The Function And Molecular Mechanism Of DEC2 In Regulating GC Progression

BackgroundGastric cancer(GC)is one of the most common malignancies and it is regarded as the second leading cause of cancer-related deaths worldwide.In recent years,according to the improvements in endoscopic detection,mortality of gastric cancer is decreasing.However,early diagnosis and effective treatment of GC remain challenging.It is therefore essential to further uncover the underlying molecular mechanisms of GC initiation and metastasis,in order to develop novel therapeutic approaches.Differentiated embryonic chondrocyte expressed gene 2(DEC2)belongs to basic helix-loop-helix(bHLH)transcription factor family,which is closely associated with developmental events,including lineage commitment,cellular differentiation and regulation of molecular clock.Importantly,recent studies indicate that DEC2 is a novel molecular marker of progression in various cancers,regulating apoptosis and epithelial mesenchymal transition progression.DEC2 may act as a tumor suppressor by inhibiting cell proliferation and metastasis in breast cancer;in addition,through competing with SP1 for the SP1 binding site of the TWIST1 promoter,DEC2 impaired epithelial-mesenchymal transition(EMT)-associated metastasis of human endometrial cancers.In contrast,some reports declared DEC2 can increase proliferation of breast cancer and invasiveness of osteosarcomas.Because of the controversial function of DEC2 in cancer malignancy,it is quite necessary to investigate whether DEC2 is involved in the initiation and metastasis of GC.AimsTo investigate the role of DEC2 in GC progression,we examined the expression of DEC2 in GC tumor tissues and adjacent non-tumor tissues by immunohistochemical(IHC)staining,and analyzed the correlations of DEC2 expression with clinicopathological characteristics and overall survival of GC patients.By manipulating DEC2 expression in GC cells,we determined the role of DEC2 in regulating cell proliferation and epithelial-mesenchymal transition(EMT)in vitro,as well as tumor growth and metastasis in vivo,and clarified the potential mechanisms underlying tumor-suppressor activity by DEC2 in GC.Methods1.Samples of paraffin-embedded GC tissues sections and clinicopathological features were collected to examine the expression of DEC2 and E-cadherin,and evaluated the correlation between DEC2 and E-cadherin and clinicopathological characteristics.Survival curve was drawn using the Kaplan-Meier method and compared by means of the Log-rank test.2.qPCR and Western blot were used to tested the expression levels of DEC2 in normal gastric cell line(GES-1)and different gastric cancer cell lines(HGC27,MGC803,BGC823,MKN-45).DEC2 vector was formed using human full length DEC2 cDNA linked with the GV166 vector to induce DEC2-overexpression in MGC803 and MKN-45 cells.Lentivirus vectors produced by 293T cells were infected into MGC803 and MKN-45 cells to establish stable transfection cell lines.Human DEC2 shRNA linked with pHAV3.1-DEC2shRNA-tGFP vector was used to induce DEC2-silence in HGC27 cells.Cells transfected with pHAV3.1-NCshRNA-tGFP vector served as negative control.3.CCK8 and EdU assays were used to measure the influence of DEC2 on cell proliferation in vitro;Annexin V-FITC/PI assay was tested by FACS to determine the effect of DEC2 on cell apoptosis in vitro;wound healing and Transwell migration and invasion assays were employed to evaluate the effect of DEC2 on cell motility and invasiveness in vitro.4.Subcutaneous tumor formation and tail vein injection model was used to measure the effect of DEC2 on tumorigenicity and metastasis in vivo.Stably transfected cell lines DEC2/MKN-45,DEC2-sh/HGC27 and their corresponding control cells were injected into right flank and the tail vein of nude mice,respectively.Tumor size,tumor weight and metastatic tumor nodes were measured.5.The effect of DEC2 on the morphology of gastric cancer cells was observed under microscope;Western blot and immunofluorescence staining assays were used to examine the expression levels of biomarkers for EMT after manipulating