The Mechanisms Of HIF-2α And Thalidomide In SBVM And Angiogenesis

Part I Effect of HIF-2αand thalidomide on the development of zebrafish subintestinal veinAim:To investigate the influence of HIF-2αand thalidomide on the development of zebrafish subintestinal vein.Methods:A overexpression plasmid was microinjected into Tg（fli1a:EGFP）^y1zebrafish embryo to up-regulate HIF-2α,the phenotype of subintestinal vein was observed,accounting the number of vascular sprout.Then,different concentration of thalidomide（200uM,400uM,800uM）was used to treat zebrafish created aforementioned,the number of vascular sprout was also recorded.Real-time PCR was used to investigate the expression of angiogenesis related pathways,such as VEGF,NOTCH and DLL4.Results:Subintestinal vascular sprouting was significantly increased under overexpression of HIF-2α（4.20±1.48,P<0.05 vs.Control）.After intervention of thalidomide,the number of subintestinal vascular sprouting was decreased gradually with increasing concentration of thalidomide,particularly at 800uM of thalidomide,the phenotype of subintestinal vein was partially similar to that of control group.Results of RT-PCR showed up-regulation of VEGF,NOTCH1a,NOTCH1b,NOTCH2,DLL4 after HIF-2αoverexpression,and could be reversed by thalidomide.Conclusion:HIF-2αcould induce abnormal sprouting of zebrafish subintestinal vein,and this effect could be reversed by thalidomide.HIF-2αpromoted the expression of VEGF,NOTCH,DLL4.And thalidomide could inhibit the activation of angiogenesis related pathways.PART II The influence of HIF-2α/thalidomide on transcriptome of zebrafish and HUVEC small non-coding RNAsAim:To investigate the influence of HIF-2αand thalidomide on transcriptome of zebrafish,the regulatory effect of thalidomide on HUVEC small non-coding RNAs.To predict the binding strength and binding site between snoRNA and nucleolin.Methods:High throughput sequencing analysis was used to find the differential expression of transcriptome of zebrafish model created aforementioned（control group,vs.HIF-2αoverexpression group vs.HIF-2αoverexpression+thalidomide 800uM group）,and to investigate the differential expression of small non-coding RNAs on HUVEC after thalidomide treatment.CatRAPID platform was used to make bioinformatics prediction of the binding strength and binding sites between snoRNAs and nucleolin.Western Blot and RT-PCR were performed to testify the results from sequences.Results:High throughput sequencing analysis of three samples of zebrafish model showed HIF-2αand thalidomide influence the expression of VEGF pathway and NOTCH pathway.We accidentally found there was differential expression in ribosome biogenesis pathway,and nucleolin was one of them.Then,13 snoRNAs was found to be differential expressed before and after thalidomide treatment,among them,SNORA31 was the only snoRNA which was up-regulated under thalidomide and expressed stably.Through bioinformatics analysis,SNORA31 could bind to nucleolin protein at relatively high strength.Conclusion:HIF-2αand thalidomide could regulate angiogenesis related pathways,and thalidomide could affect ribosome biogenesis pathway,nucleolin could also be influenced by thalidomide.SNORA31 could bind nucleolin protein at high intensity.PART III The inhibiting effect of SNORA31 on nucleolin cell surface expressionAim:To investigate the inhibiting effect of SNORA31 on nucleolin cell surface expression.Methods:IHC was used to observe the expression of nucleolin on vascular endothelial cells in small bowel vascular malformation.The tubing ability of HUVECs without nucleolin was investigated by tube formation experiment.IF was used to study the cell surface nucleolin expression after transfected by Lenti-SNORA31 and interfered by SNORA31-siRNAs,also the inhibiting effect of fragments of SNORA31.Results:Nucleolin showed higher expression in endothelial cells of small bowel vascular malformation lesions.When cell surface nucleolin was inhibited by nucleolin antibody,HUVECs could not form tubes.Cell surface nucleolin increased after interfering by SNORA31-siRNAs,and decreased by transfecting Lenti-SNORA31.Fragments containing H BOX domain could inhibit cell surface nucleolin expression.Conclusion:Nucleolin moving from nucleolus to cell surface is a key step of angiogenesis.SNORA31 could bind nucleolin,and inhibited nucleolin cell surface expression.