摘要
目的通过体外EGF作用于下调LRIG1后的GL15细胞,探讨LRIG1表达下调对p-EGFR蛋白,p-Akt蛋白和p-ERK蛋白表达的影响。
方法GL15细胞经转染LRIG1-shRNA质粒后,Western Blot检测LRIG1蛋白表达的状况。在含有G418的DMEM培养基中培养经稳定转染LRIG1-shRNA的GL15细胞,经表皮生长因子(EGF)100ng/ml体外刺激后,Western Blot检测p-EGFR,p-AKT和p-ERK表达水平随时间的效应变化。
结果Westemblot结果显示:GL15细胞转染LRIG1-shRNA质粒后,LRIG1表达较对照组降低52.32%,P<0.01有统计学差异。用EGF刺激转染了LRIG1-shRNA质粒GL15细胞,在0min时,实验组p-Akt表达较对照组升高25.12% ,P<0.01有统计学差异;在5min时,实验组p-EGFR和实验组p-Akt表达较对照组分别升高343.92%,27.34%,P<0.01有统计学差异;在30min时,实验组p-EGFR和实验组p-AKT表达较对照组分别升高70.94%,29.39%,P<0.01有统计学差异;用EGF刺激转染了LRIG1-shRNA质粒GL15细胞,在0分钟,5分钟和30分钟的时候,实验组p-ERK表达较对照组无明显变化,数据统计P>0.05无统计学差异。
结论GL15细胞经转染LRIG1-shRNA质粒后,LRIG1蛋白表达降低;用EGF刺激转染了LRIG1-shRNA质粒的和转染了pGenesil2-negative shRNA质粒的GL15细胞,前者细胞中的p-EGFR蛋白和p-AKT蛋白表达水平显著增加,而p-ERK蛋白的表达无明显变化。因此,LRIG1对p-EGFR/Akt这一信号通路存在较明显的调控作用,然而,对EGFR信号通路中的ERK通路无明显影响。
Objective Inhibition of LRIG1 gene affects the levels of p-EGFR, p-Akt and p-ERK protein of the transfected GL15 cell line.
Methods The transfected GL15 cell were cultured in medium containing G418. After EGF (100ng/ml) stimulated the cell, the levels of p-EGFR, p-Akt and p-ERK protein were detected with Western Blotting.
Results The levels of p-EGFR and p-Akt protein rised after 5 minutes and 30 minutes of EGF stimulation, but the protein levels of p-ERK weren’t changed significantly. Conclusion EGF raised the levels of p-EGFR, p-Akt protein of the transfected GL15 cell line, but don’t changed the level of p-ERK protein.
引文
[1]易伟. LRIG1抑癌基因对人脑胶质瘤细胞的作用和分子机制[D].华中科技大学, 2005.
[2]蔡明俊. LRIG3基因对脑胶质瘤细胞生物学行为差异性调控的机制研究[D].华中科技大学, 2009.
[3]刘红朝. RNA干扰LRIG1基因对人胶质母细胞瘤GL15细胞系生物学行为的影响及机制研究[D].华中科技大学, 2009.
[4]邓凡.表皮生长因子受体家族与肿瘤关系的研究进展[J].中国癌症杂志, 1999(Z1).
[5]刘家刚,刘艳辉,蔡敬,等. EGFR、PTEN在常见恶性脑肿瘤中的表达及意义[J].四川大学学报(医学版), 2006(6).
[6]薄爱华,程琳,李海峰,等.表皮生长因子受体与肿瘤发生及治疗的相关性[J].医学综述, 2006(20):1238-1240.
[7] JUNGBLUTH A A, STOCKERT E, HUANG H J, et al. A monoclonal antibody recognizing human cancers with amplification/overexpression of the human epidermal growth factor receptor[J]. Proc Natl Acad Sci U S A, 2003,100(2):639-644.
[8]滕帼英,潘泽政,吴剑,等.表皮生长因子受体靶向RNAi抑制恶性肿瘤中的研究现状[J].井冈山医专学报, 2008(6):14-16.
[9]金永健,金玉兰,李龙山. p~(16)mRNA及其蛋白、EGFR在脑胶质瘤中的表达[J].临床与实验病理学杂志, 2000(1):38-40.
