IKBKE Promoted The Expression Of YAP1 To Regulate The Malignant Progression Of Glioma By Phosphorylating Amotl2

In the latest study,IKBKE(inhibitor of nuclear factor kappa-b kinase subunit epsilon),as a oncogenic gene,is overexpressed in breast cancer,prostate cancer,non-small cell lung cancer,endometrial cancer and glioma.Our previous study confirmed that IKBKE gene was abnormally highly expressed in glioblastoma of human brain,and the expression level was negatively correlated with the prognosis of glioblastoma patients,and positively correlated with the malignant degree of human glioblastoma.Silencing the expression of IKBKE gene significantly inhibited the proliferation,invasion and migration of human glioblastoma cells.In this study,IKBKE was found to be highly expressed in glioma through CGGA,TCGA and GEO public databases,and was negatively correlated with the prognosis of patients,as to glioma malignancy,it was with positive correlation.In addition,we found that IKBKE could directly phosphorylate the Amotl2 serine 166 site by SILAC labeled phosphorylation quantitative proteomics.Phos-tag phosphorylation adhesive and protein ubiquitination experiments further confirmed that IKBKE directly phosphorylated Amotl2 and promoted its ubiquitination degradation.In addition,Amotl2 interacts with YAP1 in gliomas,inhibits YAP1 nuclear translocation and promotes its degradation.Our experiments confirmed that IKBKE could remove the inhibition of Amotl2 on YAP1,promote the expression of YAP1 and nuclear translocation,thus participate in the regulation of Hippo pathway.In vivo experiments,a model of intracranial glioma was established in nude mice,and it was found that the silencing of IKBKE gene significantly inhibited the growth of intracranial tumors and extended the survival period of tumour-bearing mice.The above results indicate that IKBKE can participate in the malignant progression of glioma through the IKBKE↑-Amotl2↓-YAP1↑ signaling pathway and provides a new research direction for targeted therapy of glioma.MethodsWe analyzed the public databases CGGA,TCGA and GEO,and determined the relationship between IKBKE and the malignant degree of glioma and the prognosis of glioma patients through the analysis of the expression of IKBKE.We collected 149 clinical samples and performed immunohistochemical staining to determine the relationship between the expression level of IKBKE and the grade of glioma.We selected primary cell G4 cultured from glioblastoma tissue and the classic glioblastoma cell line U87 MG for follow-up research.We constructed a sh IKBKE lentivirus that silenced the IKBKE gene and down-regulated the expression of the IKBKE gene in glioma cells.Then,plate cloning,CCK-8 and Ed U cell proliferation experiments were used to detect the changes in the proliferation of glioblastoma cells after silencing the IKBKE gene.In the study of molecular mechanism,we first silenced the IKBKE gene in U87 MG for quantitative proteomics detection of SILAC labeled phosphorylation,and found that the s166 site of Amotl2 was the potential phosphorylation site of IKBKE.Then we transfected wild-type IKBKE or/and wild-type Amotl2 plasmids into 293 T cells and detected the direct binding of the two exogenous proteins by protein immunoprecipitation.Next,we further detected the direct binding of endogenous IKBKE and Amotl2 in glioma cells by protein immunoprecipitation.After that,the wild-type IKBKE or/and wild-type Amotl2 plasmids were transfected into 293 T cells,and the effect of IKBKE phosphorylation on Amotl2 was detected by phos-tag phosphorylation adhesive.To further determine the relationship between IKBKE phosphorylation of Amotl2,the wild-type Amotl2 plasmid was transfected into 293 T cells,and then the IKBKE active and inactivated plasmids were transfected,respectively.The change of p-ser expression in Amotl2 was detected by in vivo kinase assay.Correlation analysis was conducted according to IKBKE and Amotl2 expressions in three public databases.After silencing or overexpression of IKBKE gene in glioma cell lines,the changes of Amotl2 gene were detected by western blot and q RT-PCR.MG132 and CHX reagent were used to treat cells in each group,and the trend of Amotl2 protein was detected by immunoprecipitation assay.Finally,IKBKE was transfected with wild-type IKBKE,wild-type Amotl2,wild-type Ub,mutant k48-ub and mutant k63-ub plasmids in 293 T cells to determine the pathway for regulating the ubiquitination degradation of Amotl2 through protein ubiquitination experiments.The sh Amotl2 lentivirus of Amotl2 was constructed to knock down the Amotl2 gene,and the changes of Amotl2,YAP1 and p-yap1(s172)were detected by western blot assay.Then the changes of Amotl2,p-yap1(s172),YAP1,CTGF and CYR61,and the changes of YAP1 intracytoplasmic proteins after IKBKE gene silencing or overexpression were detected by western blotting.The changes of YAP1 intracytoplasmic protein after IKBKE gene silencing were determined by immunofluorescence method.In the reply experiment,after simultaneous silencing or overexpression of IKBKE and Amotl2 genes,changes in YAP1 were detected by western blotting,and cell proliferation was detected by Edu.In the experiment of intracranial tumor formation in nude mice,the primary cell G4 transfected with luciferase virus was used to establish the model of intracranial tumors in nude mice with the help of stereotaxic instrument,and the size of intracranial tumors in nude mice was monitored using live imaging system every 7 days Monitor the weight and survival of tumor-bearing mice,and finally take brain slices,and then detect the changes of indicators by immunohistochemical staining.Results1.IKBKE gene is abnormally highly expressed in glioma,which is positively correlated with the malignant degree of glioma,and the higher the expression level,the worse the prognosis of patients;2.Silencing IKBKE can significantly inhibit the proliferation of glioma cells;3.IKBKE can directly bind to Amotl2,and phosphorylate Amotl2;4.IKBKE can promote the ubiquitination degradation of Amotl2 in the lysosomal pathway;5.Silencing IKBKE can significantly increase the expression of Amotl2 protein,but does not affect the expression of Amotl2 m RNA.Overexpression of IKBKE inhibits the expression of Amotl2 protein,and also does not affect the expression of Amotl2 m RNA;6.IKBKE can affect the Hippo pathway by regulating the expression of Amotl2.Silencing IKBKE can inhibit the expression of YAP1 and its downstream target genes CTGF and CYR61.Overexpression of IKBKE can increase the expression of YAP1 and its downstream target genes CTGF and CYR617.Silencing IKBKE can inhibit the transport of YAP1 from the cytoplasm to the nucleus;8.In glioma cells,Amotl2 significantly inhibits the impact of IKBKE on the Hippo pathway;9.Silencing IKBKE can significantly inhibit the growth of intracranial tumors in nude mice and prolong the survival time of tumor-bearing mice.Conclusion1.The abnormal high expression of IKBKE gene in glioma was positively correlated with the degree of glioma malignancy and negatively correlated with the prognosis of patients.2.The silencing of IKBKE gene can significantly inhibit the proliferation of human glioblastoma cells in vitro and in vivo,and significantly prolong the survival time of tumor-bearing mice;3.IKBKE directly phosphorylates the Amotl2;4.IKBKE can promote the ubiquitination of Amotl2 and inhibit the expression of Amotl2;5.IKBKE can relieve the inhibitory effect of Amotl2 on YAP1,promote YAP1 expression and nuclear transport,and thus participate in the regulation of malignant progression of glioma by Hippo pathway.In summary,IKBKE can participate in the malignant progression of glioma through the IKBKE↑-Amotl2↓-YAP1↑ signaling pathway.