2016 年第 1 期 第 14 卷

沉默Glut-1表达对食管鳞癌细胞株TE-13增殖和迁移的影响

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关键词：食管鳞癌Glut-1增殖细胞周期迁移

• 摘要：
• 【摘要】目的：沉默葡萄糖转运蛋白-1（glucose transporter protein-l，Glut-1）的表达对食管鳞癌TE-13细胞增殖、周期及迁移的影响，并探究可能的作用机制。 方法：采用慢病毒感染的方法将特异性针对Glut-1基因的Glut-1-shRNA转入TE-13细胞，建立稳定干扰Glut-1表达的TE-13细胞株，分成Glut-1-shRNA组和阴性对照组进行对比研究。采用实时荧光定量PCR和蛋白质印迹法检测Glut-1-shRNA对TE-13细胞Glut-1 mRNA及其蛋白表达的影响。CCK-8法、FCM法和Transwell小室迁移实验分别检测沉默Glut-1表达后对TE-13细胞增殖、细胞周期和迁移的影响；同时采用蛋白质印迹法检测细胞迁移以及乏氧相关蛋白的表达情况。 结果：采用慢病毒将Glut-1-shRNA转入TE-13细胞后，可明显下调Glut-1蛋白（t=6.713，P<0.01）和mRNA的表达水平（t=4.181，P<0.05）。与阴性对照组相比，Glut-1干扰后细胞的增殖至72小时（t=3.097，P<0.05）和96h（t=3.497，P<0.05）明显被抑制，同时细胞的迁移能力可见下降，但是细胞周期至24小时未见影响；基质金属蛋白酶的MMP-9（t=6.895，P<0.01）和缺氧诱导因子HIF-1α（t=13.74，P<0.001）的表达明显下调，但是MMP-2的表达没有变化（t=2.582，P>0.05）。 结论：沉默Glut-1的表达可抑制食管磷癌细胞株TE-13细胞的增殖和迁移，并明显下调细胞内MMP-9和HIF-1α蛋白的表达，为寻找靶向治疗食管癌新靶点提供了初步的实验基础。
• Methods: Cell line TE-13 were infected with a lentiviral vector carrying the Glut-1-shRNA, then the stably interference Glut-1 expression cells were selected and divided into 2 groups: Glut-1-shRNA group and negative control group. Real-time PCR and western blot analysis are used to measure the mRNA and protein levels of Glut-1 in negative control and Glut-1-shRNA infected TE-13 cells. The cell proliferation, cell cycle and migration are analyzed by CCK-8 assay, flow cytometry, transwell analysis respectively. Furthermore, the levels of proteins that involved in migration or hypoxia are analyzed by western blot. Results: Lentivirus mediated shRNA knockdown of Glut1 significantly downregulates the mRNA level (t=4.181, P<0.05) and protein level (t=6.713, P<0.01) of Glut-1. Compared with negative control, the proliferation (72h: t=3.097, P<0.05; 96h: t=3.497, P<0.05) of migration of TE-13 cells is significantly inhibited by Glut shRNA knockdown. However, the cell cycle has no significant changes at 24h after transfection. Moreover, the expression of MMP-9 (t=6.895, P<0.01) and HIF-1α (t=13.74, P<0.001) are significantly downregulated by Glut-1 shRNA knockdown. However, the expression of MMP-2 (t=2.582, P>0.05) is unchanged after Glut-1 knockdown. Conclusion: Glut-1 knockdown by shRNA significantly inhibits the proliferation and migration of TE-13 cells. And the levels of MMP-9 and HIF-1α are significantly downregulated by Glut-1 knockdown.