Study On The Angiogenesis Of Roxarsone In Vivo And The Effect On VEGFR2-mTOR Using ShRNA Targeted On Vegfr2 Gene

Roxarsone(Rox)was found of promoting growth,antibacterial and anti-coccidiosis,and was widely used as a feed additive in livestock production.However,recent studies have showed that Rox could induce angiogenesis in in vitro and in in vivo,and has the risk of inducing angiogenesis-related diseases.VEGF can bind to VEGFR2 on the surface of vascular endothelial cells and then transducts signals to downstream,and plays a regulatory role in angiogenesis.mTOR,lying the downstream of PI3K/AKT pathway,has a regulatory role in tumorigenesis and angiogenesis.Our previous study showed that Rox promoted angiogenesis in in vitro and in in vivo,and up-regulated the expressions of VEGF/VEGFR2 and PI3K/AKT.In this paper,the Vegfr2 gene-targeted shRNA mediated by lentiviral constructed by our research group before was used to transfect rat vascular endothelial cells in in vitro,and also be used in test of the mouse matrigel plug model and the mouse melanoma tumor model in in vivo.With the treatment of Rox or rapamycin,the effects on matrigel plugs,xenograft tumors and their vascular growth were studied;Western Blot was used to detect the expressions of VEGF/VEGFR2 and mTOR-related proteins to study the regulatory mechanism of VEGF/VEGFR2-mTOR in angiogenesis in vivo induced by Rox.In the experiments of mouse matrigel plug assay in in vivo,vascular endothelial cells and matrigel were injected subcutaneously in mice to establish matrigel plug models.The different treatments were divided as follows:PBS group,1.0μM Rox treatment group,shRNA interference and Rox co-treatment group(Rox+RNAi group),shRNA interference group(RNAi group).The weight,volume and hemoglobin content of the stoppers were measured,and HE staining and CD34 immunohistochemical analysis were performed on the stoppers.Western Blot was used to detect the expressions of VEGF,VEGFR2,mTOR and p-mTOR proteins.The results showed that the volume,weight and hemoglobin content of matrigel plugs increased significantly in the Rox group,which were 2.1,1.69,and 1.49 folds as the PBS group respectively;the expression levels of VEGF,VEGFR2,mTOR,and p-mTOR proteins in the plug tissue also increased significantly(P<0.05).Compared with the Rox group,the volume,weight,hemoglobin,expressions of VEGFR2,mTOR and p-mTOR proteins in matrigel plugs of the Rox+RNAi group were significantly decreased(P<0.05).Targeting Vegfr2 gene shRNA interference significantly inhibits the effects of Rox on matrigel plug growth and angiogenesis,and suppresses Rox-upregulaed mTOR signal.In the experiments of mouse B16 melanoma xenograft models,the groups of different treatments were named as follows:PBS group,25 mg/kg Rox group,shRNA interference and Rox co-treatment group(Rox+RNAi group),shRNA interference group(RNAi group),negative shRNA infection control group(NC group).Recombinant lentivirus and B16F10 cells were injected into the axilla of mice.From the second day of injection,Rox or PBS was given by gavage once a day for 7 consecutive days.The body weight of mice and the volume/weight of tumors were measured;HE staining and CD34 immunohistochemical analysis were conducted and the expressions of VEGF,VEGFR2,mTOR and p-mTOR proteins were detected by Western Blot in tumor tissues.The results showed that the volume and weight of the xenograft tumors in the Rox group were increased significantly,and the tumor weight was about 1.53 times as that of the PBS group;the tumor weight of the Rox+RNAi group was dramatically reduced to about 0.65 times as that of the Rox group.The expressions of VEGF,VEGFR2,mTOR and p-mTOR proteins in the Rox group were significantly up-regulated(P<0.05).Compared with the Rox group,the expressions of VEGF,VEGFR2,mTOR and p-mTOR proteins were significantly reduced in the tumor tissues of the Rox+RNAi group(P<0.05).Targeting Vegfr2 gene shRNA interference can significantly inhibit Rox-promoted B16F10 transplanted tumors and vascular growth,and also significantly inhibit the upregulation of mTOR signal caused by Rox.In the experiments of rat vascular endothelial cells in in vitro,different treatments were divided into PBS group,1.0 μM Rox treatment group,shRNA interference and rox co-treatment group(Rox+RNAi group),shRNA interference group(RNAi group),rox and rapamyc in co-treated group(Rox+RAPA group),rapamycin treated group(RAPA group)and shRNA negative control group(NC group).The activity of vascular endothelial cells was detected by using MTT,and the expression of VEGF,VEGFR2,mTOR and p-mTOR proteins were detected by Western Blot.The results showed that Rox promoted the growth of vascular endothelial cells and significantly up-regulated the expressions of VEGF,VEGFR2,mTOR and p-mTOR proteins(P<0.01).Compared with the Rox group,the cell viability,VEGF,VEGFR2,mTOR and p-mTOR protein levels in the Rox+RNAi group were significantly reduced(P<0.05);the difference of the expressions of VEGFR2 and mTOR proteins between the Rox+RAPA group and the Rox group were insignificant,but the expression of VEGF and p-mTOR was significantly reduced(P<0.001).RNA interference targeting Vegfr2 gene can significantly inhibit the growth-promoting effect of Rox on endothelial cells and VEGF/VEGFR2-mTOR signal;RAPA can inhibit the promotion of rox on endothelial cells and down-regulate the expressions of VEGF and p-mTOR proteins.In summary,roxarsone can significantly promote the growth and angiogenesis of matrigel plugs and mice melanoma xenografts in in vivo,which can be inhibit by using RNAi targeting Vegfr2 gene VEGF/VEGFR2-mTOR signaling plays an important regulatory role in the pro-angiogenic effect of roxarsone in in vivo.