沉默HIF-1α表达对缺氧环境下胃癌细胞GLUT-1、PKM2表达的影响
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摘要
目的观察缺氧环境下用siRNA特异性沉默胃癌细胞中缺氧诱导因子1α(HIF-1α)后,胃癌细胞葡萄糖载体蛋白1(GLUT-1)、M2型丙酮酸激酶(PKM2)表达的变化。方法将胃癌细胞BGC-823分为正常组、缺氧对照组、转染HIF-1αsiRNA组和转染对照组。用氯化钴(Co Cl2)体外模拟肿瘤细胞缺氧环境,缺氧对照组加入200μmol/L Co Cl2缺氧培养12 h;转染HIF-1αsiRNA组、转染对照组分别转染HIF-1αsiRNA、Control siRNA,48 h后加入200μmol/L Co Cl2缺氧培养12 h。采用qRT-PCR法和Western blotting法检测4组细胞内HIF-1α、GLUT-1、PKM2mRNA及蛋白表达。结果与正常组比较,缺氧对照组HIF-1αmRNA表达无明显变化(P> 0. 05),HIF-1α蛋白表达升高,GLUT-1、PKM2 mRNA及蛋白表达均升高(P <0. 05或<0. 01);与缺氧对照组比较,转染HIF-1αsiRNA组HIF-1α、GLUT-1、PKM2 mRNA及蛋白表达均降低(P <0. 05或<0. 01),转染对照组HIF-1α、GLUT-1、PKM2 mRNA及蛋白表达无明显变化(P均> 0. 05);与转染对照组比较,转染HIF-1αsiRNA组HIF-1α、GLUT-1、PKM2 mRNA及蛋白表达均降低(P <0. 05或<0. 01)。结论缺氧可促进人胃癌BGC-823细胞HIF-1α、GLUT-1、PKM2表达,沉默HIF-1α表达后BGC-823细胞HIF-1α、GLUT-1、PKM2表达下调; HIF-1α可能参与了GLUT-1、PKM2的表达调控。
Objective To investigate the expression changes of glucose transporter-1( GLUT-1) and pyruvate kinase M2( PKM2) in the gastric carcinoma BGC-823 cells after we used the small interfering RNA to silence hypoxia inducible factor-1α( HIF-1α) under hypoxia. Methods BGC-823 cells were divided into four groups: the normal group,hypoxic control group,HIF-1α siRNA-transfected group( HIF-1α siRNA group) and control siRNA-transfected group( control siRNA group). Cobalt chloride( Co Cl2) was used to simulate the hypoxic environment of tumor cells in vitro. The hypoxic control group was cultured with 200 μmol/L Co Cl2 for 12 h. The HIF-1α siRNA group and the control siRNA group were transfected with HIF-1α-siRNA and Control-siRNA for 48 h and then were cultured with 200 μmol/L Co Cl2 for 12 h. The expression levels of HIF-1α,GLUT-1,and PKM2 mRNA and protein in the cells of the four groups were detected by quantitative real-time PCR( qRT-PCR) and Western blotting. Results Compared with the normal group,the expression of HIF-1α mRNA in the hypoxia control group did not change significantly( P > 0. 05),while the expression of HIF-1α protein increased,and the mRNA and protein expression of GLUT-1 and PKM2 increased( P < 0. 05 or P < 0. 01). Compared with the hypoxic control group,the mRNA and protein expression of HIF-1α,GLUT-1,adn PKM2 in the HIF-1α siRNA group decreased( P < 0. 05 or P < 0. 01),and there was no significant change in the mRNA and protein expression of HIF-1α,GLUT-1 and PKM2 in the control siRNA group( all P > 0. 05). Compared with the control siRNA group,the mRNA and protein expression of HIF-1α,GLUT-1,and PKM2 in the HIF-1α siRNA group all decreased( P < 0. 05 or P< 0. 01). Conclusion Hypoxia can promote the expression of HIF-1α,GLUT-1 and PKM2 in the human gastric cancer BGC823 cells,the expression of HIF-1α,GLUT-1,and PKM2 is down-regulated after silencing HIF-1α,and HIF-1α maybe involved in regulating the expression of GLUT-1 and PKM2.
