The Role And Mechanism Research Of CDK5RAP3 Regulating AKT In Gastric Cancer

Objective: In China,one of the most common neoplasm is gastric cancer,ranking the third in death that caused by cancer.In China,most patients had been in advanced stage when they sought help from doctors,so they failed to obtain satisfactory curative effect in the treatment.Until now,the mechanism of gastric cancer development is still unclear.CDK5RAP3 is the binding protein of the CDK5 activator protein p35 and P39.Our previous study demonstrated that CDK5RAP3 expression was downregulated in gastric cancer,which correlated to poor prognosis,and CDK5RAP3 could inhibit Wnt/?-catenin signaling through blocking GSK-3? phosphorylation at the point of Ser9.However,it remains unclear how CDK5RAP3 suppresses GSK-3? phosphorylation.It is reported that the phosphorylation of GSK-3? is mediated by several kinases including AKT,protein kinases A(PKA)and PKC,suppressing GSK-3? activity via phosphorylation of Serine 9.In this study,we found that CDK5RAP3 might form a complex with AKT,and the prognostic value of CDK5RAP3 in gastric cancer depended on AKT activity.We also discovered that CDK5RAP3 could block AKT phosphorylation inhibiting ?-catenin signaling,which could suppress the process of epithelial to mesenchymal transition(EMT)in gastric cancer.Methods:1.Using Western blotting to detect the expression of CDK5RAP3,AKT and related protein from the packaged lentivirus to build the gastric cancer cell lines AGS with stable overexpressed CDK5RAP3 and stable silenced CDK5RAP3 2.Using CO-IP to verified that CDK5RAP3,AKT and GSK-3? can Interact with each other.3.Using gastric cancer cell lines HGC-27 with stable silenced CDK5RAP3 and AKT-si RNA to discuss the mechanism of CDK5RAP3 regulating AKT dephosphorylation in Wnt signalling pathway.4.Through CCK8,cell counting and plate clone formation experiment,disscussing the effect between CDK5RAP3 regulating AKT dephosphorylation and gastric cancer cell proliferation.5.Through Transwell little room test,investigating the correlation between CDK5RAP3 regulating AKT dephosphorylation and the invasion and migration of gastric cancer cells.6.Using gastric cancer cell lines HGC-27,NUGC-3 and MGC-803 with stable silenced CDK5RAP3,investigating the impact of CDK5RAP3 regulating AKT dephosphorylation in EMT signalling pathway by Western blotting.7.Using IHC and Western Blotting to detect the expression of CDK5RAP3 and p-AKT(Ser473),and exploring the correlation between CDK5RAP3 regulating AKT dephosphorylation and clinical pathological characteristics or prognosis in gastric cancer patients.Results:1.In immune coprecipitation assay,CDK5RAP3 could combine AKT and GSK-3?.2.CDK5RAP3 inhibited phosphorylation of GSK-3?(Ser9)through dephosphorylation of AKT(Ser473)and then inhibited the expression of ?-catenin.3.CDK5RAP3 regulated the dephosphorylation of AKT and had effect on gastric cancer cell proliferation,invasion and migration.4.CDK5RAP3 inhibited the activation of EMT pathway by suppressing AKT phosphorylation.5.In gastric cancer tissues and adjacent tissues,high expression of CDK5RAP3 showed the correlation of low expression p-AKT(Ser473),while low expression of CDK5RAP3 showed the opposite.6.CDK5RAP3 and p-AKT(Ser473)had impact on the survival of patients with gastric cancer,and it was related to CDK5RAP3 regulating AKT dephosphorylation.Conclusion: 1.CDK5RAP3 can form a complex with AKT,and it can regulate Wnt signalling pathway by suppressing AKT phosphorylation.2.CDK5RAP3 reduces the ability of gastric cancer proliferation,invasion and migration by AKT dephosphorylation.3.CDK5RAP3 was significantly related to the prognosis of patients by suppressing AKT phosphorylation.