Epstein-barr Virus Associated Gastric Carcinoma MiR-BART1-3P Transmision Of Exosome Pathway And Regulates The Expression Of Related Genes

[Background]Although Epstein-Barr virus(EBV)is known to encode over 40 different miRNAs of its own,the roles of most EBV miRNAs remain unknown.EBVaGC expresses a restricted number of EBV latent genes,including EBV-encoded small RNA(EBER)-1 and-2,EBV nuclear antigen EBNA)-1,latent membrane protein-2A(LMP2A),and BART miRNAs.There is increasing evidence that several BART miRNAs play an important role in the development of cancer.Specific miR-BART can induce the transition between latency and lytic replication.For example,miRBART20-5P can be targeted by Death-inducing factor Bcl-2 related cell death(BAD)mRNA agonist reduces apoptosis and enhances the growth of GC cells;miR-BART22 promotes EBVaGC by targeting N-myc downstream regulatory gene 1(NDRG1).However,the role of most BART miRNAs in tumorigenesis still needs further study.In recent years,it has been reported that vesicle exosomes secreted by cells participate in multiple functions including transmembrane signal transduction,substance endocytosis,and directed sorting of lipids and proteins,and play an important role in the transduction of intercellular substances and information.It has been shown that the EBV oncoprotein LMP-1 and many miRNAs are released through exosomes,and induce the proliferation of adjacent cells,inhibit apoptosis and regulate changes in biological behaviors such as viral infection.There is increasing evidence that exosome-mediated transfer of mRNA from EBV-infected cells is considered to be a possible mechanism by which viruses enter epithelial cells,but there are few relevant studies.Therefore,by exploring the role and influence of exosomes in the transmission of miR-BART1-3P in EBVrelated gastric cancer,we can further understand the occurrence and spread of tumors.[Objective]To explore the role and influence of exosomes in the miR-BART1-3P transmission pathway of EBVaGC[Material and Methods]Material:EBV positive gastric cancer cell line SNU719,EBV negative gastric cancer cell line AGS,normal gastric epithelial cell GES-1method:1.Construct a cell line overexpressing miR-BART1-3P by lentivirus infection,set AGS OE and EBV positive gastric cancer cell line SNU719 overexpressing miR-BART1-3P as experimental group cells,and transfect the empty virus EBV negative Gastric cancer cell line AGS NC and normal gastric epithelial cells GES-1 were set as control cells.2.Extract,separate and purify the exosomes of the experimental group cells by differential centrifugation,followed by cocultivation of the exosomes and the control group cells for a certain time gradient,and the experimental group cells and the control group cells for a certain time gradient Co-cultivation.3.Observed indicators(1)Morphology and EBER in situ hybridization to observe cell line differences;(2)Co-cultured exosomes were labeled by immunofluorescence staining,and EBV negative cells were taken into exosomes under laser confocal microscope;(3)Transwell migration,invasion,and scratch experiments were used to detect the effect of exosomes on EBV-negative cell function;(4)Through cellcell co-culture or exosome-cell co-culture,RT-PCR method was used to detect miR-BART1-3P and Relative quantitative expression of related target genes CXCL10,CXCL9,SST,GPER,IDO-1,PTGER3.[Resμlts]1.Successful construction of AGS over-expressing miR-BART1-3P cell line2.Successful extraction,separation,and purification of exosomes,and the successful establishment of cell-cell co-culture and exosomecell co-culture methods3.Morphologically successfully observe different cell lines and their EBER expression4.After co-cultivation of exosomes-cells,it was successfully observed under a laser confocal microscope that the exosomes were stained green and taken into the cytoplasm by EBV-negative cells.5.In the cell function experiment: CCK8 proliferation experiment showed that the OD value of the cells in the experimental group added with exosomes was lower than that of the control group over time,P<0.05,the difference was statistically significant;the cell scratch experiment showed that With the increase of time,the cell migration rate of the experimental group added with exosomes was lower than that of the control group,P <0.05,the difference was statistically significant;the cell migration and invasion experiments showed that with the increase of time,the lower cells of transwell cells added with exosomes The number was significantly lower than the control group,P <0.05,the difference was statistically significant.6.The results of RT-PCR experiments showed that after cell-cell /exosome-cell co-culture,the miR-BART1-3P and related genes CXCL10,CXCL9,SST,GPER,IDO-1,PTGER3 of 2 ^-The ΔΔCt value was higher than the control group,P <0.05,the difference was statistically significant.[Conclusions]1.Under the influence of exosomes secreted by EBV-positive cells and gastric cancer cells overexpressing miR-BART1-3P,the proliferation,invasion and migration of EBV-negative cell lines are inhibited,suggesting that this may be the prognosis of EBV-related gastric cancer One of the better reasons2.Exosomes can be taken up by EBV-negative cells,providing the basic conditions for the propagation of miR-BART1-3P carried in exosomes.Exosomes can carry EBV-miR-BART1-3P to the recipient cells and make the recipient cells highly express miR-BART1-3P and its related target genes CXCL10,CXCL9,SST,GPER,IDO-1,PTGER3.