Zhang Xiaopeng, Xiao Zhiqiang, Chen Zhuchu, Li Cui, Li Jianling, Yanhui Yu, Yang Fang, Yang YiXuan, Oyang Yongmei
From Key Laboratory of Cancer Proteomics, Ministry of Health of China 'Xiangya Hospital, and the Cancer Research Institute, Central South University, Changsha, China.
Laryngoscope. 2006 Feb;116(2):224-30. doi: 10.1097/01.mlg.0000191470.71454.a1.
A novel gene, laryngeal carcinoma-related gene 1 (LCRG1), had the characteristics of tumor-suppressor genes. It was cloned in our laboratory. The objective was to find and characterize the proteins related to LCRG1 and to elucidate the molecular mechanism of LCRG1.
We used the established cell lines of Hep-2/LCRG1 (Hep-2 cells transfected by recombinant plasmid pcDNA3.1[+]/LCRG1) and Hep-2/pcDNA3.1(+) (Hep-2 cells transfected by control vector pcDNA3.1[+]) as cell models.
Two-dimensional gel electrophoresis (2-DE) technology was performed to separate the proteins of Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cell lines, respectively. The differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization-quadruple time-of-flight MS/MS (ESI-Q-TOF MS/MS). Then the differential expression levels of partial identified proteins were determined by Western blotting analysis and quantitative real-time reverse transcriptase-polymerase chain reaction.
The results showed the attained 2-DE patterns of the two cell lines were well-resolved and reproducible. There were 1075+/-43 and 1027+/-23 protein spots observed in Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cell lines, respectively. The average matching rate of the two cell lines was 91%. Twenty-six differentially expressed protein spots were identified (twenty spots for MALDI-TOF-MS, six spots for ESI-Q-TOF MS/MS). Most of the characterized proteins were characterized as the members of enzymes (phosphoglycerate mutase, manganese superoxide dismutase, and so on), transcription proteins (rho gdp dissociation inhibitor), and so on. Those identified proteins might contribute to the tumor-suppressive function of LCRG1. The differential expression levels of the partial proteins were confirmed by real-time polymerase chain reaction and Western blotting.
We tentatively proposed those differentially expressed proteins were involved in the tumor-suppressive process related to LCRG1. These data will be helpful to elucidate the molecular mechanism of LCRG1.
一种新基因，喉癌相关基因1（LCRG1），具有肿瘤抑制基因的特征，是在我们实验室克隆得到的。目的是寻找与LCRG1相关的蛋白质并对其进行表征，阐明LCRG1的分子机制。
我们使用已建立的Hep-2/LCRG1细胞系（用重组质粒pcDNA3.1[+]/LCRG1转染的Hep-2细胞）和Hep-2/pcDNA3.1(+)细胞系（用对照载体pcDNA3.1[+]转染的Hep-2细胞）作为细胞模型。
分别采用二维凝胶电泳（2-DE）技术分离Hep-2/LCRG1和Hep-2/pcDNA3.1(+)细胞系的蛋白质。通过软件分析对差异蛋白质斑点进行分析，进行胶内酶解，然后用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和电喷雾电离-四极杆飞行时间串联质谱（ESI-Q-TOF MS/MS）进行鉴定。然后通过蛋白质印迹分析和定量实时逆转录聚合酶链反应测定部分鉴定蛋白质的差异表达水平。
结果表明，所获得的两种细胞系的二维凝胶电泳图谱分辨率良好且可重复。在Hep-2/LCRG1和Hep-2/pcDNA3.1(+)细胞系中分别观察到1075±43和1027±23个蛋白质斑点。两种细胞系的平均匹配率为91%。鉴定出26个差异表达的蛋白质斑点（20个斑点通过MALDI-TOF-MS鉴定，6个斑点通过ESI-Q-TOF MS/MS鉴定）。大多数已表征的蛋白质被表征为酶（磷酸甘油酸变位酶、锰超氧化物歧化酶等）、转录蛋白（rho GDP解离抑制剂）等成员。那些鉴定出的蛋白质可能有助于LCRG1的肿瘤抑制功能。部分蛋白质的差异表达水平通过实时聚合酶链反应和蛋白质印迹得到证实。
我们初步提出那些差异表达的蛋白质参与了与LCRG1相关的肿瘤抑制过程。这些数据将有助于阐明LCRG1的分子机制。
