NTNG1 Modulates Cisplatin Resistance In Epithelial Ovarian Cancer Cells Throuth The GAS6/AXL/Akt Pathway

BackgroundOvarian cancer is the most prevalent and lethal gynecologic malignancy.Cisplatin(DDP)-based chemotherapy is the standard strategy,and is effective at an initial time.However,the gradually increasing resistance to DDP leads to treatment failure.Increasing DNA repair and activating the survival pathways play key roles in DDP resistance.However,the mechanisms of DDP resistance are only partially understood.Therefore,it is important to understand the molecule mechanisms of DDP resistance.Here,bioinformatic analysis demonstrated a high level of NTNG1 in DDP resistant human ovarian cancer cells.NTNG1 mediates cell repulsion,attraction,and adhesion.The abnormal expression of NTNG1 is associated with the occurrence and recurrence of cancer.However,the role of NTNG1 in ovarian cancer remains unclear.The receptor tyrosine kinase AXL interacts with its ligand GAS6(growth arrest-specific 6),thereby promoting cancer cells progression,survival,and proliferation through activating the ERK or Akt pathway Recent data indicated that AXL played an important role in DDP resistance of ovarian cancer.However,the regulating mechanism was limitedly understood.Bioinformatic analyses showed that NTNG1 can interact with GAS6;these suggested that NTNG1 may correlate with GAS6/AXL/Akt pathway.The objective of this study was to explore the role of NTNG1 in DDP resistance of ovarian cancer.PART ONE THE EXPRESSION LEVEL OF NTNG1 IN OVARIAN CANCER TISSUESObjectiveTo detect the expression level of NTNG1 in epithelial ovarian cancer tissues,and then to analyzed the relationship between the NTNG1 level and clinicopathologic variable and prognostic.Methods1.The expression level of NTNG1 in DDP-resistant and DDP-sensitive cells was determined using a bioinformatic method.2.Paraffin-embedded tumor tissues and clinical data were collected.3.NTNG1 in cancer tissues was detected with an immunohistochemical assay.4.The NTNG1 level in DDP-resistant and DDP-sensitive tissues was elucidated.The relationship between NTNG1 level and survival time was analyzed.Results1.The expression level of NTNG1 in the DDP-resistant cell lines was higher than in DDP-sensitive ones,based on GSE45553 and GSE73935 datasets in GEO.2.The NTNG1 level in resistant ovarian cancer tissues was higher that in sensitive cancer tissues(p < 0.05).3.Patients with a high level of NTNG1 in cancer tissues had a shorter PFS/PFD(p < 0.05,p < 0.05).ConclusionThe expression level of NTNG1 in DDP-resistant ovarian cancer tissues was higher than that in sensitive tissues,A high level of NTNG1 related to DDP resistance,poorer therapeutic outcome,and shorter survival time.NTNG1 may promote the DDP resistance in ovarian cancer.PART TWO NTNG1 MODULATES CISPLATIN RESISTANCE IN OVARIAN CANCER CELLSObjectiveTo investigate the effects of overexpressing or silencing NTNG1 on DDP resistance in ovarian cancer cells and the molecular mechanisms of NTNG1.Methods1.The NTNG1 level in DDP-resistant and DDP-sensitive cells was determined.2.The effects of down-or up-regulating NTNG1 on apoptosis,survival and DNA repair of ovarian cancer cells was detected after DDP exposure.3.The effects of NTNG1 on activation of GAS6/AXL/Akt pathway was determined.Results1.The expression level of NTNG1 in SKOV3/DDP cells was higher than in SKOV3 cells.Therefore,SKOV3 was used for overexpression trials,and SKOV3/DDP was used for silence trials.2.Over-expressing NTNG1 increased the percentages of survival cells and decreased the percentages of apoptotic cells due to DDP in SKOV3 cells.However,silencing NTNG1 decreased the percentages of survival cells and increased the percentages of apoptotic cells in SKOV3/DDP cells.3.DDP induced the expression of ?-H2 A.X and RAD51.Overexpressing NTNG1 decreased the ?-H2 A.X level in SKOV3 cells after DDP exposure,while the RAD51 level was increased.Silencing NTNG1 increased the level of ?-H2 A.X and reduced the RAD51 level in SKOV3/DDP cells after DDP exposure.4.The phosphorylation of AXL,Akt were induced by DDP;and were further increased in SKOV3 cells after overexpressing NTNG1.The level of p-AXL and p-Akt was decreased in SKOV3/DDP cells after silencing NTNG1.COIP showed that NTNG1 can directly interacted with GAS6/AXL.ConclusionNTNG1 could directly bind to GAS6/AXL to regulate the phosphorylation of AXL and Akt,up-regulated expression of RAD51,enhanced DSB repair,and eventually resulted in DDP resistance.PART THREE NTNG1 PROMOTES CISPLATIN RESISTANCE OF OVARIAN CANCER IN VIVOObjectiveTo validate the effect of NTNG1 on DDP resistance of ovarian tumors in vivo.Methods1.NTNG1/sh NTNG1-transfected cells were injected into mice to form tumors,and then DDP was administrated.2.The volume and mass of tumors were compared.3.NTNG1 and RAD51 proteins in tumor tissues were immunohistochemically determined.Results1.In SKOV3-derived tumors,the volume/mass in group NTNG1+DDP was larger than that in group NC+DDP.In SKOV3/DDP-derived tumors,the volume/mass in group sh NTNG1+DDP was less than in group shNC+DDP.2.DDP could induce the expression of NTNG1 and RAD51 in SKOV3-and SKO3/DDP-derived tumors.In SKOV3 tumors,the RAD51 level was increased after overexpressing NTNG1;however,in SKOV3/DDP tumors,silencing NTNG1 decreased the level of RNA51.ConclusionNTNG1 modulated the capacity of DNA repair in vivo,thereby determining the action of DDP against ovarian tumors.