鼻咽癌14-3-3σ蛋白的靶向蛋白质组学研究

发布时间：2018-03-03 04:07

本文选题：鼻咽癌　切入点：14-3-3σ　出处：《中南大学》2010年博士论文　论文类型：学位论文

【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。
[Abstract]:Nasopharyngeal carcinoma (NPCs) is one of the most common malignant tumors in southern China, which is a serious threat to the lives and health of our people. The early occurrence of cervical lymph node and distant metastasis. NPC metastasis is a process in which multiple genes interact to form a key node, which plays an important role in NPC metastasis. We used proteomics method to screen differentially expressed proteins in NPC tissues and identified the differentially expressed proteins in 36 NPC tissues. Immunohistochemical staining was used to detect the expression of differential protein 14-3-3 sigma in normal nasopharyngeal epithelial tissues and NPC tissues with cervical lymph node metastasis. The results showed that there were high frequency deletion / down-regulation of 14-3-3 蟽 protein expression in NPC tissues. The deletion / down-regulation of 14-3-3 蟽 expression may play an important role in NPC metastasis, but the molecular mechanism of 14-3-3 蟽 expression deletion / down-regulation promoting NPC metastasis has not been elucidated. In order to study the mechanism of 14-3-3 蟽 in NPC transfer, we used targeted proteomics (immunoprecipitation mass spectrometry) to isolate and identify 14-3-3 蟽 interacting proteins in NPC cells. Signal pathway and protein-protein interaction protein-protein interaction (PPI) were used to analyze 14-3-3 蟽 interaction proteins and reveal their biological significance. The key interacting proteins 14-3-3 蟽 / EGFR / Keratin 8 were tested and their functions were studied. The main results are as follows:. 1. 111 14-3-30 interacting proteins were identified by targeting proteomics in NPC cells, and four of them, Keratin 8G EGFRN RAB7 and p53, were identified. 2go and cluster analysis showed that 111 14-3-3 蟽 interacting proteins could be clustered into 13-functional clusters. The functions of these proteins were mainly classified into eight aspects: cytoskeleton, transmembrane transport, molecular chaperone. Ribosomal protein GTPase, protein transporter ATPase and RNA binding. 3Analysis of the KEGG and Biocarta signaling pathways showed that the interaction protein of 1: 14-3-3 蟽 involved three Biocarta signaling pathways and six KEGG signaling pathways. Sixteen interacting proteins were involved in cytoskeletal signaling pathways and five involved in vesicular transport signaling pathways. 4. The protein interaction analysis showed that the cytoskeleton was regulated by the interaction group of 14-3-3 蟽 -p53 / RAB7. The cytoskeleton was regulated by the 14-3-3 蟽 -EGFR / CK8 interaction group, which might be related to NPC metastasis. 5. The results showed that there were 14-3-3 蟽 -EGFR / Keratin 7 interactions in NPC cells. Functional analysis showed that the down-regulation of w _ (14-3-3) 蟽 could increase the expression of EGFR and Keratin _ 8 in NPC cells and enhance the invasiveness of NPC cells in vitro. The results suggest that the down-regulation of 14-3-3 蟽 expression may promote the invasion and metastasis of NPC through 14-3-3 蟽 / EGFR / Keratin8 interaction.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.6

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