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

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 .

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

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(IPG)-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.