Tim-3对巨噬细胞葡萄糖转运体1表达的调节机制研究
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摘要
目的探讨T细胞免疫球蛋白及黏蛋白结构域分子-3(T cell immunoglobulin and mucin-domain containing molecule-3,Tim-3)对巨噬细胞葡萄糖转运体1(glucose transporter 1,GLUT1)的表达及其对细胞功能的影响,并初步探索其影响机制。方法使用Tim-3 siRNA敲低Raw264. 7细胞系观察GLUT1蛋白的表达,随后使用不同浓度Tim-3融合蛋白、Tim-3激动抗体,阻断和激活小鼠源巨噬细胞系Raw264. 7,在基因和蛋白水平观察GLUT1、甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)、Tim-3以及肿瘤坏死因子α(tumor necrosis factor-α,TNF-α) 4项指标的变化。结果 Tim-3敲低的Raw264. 7细胞系GLUT1蛋白表达水平明显升高;激活、抑制巨噬细胞Tim-3信号能分别抑制和促进GLUT1、TNF-α的基因和蛋白表达以及GAPDH基因表达,但Tim-3的基因和蛋白水平以及GAPDH蛋白水平并无显著变化。结论 Tim-3负调控巨噬细胞GLUT1表达,进一步影响TNF-α分泌,参与TNF-α转录后调控的GAPDH可能参与其中的调节机制。
Objective To explore the roles and mechanisms of T cell immunoglobulin and mucin-domain containing molecule-3( Tim-3) in regulating the expression of glucose transporter 1( GLUT1) in macrophages. Methods GLUT1 protein expressions were analyzed in Raw 264. 7 treated with Tim-3 siRNA. And then,Raw 264. 7 was administered with various concentrations of Tim-3 activating antibody and blocking fusion protein to observe the mRNA and protein characteristics of GLUT1,glyceraldehyde-3-phosphate dehydrogenase( GAPDH),Tim-3 and TNF-α. Results The mRNA expressions of GLUT1,GAPDH,TNF-α were up-regulated/down-regulated in Raw 264. 7 when treated with Tim-3 blocking fusion protein/activating antibody,while the results of Tim-3 showed changes that were not of statistical significance. The protein expressions of GLUT1、TNF-α in macrophages were found to be the same as the quantitative real-time PCR( qRTPCR) results,while the changes in GAPDH and Tim-3 protein levels were not statistically significant. Conclusion Tim-3 plays a negative regulation role in GLUT1 and TNF-α expression of macrophages. Furthermore,GAPDH binding to TNF-α mRNA which contributes to posttranscriptional repression may be involved in the process of TNF-α secretion by macrophages.
Objective To explore the roles and mechanisms of T cell immunoglobulin and mucin-domain containing molecule-3( Tim-3) in regulating the expression of glucose transporter 1( GLUT1) in macrophages. Methods GLUT1 protein expressions were analyzed in Raw 264. 7 treated with Tim-3 siRNA. And then,Raw 264. 7 was administered with various concentrations of Tim-3 activating antibody and blocking fusion protein to observe the mRNA and protein characteristics of GLUT1,glyceraldehyde-3-phosphate dehydrogenase( GAPDH),Tim-3 and TNF-α. Results The mRNA expressions of GLUT1,GAPDH,TNF-α were up-regulated/down-regulated in Raw 264. 7 when treated with Tim-3 blocking fusion protein/activating antibody,while the results of Tim-3 showed changes that were not of statistical significance. The protein expressions of GLUT1、TNF-α in macrophages were found to be the same as the quantitative real-time PCR( qRTPCR) results,while the changes in GAPDH and Tim-3 protein levels were not statistically significant. Conclusion Tim-3 plays a negative regulation role in GLUT1 and TNF-α expression of macrophages. Furthermore,GAPDH binding to TNF-α mRNA which contributes to posttranscriptional repression may be involved in the process of TNF-α secretion by macrophages.
引文
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[20]Jiang X,Zhou T,Xiao Y,et al.Tim-3 promotes tumor-promoting M2 macrophage polarization by binding to STAT1 and suppressing the STAT1-miR-155 signaling axis[J].Oncoimmunology,2016,5(9):1-12.
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[24]Zhou Y,Yi X,Stoffer JB,et al.The multifunctional protein glyceraldehyde-3-phosphate dehydrogenase is both regulated and controls colony-stimulating factor-1 messenger RNA stability in ovarian cancer[J].Mol Cancer Res,2008,6(8):1375-1384.
[25]Millet P,Vachharajani V,Mcphail L,et al.GAPDH binding to TNF-alpha mRNA contributes to posttranscriptional repression in monocytes:a novel mechanism of communication between inflammation and metabolism[J].J Immunol,2016,196(6):2541-1551.
[26]张嘉诚,韩根成.巨噬细胞代谢重塑与功能调控研究进展[J].国际免疫学杂志,2018,41(2):195-198.
[2]Mcintire JJ,Umetsu SE,Akbari O,et al.Identification of Tapr(an airway hyperreactivity regulatory locus)and the linked Tim gene family[J].Nat Immunol,2001,2(12):1109-1116.
