葡萄糖转运蛋白-1的表达对骨肉瘤细胞生物学特征的影响
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摘要
目的探讨葡萄糖转运蛋白-1(GLUT1)在骨肉瘤组织中的表达及干扰其表达对骨肉瘤细胞生物学特征的影响。方法选取择期行手术治疗的初诊骨肉瘤患者57例,利用实时荧光定量PCR技术检测骨肉瘤及癌旁组织中GLUT1基因表达;培养人骨肉瘤MG63细胞,根据转染物不同分为siRNA-GLUT1组、siRNA-NC组和空白对照组,利用实时荧光定量PCR技术和蛋白质印迹法分别检测各转染组细胞中GLUT1基因和蛋白表达,MTT法检测各转染组细胞增殖能力,利用流式细胞技术检测各转染组细胞凋亡情况,Transwell法检测各转染组细胞迁移和侵袭能力。结果骨肉瘤组织中GLUT1 mRNA相对表达量1.95±0.18,显著高于癌旁组织的1.27±0.13,差异有统计学意义(P<0.05);骨肉瘤组织中GLUT1 mRNA相对表达量与临床分期、淋巴结转移和肺部转移有关(P<0.05);MTT检测结果显示,siRNA-GLUT1组细胞吸光度A值在24、48、72、96 h时均低于siRNA-NC组和空白对照组,均差异有统计学意义(P<0.05);流式细胞检测结果显示,siRNA-GLUT1组细胞凋亡率(25.7±2.6)%,显著高于siRNA-NC组(6.3±1.4)%和空白对照组(6.9±1.7)%,差异有统计学意义(P<0.05);siRNAGLUT1组迁移细胞数和侵袭细胞数低于siRNA-NC组和空白对照,均差异有统计学意义(P<0.05)。结论 GLUT1在骨肉瘤组织中呈高表达,特异性抑制GLUT1基因表达可有效抑制人骨肉瘤MG63细胞增殖,加速细胞凋亡,减少细胞迁移及侵袭。
Objective To investigate the expression of glucose transporter-1( GLUT1) in osteosarcoma tissues and effect of interfering its expression on the biological characteristics of osteosarcoma cells. Methods 57 patients with newly diagnosed osteosarcoma undergoing elective surgery were selected. The expressions of GLUT1 genes in osteosarcoma and adjacent tissues were detected by real-time fluorescence quantitative PCR. Human osteosarcoma MG63 cells were cultured. According to the different transfection,the cells were divided into siRNA-GLUT1 group,siRNA-NC group and blank control group. The expressions of GLUT1 genes and proteins in the different transfected groups were detected by using real-time fluorescent quantitative PCR and Western blot,respectively. The cell proliferation abilities in the different transfected groups were detected by using MTT. The apoptosis of cells in the different transfected groups were detected by flow cytometry. The migration and invasion abilities of cells in the different transfected groups were detected by Transwell method. Results The relative expression levels of GLUT1 mRNA in the osteosarcoma tissues were 1. 27 ± 0. 13,which were significantly higher than 1. 95 ± 0. 18 in the adjacent tissues,the difference was statistically significant( P < 0. 05). The relative expression levels of GLUT1 mRNA in osteosarcoma tissues were related with clinical stage,lymph node metastasis and lung metastasis( P <0. 05). MTT assay showed that the absorbance A values in the siRNA-GLUT1 group at 24,48,72,96 h were significantly lower than those in the siRNA-NC group and blank control group( P < 0. 05). The apoptosis rate in the siRNA-GLUT1 group was( 25. 7 ±2. 6) %,which was significantly higher than those in the siRNA-NC group and blank control group,which were( 6. 3 ± 1. 4) % and( 6. 9 ± 1. 7) %,respectively,the difference was statistically significant( P < 0. 05). The numbers of migrating cells and invasion cells in siRNA-GLUT1 group were lower than those in the siRNA-NC group and blank control group( P < 0. 05). Conclusion GLUT1 was highly expressed in osteosarcoma tissue. Specific inhibition of GLUT1 gene expression could effectively inhibit the proliferation of human osteosarcoma MG63 cells,accelerate cell apoptosis,and reduce cell migration and invasion.
Objective To investigate the expression of glucose transporter-1( GLUT1) in osteosarcoma tissues and effect of interfering its expression on the biological characteristics of osteosarcoma cells. Methods 57 patients with newly diagnosed osteosarcoma undergoing elective surgery were selected. The expressions of GLUT1 genes in osteosarcoma and adjacent tissues were detected by real-time fluorescence quantitative PCR. Human osteosarcoma MG63 cells were cultured. According to the different transfection,the cells were divided into siRNA-GLUT1 group,siRNA-NC group and blank control group. The expressions of GLUT1 genes and proteins in the different transfected groups were detected by using real-time fluorescent quantitative PCR and Western blot,respectively. The cell proliferation abilities in the different transfected groups were detected by using MTT. The apoptosis of cells in the different transfected groups were detected by flow cytometry. The migration and invasion abilities of cells in the different transfected groups were detected by Transwell method. Results The relative expression levels of GLUT1 mRNA in the osteosarcoma tissues were 1. 27 ± 0. 13,which were significantly higher than 1. 95 ± 0. 18 in the adjacent tissues,the difference was statistically significant( P < 0. 05). The relative expression levels of GLUT1 mRNA in osteosarcoma tissues were related with clinical stage,lymph node metastasis and lung metastasis( P <0. 05). MTT assay showed that the absorbance A values in the siRNA-GLUT1 group at 24,48,72,96 h were significantly lower than those in the siRNA-NC group and blank control group( P < 0. 05). The apoptosis rate in the siRNA-GLUT1 group was( 25. 7 ±2. 6) %,which was significantly higher than those in the siRNA-NC group and blank control group,which were( 6. 3 ± 1. 4) % and( 6. 9 ± 1. 7) %,respectively,the difference was statistically significant( P < 0. 05). The numbers of migrating cells and invasion cells in siRNA-GLUT1 group were lower than those in the siRNA-NC group and blank control group( P < 0. 05). Conclusion GLUT1 was highly expressed in osteosarcoma tissue. Specific inhibition of GLUT1 gene expression could effectively inhibit the proliferation of human osteosarcoma MG63 cells,accelerate cell apoptosis,and reduce cell migration and invasion.
