The Role And Mechanism Of MiR-223-3p-mediated Notch Signaling In Regulating Endothelial Cell Migration And Sprouting

Background: Angiogenesis refers to the process by which endothelial cells(ECs)form new blood vessels through proliferation,migration,and differentiation under the action of various pro-angiogenic factors.Abnormal angiogenesis is an important event leading to the progression of age-related retinopathy and tumors.Therefore,in-depth study of the molecular regulation mechanism of angiogenesis will help to find new targets and strategies for targeting angiogenesis in the treatment of related diseases.Notch signaling is a highly conserved signaling pathway that mediates the interaction between neighboring cells.Both the literature and our research have previously found that Notch signaling can play a key role in angiogenesis by regulating EC proliferation,migration and differentiation.However,its downstream mechanisms are still unclear.Our group has used small RNA sequencing to discover a series of miRNAs regulated by Notch signaling.The expression level of miR-223-3p was significantly up-regulated after the Notch signaling activation.Whether it mediates Notch signaling to regulate EC function and thus affect angiogenesis is still unclear.In addition,the downstream regulatory mechanism of miR-223-3p remains to be further elucidated.Objective: To clarify the changes of EC phenotype after the activation of Notch signaling and its regulation on the expression of miR-223-3p;to clarify the effect of miR-223-3pmediated Notch signaling on EC phenotype;to explore the downstream molecules of miR-223-3p in regulating angiogenesis and the underlying molecular mechanism.Methods: The migration,proliferation and angiogenesis ability of EC were detected by cell Transwell assay,scratch assay,EdU incorporation assay,lumen formation assay,aortic ring sprouting assay;qRT-PCR,Western-blotting and transcriptome sequencing were used to investigate downstream molecular mechanisms.Results: Activation of Notch signaling inhibits angiogenesis and upregulates the expression of miR-223-3p.The overexpression of miR-223-3p inhibits angiogenesis.The knockdown of miR-223-3p blocks the inhibitory effect of Notch activation on EC migration and sprouting ability,but not EC proliferation ability.The miR-223-3p directly targets the 3’-noncoding region of Fbxw7,reducing its mRNA and protein levels.The overexpression of Fbxw7 can effectively rescue the inhibition of EC migration and sprouting ability by miR-223-3p.Conclusions: In conclusion,this study has confirmed that Notch signaling can inhibit EC migration and sprouting ability by upregulating miR-223-3p,which can be achieved by targeting Fbxw7.