Knockdown Of YAP Inhibits Growth In Laryngeal Cancer Via Epithelial-Mesenchymal Transition And The Wnt/?-catenin Pathway

Objective: To explore the proliferation,migration and invasion of Hep-2after YAP targeted-knockdowned via EMT program and Wnt/?-catenin signaling pathway.Methods: First,we built knockdowned YAP in Hep-2 cell line by shRNA as YAP-shRNA group.Meanwhile,we also built control(Hep-2 with no treatment),YAP-NC group(Hep-2 with non-nonspecific shRNA treatment)and YAP-shRNA+YAP-cDNA group(cDNA overexpressed after YAP knockdowned).To observe the proliferation of Hep-2 cells by CCK-8 and colony formation assay,then discussing migration and invasion in four groups via wound healing assay and transwell assay.Additionally,we examined cell cycle by flow cytometry to verify whether the expression of YAP was related to cell apoptosis.At last,we detecting the expression level of mRNA and protein by RT-PCR and western blot.Results: Downregulation of YAP lead to a obvious decrease in proliferation of Hep-2cells(P<0.01)than other groups.The scratch healing ability of Hep-2 cells was significantly decreased in YAP-shRNA group(P<0.01).The number of cells in YAP-shRNA passing through compartments was remarkable fewer than the control group,YAP-NC and YAP-shRNA+YAP-cDNA group(P<0.01).In addition,we found that downregulation of YAP increases the apoptosis and induces G2/M cell cycle arrest in Hep-2 cells.Moreover,expression of E-cadhein protein was upregulated after transfected by shRNA,whereas expression of vimentin,?-catenin and DKK1 protein was downregulated(P<0.01).Conclusion: The downregulation of YAP in Hep-2 inhibits proliferation,migration and invasion via supressing EMT program and Wnt/?-catenin signaling pathway.Objective: To explore the ability of tumourigenicity and metastasis in Hep-2 cells after YAP targeted-knockdowned.Methods: To confirm the effect of YAP in Hep-2,we generated a luciferase-labelled Hep-2 cell line(Luc-control),a stable YAP knockdown Hep-2 cell line(Luc-YAP-sh RNA)and a scrambled sh RNA Hep-2 cell line(Luc-YAP-NC).18BALB/c mice were randomly divided into 3 groups(n=6).We injected different groups with cell suspensions subcutaneously and then measured each tumour every three days.In order to detect metastasis ability of Hep-2 cells,we chose 18 BALB/c mice to randomly divided into 3 groups(n=6).Then cells of three groups were injected into the tail vein of BALB/c nude mice.Then we examined the expression level of YAP,Bcl-2and KI-67 by immunohistochemistry assay.Results: After 21 days,the size of tumours were smaller than those in the Luc-control and Luc-YAP-NC groups,suggesting that the Luc-control and Luc-YAP-NC groups grew more aggressively(P<0.01).Meanwhile,we also found fewer metastatic tumors in the pulmonary tissues of the knockdown group(P<0.01).Moreover,IHC assay exposed that the Luc-YAP-sh RNA group exhibited less YAP,Bcl-2 and KI-67 staining,indicating that YAP-downregulated suppressed the expression in vivo.Conclusion: These results demonstrated that targeted YAP knockdown repressed the tumour growth and metastasis of Hep-2 cells in vivo.