【作者】 黄卫国；

【导师】 肖志强；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 鼻咽癌(nasopharyngeal carcinoma, NPC)是我国南方最常见的恶性肿瘤之一,严重威胁我国人民的生命和健康。绝大多数NPC分化差,恶性程度高,易早期发生颈淋巴结和远处转移。NPC转移是一个多基因参与的过程,多个基因之间通过相互作用形成关键性的节点(node),这些节点在NPC转移中具有重要作用。我们前期实验采用蛋白质组学方法筛选NPC组织差异表达的蛋白质,鉴定了36个NPC组织的差异表达蛋白质,采用免疫组织化学染色检测差异蛋白质14-3-3σ(14-3-3 sigma)在正常鼻咽上皮组织、NPC组织以及颈淋巴结转移NPC组织中的表达情况。结果发现：在NPC组织中存在高频率的14-3-3σ蛋白表达缺失/下调,而且其表达缺失/下调与NPC转移相关,这提示14-3-3σ表达缺失/下调在NPC转移中可能具有重要作用,但14-3-3σ表达缺失/下调促进NPC转移的分子机制尚未阐明。为了研究14-3-3σ在NPC转移中的作用机制,本研究采用靶向蛋白质组学方法(免疫共沉淀结合质谱分析)分离鉴定NPC细胞中14-3-3σ的相互作用蛋白,采用基因本体论(GO)、功能聚类、信号通路和蛋白—蛋白相互作用(protein-protein interaction, PPI)等生物信息学方法分析14-3-3σ的相互作用蛋白,揭示其生物学意义,并对关键相互作用蛋白(14-3-3σ/EGFR/Keratin 8)进行实验验证和功能研究。主要结果如下：1、采用靶向蛋白质组学在NPC细胞中鉴定了111个14-3-30的相互作用蛋白,并对其中4个相互作用蛋白质(Keratin 8、EGFR、RAB7和p53)进行了验证。2、GO和聚类分析显示：111个14-3-3σ的相互作用蛋白可聚类成13功能相关的簇,这些蛋白的功能主要归类为8个方面：细胞骨架、跨膜转运、分子伴侣、核糖体蛋白、GTPase、蛋白转运、ATPase和RNA结合。3、KEGG和Biocarta信号通路分析显示：14-3-3σ的相互作用蛋白涉及3条Biocarta信号通路和6条KEGG信号通路,其中16个相互作用蛋白涉及到细胞骨架信号通路,5个相互作用蛋白涉及到囊泡转运信号通路。4、蛋白相互作用分析显示：14-3-3σ/p53/RAB7相互作用组(Interactome)调控囊泡转运,14-3-3σ/EGFR/CK8相互作用组调控细胞骨架,它们可能与NPC转移相关。5、实验证实在NPC细胞中存在14-3-3σ/EGFR/Keratin 7相互作用组。6、功能分析显示：14-3-3σ下调能增加NPC细胞EGFR和Keratin 8表达,增强NPC细胞的体外侵袭能力。研究结果提示,14-3-3σ表达下调可能通过14-3-3σ/EGFR/Keratin8相互作用组而促进NPC侵袭转移。更多还原

【Abstract】 Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China, and it poses one of the serious public health problems in this area. Majority of NPC are poorly differentiated and highly malignant, and usually have early cervical lymphnode and distant metastasis. It is well know that the metastasis of NPC is involved in multiple genes interaction, and there were many key nodes formed by the genes interaction. These nodes play an important role in the metastasis of NPC.In our previous study, we identified 36 differential expression proteins in NPC tissues by proteomic approach. There was a highly frequent downregulaiton of 14-3-3 a in NPC tissues, and its downregulaiton was related to the metastasis of NPC, which indicates that 14-3-3 a downregulaiton may play an important role in the metastasis of NPC. But it is unknown that the mechanism of 14-3-3σdownregulaiton-promoting NPC metastasis.To study the mechanism of 14-3-3 a downregulaiton-promoting NPC metastasis, targeted proteomics(coimmunoprecipitation coupled with MS) was used to identify 14-3-3 a-interacted proteins. Bioinformatics (Gene Ontology, cluster analysis, signal pathway analysis and protein-protein interaction analysis) was performed to analyzed the data of targeted proteomics to reveal the biological significance. Finally, experimental confirmation and functional studies on the key interactome(14-3-3σ/EGFR/Keratin 8) were performed.The main results were as following:1. One hundred and eleven 14-3-3 a-interacted proteins were identified by targeted proteomics, and four 14-3-3 a-interacted proteins (Keratin 8, EGFR, RAB7and p53) were confirmed by coimmunoprecipitation and western blot.2. Cluster analysis showed that one hundred and eleven 14-3-3 a-interacted proteins were grouped into 13 functional classes, and were further clustered into 8 functional clusters:cytoskeleton, transmembrane transporter, molecular chaperone, ribosome, Ras small GTPase(Rab type), ATPase/ATP binding, protein transport, and RNA binding.3. KEGG and Biocarta pathway analyses showed that 14-3-3σ-interacted proteins were involved in 3 Biocarta pathways, and 6 KEGG pathways. Among them,16 proteins were involved in cytoskeleton pathway, and 5 proteins were involved in vesicle transport pathway.4. Protein-protein interaction analysis showed that vesicle transport pathway was regulated by 14-3-3σ/p53/RAB7 interactome, and cytoskeleton pathway was regulated by 14-3-3σ/EGFR/keratin 8 interactome, which may is related to NPC metastasis.5.14-3-3σ/EGFR/keratin 8 interactome in NPC cells was confirmed by experiments.6. Functional studies showed that 14-3-3σdownregulation could increase the expression of EGFR and keratin 8, and enhance the in vitro invasive ability in NPC cells.The data suggest that 14-3-3σdownregulation promotes could the metastasis of NPC possibly through 14-3-3σ/EGFR/keratin 8 interactions.更多还原