[10] BESSON A, YONG V W. Mitogenic signaling and the relationship to cell cycle regulation in astrocytomas[J]. J Neurooncol, 2001,51(3):245-264.
[11]熊宗强. EGFR与LRIG1在三叉神经鞘瘤中的表达研究[D].华中科技大学, 2006.
[12] BAZLEY L A, GULLICK W J. The epidermal growth factor receptor family[J]. Endocr Relat Cancer, 2005,12 Suppl 1:S17-S27.
[13]席桂发. LRIG3对胶质母细胞瘤细胞系GL15生物学特性影响的分子生物学机制研究[D].华中科技大学, 2007.
[14]孟庆祥.表皮生长因子受体研究进展[J].国外医学.输血及血液学分册, 1999(1):34-37.
[15] ULLRICH A, COUSSENS L, HAYFLICK J S, et al. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells[J]. Nature, 1984,309(5967):418-425.
[16]刘宝全,范圣第. EGFR信号转导机制及靶向治疗[J].大连民族学院学报, 2008(1):13-16.
[17]王宝峰. LRIG2对胶质母细胞瘤细胞系GL15生物学特性的影响及其分子生物学机制研究[D].华中科技大学, 2009.
[18]王明国.表皮生长因子受体[J].国外医学(分子生物学分册), 2002(2):82-85.
[19] SEBASTIAN S, SETTLEMAN J, RESHKIN S J, et al. The complexity of targeting EGFR signalling in cancer: from expression to turnover[J]. Biochim Biophys Acta, 2006,1766(1):120-139.
[20]侯亮,姜月华,詹臻.恶性肿瘤的表皮生长因子受体靶向治疗现状[J].临床肿瘤学杂志, 2004(3):314-317.
[21]唐剑英.表皮生长因子受体家族信号转导与肿瘤治疗[J].国外医学(肿瘤学分册), 2002(2):95-97.
[22] GHIGLIONE C, CARRAWAY K R, AMUNDADOTTIR L T, et al. The transmembrane molecule kekkon 1 acts in a feedback loop to negatively regulate the activity of the Drosophila EGF receptor during oogenesis[J]. Cell, 1999,96(6):847-856.
[23] ALVARADO D, RICE A H, DUFFY J B. Bipartite inhibition of Drosophila epidermal growth factor receptor by the extracellular and transmembrane domains of Kekkon1[J]. Genetics, 2004,167(1):187-202.
[24] SUZUKI Y, SATO N, TOHYAMA M, et al. cDNA cloning of a novel membrane glycoprotein that is expressed specifically in glial cells in the mouse brain. LIG-1, aprotein with leucine-rich repeats and immunoglobulin-like domains[J]. J Biol Chem, 1996,271(37):22522-22527.
[25] NILSSON J, VALLBO C, GUO D, et al. Cloning, characterization, and expression of human LIG1[J]. Biochem Biophys Res Commun, 2001,284(5):1155-1161.
[26] GUO D, HOLMLUND C, HENRIKSSON R, et al. The LRIG gene family has three vertebrate paralogs widely expressed in human and mouse tissues and a homolog in Ascidiacea[J]. Genomics, 2004,84(1):157-165.
[27] THOMASSON M, HEDMAN H, GUO D, et al. LRIG1 and epidermal growth factor receptor in renal cell carcinoma: a quantitative RT--PCR and immunohistochemical analysis[J]. Br J Cancer, 2003,89(7):1285-1289.
[28] NILSSON J, STAREFELDT A, HENRIKSSON R, et al. LRIG1 protein in human cells and tissues[J]. Cell Tissue Res, 2003,312(1):65-71.
[29]严泽军,叶章群,杨为民,等. LRIG1和EGFR在膀胱移行细胞癌中的表达及临床意义[J].临床泌尿外科杂志, 2007(3).
[30] TANEMURA A, NAGASAWA T, INUI S, et al. LRIG-1 provides a novel prognostic predictor in squamous cell carcinoma of the skin: immunohistochemical analysis for 38 cases[J]. Dermatol Surg, 2005,31(4):423-430.
[1] MUSACCHIO M, PERRIMON N. The Drosophila kekkon genes: novel members of both the leucine-rich repeat and immunoglobulin superfamilies expressed in the CNS[J]. Dev Biol, 1996,178(1):63-76.
