MicroRNA-765 Sensitizes Osteosarcoma To Chemotherapy Through Regulating APE1 Expression

BackgroundOsteosarcoma(OS)is one of the most common bone tumor in child and adolescent and chemotherapy is a major treatment.In Osteosarcoma,tumorigenesis is positively rel ated with tumor development and progression,while negatively related with chemotherapeutic sensitivity and prognosis.One important reason for poor treatment outcomes is chemoresistance.Thus,an efficient method to prolong survival of OS patients is sens itizing tumor cells to chemotherapy.Apurimic/Apyrimidinic endonuclease 1(APE1)is a key regulation protein involved in chemosensitivity.In OS tissues,APE1 showed an elevated expression,it reducing OS chemosensitivity and resulting in poor survival thr ough increasing tumorigenesis.Mi RNAs have both anti-cancer and pro-cancer function via regulating target gene expression or activity.Through miRNA high-throughput chip technology we detected the miRNA profile in both normal and APE1 knockdown HOS cells,and discovered miR-765 showed a significantly up-regulation(> 5).Bioinformatics prediction suggested that miR-765 contained a binding site of APE1 3'-UTR.In this research,we found miR-765 could bind APE1 and inhibit its expression and DNA repair activity,combining cisplatin with miR-765 may also significantly inhibiting tumorigenesis expression such as VEGF,FGF2,TGF-? and MVD,result in an improvement of chemosensitivity.In mouse model,miR-765 could significantly inhibit tumor growth and promote cell apoptosis.Clinical research also revealed that patients with positive miR-765 showed a longer survival than those negative ones,while a reverse trend was observed for APE1 expression.In summary,our data indicated miR-765 could inhibit the expression and function of APE1 and enhance the anti-angiogenic effect of cisplatin,increase chemosensitivity in osteosarcoma.Our results would help on the identification of novel biomarker and treatment target in osteosarcoma research.Objectives:1.To identify APE1 as a down-stream target of miR-765 and its specific binding site;and miR-765 increasing chemosensitivity of osteosarcoma through inhibiting DNA repair ability of APE1.2.To confirm that miR-765 by inhibiting APE1,enhanced cisplatin in osteosarcoma cells in the anti-angiogenic effect,improve the treatment sensitivity.Methods1.Collecting clinical data and tissue samples of osteosarcoma patients(final sample size 43)2.Performing bioinformatics analysis,vector contruction,dual luciferase report genesystem and Western blot to identify that APE1 was target of miR-765.3.Conducting WB,CCK-8 and ?-H2 AX to confirm that miR-765 could inhibit APE1 expression and reduce its DNA repair ability,and finally enhance the chemosensitivity of OS cell.4.Using RT-PCR,Western blot to confirm miR-765 inhibiting APE1,VEGF,FGF2,TGF? expression via combining cisplatin;tranwell analysis and tubular forming experiments to detect the influence on tumorigenesis by combining of miR-765 and cisplatin.5.Using animal model to detect xenograft growth,and IHC to detect APE1,VEGF,FGF2,TGF?,CD34 expression in xenograft tissue.6.Conducting IHC and ISH to analyze the expression of APE1 and miR-765,evaluate their relationship and the influence on prognosis.Resutls1.MiR-765 bound APE1 in osteosarcoma cells,and negatively associates with APE1 expression.2.MiR-765 inhibited the DNA repair function of APE1,and sensitizes OS cells to cisplatin.3.MiR-765 significantly influenced tumorigenesis in osteosarcoma by reducing tumorigenesis factors expression and enhances the anti-angiogenic effect of cisplatin.4.Mice model revealed that miR-765 had a growth inhibition in xenograft.5.Clinical data suggested patients with positive miR-765 expression obtained longer survival time than those with negative one,while patients with high APE1 level showed a poor prognosis than those with low APE1.Conclusion1.We discovered that miR-765 could bind 3'-UTR of APE1,and suppress APE1 expression in vivo and vitro,regulate DNA repair in tumor cells,and inhibit tumorigenesis by combining with cisplatin and sensitize osteosarcoma to chemotherapy.This mechanism research on miRNAs regulating APE1 provided new direction for developing novel APE1 inhibitor.2.For osteosarcoma patients,miR-765 positive meant longer survival time and better prognosis,thus miR-765 may be a good prognostic factor.