IKBKE Promotes Glioblastoma Progression And Enhances Chemoresistance Through Activates The AKT/NF-KB Signaling Pathway

Purpose:This study confirmed that the expression of IKBKE protein was positively correlated with the pathological grade of glioma.Silencing IKBKE combined with temozolamide could significantly inhibit the growth of intracranial tumors in vivo.IKBKE is a potential target in the treatment of gliomas,which provides a basis for further treatment of glioma with IKBKE-specific inhibitors combined with temozolamide.Methods: Two classical glioma cell lines,LN18 and U118,were selected and then silenced or overexpressed with IKBKE,.The results were verified by WB and RT-PCR.The effects of timozolamine on the proliferation of glioma cells were studied by EDU and clone assay,and the effects of timozolamine on the migration and invasion of glioma cell line were detected by transwell.In the mechanism study,the protein changes of AKT/NF-KB signaling pathway were detected by immunoblotting after silencing or overexpressing IKBKE.In vivo experimental study The model of intracranial glioma in nude mice was established by stereotactic instrument,which was divided into three groups: NC group,NC temozolamine group,sh IKBKE group and sh IKBKE temozolamide group.The high expression of IKBKE glioma cell line LN18,was used to infect luciferase virus.The intracranial fluorescence signal of nude mice was detected by bioluminescence imager every 7 days,then the tumor size was compared according to the fluorescence signal value,and the index change was determined by immunohistochemical staining.Results: 1.LN18 and U118 were selected from multiple glioma cell lines;IKBKE sh RNA lentivirus was transfected into LK18 cell line with high expression of IKBKE,and IKBKE-plasmid lentivirus was transfected into U118 cell line with low expression of IKBKE,followed by control group Compared with the unintentional sequence group,the protein level and m RNA level of IKEKB in the IKBKE sh RNA lentivirus group were significantly decreased,and the expression of IKBKE protein and m RNA level in the IKBKE-plasmid lentivirus group were significantly increased,and the statistical difference was significant.2.In behavioral experiments,LN18/U118 and LN18 sh IKBKE/U118OE-IKBKE were divided into two groups.The experimental group was treated with temozolomide,and the experimental methods such as edu,colony formation and transwell showed that the glioma cell line was silenced after IKBKE.Temozolomide increased sensitivity;overexpression of IKBKE significantly enhanced the resistance of glioma cell lines to temozolomide.3.In the mechanism study,IKBKE can activate AKT and NF-KB pathways.Immunoblotting experiments show that silencing IKBKE can significantly reduce the expression of AKT,P65 and mgmt,and vice versa;silencing or overexpressing IKBKE in LN18 and U118 cell lines,respectively.Subsequent treatment with oxazolamide showed that IKBKE can reverse the changes in AKT/NF-KB signaling pathway proteins caused by temozolomide.4.In vivo experiments,the LN18 cell line was used to establish a nude mouse glioma model.The intracranial tumors of the nude mice with silencing IKBKE and temozolomide were significantly smaller than the other groups.The survival time and body weight were better than other groups,and the statistics were statistically significant.Conclusion: 1.In vitro experiments IKBKE can significantly enhance the tolerance of glioma cell lines LN18 and U118 to temozolomide,inhibiting its apoptosis;2.IKBKE can directly phosphorylate AKT/P65,activate AKT/NF-KB signaling pathway to participate in the development of glioma,and enhance the tolerance of glioma cells to temozolomide;3.IKBKE can participate in the regulation of mgmt expression through P65,thereby enhancing the resistance of glioma cells to temozolomide;4.Silencing IKBKE can enhance the therapeutic effect of temozolomide on intracranial glioma,lay a theoretical foundation for the further application of IKBKE specific inhibitors.