IKBKE Promotes The ZEB2-mediated EMT Process By Phosphorylating HMGA1 In GBM

IKBKE(Inhibitor of nuclear factor Kappa-B Kinase subunit Epsilon)is a non-classical member of the IκB kinase family,which plays an important role in regulating inflammatory responses,immune cell activation and proliferation,and metabolic diseases.Recent studies have shown that IKBKE,as an important oncogenic protein in a variety of tumors,can promote the growth,proliferation,invasion and drug resistance of tumors and play an important regulatory role in the occurrence and development of malignant tumors.Studies have shown that HMGA1(High Mobility Group A1)is identified as a DNA-binding protein that functions as a cofactor involved in gene transcriptional regulation and is highly expressed in tumor cells.Many studies have shown that ZEB2(Zinc finger e-box Binding homeobox 2)plays an important role in epithelial mesenchymal transformation(EMT).We found that IKBKE can regulate HMGA1 protein expression at the post-translation level,and its distribution from cytoplasm to nucleus.HMGA1 can regulate ZEB2 expression level by combining with ZEB2 promoter regions at the transcription level.Further study indicates IKBKE has an effect on the proliferation,migration and invasion of glioma cells.These studies have shown that silencing IKBKE can inhibit the malignant progression of glioma cells,and IKBKE plays the role of oncogene to regulate the malignant progression of glioma cells through the IKBKE-HMGA1-ZEB2 axis,thus proving that IKBKE is a promising target for the treatment of malignant glioma cells.Methods1.The protein expression levels and m RNA expression levels of IKBKE,HMGA1 and ZEB2 were assessed by western blot assay(WB)and q RT-PCR in six kinds of glioma cell lines.2.The protein expression levels of IKBKE,HMGA1,ZEB2 and other EMT-related biomarker proteins were verified by WB after transfection of IKBKE-sh RNA and IKBKE over-expression plasmid in U87 and SNB19 cell lines,and the protein expression levels of HMGA1,ZEB2 and other EMT-related biomarker proteins were verified by WB after the transfection of HMGA1-sh RNA in U87 and SNB19 cell lines.3.The q RT-PCR was used to detect the m RNA expression of IKBKE,HMGA1 and ZEB2 when IKBKE was knocked down or overexpressed in U87 and SNB19 cell lines,and to prove the m RNA expression of HMGA1 and ZEB2 after transfection with HMGA1-sh RNAin U87 and SNB19 cell lines.4.CCK8 assay was used to detect the effect of IKBKE knockdown or HMGA1 knockdown on proliferation of glioma cells.5.The scratch experiment and the transwell experiment was used to detect the effect of IKBKE-sh RNA or HMGA1-sh RNA transfection on the migration and invasion ability of glioma cells.6.WB and immunofluorescence experiments were used to demonstrate the cytoplasm and nucleus distribution changes of HMGA1 in U87 and SNB19 cells after IKBKE was knocked down.7.Immunoprecipitation and WB were used to prove whether there was endogenous binding between IKBKE and HMGA1,and the change of HMGA1 ubiquitination level after IKBKE was knocked down.8.The protein sequence analysis was used to predict whether IKBKE could phosphorylate HMGA1,and WB combined with phos-tag phosphorylating gel were used to prove whether IKBKE could phosphorylate HMGA1.9.Chromatin co-immunoprecipitation(Ch IP)and luciferase assay were used to test whether ZEB2 promoter could be combined by HMGA1 to regulate its expressions.10.Stereotactic technique was used to establish a model of intracranial tumor in nude mice,and the survival time of nude mice was recorded.ICH proved the changes of IKBKE,HMGA1 and ZEB2 protein expression levels.Results1.Western blot assay(WB)and q RT-PCR showed that the protein and m RNA expression levels of IKBKE,HMGA1 and ZEB2 in U87 and SNB19 cell lines were higher than those in other cell lines.2.WB showed that the protein expression levels of IKBKE,HMGA1,ZEB2 and other EMT-related indicators decreased after IKBKE was knocked down,while the protein expression levels increased when IKBKE was overexpressed.The protein expression levels of HMGA1,ZEB2 and other EMT-related biomarker proteins decreased when HMGA1 was knocked down.3.The q RT-PCR showed that the m RNA expression of IKBKE and ZEB2 decreased after IKBKE was knocked down,while the m RNA expression of HMGA1 did not change significantly.Similarly,the m RNA expression of IKBKE and ZEB2 increased after the overexpression of IKBKE,while the m RNA expression of HMGA1 did not change significantly.The m RNA expression of HMGA1 and ZEB2 decreased when HMGA1 was knocked down.4.U87 and SNB19 cells were transfected with IKBKE-sh RNA and HMGA1-sh RNA to inhibit the proliferation of glioma cells.5.U87 and SNB19 cells were transfected with IKBKE-sh RNA and HMGA1-sh RNA,respectively,to inhibit the migration and invasion of glioma cells.6.After IKBKE was knocked down,the protein expression level of HMGA1 in cytoplasm increased in U87 and SNB19 cells,while the protein expression level in nucleus decreased.7.IKBKE can directly bind to HMGA1,and IKBKE knockdown can promote ubiquitination and degradation of HMGA1.After using the proteasome inhibitor(MG132),IKBKE protein inhibited the degradation process of HMGA1 protein.8.The protein sequence analysis showed that IKBKE phosphorylated HMGA1 at the site of Ser 36 and/or Ser 44,and it was proved that IKBKE phosphorylated HMGA1 by combining WB with phos-tag phosphorylating gel.9.HMGA1 can bind to ZEB2 promoter region to confirm that HMGA1 can regulate ZEB2 expression level at the transcriptional level.10.When IKBKE or HMGA1 was knocked down in U87 cell line,the growth of intracranial tumors in nude mice was significantly inhibited and the survival time was prolonged.ICH confirmed that the protein expression levels of IKBKE,HMGA1 and ZEB2 decreased after IKBKE was knocked down,while the protein expression levels of HMGA1,ZEB2 and other EMT-related biomarker proteins decreased when HMGA1 was knocked down.Conclusion1.After the expression level of IKBKE was silenced,the proliferation,migration and invasion ability of malignant glioma cells were significantly inhibited.2.IKBKE can directly bind to HMGA1 and IKBKE phosphorylated HMGA1 at the site of Ser 36 and/or Ser 44.We concluded that IKBKE can regulate HMGA1 at the post-translation level.IKBKE knockdown can promote the ubiquitination and degradation of HMGA1 but reduce the phosphorylated level of HMGA1,and inhibit epithelial mesenchymal transformation(EMT)process,and significantly inhibit the progress of malignant glioma cells.3.HMGA1 can directly bind to the promoter region of ZEB2,and promotes the increase of ZEB2 expression levels through transcriptional regulation,and significantly inhibits the epithelial mesenchymal transformation(EMT)process when HMGA1 was knocked down.4.This study first proposes a new mechanism of IKBKE-HMGA1-ZEB2 axis regulation on the proliferation and invasion of glioma cells,providing a new direction for the treatment of glioblastoma.