Survivin siRNA联合紫杉醇对肺癌细胞株的生长抑制作用

Growth-inhibiting effects of paclitaxel combined with survivin-specific siRNA on human lung cancer cell lines

目的探讨survivin siRNA对肺癌A549细胞化疗敏感性的影响。方法将A549细胞分为siRNA转染组、N-siRNA转染组、阴性对照组、空白对照组,用MTT法检测不同浓度紫杉醇联合siRNA对细胞存活率的影响。将A549细胞分为阴性对照组、siRNA组、紫杉醇组、siRNA+紫杉醇组,采用MTT法检测siRNA联合紫杉醇作用不同时间对细胞存活率的影响;采用Western blot检测细胞内survivin、p21、PARP蛋白表达变化;采用流式细胞仪分析各组细胞周期和凋亡变化的差异。结果Survivin siRNA可增加A549细胞对不同浓度紫杉醇的敏感性,但以低浓度(0.1、1、10nmol/L)最显著(P<0.05);survivin siRNA与10nmol/L紫杉醇共同处理A549细胞48h后,对细胞增殖的抑制具有协同或相加的效果(q值分别为1.34、1.03、1.02)。siRNA不仅可以抑制A549细胞内源性survivin表达还可以抑制紫杉醇诱导的survivin表达,阴性对照组、紫杉醇组、siRNA组和siRNA+紫杉醇组中survivin蛋白条带相对强度分别为0.244、0.99、0、0;survivin siRNA和紫杉醇联合作用后,对凋亡的诱导也有轻度相加作用,阴性对照组、紫杉醇组、siR-NA组和siRNA+紫杉醇组凋亡率分别为0.47%、9.50%、4.78%、19.81%;与对照组相比,siRNA和紫杉醇联合作用对细胞周期的影响表现为G1期、G2/M期细胞增加,S期细胞比率明显减少,对PARP的裂解和p21蛋白的表达也有促进作用。结论Survivin过表达可能参与了紫杉醇获得性耐药的形成,siRNA阻抑survivin表达是增加肺癌细胞对紫杉醇敏感性的有效途径。

Objective To assess the effects of survivin-specific siRNA on chemosensitivity of lung cancer cell A549 to paclitaxel in vitro. Methods For determining if survivin-specific siRNA could enhance the responsiveness of lung cancer to paclitaxeh human lung adenocarcinoma cell lines A549 （p53 wild-type） were divided into four different treatment groups： control, survivin siRNA, paclitaxel and survivin siRNA＋paclitaxel. The effects on cell proliferation, cell cycle and apoptosis were analyzed by MTT assay and flow cytometry, respectively. The protein expression levels of survivin, p21 and PARP were evaluated by Western blot experiments. Results Survivin-specific siRNA showed a augmenting effect on the chemosensitivity of different concentrations of paclitaxel （ P〈0. 05）, especially at the low concentrations of paclitaxel （0. 1-10nmol/L） （P〈0. 05）. A combination of 100nmol/L survivin siRNA and 10nmol/L paclitaxel at 48h or more exhibited a significantly additive or synergistic inhibitory effect on the proliferation of A549 cells （P〈0. 05）. Inhibition survivin with siRNA not only silenced endogenous survivin, but also silenced the paclitaxel-induced survivin in A549 cells, the relative intensities of sur- vivin protein were 0. 244, 0, 0. 99 and 0 for the four groups （control, survivin siRNA, paclitaxel and survivin siRNA＋paclitaxel） in-order. Paclitaxel associated with survivin siRNA rnoderately increased apoptosis rate compared to either treatment alone, the apoptosis rates of the four groups were 0. 47%, 9. 5%, 4.78% and 19. 81%, respectively. Treatment with survivin-specific siRNA and paclitaxel also produced an increase in the G1 and G2/M fraction and a decrease in the S fraction and resulted in a more cleaved PARP and up-regulated the expressions of p21 protein. Conclusion The present study suggests that survivin expression plays a critical role in cell viability, and that induction of survivin by paclitaxel is a potential drug resistance factor leading to cell survival. Inhibition of paclitaxel mediated induction of survivin by siRNA significantly enhances the chemosensitivity of lung cancer cells to paclitaxel.