应用磷酸化蛋白质组学方法初步研究喉癌相关基因LCRG1的功能  

Preliminary Function Study of Laryngeal Carcinoma Related Gene LCRG1 Using Phosphorproteomics Methods

作　　者：章晓鹏[1] 肖志强[1] 李萃[1] 李建玲[1] 余艳辉[2] 欧阳咏梅[2] 冯雪萍[1] 张鹏飞[1] 陈主初[1] 

机构地区：[1]中南大学湘雅医院卫生部肿瘤蛋白质组学重点实验室,长沙410008 [2]中南大学肿瘤研究所,长沙410078

出　　处：《生物化学与生物物理进展》2005年第6期508-516,共9页Progress In Biochemistry and Biophysics

基　　金：国家重点基础研究发展规划项目(973)(2001CB510207);国家自然科学基金资助项目(30000028;30240056;30370642);教育部跨世纪优秀人才培养计划基金(教育部科技函[2002]48);湖南省科技厅重大科技专项(04XK1001);湖南省科技厅重点科研项目(02SSY2001-1);湖南省卫生厅重点科研项目(Z02-04)~~

摘　　要：mRNA差异显示技术克隆的喉癌相关基因LCRG1,对不表达该基因的喉癌细胞系(Hep-2)的生长具有明显抑制作用.软件分析推测,LCRG1可能在细胞信号传导中发挥作用.为进一步地研究LCRG1的功能,应用RT-PCR和平板克隆形成实验证实,经多次传代的Hep-2/LCRG1细胞,仍表达LCRG1,且LCRG1具有显著的抑制细胞增殖的能力.抽提Hep-2/LCRG1和Hep-2/pcDNA3.1(+)细胞系总蛋白质,应用固相pH梯度(IPG)双向凝胶电泳(2DGE),结合抗酪氨酸磷酸化抗体的免疫印迹和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS),鉴定酪氨酸磷酸化的蛋白质.得到了分辨率较高、重复性较好的Hep-2/LCRG1和Hep-2/pcDNA3.1(+)细胞系的总蛋白质双向凝胶电泳图谱;结合免疫印迹反应、软件分析和质谱技术识别并鉴定了13个差异反应的酪氨酸磷酸化的蛋白质.这些蛋白质参与了细胞信号传导和细胞代谢等过程.推测LCRG1可能是通过调节这些蛋白质的酪氨酸磷酸化、去磷酸化状态,参与细胞增殖、代谢和凋亡等过程的调控,而发挥抑瘤作用.这为全面、真实地揭示LCRG1抑瘤作用的分子机理提供了新思路.Laryngeal carcinoma related gene LCRG1, cloned by the laboratory using mRNA differential display, has the suppressive function to none expression LCRG1 Hep-2 cell line. Bioinformatics analysis using software showed LCRG1 may play function in cellular signal transduction. In order to further elucidate the function of LCRG1, RT-PCR and colony efficiency were used to identify whether LCRG1 expressed and had the tumor suppressive function in incubated Hep-2/LCRG1 cell lines, The results suggested LCRG1 was expressed in Hep-2/LCRG1 cell lines and had the significant suppressive proliferation ability. Hence, the total proteins of Hep-2/LCRG1 and Hep-2/pcDNA3.1 (+) cell lines were separated by immobilized pH gradient (FPG)-based two-dimensional gel electrophoresis(2DGE), coupled with anti-tyrosine phosphorylated antibody immunoblotting and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) identifying tyrosine-phosphorylated proteins. The well-resolved, reproducible 2DGE patterns of Hep-2/LCRG1 and Hep-2/pcDNA3.1 (+) cell lines were established and 13 differential tyrosine-phosphorylated proteins were identified using immunoblotting, analysis software and MALDI-TOF-MS methods. These proteins were involved in the signal transduction and cell cycle. So it was speculated that LCRG1 may be involved in the processes of cellular proliferation, metabolic pathways and apoptosis etc. and play tumor suppressive functions through regulating the phosphorylation/dephosphorylation status of these proteins. These data will be helpful to elucidate the molecular mechanism of LCRG1 tumor suppressive function.

关 键 词：LCRG1基因 Hep-2/LCRG1细胞系 Hep-2/pcDNA3.1(+)细胞系 双向凝胶电泳 MALDI-TOF-MS 免疫印迹 酪氨酸磷酸化蛋白质 蛋白质印迹 

分 类 号：R739.6[医药卫生—肿瘤] Q51[医药卫生—临床医学]