The Study On Colon Cancer Cell Derived Small Extracellular Vesicles Regulate Fibroblast Function And Its Mechanism

Objective:sEVs are a class of major components of extracellular vesicles.As important mediators of intercellular signal transmission,sEVs are capable of delivering various bioactive substances between cells and cells,and they are important carriers for the interaction between tumor cells and tumor microenvironment.This project aims to study how tumor cells affect the function of fibroblasts,the main element in the tumor microenvironment,through sEVs,and explore its mechanism in the occurrence and development of colorectal cancer.Methods:1.sEVs in supernatant of colon cancer cells were extracted by polyethylene glycol precipitation method,and identified by transmission electron microscopy(TEM),particle size analysis(NTA)and WB.2.sEVs derived from colon cancer cells and negative control were added into the culture supernatant of fibroblasts,respectively.The expression of activation markers of fibroblasts was detected by WB and immunofluorescence(IF)technique.Transwell migration assay,cell proliferation test CCK-8 and ELISA assay were respectively used to detect the migration ability,proliferation ability and secretion level of inflammatory cytokines of fibroblasts.3.The expression levels of integrin β4(ITGB4)in colon cancer cells and their sEVs and fibroblasts were detected by WB.The level of ITGB4 in colon cancer tissues and paracancerous tissues was detected by immunohistochemistry(IHC).WB was used to detect the level of ITGB4 in sEVs from cancer tissues and paracancerous tissues.4.pcDNA3.1-ITGB4 plasmid was transfected into fibroblasts to upregulate the protein level of ITGB4 in the cells.WB was used to detect the changes in the level of activation markers in fibroblasts.Transwell migration assay,CCK-8 assay,ELISA assay were respectively used to detect the migration ability,proliferation ability and secretion level of inflammatory factors of fibroblasts,and observe the effect of ITGB4 protein on the function of fibroblasts.5.Colon cancer cells were transfected with Si-RNA-ITGB4,and the expression of ITGB4 was down-regulated.The sEVs from the colon cancer cells was extracted by polyethylene glycol precipitation method,and the sEVs from the low level of ITGB4 and the normal level of ITGB4 were added into the supernatant of fibroblast culture respectively.WB was used to detect the expression of activation markers of fibroblasts.Transwell migration assay,CCK-8 assay and ELISA were used to detect the migration ability,proliferation ability and secretion level of inflammatory factors of cells,respectively.Result:1.Transmission electron microscopy results showed that sEVs presented a typical round discoid vesicle structure,which was wrapped by double lipid membrane.The particle diameter is about 100nm;NTA results showed that the diameter of sEVs detected ranged from 100 nm to 130 nm,with uniform distribution.WB results showed that sEVs expressed extracellular vesicle markers CD63 and TSG101,but did not express endoplasmic reticulum marker protein calnexin.2.After the addition of colon cancer cell-derived sEVs into the supernatant of fibroblast culture,the expression level of fibroblast activation markers was increased,the migration ability was enhanced,the proliferation ability was enhanced,the secretion level of IL-6 was increased,but the secretion level of IL-1β was not significantly changed.3.ITGB4 protein was highly expressed in colon cancer cell SW480 and sEVs,but not in fibroblasts.In clinical tissue specimens,the expression of ITGB4 protein was high in colon cancer tissues and their sEVs,while low in paracancerous tissues and their sEVs.4.WB results showed that the transfection of ITGB4 protein expression plasmid resulted in the overexpression of ITGB4 in fibroblasts.The high expression of ITGB4 could promote the expression of α-SMA and FAP,the activation markers of fibroblasts,enhance their migration ability,and increase the levels of IL-6,but do not significantly change the proliferation ability of fibroblasts.5.Si-RNA-ITGB4 transfection of colon cancer cells significantly reduced the expression level of ITGB4.Compared with control sEVs,sEVs containing lower levels of ITGB4 could not significantly change the expression level of fibroblast activation markers,and its promoting effect on the migration and proliferation of fibroblasts was weakened,as well as its ability to promote the secretion of inflammatory cytokine IL-6 was weakened.Conclusion:sEVs derived from colon cancer cells can promote the expression of fibroblast activation markers,enhance the ability of migration and proliferation,and promote the secretion of inflammatory cytokines,so as to change the function of normal fibroblasts.The ITGB4 protein in sEVs secreted from colon cancer cells plays an important role in promoting the functional changes of fibroblasts.