Study On The Relationship Between FRAT1 And Angiogenesis In Glioblastoma Cells

Objective:Gliomas are a kind of tumors arised from glial or precursor cells in central nervous system(CNS),accounting for 25.5% of primary CNS tumors and 80.8% of CNS malignant tumors.Among them,glioblastoma multiforme(GBM)is the most common,malignant and deadly type of primary tumor in the central nervous system.Even with remarkable advances achieved in recent years,the prognosis remains very grim.In GBM,angiogenesis is a significant prognostic factor,which is essential for the growth and metastasis of solid tumors.Vascular endothelial growth factor(VEGF),a predominant regulator of angiogenesis,is used as an important therapeutic target for angiogenesis diseases.Bevacizumab,a monoclonal antibody targeting VEGF,has received the US Food and Drug Administration(FDA)approval for the treatment of recurrent glioblastoma.However,anti-VEGF therapy has prolonged progression-free survival without significant improvement in the overall survival in clinical appliance.New biomarkers and combination therapy may improve the therapeutic effect of GBM patients.There is growing evidence supported a significant role of Wnt signaling pathway in angiogenesis both physiological and pathological conditions.This pathway affects cell proliferation,survival,differentiation,migration and apoptosis,and regulates VEGF expression.FRAT1 is a positive regulator of Wnt/β-catenin pathway,accumulating reports demonstrated that FRAT1 was highly upregulated and has a significant association with growth,invasion,migration and poor prognosis in human glioblastoma,as well as angiogenesis.However,the role of FRAT1 for angiogenesis in glioblastoma is still obscure.The objective of this study was to elucidate the relationship between FRAT1 and angiogenesis via VEGF,and determine whether downregulation of FRAT1 expression inhibits angiogenesis in human glioblastoma cells,thereby providing new research basis and consideration for treatment of GBM.Methods:U251 cells were divided into four groups,small interference RNA(siRNA)was used to transfect the first three groups(defined as siFRAT1-1,siFRAT1-2 and siFRAT1-3),and the last group of U251 cells wasn’t subject to transfection and used as negative control(defined as siNC).Real-time PCR was used to assess the relative expression levels of FRAT1 mRNA,siNC-U251 cells and the group with the lowest FRAT1 expression was used to perform subsequent experiments.Western blot and real-time PCR techniques were processed to investigated the protein and mRNA expression levels of VEGF in U251 cells,and VEGF concentration in supernatants of U251 cells was measured by Enzyme-linked immunosorbent assay(ELISA).Finally,tube formation assay of endothelial cells was conducted to assess angiogenesis ability of human umbilical vein endothelial cells(HUVECs)which were cultured with supernatants collected from U251 cells.Results:(1)Cell transfection results showed: after transfected with siRNA for 48 h,the relative expression level of FRAT1 mRNA in siFRAT1-2 group was the lowest compared with siNC group using real-time PCR technique(P<0.01).(2)The expression level of VEGF showed: in two cell groups,the relative expression levels of VEGF protein and mRNA in siFRAT1 group were dramatically inhibited as compared to siNC group using Western blot and real-time PCR techniques,respectively(mRNA: P<0.01).Meanwhile,the concentration of VEGF in supernatants collected from siFRAT1 group was drastically reduced as compared to siNC group using ELISA technique(P<0.001).(3)Tube formation assay of endothelial cells showed: after cultured with supernatants collected from U251 cells for 6h,angiogenesis ability of HUVECs in siFRAT1 group was significantly lower than in siNC group(P<0.01).Conclusion:In human glioblastoma U251 cells,after the expression of FRAT1 downregulated by siRNA: 1.the expression levels of VEGF protein and mRNA were reduced;2.The concentration of VEGF in cell supernatants was decreased;3.the angiogenesis ability of HUVECs was inhibited.Collectively,FRAT1 regulates the expression of VEGF through Wnt/β-catenin signaling pathway,overexpression of FRAT1 may promote angiogenesis of human glioblastoma.In human glioblastoma patients,FRAT1 appears to be a potential biomarker of angiogenesis and therapeutic target.