[2] GHIGLIONE C, CARRAWAY K R, AMUNDADOTTIR L T, et al. The transmembrane molecule kekkon 1 acts in a feedback loop to negatively regulate the activity of the Drosophila EGF receptor during oogenesis[J]. Cell, 1999,96(6):847-856.
[3] SUZUKI Y, SATO N, TOHYAMA M, et al. cDNA cloning of a novel membrane glycoprotein that is expressed specifically in glial cells in the mouse brain. LIG-1, a protein with leucine-rich repeats and immunoglobulin-like domains[J]. J Biol Chem, 1996,271(37):22522-22527.
[4] NILSSON J, VALLBO C, GUO D, et al. Cloning, characterization, and expression of human LIG1[J]. Biochem Biophys Res Commun, 2001,284(5):1155-1161.
[5]蔡明俊. LRIG3基因对脑胶质瘤细胞生物学行为差异性调控的机制研究[D].华中科技大学, 2009.
[6]熊宗强. EGFR与LRIG1在三叉神经鞘瘤中的表达研究[D].华中科技大学, 2006.
[7]滕帼英,潘泽政,吴剑,等.表皮生长因子受体靶向RNAi抑制恶性肿瘤中的研究现状[J].井冈山医专学报, 2008(6):14-16.
[8]唐剑英.表皮生长因子受体家族信号转导与肿瘤治疗[J].国外医学(肿瘤学分册), 2002(2):95-97.
[9]王明国.表皮生长因子受体[J].国外医学(分子生物学分册), 2002(2):82-85.
[10]刘宝全,范圣第. EGFR信号转导机制及靶向治疗[J].大连民族学院学报, 2008(1):13-16.
[11]胡兵,魏于全.表皮生长因子受体靶向肿瘤生物治疗[J].癌症, 2004(4):471-475.
[12] SEWELL J M, SMYTH J F, LANGDON S P. Role of TGF alpha stimulation of the ERK, PI3 kinase and PLC gamma pathways in ovarian cancer growth and migration[J]. Exp Cell Res, 2005,304(1):305-316.
[13]易伟. LRIG1抑癌基因对人脑胶质瘤细胞的作用和分子机制[D].华中科技大学, 2005.
[14]姚声涛,唐文渊,郭川. LRIG1诱导人胶质瘤细胞凋亡与表皮生长因子受体基因关系[J].第三军医大学学报, 2008(2):157-160.
[15]曾令成,欧一博,雷霆,等. LRIG1在脑膜瘤中的表达及与其生物学行为的关系[J].中国临床神经外科杂志, 2007(10):601-604.
[16] PI3K_AKT_PTEN在脑胶质瘤中的表达及其临床意义[J].
[17]张薇薇,苗玲. PI3K/Akt信号通路及其在神经疾病中的研究进展[J].中风与神经疾病杂志, 2007(6):755-757.
[18]夏曙,于世英. PI3K/Akt信号转导通路在恶性肿瘤中的研究进展[J].肿瘤, 2006(6):576-578.
[19] MICHL P, DOWNWARD J. Mechanisms of disease: PI3K/AKT signaling in gastrointestinal cancers[J]. Z Gastroenterol, 2005,43(10):1133-1139.
[20]夏洪平,郑芹,杨惠玲.新的抗肿瘤靶点Akt与胰腺癌分子靶向治疗[J].胰腺病学, 2006(5):315-317.
[21]谭莉莉,白文元. PI3K/AKt信号通路与肝细胞癌[J].国际消化病杂志, 2008(2):120-122.
[22] DOWNWARD J. Mechanisms and consequences of activation of protein kinase B/Akt[J]. Curr Opin Cell Biol, 1998,10(2):262-267.
[23]李小芳,姜汉国. PI_3K/AKT信号转导通路在乳腺癌转移和耐药中的研究[J].医学综述, 2008(3):355-357.
[24]赵茜,陆融,姚智.磷脂酰肌醇3激酶-蛋白质丝氨酸苏氨酸激酶途径与肿瘤的关系[J].国际肿瘤学杂志, 2006(6):401-404.
[25] SARBASSOV D D, GUERTIN D A, ALI S M, et al. Phosphorylation and regulation ofAkt/PKB by the rictor-mTOR complex[J]. Science, 2005,307(5712):1098-1101.
