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食管癌ECA109细胞中Survivin shRNA干扰对核因子κB表达的影响及调控机制
李秀梅 陈艳 姜孝芳 李卉郭琼李惠武谌宏鸣*
解剖学报 ›› 2015, Vol. 46 ›› Issue (4) : 514-520.
食管癌ECA109细胞中Survivin shRNA干扰对核因子κB表达的影响及调控机制
Expression and regulaition of survivin shRNA interference on nuclear factorκB in esophageal ECA109 cells
目的 探讨Survivin作为基因表达调控因子，对核因子κB（NF-κB）基因的转录及蛋白水平活性的影响及与NF-κB的调控关系。 方法 采用Survivin短发夹RNA（shRNA）干扰技术，脂质体转染食管癌ECA109细胞，检测Survivin沉默效果，选择最佳转染浓度；半定量RT-PCR检测Survivin shRNA干扰后食管癌ECA109细胞中NF-κB及其通路的上游调控因子核因子κB抑制蛋白α（IKKα）、IKKβ表达变化；Western blotting方法检测NF-κB蛋白的表达变化；流式细胞术检测干扰之后食管癌ECA109细胞增殖、凋亡指数的变化。 结果 Survivin shRNA干扰食管癌ECA109细胞后，Survivin mRNA和蛋白表达明显下调；NF-κB mRNA表达变化无明显差异，但其蛋白磷酸化水平降低，NF-κB 上游因子IKKα、IKKβ mRNA表达下调；食管癌ECA109细胞凋亡明显增加，G2期细胞比例增加，S期细胞减少，细胞周期受阻。 结论 Survivin通过在转录水平影响IKKα、IKKβ mRNA表达，调控NF-κB蛋白活化水平，提示Survivin在肿瘤信号调控网络中可作为一个新的基因表达调控因子。
Objective As the effect of Survivin, a gene expression regulatory factor, on the nuclear factor (NF)-kappa B gene transcription and protein activity remains unknown, this subject aims to explore the possible regulation mechanism of Survivin and NF-kappa B. Methods Survivin shRNA interference technology was applied to liposome transfect esophageal ECA109 cells, detect Survivin silence result, choose the best transfection concentration; Half quantitative RT-PCR was applied to detect genes expression of NF-kappa B and upstream regulatory factors inhibitor of NF-kappa B kinase alpha(IKK alpha), IKK beta in the same pathway; Western blotting was used to detect protein expression of the NF-kappa B. Flow cytometry was used to detect the proliferation and apoptosis of cell lines index changes after ECA109 esophageal cells transfection. Results After survivin shRNA transfection in esophageal ECA109 cells, survivin mRNA and protein expression were obviously down-regulated. NF-kappa B mRNA expression had no obvious difference, however the phosphorylated proteins level of NF-kappaB reduced and its upstream factor IKK alpha, IKK beta mRNA express reduced as well. Esophageal cell ECA109 apoptosis increased significantly, the cells ratio increased in G2 phase but decreased in S phase, and the cell cycle was blocked. Conclusion Survivin regulates NF-kappa B activation by down-regulation of IKK alpha and IKK beta mRNA expression, suggesting that survivin may be a new gene regulatory factor in the tumor signal control network.
食管癌 / Survivin / 短发夹RNA / 反转录-聚合酶链反应
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