GLUT1通过诱导自噬对食管癌细胞顺铂耐药性的作用研究

Study on the effect of GLUT1 on cisplatin resistance of esophageal cancer cells by inducing autophagy

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 blot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IC50;RT-PCR实验检测细胞中GLUT1 mRNA的表达;Western blot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:与癌旁组织比较,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细胞比较,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组比较,5、10、20、40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IC50=32.47μmol/L,si-GLUT1组细胞顺铂IC50=16.30μmol/L。与空白对照组比较,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;与顺铂+NC组比较,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 blot experiments were 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 IC50 of EC109 cells and EC109/CDDP cells;RT-PCR experiment was used to detect the expression of GLUT1 mRNA in cells;Western blot was used to detect GLUT1, LC3 B, Beclin1 and p62 protein expression 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 expression and protein expression in cancer tissues were significantly increased(P<0.05). EC109/CDDP cell line was successfully constructed. Compared with EC109 cells, the expression of GLUT1, LC3Ⅱ/LC3Ⅰ and Beclin1 protein in EC109/CDDP group were significantly increased(P<0.05), and the expression of p62 protein was significantly decreased(P<0.05). Compared with the NC group, 5 μmol/L, 10 μmol/L, 20 μmol/L, 40 μmol/L, and 80 μmol/L cisplatin-treated si-GLUT1 group significantly reduced cell viability(P<0.05), and the cisplatin IC50=32.47 μmol/L in NC group, cisplatin IC50=16.30 μmol/L in si-GLUT1 group. Compared with the blank control group, the expression of LC3Ⅱ/LC3Ⅰ and Beclin1 protein of EC109/CDDP cells in the cisplatin group was significantly increased(P<0.05), the expression of p62 protein was significantly reduced(P<0.05), there was no statistically significant difference in the number of EdU positive cells and the rate of apoptosis(P>0.05);compared with the cisplatin+NC group, the number of EdU-positive cells, LC3Ⅱ/LC3Ⅰ and Beclin1 protein expression in the cisplatin+si-GLUT1 group were significantly reduced(P<0.05), and the rate of cell apoptosis 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.