[26] ALTOMARE D A, TESTA J R. Perturbations of the AKT signaling pathway in human cancer[J]. Oncogene, 2005,24(50):7455-7464.
[27] ITOH N, SEMBA S, ITO M, et al. Phosphorylation of Akt/PKB is required for suppression of cancer cell apoptosis and tumor progression in human colorectal carcinoma[J]. Cancer, 2002,94(12):3127-3134.
[28]郭琳,王强. PI3K/Akt/mTOR信号传导通路与恶性肿瘤浸润和转移的研究进展[J].现代肿瘤医学, 2009(8):1585-1589.
[29]黄珊珊,乔玉环,郭瑞霞.丝裂原活化蛋白激酶信号转导通路与子宫内膜癌[J].国际妇产科学杂志, 2009(1):56-59.
[30] MAYO L D, DONNER D B. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus[J]. Proc Natl Acad Sci U S A, 2001,98(20):11598-11603.
[31]丘雅维,杨惠玲. PI3K通路及其抑制剂抗肿瘤的研究进展[J].中山大学学报(医学科学版), 2009(S1):223-226.
[32]章丽霞,刘钧.细胞外信号调节激酶(ERK)与宫颈癌关系的研究进展[J].川北医学院学报, 2010(5):478-482.
[33] STEINMETZ R, WAGONER H A, ZENG P, et al. Mechanisms regulating the constitutive activation of the extracellular signal-regulated kinase (ERK) signaling pathway in ovarian cancer and the effect of ribonucleic acid interference for ERK1/2 on cancer cell proliferation[J]. Mol Endocrinol, 2004,18(10):2570-2582.
[34] HILGER R A, SCHEULEN M E, STRUMBERG D. The Ras-Raf-MEK-ERK pathway in the treatment of cancer[J]. Onkologie, 2002,25(6):511-518.
[35]许瀚,李孟圈. ERK信号转导通路与乳腺癌关系的研究进展[J].肿瘤基础与临床, 2010(1):80-82.
[36]刘刚,俞薇薇. EBV、ki67、ERK1在乳腺癌中的表达及其相关性[J].实用临床医学, 2008(3):129-130.
[37] ZENG P, WAGONER H A, PESCOVITZ O H, et al. RNA interference (RNAi) for extracellular signal-regulated kinase 1 (ERK1) alone is sufficient to suppress cell viability in ovarian cancer cells[J]. Cancer Biol Ther, 2005,4(9):961-967.
[38] WANG X, MARTINDALE J L, HOLBROOK N J. Requirement for ERK activation in cisplatin-induced apoptosis[J]. J Biol Chem, 2000,275(50):39435-39443.
[39] TANG D, WU D, HIRAO A, et al. ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53[J]. J Biol Chem, 2002,277(15):12710-12717.
[40] PLOTKIN L I, AGUIRRE J I, KOUSTENI S, et al. Bisphosphonates and estrogens inhibit osteocyte apoptosis via distinct molecular mechanisms downstream of extracellular signal-regulated kinase activation[J]. J Biol Chem, 2005,280(8):7317-7325.
[41] AHMED N, PANSINO F, BAKER M, et al. Association between alphavbeta6 integrin expression, elevated p42/44 kDa MAPK, and plasminogen-dependent matrix degradation in ovarian cancer[J]. J Cell Biochem, 2002,84(4):675-686.
[42] THOMPSON N, LYONS J. Recent progress in targeting the Raf/MEK/ERK pathway with inhibitors in cancer drug discovery[J]. Curr Opin Pharmacol, 2005,5(4):350-356.
[43] KHOKHLATCHEV A V, CANAGARAJAH B, WILSBACHER J, et al. Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation[J]. Cell, 1998,93(4):605-615.
[1] SEBASTIAN S, SETTLEMAN J, RESHKIN S J, et al. The complexity of targeting EGFR signalling in cancer: from expression to turnover[J]. Biochim Biophys Acta, 2006,1766(1):120-139.
[2]刘宝全,范圣第. EGFR信号转导机制及靶向治疗[J].大连民族学院学报, 2008(1):13-16.
[3]熊宗强. EGFR与LRIG1在三叉神经鞘瘤中的表达研究[D].华中科技大学, 2006.
[4]蔡明俊. LRIG3基因对脑胶质瘤细胞生物学行为差异性调控的机制研究[D].华中科技大学, 2009.
