, （96. 1 ±2. 7）% and （96. 3 ± 1. 8）%, respectively, and decreased significantly at protein level. By MTT assay, the inhibition rate of TPT to H446 cells transfected by siRNA was lower than that of control group at same concentrations （P 〈0.01 ）. Conclusion siRNAs can silence the expression of TopⅠ and decrease the drug-sensitivity of H446 cells to TPT.