| Gastric cancer is a common malignant tumor.The incidence and death rate of gastric cancer has obvious regional characteristics,especially in Asia.Although the trend of morbidity and mortality is decreasing globally,the incidence tends is more towards to younger age pacient without improvement on nontality.Drug resistance and metastasis of gastric cancer are the main reasons for the failure treatment.Therefore,exploring the mechanism of gastric cancer development plays an important role in early diagnosis and metastasis.Exosomes are a single-layer membrane structure with a diameter of 30～150 nm.There are enriched with various biologically active substances,such as DNA,RNA,proteins,lipids,interacted with surrounding cells to achieve intercellular exchange material and signal transmission.Studies have found that tumor derived exosomes promote tumor growth and are associated with poor prognosis.Exosome plays an important role in maintaining cell development and proliferation.Therefore,it is of a great significance to study the mechanisms of tumor derived exosomes in tumor microenvironment.CD8~+T cells are the main subpopulation exerting anti-tumor effects.However,the effect of gastric cancer derived exosomes on CD8~+T cells need to be further investigation.Studies have shown that tumor derived exosomes induced T cell apoptosis through the Fas/FasL pathway and inhibit the function of CD8~+T cells by affecting cell surface molecules and cell numbers,but there was few report in gastric cancer.So,we explore the interaction between CD8~+T cells and gastric cancer derived exosomes in vitro,and further study the effect of gastric cancer derived exosomes created immunosuppressive microenvironment and promoted metastasis site formation.To identify the function of mRNA,miRNA and LncRNA in gastric cancer derived exosomes,and find potential targets and related signaling pathways,to explore its mechanism on CD8~+T cells.Therefore,this project will provide a novel strategy basis to modulate the development of tumor microenvironment.Objective:To identify the exosomes concentration,particle size,morphology and expression of exosome markers in MKN-28,MKN-45,and SGC-7901 cell lines.Effects of gastric cancer derived exosome on CD8~+T cell cycle,apoptosis,cytokine secretion and gene expression.To further explore the distribution of exosome,its impact on immunosuppressive microenvironment,and metastasis sites formation in vivo.Through genetic sequencing and bioinformatics analysis,screening differentially expressed RNA in gastric cancer exosome.Analysis of potential targets and interactions.This study aims to reveal the potential mechanism of gastric cancer derived exosome in the process of tumor immunosuppression and microenvironment formation,and to provide new ideas for the treatment of gastric cancer metastasis.Methods:Three gastric cancer cell lines exosome were obtained by ultracentrifugation,western blot was used to analyze the expression of exosome surface molecular markers,the distribution and concentration of exosome particles were detected by nanoparticle analyzer,CD8~+T cell was separated by immunomagnetic beads.The purity of CD8~+T cells was identified by flow cytometry.After co-incubation with three gastric cancer cell lines exosome,CD8~+T cell cycle was detected by flow cytometry,apoptosis was detected by Annexiv-PI staining,and cytokine secretion was detected by ELISA,gene expression level was performed by Real-Time PCR.Exosome labeled with Did fluorescent dye was injected into mice via tail vein,and bio-fluorescence imaging system was used to detect the distribution of exosome in lung,bone marrow,spleen,kidney,heart,and liver of mice.Flow cytometry was used to determine the uptake by CD45+cell subsets.Further analysis of CD45+cell types on exosome uptake,the exosomes extracted were injected into mice via tail vein every 5 day for 30 days,and on day 30,lung was digested into single cells,and the changes of immune cell subgroup were analyzed by flow cytometry.On day 30,1 × 105 MFC cells were injected through the tail vein.On 60 days,the lungs of mice were observed tumor formation by HE staining.The expression of microRNA,lncRNA,and mRNA in gastric cancer derived exosome was determined by next-generation sequencing.The interaction between different RNAs and the signaling pathways were analyzed by bioinformatics.Results:Exosomes were successfully extracted from MKN-28,MKN-45,and SGC7901.Exosomes were cup-shaped or dish-shaped with 50-100 nm in diameter and expressed exosome biomarker Alix and TSG101.There is not much difference in size of exosomes between different cells line.CD8~+T cells were successfully extracted with a purity of over 98%.After incubation with exosome,CD8~+T cells was induced apoptosis with had a dose-dependent in MKN-28-exo group.MKN-28-exo,MKN-45-exo,and SGC-790-exo altered CD8~+T cytokine secretion,especially IL-10,exosome in CD8~+T cells,IL-10,FOXP3 and IFN-y gene expression were significantly upregulated in three groups handling CD8~+T cells.MKN-28-exo and MKN-45-exo are mainly distributed in the lungs at 4 hours.Flow cytometry analysis shows that 62.77%CD45+cells uptanken MKN-45-exo.The uptake capacity of exosome by different cell subgroups is different.Among them,NK and Mφcells absorbed more MKN-45-exo than other cell subset.After long-term exosome stimulation,the exosome from MKN-28 and MKN-45 groups created an immunosuppressive microenvironment in the lungs and three gastric cancer derived exosome all form metastatic sites in the lungs.Gene sequencing and bioinformatics analysis revealed ceRNA interactions in MKN28-exo,MKN-45-exo,and SGC-790-exo,mainly LncRNA-microRNA-mRNA.LncRNA-DHX9,LncRNA-RUFY2,miRNA-148,miRNA-16,miRNA-let-7b,PPP3R1,TRAM1,PPP2CA,HMGA1,RBM15,GTF2A2,etc.It will provid a novel perspective for the mechanism of gastric cancer and immune cells research.Conclusion:Exosome was successfully extracted,interaction with CD8~+T cells was found to have effects on CD8~+T cell cycle,apoptosis,cytokine secretion and gene expression.Different cell lines derived exosome had different effects on CD8~+T cells,exosome firstly distributed in the lung,and then scattered in other organs,gastric cancer derived exosomes were mainly uptaken by NK and Mφ.After long-term stimulation of gastric cancer derived exosome produced an immunosuppressive microenvironment and metastasis sites formation in the lung,after analyzing the RNA in exosome by bioinformatics,it was found that miR-16,miR-148 and miRNA-let-7b are related to LncRNA and mRNA expression in exosome,which provide new option for the study of mechanism for exosome interaction with tumor cells and CD8~+T cells. |
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