Protein Composition And Function Of Adverse Pathological Features Of Colorectal Cancer (peripheral Nerve Invasion And Pathological Mucinous Adenocarcinoma

Backgroud:As the most common malignant tumor,the incidence of colorectal cancer(CRC)is increasing year by year,which brings heavy economic burden to the country and society.With deepening research on CRC,it has been found that the prognosis is poor for CRC patients with adverse pathological features such as perineural invasion(PNI)and mucinous carcinoma.The molecular mechanisms leading to the differences in prognosis remain unknown.The purpose of this study is to gain a deeper understanding of PNI and mucinous carcinoma,two adverse pathological features of CRC,at the protein level.Methods:Part Ⅰ:Extracellular vesicles(EVs)were isolated from the serum of healthy volunteers,PNI CRC patients and non-PNI(NPNI)CRC patients by size exclusion chromatography and Total Exosome Isolation Reagent and the isolated EVs were identified by transmission electron microscopy,nanoparticle tracking analysis and immunoblot analyses of exosome markers.The EVs were used to stimulate CRC cancer lines SW480 and HCT116 respectively,and phenotypic experiments such as proliferation,invasion and migration were carried out.Principal component analysis,Venn map,cluster analysis,GO(Gene ontology)enrichment analysis,KEGG(Kyoto encyclopedia of genes and genomes)pathway analysis,construction of protein-protein interaction network and other methods were used to make an exploratory analysis of proteomic data.In order to further validate the EVs protein SFN and SAA1 in CRC patients,SFN and SAA1 were quantitatively detected by ELISA.The clinical significance of EVs in the diagnosis of PNI CRC was evaluated by the ROC curve.The significance of EVs protein SFN in predicting the prognosis of CRC was evaluated by multivariate analysis of Cox risk regression model based on long term follow up data.Part Ⅱ:A tandem mass tag proteomics approach was used to identify differentially expressed proteins(DEPs)in colon carcinoma tissues from different locations and with different histological types to reveal the underlying mechanisms of these differences at the protein level.In this study,the DEPs between right colon mucinous adenocarcinoma(RMC)and non-mucinous adenocarcinoma(RNMC)and the DEPs between left colon mucinous adenocarcinoma(LMC)and non-mucinous adenocarcinoma(LNMC)were compared.Bioinformatics analysis was used to explore the DEPs.Gene Ontology(GO)functional enrichment analysis,and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis of the DEPs were performed.Protein-protein interaction(PPI)analysis was performed and visualized using the string APP plugin in Cytoscape.The correlation analysis between the expression levels of core genes and their clinical prognosis was verified in combination with the TCGA database.Results:Part Ⅰ:EVs were successfully isolated from the serum of patients and healthy controls.Phenotypic experiments showed that EVs in the PNI group could promote the invasion and migration of CRC cancer cell lines,but had no significant effect on proliferation of CRC cell lines.Compared with serum EVs in the non-PNI(NPNI)group,the effect of EVs from the PNI group on tumor cell metastasis was more significant.According to the bioinformatics analysis of proteomic data,there was a significant difference in the protein expression profile of EVs in serum between PNI and NPNI groups.The differentially expressed proteins were mainly enriched into the category of innate immune response and positively regulate cell growth.Western blot(WB)experiment confirmed that serum EVs from the PNI group promote tumor metastasis by upregulating EMT pathway-related proteins to induce epithelial-mesenchymal transformation of tumor cells.Compared with the NPNI group,the expression of SFN in serum EVs of PNI group was significantly up-regulated.EVs SFN expression levels demonstrated AUC values of 0.84 for discriminating patients with PNI from NPNI.The expression level of SFN in EVs was an independent risk factor for disease free survival(DFS).Part Ⅱ:Proteomic profiles were significantly different in different tumor locations(left-side colon/right-side colon)and histological types(mucus/non-mucus).In right-sided colon cancer,the GO analysis showed the mucinous specific DEPs were enriched in extracellular matrix-related proteins,and most of them were up-regulated,indicating that extracellular matrix-related remodeling played an important role in rightside mucinous carcinomas,and KEGG pathway analysis showed that DEPs were mainly enriched in IL-17 signal pathway.In left-sided colon cancer,the GO showed the mucinous specific DEPs were mainly enriched in the renin-angiotensin system,suggesting that the abnormal expression of renin-angiotensin system may lead to tumor progression.Conclusions:Part Ⅰ:We identified differences in EVs protein profiles between PNI and NPNI groups.The EVs in PNI group may promote tumor metastasis by up-regulating EMT-related proteins.The expression of SFN in serum EVs was related to the prognosis of CRC,and may have the potential to become a diagnostic and prognostic marker in colon cancer.Part Ⅱ:The proteomics profiles of colon cancers showed distinct differences related to locations and histological types.In right-sided colon cancer,the mucinous specific DEPs were mainly enriched in extracellular matrix-related proteins,and most of them were upregulated,indicating that ECM and extracellular matrix-related remodeling played an important role.In left-sided colon cancer,the mucinous specific DEPs were mainly enriched in the renin-angiotensin system.These results suggested a distinct mechanism underlying the diverse subtypes of colon cancers.