IKBKE Regulates Cell Proliferation And Epithelial–mesenchymal Transition Of Malignant Glioma Via The Hippo Pathway

objectiveIn this article,we demonstrated that IKBKE is overexpressed in several glioma cell lines and can promote glioma cell proliferation,migration and invasion.What's more,we verified IKBKE promotes EMT through enhancing the expressions of YAP1 and TEAD2.we verified that silencing IKBKE can inhibit glioblastoma progression in vitro and in vivo.Methodswe selected U87-MG and LN-229 as representatively investigative cells.We used the CCK-8 assay and cell clone formation experiment to evaluate the different proliferation abilities of U87-MG and LN-229 in knockdown IKBKE and scrambled control cells.To test whether the downregulation of IKBKE in U87-MG and LN-229 cells affected their migration abilities,we adopted the wound healing assay.Meanwhile,a transwell assay was applied to assess cell invasion ability.To further elucidate the detailed mechanism,we assessed the protein level changes of MMP2 and MMP9 by real-time RT-PCR and western blot.we investigated whether IKBKE expression level impacts EMT markers,real-time RT-PCR,western blot and immunofluorescence techniques were used for analysis.What's more,we overexpressed IKBKE and then testified YAP1,TEAD2,and EMT markers expression in malignant glioma cells.We investigated detailed effects between IKBKE and YAP1,TEAD2.To further explore whether the Hippo pathway could influence epithelial-mesenchymal transition(EMT)in glioma cell lines,we first designed siRNA to respectively downregulate the expression of YAP1 and TEAD2.After silencing YAP1,we tested the expression level of E-cadherin and vimentin using western blot and the mRNA expression according to real-time RT-PCR.Furthermore,we discovered that the expression of N-cadherin,?-catenin,and vimentin adopted western blot analysis and the mRNA levels found by real-time RT-PCR after knocking down TEAD2 expression.Additionally,we respectively overexpress YAP1 and TEAD2 in glioblastoma cell lines transfected with IKBKE-shRNA,and we tested the expression of EMT markers is the same as that silencing respectively YAP1 and TEAD2 experiment.We further investigated U87-MG tumourigenesis in vivo.First,the nude mice were subcutaneously inoculated with scrambled vector U87-MG cells and IKBKE-knocked down U87-MG cells.Then,we respectively implanted scrambled/luciferase lentivirus and IKBKE-shRNA/luciferase lentivirus U87-MG cells into nude mouse cerebrums.Furthermore,we stained the intracranial tumours with IKBKE,YAP1,MMP-2,MMP-9,E-cadherin,N-cadherin,?-catenin,vimentin,and snail through the immunohistochemistry experiment.ResultsThe CCK-8 assay showed that U87-MG and LN-229 proliferation capacities were significantly impaired as time passed after knockdown IKBKE.What's more,cells treated with IKBKE-shRNA exhibited decreased clone formation and smaller clone diameter compared with scrambled group.The wound healing assay demonstrated that silencing IKBKE inhibited cells' wound healing.The transwell experimental results demonstrated that the average number of cells with IKBKE-shRNA across chambers was decreased significantly.Significant decreases of MMP2 and MMP9 in mRNA and protein level after knocking down IKBKE.The epithelial marker E-cadherin level increased,while mesenchymal markers N-cadherin,vimentin,Snail,Slug and twist levels decreased in IKBKE-silenced tumour cells.Meanwhile,the expression levels of YAP1 and TEAD2 obviously decreased after silencing IKBKE.After overexpressing IKBKE,the expression levels of EMT markers,YAP1 and TEAD2 is contrary to the silencing IKBKE experimental results.While YAP1 and TEAD2 mRNA levels remained unchanged after knocking down IKBKE and overexpressing IKBKE.We certified that IKBKE directly interacted with YAP1 and TEAD2.After silencing YAP1 in U87-MG and LN-229,the expression level of vimentin was decreased and the expression level of E-cadherin was increased.Furthermore,we discovered that the expression of N-cadherin,?-catenin,and vimentin was decreased after knocking down TEAD2 expression.The E-cadherin expression was first increased after transfection with IKBKE-shRNA and then decreased as YAP1 was overexpressed.However,the changes of vimentin expression was contrary to the tendency of E-cadherin expression.Similarly,the expression levels of N-cadherin,?-catenin and vimentin were recovered after overexpressing TEAD2 in glioblastoma cell lines transfected with IKBKE-shRNA.Tumour size,growth and weight from mice infected with scrambled lentivirus obviously increases over the tumours from mice transfected with IKBKE-shRNA.Intracranial tumours transfected with IKBKE-shRNA were significantly smaller.Likewise,IKBKE downregulation was associated with a longer survival rate and a decreased loss of weight in mice.Immunohistochemistry showed increased expression of E-cadherin and decreased expressions of MMP-2,MMP-9,N-cadherin,?-catenin,vimentin and snail in the tumours transfected with IKBKE-shRNA.ConclusionSilencing IKBKE inhibits malignant glioma cells proliferation,migration and invasion.IKBKE promotes epithelial-mesenchymal transition(EMT),and IKBKE could induce increase expression of YAP1 and TEAD2 through inhibits the Hippo pathway.Furthermore,YAP1 and TEAD2 also promotes EMT.While IKBKE directly interacts with YAP1 and TEAD2.As a result of all this,IKBKE promotes epithelial-mesenchymal transition(EMT)through effects on the Hippo pathway.Knockdown of IKBKE inhibits malignant glioma formation in mouse subcutaneous and intracranial models.