MiRNA Expression Of Exosomes In EBV-positive Gastric Carcinoma Cell

[Objective] To establish an isolation and purification method for exosomes in culture supernatant of EBV-associated gastric cancer cell lines.To investigate the expression of miRNA in exosome of EBV-positive gastric carcinoma cells.[Methods] The exosomes of EBV-positive and negative gastric carcinoma cells were separated by ultracentrifugation,the morphology of exosomes was identified by transmission electron microscopy,the exosome size was analyzed by Nanosight,and the expression of exosome membrane protein CD63 and CD81 was detected by Western blot.High-throughput sequencing was used to detect miRNA expression profiles in gastric cancer cell lines and their exosomes.[Results] 1.Under the ultra-microscopic electron microscope,the exosomes are seen as a typical translucent cup-like structure or a flat spherical structure.2.The nanoparticle tracking analyzer(Nanosight)showed that the exosomes were between 30 and 150 nm in diameter.3.Western blot(WB)assays showed that exosomes secreted by EBVaGC and EBVnGC cells expressed specific exosome membrane-associated proteins CD63 and CD81 4.High-throughput sequencing revealed that EBVaGC(SUN-719)and EBVnGC(AGS)and their secreted exosomes were highly expressed with certain human miRNAs,among which AGS-Exo was highly expressed with hsa-miR-371a-5p,hsa-miR-7-5p and hsa-miR-181b-5p.SNU-719 Exo was highly expressed as hsa-miR-26a-5p,hsa-miR-92a-3p and hsa-let-7f-5p.Nearly all EBV-related miRNAs(EBV-miRNA)were expressed in SUN-719 cells and their exosomes,among which EBV-miR-BART22,EBV-miR-BART10-3p and EBV-miR-BART11-5p were the highest in SUN-719 cells,and EBV-miR-BART9-5p and EBV-miR-BART11-5p were the highest in SUN-719 Exo.[Conclusion] 1.Both EBVaGC and EBVnGC cells can secrete exosomes,and the secreted exosomes express exosome membrane-associated proteins such as CD63 and CD81.2.Exosomes secreted by EBVnGC cells express human related miRNA.Exosomes secreted by EBVaGC cells express human-related miRNA and EBV-related miRNA,which may be involved in regulating the expression of virus and cell proteins related to cell proliferation,transformation,apoptosis,survival and immune escape,and participate in the occurrence and development of EBVaGC.