Study On The Proteomics Of Exosomes From Colorectal Cancer Tissue And The Function Of Exosome Protein TRIM2

Part Ⅰ Screening of Colorectal Cancer Diagnostic Biomarkers in Exosomal ProteinsBackground:Colorectal cancer(CRC)is the third most common malignancy in the world and has the second highest mortality rate.Early diagnosis is effective in improving patient prognosis,so it is clinically important to find reliable diagnostic biomarkers for CRC.Meanwhile,the era of molecular omics has been emerging in tumor research,and proteomics can clearly show epigenetic information,which is closely related to the pathological and physiological status of the human body.Objective:To characterize the proteomic profile of exosomes of CRC tissue origin and to search for exosomal proteins as biomarkers for the diagnosis of CRC.Methods:Ten colorectal cancer patients aged 50 years or older with a histopathological diagnosis of moderately differentiated adenocarcinoma were recruited,and CRC tissue and adjacent intestinal tissue(>5 cm)were collected and paired to form the experimental and control groups.Exosomes were extracted from each tissue sample separately and exosome identification was performed.Data-independent acquisition(DIA)mass spectrometry analysis was carried out in 8 matched pairs of tissue exosome samples to explore the proteomic expression profile of CRC.Differentially expressed proteins between CRC tissue exosomes and adjacent intestinal tissue exosomes were screened by bioinformatics analysis.Parallel reaction monitoring(PRM)analysis was applied to validate some of the differentially expressed exosomal proteins in all 10 pairs of tissue exosome samples.Results:1393 and 1304 proteins were identified in CRC and adjacent tissue exosomes,respectively,of which 283 proteins were differentially expressed in the two groups.Functional and pathway enrichment analysis revealed that these differentially expressed exosomal proteins were involved in multiple biological processes related to cytoskeleton construction,cell motility and migration,immune response,tumor growth and telomere metabolism,as well as pathways such as ECM-receptor interactions,focal adhesion and mTOR signaling pathways.Differentially expressed exosomal proteins TRIM28,NHP2,OLFM4,TOP1,SAMP and TAGL were validated by PRM analysis.Conclusion:This study comprehensively characterized the proteomic profile of exosomes of CRC tissue origin,and exosomal proteins TRIM28,NHP2,OLFM4,TOP1,SAMP and TAGL may be potential CRC diagnostic biomarkers.Part Ⅱ Effects of Exosomal Protein TRIM28 on the Proliferation,Migration and Invasion of Colorectal Cancer CellsBackground:Exosomes are an important component of the tumor microenvironment and are involved in a variety of pathological processes,and the mechanism of exosomes in colorectal cancer is still unclear.Meanwhile,TRIM28 protein was found to be abnormally expressed in a variety of tumors,such as hepatocellular carcinoma,gastric cancer,prostate cancer,ovarian cancer and breast cancer,however,the function of TRIM28 in the development of CRC is not clear.Objective:The aim of this study was to explore the effect of exosomal protein TRIM28 on the biological behavior of CRC cells and the possible mechanism.Methods:Quantitative Real-time PCR(qRT-PCR)and Western blot were applied to detect the expression levels of TRIM28 mRNA and protein in CRC cells and normal intestinal epithelial cells.The supernatants of CRC cells and normal intestinal epithelial cells were collected,exosomes were extracted and identified,and the mRNA and protein levels of TRIM28 in exosomes were detected by qRT-PCR and Western blot.TRIM28 siRNA was transfected into CRC cells,and cell scratch,CCK-8,Transwell migration and invasion assays were applied to explore the effects of knockdown of TRIM28 in CRC cells and exosomes on proliferation,apoptosis,migration and invasion of CRC cells,respectively.Expression changes of E-cadherin,N-cadherin and Vimentin were detected by Western blot after knockdown of TRIM28 in CRC cells and exosomes.Results:TRIM28 mRNA and protein were significantly elevated in CRC cells,while expression was elevated in CRC cell supernatant exosomes.In vitro functional assays showed that knockdown of TRIM28 expression in CRC cells could effectively reduce cell proliferation,migration and invasion ability,while knockdown of TRIM28 expression in exosomes could also reduce cell proliferation,migration and invasion ability.Knockdown of TRIM28 in CRC cells and exosomes resulted in elevated expression of E-cadherin and decreased expression of N-cadherin and Vimentin.Conclusion:TRIM28 promotes proliferation,migration and invasion of colorectal cancer cells.It is a CRC pro-oncogenic factor,and may exert biological effects through exosomes and regulation of epithelial-mesenchymal transition pathway.Part Ⅲ Potential Value of Serum Exosomal Protein TRIM28 in the Screening of Colorectal CancerBackground:Previous studies have demonstrated that exosomal TRIM28 expression is elevated in CRC tissues and is also a pro-cancer factor in CRC.However,the expression level of exosomal TRIM28 in serum is unknown and whether it can be translated into clinical applications has yet to be investigated.Objective:The aim of this study was to verify the potential of exosomal protein TRIM28 as a biomarker for the screening of colorectal cancer.Methods:Sera from 47 CRC patients and 17 healthy control(HC)subjects were collected,exosomes were extracted,and the expression of exosomal protein TRIM28 was measured by enzyme-linked immunosorbent assay(ELISA).The receiver operating characteristic(ROC)was applied to evaluate the efficacy of exosomal protein TRIM28 and exosomal protein TRIM28 in combination with carcinoembryonic antigen(CEA)and carbohydrate antigen 199(CA199)for the diagnosis of CRC,respectively.Sera from 15 CRC patients before and after surgery were collected,exosomes were extracted,and the changes in the expression of exosomal protein TRIM28 in serum before and after surgery were detected by ELISA.Results:The expression level of exosomal protein TRIM28 was significantly higher in serum of CRC patients than in HC.Exosomal protein TRIM28 had a higher diagnostic efficacy for CRC,with an area under the curve of ROC of 0.895,which was higher than that of CEA(0.761)and CA199(0.791).Combining exosomal protein TRIM28 with CEA and CA199 to construct a binary regression model,the area under the curve of ROC was 0.962,with higher diagnostic efficacy than individual indicators.Meanwhile,the expression of serum exosomal protein TRIM28 was significantly lower in postoperative water serum of CRC patients compared with preoperative.Conclusion:The exosomal protein TRIM28 was significantly elevated in peripheral blood of colorectal cancer and has the potential to become a non-invasive biomarker for the screening of colorectal cancer,which needs to be further investigated by expanding the sample size.