Research On The Malignant Phenotypes Mediated By Esophageal Cancer Exosomes And Quantitative Proteomic Analysis Of MTA1-associated Exosomes

Objective To investigate the phenotypic transmission mediated by esophageal cancer-derived exosomes between cancer cells and the effect of MTA1 on exosomal contents of esophageal cancer cells.Methods The KYSE410-derived exosomes were isolated by differential centrifugation method;the morphology and biological markers of exosomes were separately defined by transmission electron microscopy and Western blotting;KYSE410,KYSE510 and YES2 cells were able to uptake the exosomes labeled with fluorescent dye,which can be tracked under confocal microscopy;transwell chambers assay,wound healing experiment,cell proliferation recording and clone formation assay were separately conducted to test how KYSE410-derived exosomes influence the migration,invasion,proliferation and clonal formation of three esophageal cancer cells;the changes of proteins involved in Wnt/?-catenin and PI3K/Akt signaling pathway were detected by immunoblotting.Co-expressing genes of MTA1 and data from MTA1 Co-IP mass spectrometry were used to conduct Gene Ontology analysis;Nanosight was used to assess the average diameter of exosomes from esophageal cancer cells with different MTA1 expression;label-free quantitative proteomic analysis was used to detect the exosomal proteins from esophageal cancer cells with forced MTA1 expression or MTA1 knockout and we also conducted GO cluster analysis on the proteins subsets regulated by MTA1 in exosomes;the correlation between MTA1 and target differential proteins at tissue-and cell-level was analyzed using CCLE database and cBioPortal platform;The relationships between target differential proteins and clinicopathological features were also assessed.Results Exosomes with membrane structure could be defined by transmission electron microscopy and exosomal marker proteins CD63 and CD81 could be detected by immunoblotting;the three esophageal cancer cells were visualized to successfully uptake the KYSE410 exosomes labelled by fluorescent dye;The invasion and migration abilities of three esophageal cancer cells were listed as:KYSE410>KYSE510>YES2.KYSE410-origined exosomes promoted the migration and invasion of KYSE410,KYSE510,YES2 cells;KYSE410 exosomes promoted the colony formation of KYSE410,KYSE510,YES2 cells,while we did not see KYSE410-exosomes promote the proliferation of these cells;p-catenin and p-AKT were upregulated when KYSE410 exosomes were exerted on the three esophageal cancer cells.GO cluster analysis of MTA1 co-expression genes showed that MTA1 affected vesicle organization and biogenesis,cluster analysis of MTA1 Co-IP interacting proteins showed that most of the MTA1 interacting proteins were located in exosomes;neither the average diameter of exosomes nor the quantitation of exosomal proteins showed significant difference in esophageal cancer cells with MTA1 manipulation;25 exosomal proteins were firstly identified;analysis using Cyto Scape software showed that the highest degree of connectivity is UBC;Cell Component analysis showed that the exosomal proteins from KYSE410 cells and KYSE450 cells mainly located in the cytoplasm,nucleus,lysosome,plasma membrane and other parts;the main molecular functions included catalytic activity,molecular chaperone activity,cytoskeletal protein binding,GTPase activity and so on;the main biological process involved protein metabolism,energy pathway,cell growth and maintenance,immune response and so on;the main biological pathway involved in integrin family cell surface interactions,?1 integrin cell surface interactions,proteoglycan syndecan-mediated signaling events and so on;differential proteins regulated by MTA1 from KYSE410-derived exosomes and KYSE450-derived exosomes both involved in negative regulation of macromolecule metabolism,phosphate metabolism,macromolecule catabolism and so on;MYH9,SLC7A5 and CCT4 were all moderately correlated with MTA1 at the tissue level;MYH9,CCT4 showed relevance with T stage and clinical stage of esophageal cancer patients;esophageal cancer patients with high SLC7A5,CCT4 expression showed shorter overall survival time.Conclusions Concentrated exosomes from KYSE410 promoted the migration and invasion,colony formation of KYSE410 and esophageal cancer cell lines KYSE510 and YES2,possibly exerting the effects by activating Wnt/?-catenin and PI3K/Akt signaling pathways.Cluster analysis showed that MTA1 may be involved in the regulation of exosomes;MTA1 exerted no effect on the size and quantitation of exosomes;but MTA1 exerted effects on the content of exosomes;key differential proteins MYH9,SLC7A5,CCT4 may serve as prognostic factors for esophageal cancer.