The Mechanism Of Cancer Exosome-Derived Integrin ?6-and ?4-Mediated Promotion Of Pulmonary Metastasis Of Colorectal Cancer

Part ? Bioinformatics Analysis of Integrins ?6 and ?4 in Colorectal CancerObjective: To determine the distribution of integrin ?6(ITGA6)and integrin ?4(ITGB4)in colorectal cancer(CRC)and normal tissues using bioinformatics analysis.Materials and methods: In the first part of this study,data regarding colon and rectal cancer from The Cancer Genome Atlas(TCGA)database were analyzed using information regarding ITGA6 and ITGB4 in the tumor and normal colonic tissues from the gene titanium expression(GTEX)database.The relationship between the expression of ITGA6 and ITGB4 with the clinical characteristics of CRC were also studied.The survival analysis of ITGA6 and ITGB4 based on gene expression level was carried out using the online Star Base Pancancer tool and the GEPIA website.The target genes enrichment were carried out with the Kyoto Encyclopedia of genes and genes(KEGG)to screen the common pathways.The protein-protein interaction network(PPI network)of ITGA6 and ITGB4 were analyzed using the String website.Results: Expression levels of ITGA6 and ITGB4 in CRC were significantly higher than those in normal colon(P < 0.05;P < 0.05).Expression levels of ITGA6 were inconsistent across various stages of CRC,with the expression of ITGA6 in stage IV CRC being significantly lower(P < 0.05).There was no significant difference in the expression levels of ITGB4 in various stages of CRC(P > 0.05).The expression of ITGA6 and ITGB4 significantly correlated in CRC(R = 0.64,P < 0.001).KEGG pathway analysis of the coexpressed genes of ITGA6 and ITGB4 showed that the target genes were primarily concentrated in the ECM receiver interaction pathway.The overall survival rate of patients with high expression of ITGA6 was significantly higher than that of patients with low expression of ITGA6(HR = 0.60,P <0.05).The expression of ITGB4 was not related to the prognosis of CRC(HR = 0.94,P > 0.05).Conclusion: High expression levels of ITGA6 was closely related to the progression of CRC.Part ? The Isolation,Extraction and Identification of Exosomes from Colorectal Cancer cellsObjective: To explore the mechanism of action of CRC exosomes in tumor metastasis,we isolated and identified exosomes of CRC.Materials and methods: High metastatic potential CRC cell line(SW620/HCT116)and low metastatic potential CRC cell line(SW480/Caco2)were cultured and the supernatant was collected.The exosomes of CRC cells were obtained by ultracentrifugation.The protein marker molecules(CD9,CD63 and TSG101)of exosomes were identified by western blot(WB).The size and morphology of exosomes were measured using transmission electron microscopy(TEM),and the concentration and particle size distribution of exosomes were measured using nanoparticle tracking analyzer(NTA).Results: WB results showed that CD9,CD63 and TSG101 were expressed on the surface of vesicles in the supernatants of CRC cell cultures.NTA analysis showed that the size of vesicles secreted by CRC cells was about 100 nm.The secretory vesicles of CRC cells were further examined using TEM.We found that the vesicles were discoid and double-membranous,consistent with the morphological characteristics of exosomes.Conclusion: Exosomes from the supernatants of CRC cell culture can be obtained and purification by ultracentrifugation,retaining the typical biological characteristics of exosomes.Part ? Exosomal ITGA6 and ITGB4 of CRC Prompt the Proliferation and Tube Formation Capacity of Endothelial CellsObjective: In the third part of the study,we explored the role of exosomal ITGA6 and ITGB4 in the invasion of CRC in vitro,so as to lay the foundation for a further study of the molecular mechanisms related to organophilic metastasis of CRC.Materials and methods: WB was used to measure expression levels of ITGA6 and ITGB4 with the high metastatic potential CRC cell line(SW620/HCT116)and low metastatic potential CRC cell line(SW480/Caco2).The protein levels of ITGA6 and ITGB4 in the supernatant of HCT116/SW620 and SW480/Caco2 cells were measured using enzyme-linked immunosorbent assay(ELISA).The short hairpin RNA(sh RNA)vectors of ITGA6 and ITGB4 were synthesized in SW620 and HCT116 cell lines.The stable and low expression cell lines were constructed using lentivirus package