下调DNA拓扑异构酶Ⅰ基因提高小细胞肺癌细胞株对足叶乙甙的敏感性

Downregulation the expression of Top Ⅰ gene can improve sensitivity of Top Ⅰ inhibitors in small cell lung cancer cell lines

目的检测DNA拓扑异构酶Ⅰ(TopⅠ)在小细胞肺癌细胞株中的表达与TopⅡ抑制剂(足叶乙甙)敏感性的关系,为临床个体化治疗提供依据。方法Western-blot检测TopⅠ在小细胞肺癌细胞株H446蛋白水平的表达;应用人工合成并荧光标记的siRNA通过脂质体瞬时转染H446细胞,流式细胞仪检测转染效率,分别应用定量RT-PCR及Western-blot检测各干扰序列在mRNA及蛋白水平的干扰效果,筛选最佳干扰序列;并对转染后的细胞株进行足叶乙甙(VP16)药物敏感性试验,观察TopⅠ的表达水平与VP16敏感性的关系。结果TopⅠ基因在H446中有较高表达。siRNA干扰效果满意,流式细胞仪显示转染效率可达86.67%左右,RT-PCR显示干扰序列在mRNA水平干扰效率可达(96.33±1.87)%,Western-blot显示在蛋白水平也有明显的干扰效果。MTT结果显示,相同浓度的VP16对TopⅠ下调后H446细胞的抑制率明显高于转染前的H446细胞(P<0.01),IC50值分别为66.94μmol/L(1.97×PPC)及338.79μmol/L(9.97×PPC)。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;TopⅠ表达下调明显提高了对VP16的敏感性,为临床个体化治疗提供实验依据。

Objective To investigate the expressions of Top Ⅰ gene in small cell lung cancer cell line H446, and explore the relationship between the expression level of Top Ⅰ and the sensitivity of small cell lung cancer cell lines to Top Ⅱ inhibitors（VP16）. Methods Total protein was extracted and Western-blot was performed to detect TopⅠ expressions in H446;Design and synthesize 4 pairs of siRNA, Lipofectamine 2000 was used for the transient transfection of H446 by siRNA. Top Ⅰ mRNA was analyzed by quantitative RT-PCR and TopⅠ protein was detected by Western-blots. The transfeeted cells were treated with VP16, inhibition ratio were evaluated by MTr assay. Results TopⅠ gene expresses to be positive in H446 cells ; Lipofectamine 2000 can mediate siRNA effectively. TopⅠ mRNA of transfected cells reduced by （96. 33 ± 1.87 ） % compared to its parental cells, and decreases significantly at protein levels. By MTT assay, it was discovered that the inhibition ratio of VP16 to H446 was lower than that of H446 transfected by siRNA at same concentrations （P 〈 0. 01 ）; IC50 value were 66. 94 μmol/L and 338. 79 μmol/L respectively. Conclusions siRNAs can silence the expression of TopⅠ .Downregulating the TopⅠ levels of H446 can increase the sensitivity of VP16 significantly.