Objective To investigate the expression changes of glucose transporter-1( GLUT-1) and pyruvate kinase M2( PKM2) in the gastric carcinoma BGC-823 cells after we used the small interfering RNA to silence hypoxia inducible factor-1α( HIF-1α) under hypoxia. Methods BGC-823 cells were divided into four groups: the normal group,hypoxic control group,HIF-1α siRNA-transfected group( HIF-1α siRNA group) and control siRNA-transfected group( control siRNA group). Cobalt chloride( Co Cl2) was used to simulate the hypoxic environment of tumor cells in vitro. The hypoxic control group was cultured with 200 μmol/L Co Cl2 for 12 h. The HIF-1α siRNA group and the control siRNA group were transfected with HIF-1α-siRNA and Control-siRNA for 48 h and then were cultured with 200 μmol/L Co Cl2 for 12 h. The expression levels of HIF-1α,GLUT-1,and PKM2 mRNA and protein in the cells of the four groups were detected by quantitative real-time PCR( qRT-PCR) and Western blotting. Results Compared with the normal group,the expression of HIF-1α mRNA in the hypoxia control group did not change significantly( P > 0. 05),while the expression of HIF-1α protein increased,and the mRNA and protein expression of GLUT-1 and PKM2 increased( P < 0. 05 or P < 0. 01). Compared with the hypoxic control group,the mRNA and protein expression of HIF-1α,GLUT-1,adn PKM2 in the HIF-1α siRNA group decreased( P < 0. 05 or P < 0. 01),and there was no significant change in the mRNA and protein expression of HIF-1α,GLUT-1 and PKM2 in the control siRNA group( all P > 0. 05). Compared with the control siRNA group,the mRNA and protein expression of HIF-1α,GLUT-1,and PKM2 in the HIF-1α siRNA group all decreased( P < 0. 05 or P< 0. 01). Conclusion Hypoxia can promote the expression of HIF-1α,GLUT-1 and PKM2 in the human gastric cancer BGC823 cells,the expression of HIF-1α,GLUT-1,and PKM2 is down-regulated after silencing HIF-1α,and HIF-1α maybe involved in regulating the expression of GLUT-1 and PKM2.
引文
[1]Bensinger SJ,Christofk HR. New aspects of the Warburg effect incancer cell biology[J]. Semin Cell Dev Biol,2012,23(4):352-361.
[2]Guo Y,Scheuermann TH,Partch CL,et al. Coiled-coil coactiva-tors play a structural role mediating interactions in hypoxia-induc-ible factor heterodimerization[J]. J Biol Chem,2015,290(12):7707-7721.
[3]Yang W,Lu Z. Nuclear PKM2 regulates the Warburg effect[J].Cell Cycle,2013,12(19):3154-3158.
[4]Zhang TB,Zhao Y,Tong ZX,et al. Inhibition of glucose-trans-porter 1(GLUT-1)expression reversed Warburg effect in gastriccancer cell MKN45[J]. Int J Clin Exp Med,2015,8(2):2423-2428.
[5]Zhang ZG,Zhang QN,Wang XH,et al. Hypoxia-inducible factor1 alpha(HIF-1α)as a prognostic indicator in patients with gastrictumors:a meta-analysis[J]. Asian Pac J Cancer Prev,2013,14(7):4195-4198.
[6]Wang Y,Li Z,Zhang H,et al. HIF-1αand HIF-2αcorrelate withmigration and invasion in gastric cancer[J]. Cancer Biol Ther,2010,10(4):376-382.
[7]Yi L,Cao Y,Zhang W,et al. A small-molecule inhibitor of glu-cose transporter 1 downregulates glycolysis,induces cell-cycle ar-rest,and inhibits cancer cell growth in vitro and in vivo[J]. MolCancer Ther,2012,11(8):1672-1682.
[8]Mao ZP,Zhao LJ,Zhou SH,et al. Expression and significance ofglucose transporter-1,P-glycoprotein,multidrug resistance-associ-ated protein and glutathione S-transferase-πin laryngeal carcinoma[J]. Oncol Lett,2015,9(2):806-810.
[9]张鹏飞,吴继锋,张红,等.胃癌中HIF-1α、VEGF及Glut1的表达[J].安徽医科大学学报,2010,45(1):86-89.
[10]Luo W,Hu H,Chang R,et al. Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1[J]. Cell,2011,145(5):732-744.
