Study On The Role And Mechanism Of IKBKE In Mesenchymal Glioblastoma

Objective: Both NF-κB and STAT3 pathways are significantly activated in glioblastoma especially in mesenchymal GBM.IKBKE,as an upstream kinase in NF-κB pathway,may be involved in the carcinogenesis of mesenchymal GBM.In this study,we will clarify the expression pattern of IKBKE in glioma and its relationship with prognosis.We also aim to clarify the expression characteristics and role of IKBKE in mesenchymal GBM,and the specific mechanism of IKBKE involved in the crosstalk between NF-κB and STAT3 pathways in the immunosuppressive microenvironment of mesenchymal GBM.Finally,we will explore the therapeutic strategy of GBM targeted by IKBKE.Content and Methods: To analyze the expression pattern of IKBKE in different grades of glioma,newly diagnosed and recurrent gliomas as well as its relationship with patient’s prognosis,we integrated the whole gene transcriptome and related clinical data of TCGA,CGGA and GEO databases.Western blot and immunohistochemistry were used to detect the protein expression of IKBKE in different grades of gliomas and different regions of glioblastoma.The tumor mass was removed based on Brain MRI which showed the central necrosis,tumor border and edge enhancement areas of glioblastoma,and the expression of IKBKE in above areas was detected by immunohistochemistry of tissue microarray.TCGA and CGGA databases were employed to analyze the expression of IKBKE in each subtype,as well as the relationship between IKBKE and mesenchymal subtype markers.ROC curve was used to predict the specificity and sensitivity of IKBKE as a mesenchymal subtype marker.Based on TCGA database,the immunosuppressive characteristics of mesenchymal GBM were verified by bioinformatics analyses.GSEA and Pearson correlation analysis were used to verify the correlation between IKBKE and immune cell response and immune related signaling pathway in mesenchymal GBM.Bioinformatics analysis(Differential gene analysis,KEGG analysis,GSEA)was used to explore the IKBKErelated pathways in mesenchymal subtype.The co-expression and co-localization pattern of IKBKE,STAT3 and PD-L1 in GBM were verified by immunofluorescence staining.Western blot was used to verify the regulatory effect of IKBKE on STAT3 and PD-L1 protein.The protein interaction between IKBKE and STAT3 was confirmed by immunoprecipitation and immunofluorescence.The regulatory mechanism of transcription factor STAT3 on PD-L1 expression was verified by chromatin immunoprecipitation and luciferase reporter assay.The effect of inhibiting IKBKE on glioma growth and the patient’s prognosis were verified in vivo study.Results: IKBKE was increasingly expressed in gliomas as the pathological grades increase,and increased IKBKE expression was also detected after tumor recurrence,IKBKE expression was negatively correlated with the prognosis of patients with gliomas.The expression of IKBKE in the necrotic area of the center of glioblastoma is higher than that of the tumor border and tumor edge.The Ivy Glioblastoma Atlas database showed that IKBKE and mesenchymal subtype markers are mostly expressed around necrotic areas and microvascular proliferation areas,while proneural markers are more likely expressed at tumor margins and infiltrated areas.IKBKE has the highest expression in mesenchymal GBM,and it is positively correlated with mesenchymal markers and negatively correlated with proneural markers.IKBKE is a potential predictor for mesenchymal subtypes of glioblastoma.Compared with proneural GBM,mesenchymal GBM confers much more immunosuppressive signatures.Compared with other subtypes,immune checkpoints are enriched in mesenchymal GBM and have certain predictive value for mesenchymal subtypes.In the mesenchymal subtype GBM,IKBKE is related to immune response and immune-related signal pathways,and is positively related to immune checkpoints,inflammatory factors and immune regulatory molecules.A series of bioinformatics analysis showed that in mesenchymal GBM,IKBKE has the closest relationship with JAK/STAT pathway.In glioblastoma tissues,IKBKE,p STAT3,PD-L1 have a co-expression and co-localization pattern and the fluorescence intensity in the tumor center is significantly higher than that in the peritumor tissues.IKBKE can phosphorylate STAT3 to promote its nuclear translocation and bind to the PD-L1 promoter,thereby transcriptionally promoting the expression of PD-L1.Intracranial glioma mouse model showed that knocking down IKBKE can slow down the growth of tumors and prolong the survival time of mice.In addition,knocking down IKBKE inhibited the protein expression of downstream p STAT3 and PD-L1 in vivo.Conclusion: IKBKE is highly expressed in glioblastoma and confers poor clinical prognosis.IKBKE is enriched in mesenchymal GBM and could serve as potential marker for mesenchymal GBM.Mesenchymal GBM shows immunosuppressive features,IKBKE can regulate the immunosuppressive microenvironment of glioblastoma through the STAT3/PD-L1 pathway,and knocking down IKBKE can improve the survival and prognosis of glioma bearing mice.