DEC2 expression.6.Western blot assay was used to detect the phosphorylation of ERK and NF-?B,after manipulating DEC2 expression.ERK inhibitor U0126 was applied to corroborate the effect of ERK/NF-?B pathway in DEC2-mediated tumor malignant behavior.Results1.Expression of DEC2 is significantly downregulated in GC and associated with poor prognosis of GCIHC staining showed DEC2-high expression tissues accounted for 67.35%and 22.45%in adjacent normal tissues and tumor tissues,respectively.DEC2 expression was significantly correlated with tumor invasion(p=0.001),lymph node metastasis(p=0.033),and TNM stage(p=0.001);the lower DEC2 expression was correlated with decreased E-cadherin expression(r=0.563,p<0.001).Kaplan-Meier analysis showed that the overall survival of patients with DEC2+/E-cadherin+ expression was much higher than that of group with DEC2-/E-cadherin-expression(p=0.003).2.DEC2 inhibits GC cell proliferation and migration and invasion in vitroTo determine the functional role of DEC2 in GC cells,we examined DEC2 expression in normal gastric cell line(GES-1)and GC cell lines(HGC27,MGC803,BGC823,MKN-45).MGC803 and MKN-45 cells with endogenous low DEC2 expression were selected to be transfected with DEC2 vector,leading to significantly up-regulation of DEC2.Up-regulation of DEC2 inhibited the proliferation,migration and invasion of MGC803 and MKN-45 cells,but promoted apoptosis in these cells.Next,we silenced the expression of DEC2 in HGC27 cells by shRNA.Silence of DEC2 promoted cell proliferation,migration and invasion of HGC27 cells,but impaired apoptosis in these cells.3.DEC2 suppresses GC tumor growth and metastasis in vivoTo address whether DEC2 inhibits tumor proliferation and metastasis in vivo,Stably transfected cell lines DEC2/MKN-45,DEC2-sh/HGC27 and their corresponding control cells were injected into right flank and the tail vein of nude mice,respectively.the tumor volumes and weights were lower in mice with injection of DEC2/MKN-45 cells,but tumor volumes and weights were higher in mice with injection of DEC2-sh/HGC27 cells at the designated time points.Hematoxylin-eosin(HE)staining showed that less metastatic foci were observed in the lungs of DEC2/MKN-45-treated mice and more metastatic foci were observed in the lungs of DEC2-sh/HGC27-treated mice,compared with their corresponding control cells.4.DEC2 inhibits EMT progress in GC cellsDEC2 impaired the mesenchymal morphology in stable transfected DEC2/MGC803 and DEC2/MKN-45 cells,while the mesenchymal morphology was not markedly altered in DEC2-sh/HGC27 cells.Immunofluorescence staining and Western blot results showed that DEC2 overexpression significantly reduced N-cadherin and Vimentin expression but increased E-cadherin expression in MGC803 and MKN-45 cells,whereas silence of DEC2 in HGC27 cells got the opposite results.5.DEC2 inhibits EMT-associated metastasis via inactivation of ERK/NF-?B pathwayWe detected the activation of ERK/NF-?B pathway and found that the phosphorylation of ERK and NF-?B were decreased in DEC2/MGC803 and DEC2/MKN-45 cells,but enhanced in DEC2-sh/HGC27 cells.Meanwhile,we detected the expression level of E-cadherin is higher in DEC2/MGC803 and DEC2/MKN-45 cells,but lower in DEC2-sh/HGC27 cells.U0126 treatment further inhibited the expression of NF-?B p65 and the EMT marker and the migration and invasion ability in stable transfected DEC2/MGC803 and DEC2/MKN-45 cells.In contrast,pretreatment of the cells with U0126 blocked activating effects of DEC2 knockdown on ERK/NF-?B,migration and EMT in DEC2-sh/HGC27 cells.Conclusion1.DEC2 expression is downregulated in GC tissues,and this downregulation appears to correlate with poor prognosis.Therefore,DED2 may be a useful biomarker to predict tumor progression.2.DEC2 inhibits GC cell proliferation and metastasis in vitro and in vivo,the inhibiting effects are attributed to a negative influence on EMT-associated metastasis through inactivation of ERK/NF-?B/EMT pathway in GC,indicating that targeting the DEC2/ERK/NF-?B/EMT axis may serve as a novel diagnostic and therapeutic approach for managing GC patients.