Study Of The Effect On Cell Migration And Invasion Of Dickkopf 1 In Non-small Cell Lung Cancer

Background:Non-small cell lung cancer(NSCLC)accounts for 80–85% of all lung cancers,the leading cause of cancer-related deaths worldwide.Most patients have advanced or metastatic disease at the time of diagnosis,and poor clinical outcome is common.Although several approaches,such as radiotherapy,chemotherapy,and surgical intervention can improve the clinical symptoms,the prognosis of NSCLC is still poor.NSCLC progression and metastasis have been the primary causes for poor clinical outcomes of patients.Recently,multiple studies have shown that the expression of Dickkopf 1(DKK1)is closely related to the development of lung cancer.However,the specific mechanism how DKK1 functions still remains unclear.Objective:Investigate the role of DKK1 in NSCLC cell migration and invasion and explore the mechanism.Methods: DKK1 was knocked out by the lentivirus-mediated short hairpin RNA interference(sh RNA)approach in H1299 and 95 C NSCLC cell lines.Subsequently,the migration and invasion ability were assessed by wound healing and transwell assays.In addition,epithelial-mesenchymal transition(EMT)markers and ?-catenin were examined by Western blot.The signaling pathway downstream of DKK1 was characterized using the Wnt signaling pathway inhibitor,IWP2,and GSK3? inhibitor,Li Cl.Immunofluorescence analysis investigated the subcellular localization of ?-catenin.Results: 1.Considering that the green fluorescence protein(GFP)sequence was fused in the recombinant virus,we performed a fluorescence analysis to examine the transfection efficiency.The results revealed that >85% cells were successfully transfected with DKK1-sh RNA or non-silencing-sh RNA(NS-sh RNA).Western-blot results demonstrated that the expression of the DKK1 protein was dramatically decreased in H1299-DKK1-KD and 95C-DKK1-KD,further confirming the successful construction of cell models of DKK1 knockdown(DKK1-KD)and negative controls(NC).2.As the wound healing assay illustrated,knockdown of DKK1 decreased the wound closure area.The migration transwell assay showed that fewer cells migrated to the other side of chamber membrane in H1299-DKK1-KD cell group compared with H1299 and H1299-NC.The similar results were found in 95 C cell line.3.The cell invasion ability was explored using invasion transwell assay with Matrigel.The results showed that,compared with H1299 and H1299-NC,the number of cells that passed through Matrigel was lower in H1299-DKK1-KD.Similarly,fewer cells passed through Matrigel and invaded to the other side of chamber membrane in 95C-DKK1-KD cell group.4.Western-blot analysis revealed that the downregulation of DKK1 decreased the expression of ZEB1 and Snail as compared to the control groups both in H1299 and 95 C cell lines.The result indicated that DKK1 induced the occurence of EMT.5.Western-blot analysis revealed that the downregulation of DKK1 decreased the expression of ?-catenin,in the meanwhile,promoted the phosphorylation of ?-catenin.6.IWP2,the inhibitor of Wnt/?-catenin signaling pathway,was applied to inhibit the expression of ?-catenin.Then we examined the influence of IWP2 on EMT,migration and invasion.The results showed that,in H1299 and H1299-NC cell groups,the expression of EMT-related proteins,cell migration and invasion ability were decreased dramatically after being treated with IWP2.Consistently,the expression of EMT-related proteins,cell migration and invasion ability in 95 C and 95C-NC groups were also inhibited after being treated with IWP2.7.Li Cl,the inhibitor of GSK3?,was applied to inhibit the phosphorylation of ?-catenin,rescuing the expression of ?-catenin.After treatment with Li Cl,the expression of ZEB1 and Snail increased as compared to the corresponding cells without Li Cl,especially in the DKK1-KD cell group.Migration and invasion results demonstrated that after using Li Cl,more cells migrated and invaded to the other side of chamber membrane,especially in the DKK1-KD group.These findings reconfirmed the key role of DKK1 in regulating the expression of ?-catenin by inhibiting its phosphorylation.8.Cell immunofluorescence analysis was applied to determine the effect of DKK1 on subcellular localization of ?-catenin,and found that ?-catenin localized in the cytoplasm,nucleus,and cytomembrane.Furthermore,we also observed that nuclear ?-catenin was significantly decreased in DKK1-KD groups than in NC groups.Conclusions: 1.Knockdown of DKK1 decreases the expression of EMT-related proteins,and also inhibits the cell migration and invasion abilities.2.DKK1 is positively correlated to the expression of ?-catenin,and downregulation of DKK1 promotes the phosphorylation of ?-catenin.3.?-catenin plays a vital role in DKK1-induced EMT,cell migration and invasion.4.DKK1 regulates the expression of ?-catenin by inhibiting its phosphorylation.5.DKK1 inhibits phosphorylation of ?-catenin,resulting in the stabilization of nuclear ?-catenin,realizing the control on EMT,migration and invasion afterwards.