[3]Zhu C,Anderson AC,Schubart A,et al.The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity[J].Na Immunol,2005,6(12):1245-1252.
[4]Sanchez-Fueyo A,Tian J,Picarella D,et al.Tim-3 inhibits Thelper type 1-mediated auto-and alloimmune responses and promotes immunological tolerance[J].Nat Immunol,2003,4(11):1093-1101.
[5]Sabatos CA,Chakravarti S,Cha E,et al.Interaction of Tim-3and Tim-3 ligand regulates T helper type 1 responses and induction of peripheral tolerance[J].Nat Immunol,2003,4(11):1102-1110.
[6]Liu J,Zhang S,Hu Y,et al.Targeting PD-1 and Tim-3 pathways to reverse CD8 T-cell exhaustion and enhance ex vivo T-cell responses to autologous dendritic/tumor vaccines[J].J Immunother,2016,39(4):171-180.
[7]Zhang Y,Ma CJ,Wang JM,et al.Tim-3 negatively regulates IL-12 expression by monocytes in HCV infection[J].PLo S One,2011,6(5):1-14.
[8]Zhang Y,Ma CJ,Wang JM,et al.Tim-3 regulates pro-and anti-inflammatory cytokine expression in human CD14+monocytes[J].J Leukoc Biol,2012,91(2):189-196.
[9]Mueckler M,Thorens B.The SLC2(GLUT)family of membrane transporters[J].Mol Aspects Med,2013,34(3):121-138.
[10]Pandeti S,Arha D,Mishra A,et al.Glucose uptake stimulatory potential and antidiabetic activity of the Arnebin-1 from Arnabia nobelis[J].Eur J Pharmacol,2016,789(3):449-457.
[11]Yan J,Young ME,Cui L,et al.Increased glucose uptake and oxidation in mouse hearts prevent high fatty acid oxidation but cause cardiac dysfunction in diet-induced obesity[J].Circulation,2009,119(2):2818-2828.
[12]Ancey PB,Contat C,Meylan E.Glucose transporters in cancer:from tumor cells to the tumor microenvironment[J].FEBS J,2018,12(6):21-25.
[13]Wegiel B,Vuerich M,Daneshmandi S,et al.Metabolic switch in the tumor microenvironment determines immune responses to anti-cancer therapy[J].Front Oncol,2018,8(2):284.
[14]Yang M,Mckay D,Pollard JW,et al.Diverse functions of macrophages in different tumor microenvironments[J].Cancer Res,2018,78(19):5492-5503.
[15]Martinez FO,Helming L,Gordon S.Alternative activation of macrophages:an immunologic functional perspective[J].Annu Rev Immunol,2009,27:451-483.
[16]Pelgrom LR,Everts B.Metabolic control of type 2 immunity[J].Eur J Immunol,2017,47(8):1266-1275.
[17]Wenes M,Shang M,Di Matteo M,et al.Macrophage metabolism controls tumor blood vessel morphogenesis and metastasis[J].Cell Metab,2016,24(5):701-715.
[18]Rodriguez-Fandino OA,Hernandez-Ruiz J,Lopez-Vidal Y,et al.Maturation phenotype of peripheral blood monocyte/macrophage after stimulation with lipopolysaccharides in irritable bowel syndrome[J].J Neurogastroenterol Motil,2017,23(2):281-288.
[19]Jahangier ZN,Jacobs JW,Kraan MC,et al.Pretreatment macrophage infiltration of the synovium predicts the clinical effect of both radiation synovectomy and intra-articular glucocorticoids[J].Ann Rheum Dis,2006,65(10):1286-1292.
[20]Jiang X,Zhou T,Xiao Y,et al.Tim-3 promotes tumor-promoting M2 macrophage polarization by binding to STAT1 and suppressing the STAT1-miR-155 signaling axis[J].Oncoimmunology,2016,5(9):1-12.
[21]Verdeguer F,Aouadi M.Macrophage heterogeneity and energy metabolism[J].Exp Cell Res,2017,360(1):35-40.
[22]Nagy E,Rigby WF.GAPDH selectively binds AU-rich RNA in the NAD(+)-binding region[J].J Biol Chem,1995,270(6):2755-2763
[23]Ikeda Y,Yamaji R,Irie K,et al.Glyceraldehyde-3-phosphate dehydrogenase regulates cyclooxygenase-2 expression by targeting mRNA stability[J].Arch Biochem Biophys,2012,528(2):141-147.
[24]Zhou Y,Yi X,Stoffer JB,et al.The multifunctional protein glyceraldehyde-3-phosphate dehydrogenase is both regulated and controls colony-stimulating factor-1 messenger RNA stability in ovarian cancer[J].Mol Cancer Res,2008,6(8):1375-1384.
[25]Millet P,Vachharajani V,Mcphail L,et al.GAPDH binding to TNF-alpha mRNA contributes to posttranscriptional repression in monocytes:a novel mechanism of communication between inflammation and metabolism[J].J Immunol,2016,196(6):2541-1551.
[26]张嘉诚,韩根成.巨噬细胞代谢重塑与功能调控研究进展[J].国际免疫学杂志,2018,41(2):195-198.