Exosomes Carrying MiR-155 Target FOXO3 Of Endothelial Cells And Promote Angiogenesis In Gastric Cancer

Background and objective:Gastric cancer(GC)is a disease with high incidence and high mortality.According to the latest statistics in 2018,GC is the fifth most common cancer and ranks as the second for cancer-related deaths worldwide.An important reason for the rampancy of GC lies in its powerful ability of angiogenesis,which leads to continuous proliferation and metastasis of GC,the rapid progress of the disease,and ultimately the death of patients.Hence,if the angiogenesis of GC could be effectively blocked,the growth and spread of tumor cells would be correspondingly inhibited,and GC progression would be limited or delayed,which results in the extension of survival time for GC patients.In fact,there have been many targeted drugs based on angiogenesis of GC,but their specific efficacy has not reached people’s ideal expectations.Moreover,most anti-angiogenic drugs are very expensive without universal applicability,adding a heavy economic burden to numerous patients.Therefore,further researches are needed to explore the mechanism of angiogenesis in GC and anti-angiogenesis therapy.Exosomes have been the research hotspot in the field of cell biology in recent decades.They are kind of tiny vesicles secreted by various cells with the diameter of40-100 nm,which exist in blood,urine,lymph and other body fluids.And exosomes can package and transport proteins,m RNAs,micro RNAs(miRNAs)and other biological small molecules to participate in the cellular communication.One of the exosomes carriers,miRNAs are single non-coding RNA molecules with length of about 20 nucleotides that specific bind to the 3’-UTR non-coding regions of target genes.By cutting off m RNA molecule of target genes,or inhibiting the translation of target genes,or the combination of the above two functions,exosomes exert negative regulation of target gene expression level,and participate in many physiological or pathological process,such as cell proliferation,cell differentiation,antigen-presenting and tumor invasion.However,the research of exosomes in tumor angiogenesis is not thorough,and further exploration is needed.In this study,according to the delivery function,combined with the characteristics of relatively stable exists in human body,we discussed the effcts of exosomes enveloping specific miRNAs on angiogenesis of GC,and the possibility of anti-angiogenesis therapy,which provides a new way aiming at the treatment of GC angiogenesis in the future.Methods:1.The expression level of FOXO3 protein was detected by Western blot in GCtissues and adjacent normal tissues.And verifying the effect of FOXO3 onangiogenesis.The upstream potential miRNA of FOXO3 was predicted bybioinformatics software Target Scan,etc.Then,the expression of upstreammiRNA in GC cells was up-regulated or down-regulated by cell transfection,andthe regulatory relationship between upstream miRNA and FOXO3 in GC cellswas verified by Western blot.2.The expression of FOXO3 was up-regulated or down-regulated by lentivirus orsmall interfering RNAs(si RNAs).Edu proliferation assay,cell wound healingassay and endothelial tube formation assay were used to test the effect of FOXO3in proliferation,migration,and ring formation.The expression of upstreammiRNA in GC cells was up-regulated or down-regulated by cell transfection,andthe above functional experiments were performed to complete the verification.3.The level of miRNA encapsulated in exosomes was up-regulated ordown-regulated by cell transfection.Exosomes in GC cell culture medium wereisolated by differential centrifugation,and verified by electron microscope andWestern blot.The content of miRNA enveloped by exosomes was analyzed byq RT-PCR.Then,in vitro and in vivo,Edu proliferation assay,cell wound healingassay and endothelial tube formation assay were used to verify that exosomes canbe worked as carriers to encapsulate and transport miRNA.And they inhibitedthe angiogenesis of GC by regulating the expression level of FOXO3,thereby limiting the tumor progression.Results:1.We found the expression characteristics of mi R-155 and FOXO3 in serum and GC tissues at the clinical level,which is the high expression of mi R-155 and the low expression of FOXO3.2.We found a cross-cell signaling pathway between mi R-155 transported by exosomes and FOXO3 of vascular endothelial cells in the tumor microenvironment of GC.3.The biological phenomenon of GC exosomes carrying miRNAs to promote angiogenesis and tumor growth was elucidated,which provided new targets and strategies for anti-angiogenesis therapy of GC and other tumors.Conclusion:Through the above experiments,we found that the mi R-155 /FOXO3 axis with exosomes as carriers in GC contributes to the vascular proliferation,which leads to the continuous development and spread of GC.Therefore,using mi R-155 inhibitors enveloped by exosomes can play a targeted inhibitory effect,and reduce the proliferation as well as migration of vascular endothelial cells,thus limit the progression of GC.