[5] BAZLEY L A, GULLICK W J. The epidermal growth factor receptor family[J]. Endocr Relat Cancer, 2005,12 Suppl 1:S17-S27.
[6]滕帼英,潘泽政,吴剑,等.表皮生长因子受体靶向RNAi抑制恶性肿瘤中的研究现状[J].井冈山医专学报, 2008(6):14-16.
[7]唐剑英.表皮生长因子受体家族信号转导与肿瘤治疗[J].国外医学(肿瘤学分册), 2002(2):95-97.
[8]修霞,吕志民.泛素介导的表皮生长因子受体的降解[J].中国医学科学院学报, 2005(1):120-127.
[9]王明国.表皮生长因子受体[J].国外医学(分子生物学分册), 2002(2):82-85.
[10]曾令成,欧一博,雷霆,等. LRIG1在脑膜瘤中的表达及与其生物学行为的关系[J].中国临床神经外科杂志, 2007(10):601-604.
[11]易伟. LRIG1抑癌基因对人脑胶质瘤细胞的作用和分子机制[D].华中科技大学, 2005.
[12] MUSACCHIO M, PERRIMON N. The Drosophila kekkon genes: novel members of both the leucine-rich repeat and immunoglobulin superfamilies expressed in the CNS[J]. Dev Biol, 1996,178(1):63-76.
[13] SUZUKI Y, SATO N, TOHYAMA M, et al. cDNA cloning of a novel membraneglycoprotein that is expressed specifically in glial cells in the mouse brain. LIG-1, a protein with leucine-rich repeats and immunoglobulin-like domains[J]. J Biol Chem, 1996,271(37):22522-22527.
[14] NILSSON J, VALLBO C, GUO D, et al. Cloning, characterization, and expression of human LIG1[J]. Biochem Biophys Res Commun, 2001,284(5):1155-1161.
[15] GOLDONI S, IOZZO R A, KAY P, et al. A soluble ectodomain of LRIG1 inhibits cancer cell growth by attenuating basal and ligand-dependent EGFR activity[J]. Oncogene, 2007,26(3):368-381.
[16] GUR G, RUBIN C, KATZ M, et al. LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation[J]. EMBO J, 2004,23(16):3270-3281.
[17] EKSTRAND A J, JAMES C D, CAVENEE W K, et al. Genes for epidermal growth factor receptor, transforming growth factor alpha, and epidermal growth factor and their expression in human gliomas in vivo[J]. Cancer Res, 1991,51(8):2164-2172.
[18] VON BOSSANYI P, SALLABA J, DIETZMANN K, et al. Correlation of TGF-alpha and EGF-receptor expression with proliferative activity in human astrocytic gliomas[J]. Pathol Res Pract, 1998,194(3):141-147.
[19] PENAR P L, KHOSHYOMN S, BHUSHAN A, et al. Inhibition of epidermal growth factor receptor-associated tyrosine kinase blocks glioblastoma invasion of the brain[J]. Neurosurgery, 1997,40(1):141-151.
[20]甄海宁,章翔,张志文,等.原癌基因c-erbB-1和c-erbB-2在人脑胶质瘤中的表达[J].第四军医大学学报, 1999(2):106-109.
[21]刘艳辉,何能前,毛伯镛,等. EGFR在髓母细胞瘤中的表达研究[J].四川医学, 2000(10):918-919.
[22]郭琳,王强. PI3K/Akt/mTOR信号传导通路与恶性肿瘤浸润和转移的研究进展[J].现代肿瘤医学, 2009(8):1585-1589.
[23]侯亮,姜月华,詹臻.恶性肿瘤的表皮生长因子受体靶向治疗现状[J].临床肿瘤学杂志, 2004(3):314-317.
[24]胡兵,魏于全.表皮生长因子受体靶向肿瘤生物治疗[J].癌症, 2004(4):471-475.
[25]高艳景,刘晔,袁孟彪.表皮生长因子受体与肿瘤治疗研究进展[J].国外医学(肿瘤学分册), 1999(2).
[26]姚声涛,唐文渊,郭川. LRIG1诱导人胶质瘤细胞凋亡与表皮生长因子受体基因关系[J].第三军医大学学报, 2008(2):157-160.