| Background:Colorectal cancer(CRC)is one of the most common gastrointestinal tract malignant tumors.Over the past few years,distinction between left-and right-sided colon cancer(LCC and RCC)has been brought into focus due to their diversities anatomy,clinical characteristics,biology,prognosis and treatment response.The cause of the difference between LCC and RCC is still controversial.The present study aimed to perform a detailed analysis of the diversity of exosome between LCC and RCC using quantitative proteomics,in order to investigate the molecular differences between LCC and RCC.Methods:We isolated exosomes from patients with LCC,RCC and healthy volunteers by ultracentrifugation or Total Exosome Isolation Reagent.And then serum-derived exosomes were identified by transmission electron microscopy,nanoparticle tracking analysis and immunoblot analyses of exosome markers.CRC cancer cell lines were treated with serum-derived exosomes,and phenotypic assays such as proliferation,invasion and migration were conducted.Quantitative proteomics analyses were performed on serum-derived exosomes and CRC cancer cell line SW480 treated with exosomes,respectively.In-depth analysis of proteomic data was performed through principal component analysis,Venn map,Hierarchical Ward-linkage clustering,Gene Ontology enrichment analysis,Wikipathway analysis,and protein-protein interaction network construction.The results of bioinformatics analyses prompted us to investigate the contribution of exosomal SPARC or LRG1 to the clinical characteristics or progression of colon cancer.To address this issue,serum samples from 125 patients with malignancies including colon cancer,thyroid cancer,cervical cancer,and gastric cancer and 25 healthy volunteers were analyzed by ELISA.AUC value was calculated by ROC curve to evaluate the potential of exosomal SPARC or LRG1 in the diagnosis of colon cancer.And the significance of exosomal SPARC or LRG1 in predicting the prognosis of colon cancer was evaluated by Cox regression model based on long-term follow-up data.Result:It was confirmed that exosomes were successfully isolated from the serum of patients and healthy controls in this study by means of transmission electron microscopy,nanoparticle tracking analysis and immunoblot analyses of exosome markers.Trans well assays showed that serum-derived exosomes from both LCC and RCC patients promoted the cellular motility of SW480 and HCT116 cells,although RCC-derived exosomes had a greater effect than those obtained from LCC patients.However,neither RCC-derived exosomes nor LCC-derived exosomes promoted the proliferation of CRC cell lines.These results indicated that exosomes derived from RCC contain more factors that are capable of promoting metastasis.Proteomic data are available via ProteomeXchange with the identifiers PXD012283 and PXD012304.In-depth analysis of proteomic data was performed.The expression profile of the serum exosomal proteome in patients with RCC was different from that of patients with LCC.Gene Ontology analysis revealed that differentially expressed proteins between RCC and LCC are related mostly to biological processes of extracellular matrix(ECM)remodeling,epithelial cell migration,and regulation of angiogenesis.And the sidedness differentially expressed proteins were predominantly enriched in multiple cancer-related pathways,such as the PI3K-Akt signaling pathway,TGF-? signaling pathway,senescence and autophagy in cancer.Compared with LCC,the expression of exosomal SPARC and LRG1 was significantly up-regulated in RCC.Proteomic data suggest that cell line treated with exosomes derived from LCC or RCC underwent epithelial-mesenchymal transition(EMT).It can be inferred that serum-derived exosomes from RCC may activate the EMT signaling pathway of tumor cells by upregulating ECM-related proteins such as SPARC and LRG1,thereby promoting tumor invasion and migration.Exosomal SPARC and LRG1 expression levels demonstrated AUC values of 0.95 and 0.93 for discriminating patients with colon cancer from healthy controls.Moreover,the expression levels of SPARC and LRG1 correlated with tumor sidedness and were predictive of tumor recurrence.Conclusion:We identified differences in exosomal protein profiles between LCC and RCC.Higher levels of ECM-related proteins,especially SPARC and LRG1,were found in exosomes derived from RCC.Serum-derived exosomes of RCC may promote metastasis via upregulation of ECM-related proteins,especially SPARC and LRG1,which may serve as diagnosis and prognosis biomarkers in colon cancer. |
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