Study On The Mechanism Of VEGFR2 In Angiogenesis Induced By Roxarsone In Vitro Using RNA Interfere Target On Vegfr2 Mediated By Lentivirus

Background:Angiogenesis plays an extremely important role in the pathogenesis of diseases.If abnormalities occur,the body will develop diseases such as tumors,stroke,coronary heart disease,various eye diseases and so on.VEGF/VEGFR2 signaling pathway participates in the growth and reconstruction of new blood vessels during angiogenesis.Angiogenesis factors regulate the activation and expression of VEGFR2 on the surface of vascular endothelial cells,thereby activating or inhibiting the signal transduction pathways downstream of related signal transduction pathways,thereby exerting the functions of regulating vascular endothelial growth and proliferation and vascular formation of the body.In our previous study,the angiogenesis was promoted by roxarsone,and the expression of VEGF/VEGFR2 increased in vitro or in vivo.In this paper,the RNA interference mediated by lentivirus target on vegfr2 gene was developed to silence the expression of vegfr2 gene.The recombinant lentivirus was used to infect rat endothelial cells to silence the expression of the VEGFR2 receptor in the proliferation,migration,and tube formation assay,thereby the role of VEGF/VEGFR2 signaling pathway in the angiogenic effects induced by Roxarsone was studied.Objective:The VEGF/VNEGFR2 signaling involved in the angiogenesis effect promoted by roxarsone was investigated by RNA interference mediated by lentivirus target on rat vegfr2 gene.Methods:?Construction of recombinant lentivirus vectors:The lentiviral vector expressed shRNA target on rat vegfr2 gene and the negative RNA were developed separately.Then the infection condition of the recombinant lentivirus on rat endothelial cells was screened.The interference efficiency of established lentiviral vectors on the vegfr2 gene was evaluated based on the VEGFR2 expression and its mRNA by RT-PCR and Western-Blot method.?Effect of targeting vegfr2 lentiviral vector interference on ROX-induced growth of rat vascular endothelial cells:The endothelial cells were treated at ROX and/or lentiviral vector in BrdU proliferation assay,scratch test and tube formation assays.The different treatment were designed as such groups as PBS,1.0?M ROX,NC,sh-vegfr2 and sh-vegfr2+ROX.The rate of BrdU positive cells,migration distant,number of nodes and length of tube-like of rat vascular endothelial cells were determined,the expression of VEGFR2 was detected via Western Blot.Results:?The recombinant lentiviral vector constructed can express the expected sequence of shRNA of vegfr27 the titer of lentivirus vectors was 1×109TU/ml.The optimized conditions for recombinant lentivirus infection of endothelial cells were 40 MOI and 0.5 ?g/ml Polybrene,the infection efficiency was up to 90%.The silencing effect of recombinant lentivirus on VEGFR2 in endothelial cells was 70%or more by RT-PCR and Western blot detection.?In tests of ROX and or not co-treated with recombinant lentivirus in endothelial cells,results showed that the number of BrdU-positive cells,the distance of migration,the number of nodules and the length of tubular branches in the ROX treatment along group were significantly higher than those in the control group(P<0.01).Compared with PBS control group,the number of BrdU positive cells,migration distance,number of nodules and length of tubular branch were significantly decreased in the sh-vegfr2 treatment group(P<0.01),the expression of VEGF and VEGFR2 protein in the sh-vegfr2 lentivirus interfering group was significantly lower(P<0.01).The cell proliferation,migration and tube formation in the sh-vegfr2+ROX group were significantly decreased compared with the ROX treatment single group(P<0.01).Conclusion:?The lentiviral vector targeting the rat vegfr2 gene was successfully constructed at a higher virus titer,and the silencing effect on the VEGFR2 expression in endothelial cells was over 70%.?The proliferation,migration and tube-like formation of endothelial cells promoted by roxarsone can be effectively inhibit by the vegfr2 shRNA in lentiviral vector-mediated,indicating that the VEGFR2 mechanism plays an important regulatory role in the pro-angiogenic effect of roxarsone on endothelial cells in vitro.