The Effect Of Gastric Cancer Cell-Derived Exosomes On The Phenotypic Transformation Of Hepatic Stellate Cells And Effect Of Transformation On The Malignant Biological Behavior Of Gastric Cancer Cells

Objective:To study the effect of gastric cancer cell-derived exosomes(Exo)on the phenotypic transformation of hepatic stellate cells(HSCs),and to explore the role of hepatic stellate cell activation on the malignant biological behaviors of gastric cancer cells and the underlying molecular mechanisms.Methods:1.Exosomes secreted by gastric cancer cells(AGS)were extracted and purified by polymer precipitation combined with ultrafiltration.The marker proteins were detected by Western blot(WB),followed by transmission electron microscopy(TEM)to observe the morphological characteristics,and Nanoparticle tracking analysis(NTA)to detect particle diameter to verify whether it meets the exosomes metrics.2.The fluorescent dye PKH26 was used to label exosomes,and DAPI to label hepatic stellate cells(LX-2),and the uptake of exosomes by hepatic stellate cells(LX-2)was assessed by the fluorescence tracing method.3.To study the phenotypic transformation of hepatic stellate cells and its association with tumor associated fibroblasts(CAFs),gastric cancer cell-derived exosomes were incubated with hepatic stellate cells(LX-2),and real-time quantitative PCR(RT-qPCR)was used to detect the mRNA expression levels of α-smooth muscle actin(α-SMA)and fibroblast activation protein(FAP)in hepatic stellate cells(LX-2)after exosome intervention.4.Cell counting kit-8(CCK8)assay,Colony formation assay,Transwell invasion assay and Wound healing assay were applied to detect the effect of activated hepatic stellate cell(LX-2)on the malignant biological behavior of gastric cancer cells.5.Western blot was applied to detect the effect of activated hepatic stellate cell(LX-2)on the expression of proteins related to epithelial mesenchymal transition(EMT)in gastric cancer cells.Results:1.Gastric cancer cell-derived exosomes were successfully extracted and purified using polymer precipitation combined with ultrafiltration,and identified by transmission electron microscopy(TEM),Nanoparticle tracking analysis(NTA)and Western blot(WB).The results indicated that the extracted particles meet exosomes metrics.2.We used fluorescent dye to label gastric cancer cell-derived exosomes and hepatic stellate cells(LX-2),exosomes were successfully observed to be enriched within hepatic stellate cells(LX-2)by a fluorescence tracing method,indicating that hepatic stellate cells(LX-2)can take up exosomes in large amounts.3.Gastric cancer cell-derived exosomes co-incubated with hepatic stellate cells,the mRNA expression levels of tumor associated fibroblast markers α-SMA and FAP were measured by RT-qPCR,and the results indicated that the expression levels of both were significantly increased,indicating that the phenotype of hepatic stellate cells transformed into that of tumor associated fibroblasts.4.The results of CCK8 cell proliferation assay and Colony formation assay indicated that activated hepatic stellate cells significantly enhanced gastric cancer cell proliferation ability,and the results of Transwell invasion assay and Wound healing assay indicated that activated hepatic stellate cells significantly promoted gastric cancer cell invasion,migration ability.5.After incubation with activated hepatic stellate cells,the expression of epithelial markers E-cadherin in Gastric cancer cells was significantly down-regulated,while the expression of mesenchymal markers including N-cadherin and Vimentin was significantly up-regulated.Conclusion:Gastric cancer cell-derived exosomes can promote the phenotype transformation of hepatic stellate cells,and induce hepatic stellate cells to activate into tumor associated fibroblasts.Gastric cancer cell-derived exosomes can significantly induce epithelial mesenchymal transition of gastric cancer cells,and significantly enhance the proliferation,invasion and migration of gastric cancer cells.