[11]Gao Y,Xu D,Yu G,et al. Overexpression of metabolic markersHK1 and PKM2 contributes to lymphatic metastasis and adverseprognosis in Chinese gastric cancer[J]. Int J Exp Pathol,2014,8(8):9264-9271.
[12]Kwon OH,Kang TW,Kim JH,et al. Pyruvate kinase M2 pro-motes the growth of gastric cancer cells via regulation of Bcl-xl ex-pression at transcriptional level[J]. Biochem Bioph Res Commun,2012,423(1):38-44.
[13] Wang HJ,Hsieh YJ,Cheng WC,et al. JMJD5 regulates PKM2nuclear translocation and reprograms HIF-1α-mediated glucose me-tabolism[J]. Proc Natl Acad Sci,2014,111(1):279-284.
[14]Yang W,Xia Y,Ji H,et al. Nuclear PKM2 regulatesβ-catenintransactivation upon EGFR activation[J]. Nature,2011,480(7375):118-122.
[15] Oronsky BT,Oronsky N,Fanger GR,et al. Follow the ATP:tumor energy production:a perspective[J]. Anticancer AgentsMed Chem,2014,14(9):1187-1198.
[2]Guo Y,Scheuermann TH,Partch CL,et al. Coiled-coil coactiva-tors play a structural role mediating interactions in hypoxia-induc-ible factor heterodimerization[J]. J Biol Chem,2015,290(12):7707-7721.
[3]Yang W,Lu Z. Nuclear PKM2 regulates the Warburg effect[J].Cell Cycle,2013,12(19):3154-3158.
[4]Zhang TB,Zhao Y,Tong ZX,et al. Inhibition of glucose-trans-porter 1(GLUT-1)expression reversed Warburg effect in gastriccancer cell MKN45[J]. Int J Clin Exp Med,2015,8(2):2423-2428.
[5]Zhang ZG,Zhang QN,Wang XH,et al. Hypoxia-inducible factor1 alpha(HIF-1α)as a prognostic indicator in patients with gastrictumors:a meta-analysis[J]. Asian Pac J Cancer Prev,2013,14(7):4195-4198.
[6]Wang Y,Li Z,Zhang H,et al. HIF-1αand HIF-2αcorrelate withmigration and invasion in gastric cancer[J]. Cancer Biol Ther,2010,10(4):376-382.
[7]Yi L,Cao Y,Zhang W,et al. A small-molecule inhibitor of glu-cose transporter 1 downregulates glycolysis,induces cell-cycle ar-rest,and inhibits cancer cell growth in vitro and in vivo[J]. MolCancer Ther,2012,11(8):1672-1682.
[8]Mao ZP,Zhao LJ,Zhou SH,et al. Expression and significance ofglucose transporter-1,P-glycoprotein,multidrug resistance-associ-ated protein and glutathione S-transferase-πin laryngeal carcinoma[J]. Oncol Lett,2015,9(2):806-810.
[9]张鹏飞,吴继锋,张红,等.胃癌中HIF-1α、VEGF及Glut1的表达[J].安徽医科大学学报,2010,45(1):86-89.
[10]Luo W,Hu H,Chang R,et al. Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1[J]. Cell,2011,145(5):732-744.
[11]Gao Y,Xu D,Yu G,et al. Overexpression of metabolic markersHK1 and PKM2 contributes to lymphatic metastasis and adverseprognosis in Chinese gastric cancer[J]. Int J Exp Pathol,2014,8(8):9264-9271.
[12]Kwon OH,Kang TW,Kim JH,et al. Pyruvate kinase M2 pro-motes the growth of gastric cancer cells via regulation of Bcl-xl ex-pression at transcriptional level[J]. Biochem Bioph Res Commun,2012,423(1):38-44.
[13] Wang HJ,Hsieh YJ,Cheng WC,et al. JMJD5 regulates PKM2nuclear translocation and reprograms HIF-1α-mediated glucose me-tabolism[J]. Proc Natl Acad Sci,2014,111(1):279-284.
[14]Yang W,Xia Y,Ji H,et al. Nuclear PKM2 regulatesβ-catenintransactivation upon EGFR activation[J]. Nature,2011,480(7375):118-122.
[15] Oronsky BT,Oronsky N,Fanger GR,et al. Follow the ATP:tumor energy production:a perspective[J]. Anticancer AgentsMed Chem,2014,14(9):1187-1198.