An In Vitro Study On The Anti-angiogenic Activity Of CRGD-siVEGFR2 Molecule

Tumor growth depends on new blood vessel formation,tumor cells immediately get capabilities of metastasis and invasion since tumor tissue has an independent vascular supply.RNA interference(RNAi),a new technology developed in recent years,is widely used as a tool for cancer gene therapy.This study designed and synthesized cRGD-siVEGFR2 molecule,and studied its biological function in vitro,explored its anti-angiogenesis effect and applied feasibility as a novel tumor-selective small-molecule drug.Methods1.Establishment of primary umbilical vein endothelial cells(HUVECs)HUVECs harvested by collagenase ? enzyme digestion method.Cells were identified with factor ? antibody by immunofluorescence.The purity was detected using flow cytometry(FCM)via surface protein CD31.And to explore the optimal serum starvation conditions by FCM.2.Synthesis of cRGD-siRNAcRGD was conjugated to the 5' end of siRNA sense strand via a linker to prepare cRGD-siRNA molecules.The structural identification was characterized by LC-MS analysis and the purity was determined by RP-HLPC.3.Biological function of cRGD-siVEGFR2 molecule(1)Study of serum stability:siVEGFR2,cRGD-siVEGFR2 and cRGD-siNC were mixed with mice blood serum respectively and incubated.Serum stability was analyzed by agarose gel electrophoresis.(2)Study of cellular toxicity:cells were transfected with 1000nM,2000nM,3000nM RPM or cRGD-siNC respectively.Cell vitality was detected by CCK8 kit.(3)Study of silencing effect:cells were transfected with 100nM,500nM,800nM,1000nM cRGD-siVEGFR2 molecule,positive control Lipo/siVEGFR2(100nM),Lipo/cRGD-siVEGFR2(100nM),negative control cRGD-siNC(1000nM).Then the specific silencing effects were examined by RT-PCR assay and western-blot assay.(4)Effects on cell proliferation and migration:cells were transfected with 100nM,500nM,800nM,1000nM cRGD-siVEGFR2,Lipo/siVEGFR2,cRGD-siNC.The effects on cell proliferation was detected by CCK8 assay and EdU assay,and the effect on cell migration was detected by wound healing assay and transwell assay.(5)Effect on tube formation in vitro:cells transfected by 1000nM cRGD-siVEGFR2,Lipo/siVEGFR2,cRGD-siNC were seeded in Matrigel conducting cell differentiation assayResults1.HUVECs could be harvested by collagenase ? enzyme digestion method.Cells were polygonal and arranged like a stone pavement.Immunocytochemistry showed that HUVECs highly expressed ?-related antigen.The purity of HUVECs reached 99.67%.Cell culture in the presence of different concentrations of FBS for 6h resulted in 70%G0/G1 phase cells,which increased to 80%-90%at 12h,and further to around 95%at 18h and 24 h.2.cRGD-siRNA molecules were synthesized successfully.The measured molecular mass of cRGD-siRNA were basically the same as theoretical mass,the purity of them were about 80%.3.Biological function of cRGD-siVEGFR2 molecule(1)Serum stability:Agarose electrophoresis showed siVEGFR2 was mostly being absolutely degraded at 24h.cRGD-siVEGFR2 and cRGD-siNC were slightly being degraded at 72h.(2)Cellular toxicity:O.D.value of RPM group was about 1.0;O.D.value of cRGD-siNC group remained above 0.85,and at 1000nM was about 1.0.(3)Silencing effect:cRGD-siVEGFR2 could down-regulate the expression of VEGFR2 gene and protein.The silence efficacy was concentration dependent.(4)Effects on cell proliferation and migration:cRGD-siVEGFR2 could inhibit the ability of cell proliferation and migration in time-and concentration-dependent manners.(5)Effect on tube formation in vitro:HUVECs could form typical funicular structures,create multiple branches then close into tubes when in matrigel for 6h.And tube was effectively reduced after transfected with cRGD-siVEGFR2.Conclusions1.High quality HUVECs can be harvested by digestion with collagenase.Serum-free culture for 12h is the best serum starvation condition.2.To prepare high purity,good stability,nontoxic cRGD-siRNA molecular,cRGD is covalently conjugated to 5'-end of siRNA sense strand.3.cRGD-siVEGFR2 silences the mRNA expression of VEGFR2 gene then reduces the expression of VEGFR2 protein,and effectively inhibits cell proliferation,migration and tube formation in vitro in a concentration and time-dependent manner.