Proteomic Profiling And Functional Characterization Of Serum-Derived Extracellular Vesicles In The Mucinous And Non-Mucinous Colon Adenocarcinoma

Background:Mucinous adenocarcinoma is a distinct pathological subtype of colon adenocarcinoma with a worse prognosis compared to non-mucinous adenocarcinoma.However,a clear distinction between mucinous and non-mucinous adenocarcinomas remains unknown.Extracellular vesicles are a class of enclosed vesicles containing proteins,lipids,and nucleic acids that are secreted by cells into surrounding tissues or serum.Extracellular vesicles can promote tumorigenesis by regulating tumor cell proliferation,invasion,metastasis,angiogenesis,and evasion of immune surveillance.In this study,we investigated the differences between mucinous and non-mucinous adenocarcinoma patients at the molecular level by cutting through the serum extracellular vesicles.Methods:Extracellular vesicles were isolated from sera of healthy volunteers and patients with mucinous and non-mucinous colon cancer by ultracentrifugation and reagent precipitation,and identified by transmission electron microscopy,nanoparticle tracking analysis,and detection of extracellular vesicle markers,respectively.Quantitative proteomics studies were performed on serum extracellular vesicles and extracellular vesicle-treated cell line SW480,respectively.In-depth analysis of proteomics data was performed by principal component analysis,Wayne diagram,cluster analysis,Gene Ontology enrichment analysis,Wikipathway pathway analysis,and construction of protein-protein interaction networks.The clinical significance of PLA2G2A protein was explored by stratifying the analysis of mucinous and non-mucinous adenocarcinoma patients through TCGA,GEO,and other database colon cancer cohorts.To further explore the action mechanism of PLA2G2A protein on tumor cells,in vitro experiments were performed to verify the effect of PLA2G2A on Wnt/beta-catenin signaling pathway by overexpressing PLA2G2A.Results:This study successfully isolated Extracellular vesicles from patient and healthy control sera.Proteomics data of extracellular vesicles and cell lines were uploaded to ProteomeXchange,and bioinformatics analysis was performed by two sets of protein profiling data,and the results of the study confirmed significant differences between mucinous and non-mucinous adenocarcinoma colon cancer at the level of protein expression profiles of serum extracellular vesicles.The differentially expressed proteins between the two could be enriched in biological processes such as endothelial cell migration,extracellular matrix remodeling,and signaling pathways such as metabolic reprogramming and VEGFA-VEGFR2 in colon cancer.PLA2G2A expression was significantly upregulated in mucinous colon cancer serum exosomes compared to nonmucinous adenocarcinomas.High PLA2G2A expression was associated with poor DFS in colon cancer patients carrying BRAF mutations as externally validated by TCGA and GEO data cohorts.In vitro experiments showed that PLA2G2A overexpression could lead to intracellular aggregation of beta-catenin,making it clear that PLA2G2A may influence the malignant biological behavior of colon cancer cells through the beta-catenin signaling pathway.Conclusion:The serum extracellular vesicles of mucinous and non-mucinous colon cancer patients reflected the differences between mucinous adenocarcinoma and non-mucinous colon cancer in terms of protein expression profile and function.Mucinous colon cancer extracellular vesicles contained higher levels of extracellular PLA2G2A protein.The extracellular vesicles of mucinous colon cancer may promote tumor metastasis by upregulating PLA2G2A.PLA2G2A is significantly highly expressed in mucinous colon cancer,correlates with colon cancer prognosis,and has the potential to become a diagnostic and prognostic predictive marker for colon cancer.