Flot2 Promotes Angiogenesis In Nasopharyngeal Carcinoma Microenvironment Through Exosomal MiR-23b-3p/ZO-1 Axis

Background:Nasopharyngeal carcinoma(NPC)is a malignancy derived from the nasopharynx epithelium.The incidence of NPC is characterized by obvious ethnic aggregation and unbalanced geographical distribution,with high incidence in South China,Southeast Asia,and North Africa.Tumor metastasis is the key factor for the clinical treatment failure of NPC patients.Previous studies in our lab have identified that Flotillin 2(Flot2),a key gene in nasopharyngeal carcinoma metastasis,is highly expressed in NPC and promotes the progression of NPC.However,the underlying molecular mechanism of Flot2 in promoting the progression of NPC has not been fully elucidated.The purpose of this study was to explore whether Flot2 regulates angiogenesis in the microenvironment of NPC through the exosome pathway,to further reveal the molecular mechanism of Flot2 in promoting nasopharyngeal carcinoma metastasis,and to provide new idea and experimental basis for NPC targeted therapy with Flot2 as the core.Methods:1.Nasopharyngeal carcinoma cell lines stably interfering Flot2 and control cell lines(5-8F-shctr,5-8F-shFlot2,C666-1-shctr and C666-1-shFlot2)were constructed by using lentiviral vectors.2.The Transwell chamber simulated co-culture system and exosome secretory inhibitor GW4869 were used to identify whether there existed intercellular cross-talk mediated by exosomes between NPC cells and Human umbilical vein endothelial cells(HUVECs).3.Ultracentrifugation was used to extract exosomes derived from supernatant of 5-8F-shctr,5-8F-shFlot2,C666-1-shctr and C666-1-shFlot2 cell medium(named as Exo-5-8F-shctr,Exo-5-8F-shFlot2,Exo-C666-1-shctr,Exo-C666-1-shFlot2).Transmission electron microscopy(TEM),Nanoparticle tracking analysis(NTA),and Western blot(WB)were used to identify the morphology,particle size distribution,and marker proteins of the exosomes.4.Lipophilic carbonyl cyanine dye DIO was used to label the exosomes derived from NPC Cells and to trace the uptake of exosomes by HUVEC cells.5.Cell proliferation,migration,tube formation,vascular permeability,and transendothelial migration of tumor cells were assayed to detect the effect of exosomes derived from NPC cells interfering with Flot2 on angiogenesis of HUVEC cells in vitro.Matrigel plug assay was performed to detect the effect of exosomes on angiogenesis in vivo.6.qPCR was used to detect the differentially expressed miRNAs in exosomes and corresponding miRNAs in HUVEC cells treated with Exo-5-8F-shctr,Exo-5-8F-shFlot2,Exo-C666-1-shctr,Exo-C666-1-shFlot2.7.Starbase database was used to predict candidate target genes of miR-23b-3p,Luciferase reporter gene assay,qPCR,and WB were performed to identify whether ZO-1 was one of the target genes of miR-23b-3p.8.Cell migration,tube formation,and vascular permeability experiments were performed to determine whether Flot2 promoting in vitro endothelial cell angiogenesis was dependent on exosomal miR-23b-3p.Results:1.The co-culture system simulated by Transwell chamber showed that exosomes could mediate the cross-talk between NPC cells and HUVEC cells,and promote the angiogenesis of HUVEC cells in vitro.2.The results of TEM,NTA,and WB showed that the vesicles extracted by ultracentrifugation from the supernatant of NPC cell medium had exosomal "teacup" structure.The particle size of vesicles was within the defined range of exosome,and the vesicles carried exosomal biomarker proteins such as CD63,CD9,and TSG101.3.The results of cell proliferation,migration,tube formation,vascular permeability,transendothelial cell migration,and matrigel plug assays showed that compared with Exo-5-8F-shFlot2 or Exo-C666-1-shFlot2,Exo-5-8F-shctr or Exo-C666-1-shctr significantly promoted angiogenesis of HUVECs in vitro.Compared with Exo-5-8F-shFlot2,Exo-5-8F-shctr significantly promoted angiogenesis of HUVECs in vivo.4.qPCR results showed that knocking down the expression of Flot2 in NPC cells significantly reduced the content of miR-21-5p,miR-23b-3p,and miR-25-3p in exosomes derived from corresponding NPC cells.In addition,after incubating HUVEC cells with exosomes,compared with Exo-5-8F-shctr or Exo-C666-1-shctr groups,the expression of miR-21-5p,miR-23b-3p,and miR-25-3p was significantly lower in HUVEC cells incubated with Exo-5-8F-shFlot2 or Exo-C666-1-shFlot2.5.miR-23b-3p could promote the migration of HUVEC cells,induce the tube formation of HUVEC cells,and enhance the permeability of vascular endothelial cells and trans-endothelial cell migration of tumor cells.Moreover,miR-23b-3p targeted ZO-1 m RNA and reduced the expression of ZO-1,which resulted in increased angiogenesis of HUVEC cells in vitro.6.Rescue experiments showed that miR-23b-3p inhibitors could partially inhibit the promoting effect of exosomes derived from 5-8F or C666-1(Exo-5-8F-shctr or Exo-C666-1-shctr)on the angiogenesis of HUVEC cells in vitro.Conclusions:1.There exists exosome-mediated cross-talk between NPC cells and endothelial cells in the tumor microenvironment.2.Flot2 can promote the angiogenesis of HUVEC cells in vitro and in vivo through exosomes.3.The role of Flot2 in promoting angiogenesis partly depends on the miR-23b-3p/ZO-1 axis through exosomes.