MicroRNA-223 Antagonizes Angiogenesis By Targeting Septin2

Objective:The aim of this study was to investigate the role of SEPT2?Septin2?in angiogenesis and to enrich the molecular mechanism by which miR-223 inhibits angiogenesis.Methods:A method for isolation and culture of umbilical vein endothelial cells?HUVECs?was established.Real-time PCR and Western-Blot were used to detect the different expression level of SEPT2 in HUVECss between the control group and miR-223 over-expression group.The luciferase reporter gene assay system verified whether SEPT2 was directly regulated by miR-223.The role of SEPT2 in angiogenesis was studied on the aspects of gain-and loss-of-function.Experimental animals included miR-223^f/ymice,miR-223?EC mice and miR-223^-/y.The angiogenesis ability of three genotype mice was evaluated by hindlimb surgery and immunofluorescence staining.Results:The expression of SEPT2 in HUVECs was detected by both Real-time PCR and Western-Blot.Compared with the control group,the expression level of SEPT2 in the miR-223 over-expression group was decreased.Our results demonstrate that SEPT2 mRNAs have miR-223 target sites,and the interaction of miR-223 with 3'UTRs of septin-2 leads to downregulation of luciferase expression in luciferase reporter gene system.Knockdown of SEPT2 inhibits the ability of HUVECs to form tubular structures on the surface of the substrate or in a three-dimensional environment,while over-expression of SEPT2 can promote this ability.Over-expression of SEPT2 rescues impaired angiogenesis in miR-223–expressing cells.Compared with miR-223^f/ymice,miR-223?EC mice were detected increased blood perfusion on day 7 after hindlimb,which was similar to miR-223^-/ymice.Gastrocnemius immunofluorescence staining showed that the vascular density of miR-223?EC genotype mice were higher than that of the miR-223^f/y/y mice in the gastrocnemius muscle,which was the same to the density of miR-223^-/y mice.Conclusion:1.SEPT2 is the direct target of miR-223.2.The expression of SEPT2 promotes angiogenesis.3.miR-223?EC mice have enhanced angiogenesis capacity,which is consistent with miR-223^-/y mice.