RNA干扰Akt对食管癌细胞株Eca109生物学特性的影响

RNA interference-mediated down-regulation of Akt attenuates cell proliferation and migration in human esophageal squamous cell carcinoma cell line Eca109

目的:探讨RNA干扰沉默Akt对人食管鳞癌细胞体外增殖、迁移及血管生成拟态(VM)形成的影响.方法:应用倒置荧光显微镜观察Akt的干扰质粒转染食管癌细胞Eca109后绿色荧光蛋白的表达;采用Western blot方法检测Akt蛋白的表达;四甲基偶氮唑蓝(MTT)法检测转染前后细胞增殖能力的变化;Transwell方法检测干扰前后细胞迁移能力的变化;取24孔培养板每孔加入300μL Matrigel原液,待其凝固,每孔内接种1mL浓度为5×105/mL的各组细胞悬液,培养12h后观察并计数转染前后各组细胞的管状结构数量.结果:Eca109/8单克隆干扰效果好,Western blot结果显示几乎无Akt表达.Akt被干扰后,细胞增殖能力明显减弱(P<0.05),运动迁移能力显著下降,与未转染的细胞相比穿过人工基底膜的细胞数明显减少(59.33±2.87 vs 130.5±2.22,P<0.05).三维培养提示干扰后细胞的管状结构数量明显减少(P<0.05).结论:RNA干扰可引起Eca109中Akt的沉默,而Akt表达下调后食管癌细胞株Eca109的增殖与迁移能力及VM能力均减弱.推测阻断Akt通路有可能成为治疗人食管鳞癌的新靶点.

AIM：To investigate the effect of RNA interference-mediated down-regulation of Akt on cell proliferation,migration,and vasculogenic mimicry formation in human esophageal squamous cell carcinoma cell line Eca109.METHODS：Plasmids harboring small interfering RNA targeting the Akt gene were introduced into Eca109 cells by liposome-mediated transfection.The expression of Akt protein was measured by Western blot.The proliferation of Eca109 cells was determined by methyl thiazolyl tetrazolium（MTT） assay.The migration of Eca109 cells was evaluated by Transwell migration assay.Vasculogenic mimicry was evaluated by counting the number of capillary structures.RESULTS：Akt expression was markedly downregulated in Eca109/8 cell clone（transfected with the plasmid harboring small interfering RNA targeting the Akt gene）.The expression of Akt protein in Eca109/8 cells could not be detected by Western blot.The proliferation of Eca109/8 cells decreased significantly compared with untransfected Eca109 cells（P 0.05）.Transwell migration assay showed that less Eca109/8 cells could move through the Transwell membrane when compared with untransfected Eca109 cells（59.33 ± 2.87 vs 130.5 ± 2.22,P 0.05）.Eca109 cells were capable of forming vasculogenic mimicry patterns in vitro.Silencing of the Akt gene could significantly suppress vasculogenic mimicry formation in Eca109 cells.CONCLUSION：RNA interference-mediated down-regulation of Akt can attenuate the proliferation,migration,and vasculogenic mimicry formation of Eca109 cells.Blockade of the Akt pathway may provide a new approach to the treatment of human esophagus squamous cell carcinoma.