引文
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[15]郭慧玲,刘睿,王红,等.GLUT1和PKM2在二甲双胍抑制人乳头状甲状腺癌细胞增殖中的作用[J].现代肿瘤医学,2015,23(7):900-904.
[16]VAZ CV,MARQUES R,ALVES MG,et al.Androgens enhance the glycolytic metabolism and lactate export in prostate cancer cells by modulating the expression of GLUT1,GLUT3,PFK,LDH and MCT4 genes[J].J Cancer Res Clin Oncol,2016,142(1):5-16.
[17]TANG Y,CAO K,WANG Q,et al.Silencing of Cer S6 increases the invasion and glycolysis of melanoma WM35,WM451 and SK28 cell lines via increased GLUT1-induced downregulation of WNT5A[J].Oncol Rep,2016,35(5):2907-2915.
[2]WANG JG,LIU B,GAO H,et al.Primary cardiac osteosarcoma[J].Heart Lung Circ,2016,25(7):698-704.
[3]FRIEBELE JC,PECK J,PAN X,et al.Osteosarcoma:A meta-analysis and review of the literature[J].Am J Orthop(Belle Mead NJ),2015,44(12):547-553.
[4]ORONSKY BT,ORONSKY N,FANGER GR,et al.Follow the ATP:tumor energy production:a perspective[J].Anticancer Agents Med Chem,2014,14(9):1187-1198.
[5]余苏云,刘兆国,贾琦,等.葡萄糖转运蛋白1与肿瘤能量代谢关系的研究进展[J].中国药理学通报,2016,32(7):906-909.
[6]LABAK CM,WANG PY,ARORA R,et al.Glucose transport:meeting the metabolic demands of cancer,and applications in glioblastoma treatment[J].Am J Cancer Res,2016,6(8):1599-1608.
[7]孙志宏,齐莹.RNAi干扰Twist基因对喉鳞状细胞癌Hep-2细胞株增殖与侵袭力及E-钙黏蛋白甲基化的影响[J].中国耳鼻咽喉颅底外科杂志,2016,22(6):467-471.
[8]钟玲.miR-34b在老年鼻咽癌组织中表达及对细胞增殖、侵袭的影响[J].中国老年学杂志,2017,37(7):1668-1670.
[9]HEYMANN MF,BROWN HK,HEYMANN D.Drugs in early clinical development for the treatment of osteosarcoma[J].Expert Opin Investig Drugs,2016,25(11):1265-1280.
[10]HOWELL D,MOLLOY S,WILKINSON K,et al.Patient-reported outcomes in routine cancer clinical practice:a scoping review of use,impact on health outcomes,and implementation factors[J].Ann Oncol,2015,26(9):1846-1858.
[11]SCHAAL C,PILLAI S,CHELLAPPAN SP.The Rb-E2F transcriptional regulatory pathway in tumor angiogenesis and metastasis[J].Adv Cancer Res,2014,121(7):147-182.
[12]庾光丹,牛建钦,肖岚,等.胶质细胞中葡萄糖转运机制研究进展[J].中国组织化学与细胞化学杂志,2016,25(1):80-83.
[13]RAGONA F,MATRICARDI S,CASTELLOTTI B,et al.Refractory absence epilepsy and glut1 deficiency syndrome:a new case report and literature review[J].Neuropediatrics,2014,45(5):328-332.
[14]陆凯强,徐秋蓉,谢伟全,等.葡萄糖转运蛋白1在癌症中的表达及其调控[J].临床与病理杂志,2016,36(9):1413-1417.
[15]郭慧玲,刘睿,王红,等.GLUT1和PKM2在二甲双胍抑制人乳头状甲状腺癌细胞增殖中的作用[J].现代肿瘤医学,2015,23(7):900-904.
[16]VAZ CV,MARQUES R,ALVES MG,et al.Androgens enhance the glycolytic metabolism and lactate export in prostate cancer cells by modulating the expression of GLUT1,GLUT3,PFK,LDH and MCT4 genes[J].J Cancer Res Clin Oncol,2016,142(1):5-16.
[17]TANG Y,CAO K,WANG Q,et al.Silencing of Cer S6 increases the invasion and glycolysis of melanoma WM35,WM451 and SK28 cell lines via increased GLUT1-induced downregulation of WNT5A[J].Oncol Rep,2016,35(5):2907-2915.