Apurinic/Apyrimidinic Endonuclease 1 Regulates Angiogenesis In A Transforming Growth Factor β-Dependent Manner In Human Osteosarcoma

Background:Angiogenesis plays a vital role in tumor growth and metastasis.It has been reported to be inversely correlated with overall survival of osteosarcoma patients.There were studies showed that apurinic/apyrimidinic endonuclease 1(APE1),a dually functional protein having both base excision repair(BER)and redox activities,is involved in tumor angiogenesis,although these mechanisms are obscure.Our previous study revealed that the expression of transforming growth factor β(TGFβ)was significantly reduced in APE1 silencing osteosarcoma cells.Transforming growth factor β promotes cancer metastasis through various mechanisms,immunosuppression,angiogenesis,and invasion are included.In the current study,we initially demonstrated that APE1,TGFβ,and microvessel density(MVD)have pairwise correlation in osteosarcoma tissue samples.In addition,TGFβ,tumor size,and MVD were inversely related to the prognosis of osteosarcoma patients.We found that knocking down APE1 in osteosarcoma cells resulted in downregulation of TGFβ.Besides,APE1-siRNA led to suppression of angiogenesis in vitro according to HUVECs in Transwell and Matrigel tube formation assays.APE1-siRNA also reduced secretory protein level of TGFp of culture medium supernatant,and then resulted in decreased phosphorylation of Smad3 of HUVECs.Moreover,exogenous purified TGFβ protein reversed the reduced angiogenic capacity.In a mouse xenograft model,siRNA-mediated knocking down of APE1 downregulated TGFβ expression,tumor size,and MVD.Collectively,current evidences indicate that APE]regulates angiogenesis in osteosarcoma through a TGF(3-dependent signaling pathway,suggesting a novel target for anti-angiogenesis therapy in human osteosarcoma.Objectives:1.To reveal the expression of APE1,TGFP,CD34 in osteosarcoma tissues and their relationship with prognosis,as well as the correlation between APE]and TGFβ.2.To confirm the effect of APE1 and TGFβ in angiogenesis of osteosarcoma.Methods:1.Clinical data and tissue samples were collected from 80 osteosarcoma patients with confirmed pathological diagnosis.2.Immunohistochemistry were carried out to investigate the expression of APE1,TGFβ,MVD in human osteosarcoma.3.APE1-siRNA transfection,Western Blot and RT-PCR were used to test the expression of APE1,TGFβ in protein level and mRNA level of osteosarcoma cells,as well as protein level of Smad3/pSmad3 in HUVECs.4.Enzyme-linked immunosorbent assay(ELISA),Transwell and Tube Formation assays were performed to evaluate the effect of APE1 and TGFβ on the capability of angiogenesis.5.Data was analysed using SPSS 20.0(IBM,Armonk,NY,USA).The χ2-test or Fisher’s exact probability were carried out for count data.Student’s t-test or the Kruskal-Wallis test was conducted for measurement data.The Kaplan-Meier survival test was used to compare the overall survival rate between TGFβ-positive and TGFβ-negative groups.Spearman’s rank correlation coefficient was performed to evaluate the association between MVD,APE 1,and TGFβ expression in osteosarcoma patients.Results:1.High expression of APE1 and TGFβ correlated with high MVD in osteosarcoma tissues.2.Patients with high expression of APE1,TGFβ and MVD showed poorer overall survival(OS)compared with those of low expression of APE1,TGFβ and MVD.3.TGF-β,tumore size and MVD were important prognostic factors for osteosarcoma.4.APE1-siRNA decreased the protein level and mRNA level of TGFβ,as well as the secretion of TGFβ in the supernatant.5.Lower TGFβ resulted in decreased phosphorylation level of Smad3 in HUVECs.Conclusions:1.The expression of APE1,TGFβ,MVD was positively correlated to each other in osteosarcoma.2.Expression of APE1,TGFβ and MVD were negatively correlated with OS of osteosarcoma patients.3.TGFβ,tumor size,and MVD were inversely related to the prognosis of osteosarcoma,and the ran k of effectiveness decreased.4.APE1-siRNA regulates expression of TGFβ positively in both protein level and mRNA level in human osteosarcoma.5.APE1-siRNA inhibits the capability of angiogenesis through the TGFβ/Smad3 signaling pathway in human osteosarcoma.