The Role And Mechanism Of Exosomal MiR-210-3p Derived From Pancreatic Cancer Cells On Angiogenesis

Objective: To investigate the effect and mechanism of miR-210-3p derived from exosomes of pancreatic cancer on angiogenesis in pancreatic cancer.Methods: Differentially expressed micro RNAs were screened in six pancreatic cancer and five normal pancreatic microarray from GSE43796,and miR-210 was found out.Based on the result from GEO,TCGA database and q RT-PCR of pancreatic tissue and cancer cells was analyzed for the expression of miR-210 subtype.Then,the expression of miR-210-3p was further verified in 22 pair of pancreatic cancer tissues with adjacent tissues,the correlation was analyzed by TCGA between miR-210-3p and angiogenesis,clinical features in pancreatic cancer.For detecting the mechanism of miR-210-3p in angiogenesis,exosomes were extracted from pancreatic cells and identified by electron microscopy,NTA,WB(CD63,TSG101,GRP94).Next,umbilical vein endothelial cells(HUVECs)was co-cultured with pancreatic cancer cell exosomes.the ability of angiogenesis was measured by CCK8,formation of tubule,transwell assay,the expression of MMP9,VEGFA,FGF2,ANG,ANGPTL4,PDGFB was valued by q RTPCR and expression of VEGFA was detected by WB.q RT-PCR was implemented for the expression of miR-210-3p in the exosomes of pancreatic cancer cells and serum exosomes of 18 healthy subject and 20 pancreatic cancer patients,and further verified the expression of miR-210-3p in HUVECs co-cultured with pancreatic cancer cells exosomes or exosomes inhibitors(GW4869).After transfecting miR-210-3p mimics or miR-210-3p inhibitors in HUVECs,the effects of exosomes on angiogenesis was measured by CCK-8,formation of tubule,transwell assay,the expression of MMP9,VEGFA,FGF2,ANG,ANGPTL4,PDGFB was valued by q RT-PCR and expression of VEGFA was detected by WB.Finally,HUVECs co-cultured with pancreatic cancer exosome was transfected with miR-210-3p inhibitor,we measured ability of angiogenesis and the expression of angiogenic molecules.Results: MiR-210 was highly expressed in the pancreatic cancer tissues from GSE43796.Compared with no significantly difference of miR-210-5p subtype,miR-210-3p analyzed in TCGA database and q RT-PCR of pancreatic cancer cells was highly expressed in pancreatic cancer,and it was further verified in 22 pairs of pancreatic cancer and adjacent tissues.TCGA database analyzing showed significantly positively correlated between miR-210-3p and angiogenic molecules(VEGFA,ANG,ANGPTL4),the shorter overall survival in pancreatic cancer patients.Then,exosomes were extracted from pancreatic cancer cells.Transmission electron microscopy,NTA analysis and highly expressions of CD63 and TSG101 suggested that exosomes were extracted successfully.HUVECs was co-cultured with exosomes from pancreatic cancer cell of highly expressed miR-210-3p.CCK8,formation of tubule,transwell assay showed the ability of angiogenesis were enhanced.Meanwhile,q RT-PCR and WB showed the expression of VEGFA,ANG,PDGFB,ANGPLT4,MMP9 gene and VEGFA protein was elevated.q RT-PCR analyzing showed highly expression of miR-210-3p in the pancreatic cancer cell lines CFPAC-1,PANC-1 with their exosomes,and it was further confirmed in serum exosomes of 20 pancreatic cancer and 18 healthy persons.After HUVECs was cocultured with the exosomes of CFPAC-1 and PANC-1 cells,the expression of miR-210-3p was elevated and decreased by the exosome inhibitor(GW4896),indicating that pancreatic cancer cells delivered miR-210-3p to HUVECs by exosomes.HUVECs was transfected with miR-210-3p mimics or inhibitors,and CCK8,formation of tubule,transwell assay,q RT-PCR,WB reveled miR-210-3p enhanced ability of angiogenesis and elevated the expression of angiogenic molecules.However,inhibition of miR-210-3p could reverse the impact of exosomes in promoting angiogenesis of HUVECs.Conclusion: MiR-210-3p was highly expressed in pancreatic cancer tissues and cell lines,and was positively associated with shorter overall survival and angiogenesis;MiR-210-3p was highly expressed in pancreatic cancer exosomes and was transferred to HUEVCs.MiR-210-3p promoted the ability of angiogenesis,migration and proliferation in HUVECs,so as to promote tumor progression.So that it provided a potential target for anti-angiogenesis therapy.