GLUT1对人结肠癌细胞5-氟尿嘧啶耐药性的调控作用及其机制研究

Regulatory Effects and Mechanism of GLUT1 on 5-Fu Resistance in Human Colon Cancer Cells

该研究探究葡萄糖转运蛋白1(GLUT1)对人结肠癌细胞5-氟尿嘧啶(5-Fu)耐药性的调控作用及其机制。实验收集17例5-Fu化疗敏感结肠癌患者的结肠癌组织及癌旁组织、13例5-Fu化疗耐药结肠癌患者的结肠癌组织和癌旁组织,实时荧光定量PCR(RT-qPCR)法和Western blot法检测结肠癌组织和癌旁组织中GLUT1基因和蛋白的表达水平。采用5-Fu浓度递增间断刺激法诱导建立5-Fu耐药HT-29细胞(HT-29/5-Fu)。体外培养HT-29/5-Fu细胞及其亲本HT-29细胞,MTT法检测5-Fu对2种细胞增殖的抑制作用,RT-qPCR法和Western blot法检测HT-29/5-Fu细胞及其亲本HT-29细胞中GLUT1基因和蛋白的表达水平。将HT-29/5-Fu细胞分为正常对照组(NC)、无义对照质粒组(pRS-scrambled)、GLUT1干扰质粒组(pRS-GLUT1),RT-qPCR法和Western blot法检测各组GLUT1基因和蛋白的表达,MTT法检测细胞增殖能力变化,流式细胞术检测细胞周期和细胞凋亡情况,RT-qPCR法和Western blot法检测MRP1、ABCB1、GST-π、Bcl-2和Bax mRNA和蛋白的表达。该实验显示:(1)5-Fu化疗耐药结肠癌组织中GLUT1基因和蛋白表达水平均较5-Fu化疗敏感结肠癌组织升高(P<0.05);(2)培养24 h或48 h时5-Fu(5~100 pg/mL)对亲本HT-29细胞的抑制率较HT-29/5-Fu显著升高(P<0.05);(3)HT-29/5-Fu细胞中GLUT1基因和蛋白的表达水平较其亲本HT-29细胞显著升高(P<0.05);(4)GLUT1干扰质粒组细胞中GLUT1 mRNA和蛋白表达水平较正常对照组和无义对照质粒组显著降低(P<0.05);(5)GLUT1干扰质粒组48 h细胞吸光度值均较正常对照组和无义对照质粒组显著降低(P<0.05);(6)GLUT1干扰质粒组G_(0)/G_(1)期细胞比例较正常对照组和无义对照质粒组显著升高,而S期细胞比例显著减少(P<0.05);(7)GLUT1干扰质粒组细胞凋亡率较正常对照组和无义对照质粒组显著升高(P<0.05);(8)GLUT1干扰质粒组细胞中MRP 1、GST-π、ABCB 1和Bcl-2 mRNA和蛋白表达较正常对照组和无义对照质粒组显著降低(P<0.05),Bax mRNA和蛋白水平较正常对照组和无义对照质粒组显著升高(P<0.05)。该实验证明,GLUT1基因沉默可增加耐药结肠癌细胞对5-Fu的敏感性,这可能与其调节细胞周期、促进细胞凋亡、抑制结肠癌细胞中耐药相关蛋白表达以及调节Bcl2/Bax凋亡通路有关。

This study was to investigate the regulatory effect and mechanism of GLUT1(glucose transporter 1)on 5-Fu(5-fluorouracil)resistance in human colon cancer cells.In this study,the gene and protein expression of GLUT1 in colon cancer tissues and adjacent tissues in 17 patients with 5-Fu chemo-sensitive colon cancer and 13 patients with 5-Fu chemo-resistant colon cancer was detected by RT-qPCR(real-time fluorescence quantitative polymerase chain reaction)and Western blot.5-Fu-resistant HT-29 cells(HT-29/5-Fu)were induced by increasing 5-Fu concentration intermittently.HT-29/5-Fu cells and their parents HT-29 cells were cultured in vitro.The inhibitory effect of 5-Fu on the proliferation of HT-29/5-Fu cells and HT-29 cells was detected by MTT assay.The gene and protein expression of GLUT1 in HT-29/5-Fu cells and HT-29 cells was detected by RT-qPCR and Western blot.The HT-29/5-Fu cells were divided into normal control group(NC),nonsense control group(pRS-scrambled)and GLUT1 interfering group(pRS-GLUT1).The gene and protein expression of GLUT1 was detected by RT-qPCR and Western blot.The cell cycle and apoptosis were detected by flow cytometry.The gene and protein expression of MRP1,ABCB1,GST-π,Bcl-2 and Bax in HT-29/5-Fu cells and HT-29 cells was detected by RT-qPCR and Western blot.This study showed as below:(1)The gene and protein expression of GLUT1 in 5-Fu-resistant colon cancer tissues was significantly increased than that in 5-Fu-sensitive colon cancer tissues(P<0.05);(2)The inhibitory rate of 5-Fu(5-100 pg/mL)on HT-29 cells was significantly increased than that of HT-29/5-Fu(P<0.05);(3)The gene and protein expression of GLUT1 in HT-29/5-Fu cells was significantly increased than that in HT-29 cells(P<0.05);(4)The mRNA and protein expression of GLUT1 in pRS-GLUT1 group was significantly decreased than that in NC group and pRS-scrambled group(P<0.05);(5)The absorbance in pRS-GLUT1 group was significantly decreased than that in NC group and pRS-scrambled group(P<0.05);(6)The proportion of cells in G_(0)/G_(1) phase in pRSGLUT1 group was significantly increased,and the proportion of cells in S phase in pRS-GLUT1 group was significantly decreased than that in NC group and pRS-scrambled group(P<0.05);(7)The apoptosis rate in pRS-GLUT1 group was significantly increased than that in NC group and pRS-scrambled group(P<0.05);(8)The mRNA and protein expression of MRP 1,GST-π,ABCB 1 and Bcl-2 in pRS-GLUT1 group was significantly decreased than that in NC group and pRS-scrambled Group(P<0.05),and the mRNA and protein level of Bax in pRS-GLUT1 group was significantly higher than that in NC group and pRS-scrambled group(P<0.05).This study proved that GLUT1 gene silencing can increase the sensitivity of resistant colon cancer cells to 5-Fu,which may be related to regulating cell cycle,promoting apoptosis,inhibiting the expression of drug resistance related proteins,and regulating Bcl2/Bax apoptosis pathway in colon cancer cells.