Zhang Xiao-Peng, Xiao Zhi-Qiang, Chen Zhu-Chu, Li Cui, Li Jian-Ling, Yu Yan-Hui, Ouyang Yong-Mei, Feng Xue-Ping, Zhang Peng-Fei
Health Ministry Key Laboratory of Cancer Proteomics, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P. R. China.
Ai Zheng. 2006 Jan;25(1):22-8.
BACKGROUND & OBJECTIVE: Laryngeal carcinoma-related gene 1 (LCRG1), a novel laryngeal carcinoma-related tumor suppressor gene, was cloned with mRNA differential display method. Previous researches showed LCRG1 might inhibit cell growth, proliferation, colony formation in soft agar, and tumorigenesis of laryngeal carcinoma cell line Hep-2. This study was to screen the proteins associated with the tumor suppressive function of LCRG1 by comparative proteomics method.
The whole cellular proteins of Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cells were extracted and separated by two-dimensional gel electrophoresis (2-DE) technology. After electrophoresis, the gels were stained by Coomassie Brilliant Blue G-250, and analyzed using PDQuest software. The differentially expressed proteins were cut from the gels, analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), or electrospray ionization-quadrupole time-of-flight MS/MS (ESI-Q-TOF MS/MS), and identified through searching database with Mascot software.
The well-resolved, reproducible 2-DE patterns of Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cells were established. The total protein spots were 1,075+/-43 in Hep-2/LCRG1 cells and 1,027+/-23 in Hep-2/pcDNA3.1(+) cells, with an average matching rate of 91%. Using mass spectrometry technology, 20 differential protein spots between the 2 cell lines were identified. Among them, 16 were identified by MALDI-TOF-MS, and 4 were identified by ESI-Q-TOF MS/MS. Some of the identified proteins were characterized as members of cellular transcription and metabolism enzymes.
In this study, 2-DE gels of Hep-2/LCRG1 cell line with high expression of LCRG1 mRNA and vector control Hep-2/pcDNA3.1(+) cell line were established; some differential proteins related to LCRG1 were identified by mass spectrometry. These data will help to illustrate the molecular mechanism of LCRG1.
喉癌相关基因1（LCRG1）是通过mRNA差异显示方法克隆出的一种新型喉癌相关抑癌基因。以往研究表明，LCRG1可能抑制喉癌细胞系Hep-2的细胞生长、增殖、软琼脂集落形成及肿瘤发生。本研究采用比较蛋白质组学方法筛选与LCRG1肿瘤抑制功能相关的蛋白质。
提取Hep-2/LCRG1和Hep-2/pcDNA3.1(+)细胞的全细胞蛋白质，采用二维凝胶电泳（2-DE）技术进行分离。电泳后，凝胶用考马斯亮蓝G-250染色，并用PDQuest软件进行分析。从凝胶上切下差异表达的蛋白质点，用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）或电喷雾电离-四极杆飞行时间串联质谱（ESI-Q-TOF MS/MS）进行分析，并用Mascot软件检索数据库进行鉴定。
建立了Hep-2/LCRG1和Hep-2/pcDNA3.1(+)细胞分辨率良好、可重复的2-DE图谱。Hep-2/LCRG1细胞中的总蛋白点数为1075±43，Hep-2/pcDNA3.1(+)细胞中的总蛋白点数为1027±23，平均匹配率为91%。采用质谱技术，鉴定出2种细胞系之间的20个差异蛋白质点。其中，16个通过MALDI-TOF-MS鉴定，4个通过ESI-Q-TOF MS/MS鉴定。一些鉴定出的蛋白质被表征为细胞转录和代谢酶的成员。
本研究建立了LCRG1 mRNA高表达的Hep-2/LCRG1细胞系和载体对照Hep-2/pcDNA3.1(+)细胞系的2-DE凝胶图谱；通过质谱鉴定了一些与LCRG1相关的差异蛋白质。这些数据将有助于阐明LCRG1的分子机制。
