DDAH1 Mediates Gastric Cancer Cell Invasion And Metastasis Via WNT/β-catenin Signaling Pathway

Objective: To investigate whether Dimethylarginine dimethylaminohyrolase I(DDAH1)suppresses gastric cancer cell migration and invasion via down-regulating WNT/ β-catenin signaling.Methods: 1.The expression of DDAH1 in 7 gastric cancer cell lines were detected by Western blot method;DDAH1 over-expression and knockdown stable GC cell lines were established,including AGS and MGC803.Then the expression DDAH1 in over-expression and knockdown stable cells was validated through Western blot method;2.CCK8 and colony formation assays were used to detect the effect of DDAH1 on GC cell proliferation.Wound-healing and transwell assay were to detected the influence of DDAH1 on GC cell migration and invasion,then animal assay was implemented to identify the influence of DDAH1 on metastasis in vivo.3.We used RT-PCR and Western blot method to detect the influence of DDAH1 on epithelial-mesenchymal transition(EMT)related markers,such as E-cadherin,ZO-1,Vimentin,N-cadherin and snail.4.We used immunofluorescent staining,fractionation experiments and immunohistochemistry to detect how DDAH1 expression influence on β-catenin location in GC cells and tissues.In addition,DDAH1 knockdown stable cell and their negative control cell were transfected with β-catenin siRNA,then the change of EMT related protein expression,migration and invasion of GC cells were detected;5.Finally we investigate the effect of DDAH1 on Wnt1,GSK3β,p-GSK3β(Ser9),p-β-catenin(Ser33/37 / Thr41)and β-catenin expression by Western blot method,then we detected the effect of WNT activator on DDAH1 over-expression cell and WNT inhibitor on DDAH1 silence cell using transwell experiment.Results: 1.DDAH1 has no effect on AGS and MGC803 GC cells proliferation.2.DDAH1 suppresses AGS and MGC803 GC cells migration and invasion;3.DDAH1 inhibites GC cells EMT process;4.DDAH1 negatively regulates β-catenin in vitro and its expression is also reversely related with β-catenin in 150 GC tissues.shDDAH1 regulates EMT,migration and invasion of GC cells via β-catenin.5.DDAH1 negatively mediates wnt1 and down-regulates β-catenin via the axis of wnt1-GSK3β-β-catenin.In addition,DDAH1 inhibites GC cells EMT,migration and invasion through WNT/β-catenin signaling pathway.Conclusion: DDAH1 inhibits of GC cell invasion and metastasis by negatively regulating WNT/β-catenin signaling pathway.