GLUT1蛋白对食管癌细胞顺铂耐药性的作用及其机制研究

Effect and mechanism of GLUT1 protein on cisplatin resistance of esophageal cancer cells

目的:探究葡萄糖转运蛋白1(GLUT1)对食管癌细胞顺铂耐药性的作用及其可能的作用机制。方法:收集29例食管癌组织及其癌旁组织,RT-PCR和Western blotting实验检测组织中GLUT1的表达;以EC109细胞株为亲本细胞,采用顺铂间歇冲击的方法构建顺铂耐药EC109/CDDP细胞株;CCK-8试剂盒测定EC109细胞和EC109/CDDP细胞顺铂IC_(50);RT-PCR实验检测细胞中GLUT1 mRNA的表达;Western blotting检测细胞中GLUT1、LC3B、Beclin1和p62蛋白表达;EdU实验检测细胞增殖;流式细胞术检测细胞凋亡。结果:与癌旁组织相比,癌组织中GLUT1 mRNA表达和蛋白表达明显升高(P<0.05)。EC109/CDDP细胞株构建成功。与EC109细胞相比,EC109/CDDP组细胞中GLUT1表达、LC3Ⅱ/LC3Ⅰ和Beclin1蛋白表达明显升高(P<0.05),p62蛋白表达明显降低(P<0.05)。与NC组相比,5μmol/L、10μmol/L、20μmol/L、40μmol/L和80μmol/L顺铂处理si-GLUT1组细胞活力明显降低(P<0.05),其中NC组细胞顺铂IC_(50)=32.47μmol/L,si-GLUT1组细胞顺铂IC_(50)=16.30μmol/L。与空白对照组相比,顺铂组EC109/CDDP细胞中LC3Ⅱ/LC3Ⅰ和Beclin1蛋白表达明显升高(P<0.05),p62蛋白表达明显降低(P<0.05),EdU阳性细胞数和凋亡率差异无统计学意义(P>0.05);与顺铂+NC组相比,顺铂+si-GLUT1组EdU阳性细胞数、LC3Ⅱ/LC3Ⅰ和Beclin1蛋白表达明显降低(P<0.05),细胞凋亡率和p62蛋白表达明显升高(P<0.05)。结论:食管癌中GLUT1蛋白通过上调自噬促进食管癌细胞顺铂耐药性。

Objective:To explore the effect of glucose transporter 1(GLUT1)on the cisplatin resistance of esophageal cancer cells and its possible mechanism.Methods:Twenty-nine cases of esophageal cancer tissues and their adjacent tissues were collected.RT-PCR and Western blottingwere used to detect the expression of GLUT1 in the tissues;EC109 cell line was used as the parent cell,and the cisplatin intermittent shock method was used to construct cisplatin-resistant EC109/CDDP cell line.CCK-8 kit was used to determine the cisplatin IC_(50) of EC109 cells and EC109/CDDP cells.RT-PCR was used to detect the expression of GLUT1 mRNA in cells.Western blotting was used to detect the protein expressions of GLUT1,LC3B,Beclin1 and p62 in cells.EdU test was used to detect cell proliferation.Flow cytometry was used to detect cell apoptosis.Results:Compared with adjacent tissues,GLUT1 mRNA and protein expression in cancer tissues were significantly increased(P<0.05).EC109/CDDP cell line was successfully constructed.Compared with EC109 cells,the expressions of GLUT1,LC3Ⅱ/LC3Ⅰand Beclin1 protein in EC109/CDDP cells were significantly increased,and the expression of p62 protein was significantly decreased(P<0.05).Compared with NC group,5μmol/L,10μmol/L,20μmol/L,40μmol/L,and 80μmol/L cisplatin-treated si-GLUT1 group significantly reduced cell viability(P<0.05),and the cisplatin IC_(50) was 32.47μmol/L and 16.30μmol/L in NC group and si-GLUT1 group,respectively.Compared with the blank control group,the LC3Ⅱ/LC3Ⅰand Beclin1 protein level of EC109/CDDP cells in the cisplatin group were significantly increased,while the expression of p62 protein was significantly reduced(P<0.05).There was no significant difference in the number of EdU positive cells and apoptosis ratebetween the blank control group and cisplatin group(P>0.05).Compared with cisplatin+NC group,the number of EdU-positive cells,LC3Ⅱ/LC3Ⅰand Beclin1 protein expression in cisplatin+si-GLUT1 group were significantly reduced,and the cell apoptosis rate and p62 protein expression were significantly increased(P<0.05).Conclusion:GLUT1 protein in esophageal cancer promotes cisplatin resistance in esophageal cancer cells by up-regulating autophagy.