Targeted Proteomic Analysis Of 14-3-3σ In Nasopharyngeal Carcinoma

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.