沉默GLUT-1表达对食管鳞癌细胞株TE-13增殖和迁移的影响

The effects of GLUT-1 knockdown on proliferation and migration of esophageal squamous cell carcinoma cell line TE-13

目的研究沉默葡萄糖转运蛋白-1(glucose transporter protein-1,GLUT-1)的表达对食管鳞癌TE-13细胞增殖、周期及迁移的影响及可能的作用机制。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-sh RNA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-sh RNA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-sh RNA对TE-13细胞GLUT-1 m RNA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-sh RNA转入TE-13细胞后,可明显下调GLUT-1蛋白(t=6.713,P<0.01)和m RNA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 h(t=3.097,P<0.05)和96 h(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 h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。

Objective To investigate the effects of glucose transporter protein-1 （GLUT-l） knockdown on cell proliferation, cycle and migration of the esophageal squamous carcinoma cell line TE-13, and to study the background mechanism. Method Cell line TE- 13 were transfected with a lentiviral vector carrying the GLUT- 1-shRNA which is specific to GLUT-1 gene, then the stably interfered GLUT-1 expression cells were selected and all ceils were divided into 2 groups： GLUT-1-shRNA group and negative control group. Real-time PCR and western blot analysis were used to determine the mRNA and protein levels of GLUT-1 in negative control and GLUT-1-shRNA transfected TE-13 cells. The cell proliferation, cell cycle and migration were analyzed by CCK-8 assay, flow cytometry, transwell analysis, respectively. Furthermore, the levels of proteins that were involved in migration or hypoxia were analyzed by western blot. Result Lentivirus mediated shRNA knockdown of GLUT-1 significantly downregulated 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 （72 h： t=-3.097, P〈0.05; 96 h： t=3.497, P〈0.05） of migration of TE-13 cells was significantly inhibited by GLUT-1 shRNA knockdown. However, the cell cycle showed no significant changes in 24 h after transfection. Moreover, the expression of MMP-9 （t=-6.895, P〈0.01） and HIF- 1α （t= 13.74, P〈0.001） were significantly downregulated by GLUT- 1 shRNA knockdown, while the expression of MMP-2 （t=-2.582, P〉0.05） remained unchanged before and 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 signifi- cantly downregulated by GLUT-1 knockdown, which provides preliminary theoretical basis in searching for targeted therapy for esophageal carcinoma.