Identification Of Annexin A1 Interacting Proteins And Drawing The Network Map Of Regulation-protein Molecule

[Objective] For screening and identification Annexin A1(ANXA1) interaction proteins and drawing the network map of regulation-protein molecule in nasopharyngeal carcinoma(NPC) cells, and to study the molecular mechanism of Annexin A1 in NPC invasion and metastasis.[Methods] In this research, we collected the total proteins of NPC cell line 6-10 B which has ability of the low metastatic potential but high expression Annexin A1, and used co-immunoprecipitation(co-ip) with gel electrophoresis to separate Annexin A1 interaction proteins, then applied two-dimensional liquid chromatography tandem mass spectrometry(2DLC-MS/MS) to identified proteins that associated with Annexin A1 in NPC 6-10 B cell. Next, we performed bioinformatics methods such as Gene ontology, function clustering, signaling pathways and protein- protein interaction network analysis to draw Annexin A1 interacting proteins molecular network in invasion and metastasis of nasopharyngeal carcinoma. Finally, we verified S100A9 and Vimentin, two key proteins of these Annexin A1 interaction proteins, by co-ip combined with western blot.[Results] 1. Co-ip combined with mass spectrometry were applied and identified 436 Annexin A1 interaction proteins of NPC cell line 6-10 B. 2. GO_BP analysis showed the protein biological process of the Annexin A1 interaction proteins mainly involves response to stimulus, localization, cellular metabolic process, etc. GO_MF analysis showed the molecular function of the Annexin A1 interaction proteins mainly involves catalytic activity, cation binding, protein binding, nucleotide binding, cytoskeleton, ATP binding, etc. GO_CC analysis showed the Annexin A1 interaction proteins localization mainly involves in cytoplasm, organelle, plasma membrane, cytoskeleton, etc. 3. Gene Functional Classification showed the Annexin A1 interaction proteins were grouped into 21 functional classes. The function of these proteins mainly involves: calcium-dependent phospholipid binding, signal peptide, integral to membrane, transmembrane transport, protein kinase activity, cytoplasmic vesicle part, cytoskeleton organization, etc. 4. KEGG and Biocarta signaling pathways analysis showed the Annexin A1 interaction proteins were involved in 35 KEGG signaling pathways, and 7 Biocarta signaling pathways. Among them, 4 proteins were involved in cytoskeleton signaling pathway, 9 proteins were involved in adherens junction signaling pathway, and 12 proteins were involved in transmembrane transport signaling pathway. 5. Protein-protein interaction network analysis showed S100A9, Vimentin and Annexin A1 constitute interactome. 6. S100A9 protein and Vimentin protein interact with Annexin A1 protein that was verified by co-ip combined with western blot, which was consistent with mass spectrometry results.[Conclusions] S100A9, Vimentin and Annexin A1 constitute interactome, which may affect the invasion and metastasis of NPC through the Annexin A1/S100A9/Vimentin related signaling pathways.