infection.The exosomes of ITGA6 and ITGB4 in SW620/HCT116 and their control cell lines were co-cultured with vascular endothelial cells.The CCK-8 method was used to explore the effect of exosomes on cell proliferation,and the tubule formation experiment was used to explore the effect of exosomes on angiogenesis.The stable over-expression cell lines of ITGA6 and ITGB4 were constructed in SW480 and Caco2.The exosomes of SW480/Caco2 and their control cell lines over-expressing ITGA6 and ITGB4 were co-cultured with vascular endothelial cells.The CCK-8 method was used to detect the effect of exosomes on cell proliferation,and the tubule formation experiment was used to detect the effect of exosomes on angiogenesis.Results: Expression levels of m RNA of ITGA6 and ITGB4 in SW620/HCT116 were significantly higher than that in SW480/Caco2(P < 0.01).Expression levels of ITGA6 and ITGB4 in the supernatants of cultured SW620/HCT116 cells were significantly higher than those of SW480/Caco2 cells(P < 0.01).WB results showed that the expression of ITGA6 and ITGB4 in SW620/HCT116 was significantly higher than that in SW480/Caco2 cells.Compared with SW480/Caco2,SW620/HCT116 significantly promoted the proliferation of vascular endothelial cells.Compared with SW480/Caco2-derived exosomes,HCT116/SW620 cell-derived exosomes co-cultured with vascular endothelial cells significantly increased the ability of tubule formation(P < 0.05;P < 0.05).After the knockdown of ITGA6 and ITGB4,the ability of SW620/HCT116-derived exosomes in promoting vascular endothelial cell proliferation and tubule formation was significantly reduced.After overexpression of ITGA6 and ITGB4,the ability of SW480/Caco2-derived exosomes in promoting proliferation and tubule formation of vascular endothelial cells was significantly enhanced(P < 0.01;P < 0.05).Conclusion: CRC cell-derived exosomal ITGA6 and ITGB4 promoted proliferation and tube formation capacity of endothelial cells.Part IV The Role of ITGA6 and ITGB4 in Promoting Lung Metastasis of CRCObjective: In the fourth part of this study,we explored the molecular mechanisms of exosomal ITGA6 and ITGB4 in CRC metastasis through experiments in vivo,so as to provide effective prevention and treatment targets for CRC metastasis related researches.Materials and methods: The low metastatic potential CRC cell line SW480 was transfected by luciferase labeled lentivirus and injected into the caudal vein of nude mice.The exosomes derived from SW620/HCT116 cells were extracted and injected into the caudal vein of nude mice.One month after inoculation,the effect of exosomes on lung metastasis was observed by the bioluminescence imaging using in vivo imaging system.Using luciferase-labeled lentivirus to infect SW620 cells with high metastatic tendency,the animal model of lung metastasis was established by injecting them into the caudal vein of nude mice.GW4869,a specific inhibitor of exocrine secretion,was used to treat nude mice for 2 weeks.One month after inoculation,the effect of inhibitors on lung metastasis was observed using the in vivo imaging system.The low metastatic potential CRC cell SW480 was transfected by luciferase-labeled lentivirus and injected into the caudal vein of nude mice to establish the animal model of lung metastasis.Nude mice were treated with SW620/HCT116 cells with ITGA6/ITGB4 knockdown and its control group.One month after inoculation,the effect of exosomes on lung metastasis was measured using the in vivo imaging system.Results: There was no significant lung metastasis in the mice injected with SW480 cells alone,but there was metastasis in the abdominal cavity.Nude mice injected with SW480 cells and SW620 cell-derived exosomes significantly increased lung metastasis induced by SW480 cells.Nude mice injected with SW480 cells and HCT116 cell-derived exosomes did not increase lung metastasis;however,there were metastases in other parts.After treatment with GW4869,the metastases to lung were significantly inhibited(P<0.05).Compared with the control group,SW620-sh ITGA6/ITGB4 cell-derived exosomes significantly reduced the proportion of lung metastases in mice(P < 0.05).HCT116-sh ITGA6/ITGB4 cell-derived exosomes did not significantly change the number of lung metastases in mice(P > 0.05).Conclusion: CRC cell-derived exosomal ITGA6 and ITGB4 promote lung organotropic metastasis of CRC.