EGFR VⅢ Downregulated H2AZK4/7AC Though The PI3K/AKT-HDAC2 Pathway Regulating Cell Cycle Progression In Glioma

Objectives:The EGFR vⅢ mutation is the most common malignant event in glioma and is involved in multiple malignant progressions of glioma.We extracted U87-vⅢ and U87 cell nuclear proteins for mass spectrometry analysis and found that when the EGFR vⅢ mutation was activated,histone H2AZK4/7AC expression was reduced.Histone acetylation is usually associated with gene transcription.We hypothesize that EGFR vⅢ affects the transcription of tumor suppressor genes by down-regulating H2AZK4/7AC,thereby accelerating the malignant progression of glioma.We explored the mechanism by which EGFR vⅢ downregulates H2AZK4/7AC and the possible mechanism of H2AZK4/7AC involved in the regulation of glioma cell cycle by constructing glioma cells that continuously overexpress EGFR vⅢ and using small molecule inhibitors.From the perspective of epigenetic disorders,we explored potential drug targets for EGFR vⅢ mutant gliomas and the therapeutic effect of HDAC inhibitor FK228.We strived to provide new and promising treatments for EGFR vⅢ mutant gliomas.Methods:1.The U87,U251 and N9 cells were transfected with lentivirus to obtain glioma cells stably expressing EGFR vⅢ.The expression of H2AZK4/7AC was detected by mass spectrometry after EGFR vⅢ mutation.The effect of EGFR vⅢ mutation on H2AZK4/7AC expression was verified by Western blotting and immunofluorescence in vitro.Changes in H2AZK4/7AC expression were observed after treatment of U87-vⅢ,U251-vⅢ,N9-vⅢ cells with a broad spectrum of HDAC inhibitor TSA.IP experiments explored the interaction between HDAC1/2 and H2 AZ,immunofluorescence experiments were performed to see if there was colocalization between HDAC2 and H2 AZ.Then,treated U87-vⅢ,U251-vⅢ,N9-vⅢ cells with Si RNA against HDAC1 and HDAC2 to observe changes in H2AZK4/7AC expression.The PI3K/AKT pathway inhibitor LY294002 was used to treat the cells to block the PI3K/AKT pathway induced by EGFR vⅢ mutation,and the expression changes of HDAC2,H2AZK4/7AC,H3K27 AC and H3K27me3 were observed.Since HDAC1/2 usually forms a complex,the downstream H2AZK4/7AC,H3K27 AC,H3K27me3,and H3K4me3 changes are observed after knocking out both HDAC1 and HDAC2 by Si RNA.2.Among the total protein profiles of U87-vⅢ and U87 cells,106 genes that were down-regulated after EGFR-vⅢ mutation were selected and brought into TCGA and CGGA database to predict their correlation with EGFR,survival,tumor grade,tissue typing.We screened the candidate genes.To verify whether EGFR vⅢ can down-regulate the transcription and translation of USP11 via the PI3K/AKT-HDAC1/2 axis,and detect the expression changes of P-AKT,HDAC1,HDAC2,H3K27 AC,H2AZK4/7AC,and USP11,respectively.LY294002 and si RNA against HDAC1 and HDAC2 were used to observe the recovery of USP11.Chip-q PCR verified whether EGFR vⅢ could down-regulate the transcription and translation of USP11 by reducing the enrichment of H3K27 AC and H2AZK4/7AC in the USP11 promoter region.3.To investigate the effect of FK228(HDAC1/2 blocker)on the cell cycle of EGFR vⅢ mutant glioblastoma.FK228 pretreated cells and we detected cell cycle changes by flow cytometry.WB was performed to detect Cyclin D1,CDK4,CDK6,P21 expression changes.The intracranial models of U87-vⅢ and U87 were constructed to investigate the effects of FK228 on tumor growth and the in vivo expression changes of downstream Cyclin D1,CDK4,CDK6,P21 and USP11.Results:1.H2AZK4/7AC expression was down-regulated after EGFR vⅢ mutation.EGF stimulation of U87 cells also caused a down-regulation of H2AZK4/7AC.The expression of H2AZK4/7AC was up-regulated after TSA treatment,suggesting that HDACs may be involved in H2AZK4/7AC expression regulation.IP experiments confirmed that H2 AZ interacts with HDAC1 and HDAC2,respectively.H2AZK4/7AC expression is increased after knockdown of HDAC2,and confocal display shows HDAC2 and H2 AZ has nuclear colocalization.After EGFR vⅢ mutation,HDAC1/2 transcription and translation levels increased,EGFR vⅢ mutation activated PI3K/AKT pathway,HDAC2 expression decreased after LY294002 treatment,and H2AZK4/7AC,H3K27 AC expression increased.There was no significant change in H3K27me3 and H3K4me3 expression.After knocking out HDAC1/2,the expression of H2AZK4/7AC and H3K27 AC increased.There was no significant change in H3K27me3 expression.2.Bioinformatics analysis showed that USP11,SELK,HIP1 R,CYFIP2,ALAD were negatively correlated with EGFR expression,negatively correlated with glioma grade,and was associated with glioma tissue typing.Prognostic analysis showed a better survival for high expression of USP11,SELK,HIP1 R,CYFIP2,ALAD in glioma.EGFR vⅢ down-regulates the transcription and translation of USP11 by the epigenetic approach via the PI3K/AKT-HDAC1/2 axis.3.In vitro experiments demonstrated that FK228 can induce G1/S phase arrest in EGFR vⅢ mutant glioblastoma.The intracranial model confirmed that FK228 can slow the growth of glioblastoma carrying the EGFR vⅢ mutation by blocking the G1/S phase transition.In vivo experiments,P-AKT,HDAC1,and HDAC2 expression were up-regulated and H3K27 AC,H2AZK4/7AC,and USP11 expression was down-regulated after EGFR vⅢ mutation.FK228 treatment partially reversed the down-regulation of USP11 caused by the EGFR vⅢ mutation.Conclusions:1.The EGFR vⅢ mutation can down-regulate the expression of H2AZK4/7AC via the PI3K/AKT-HDAC2 axis.2.HDAC2,but not HDAC1,can specifically bind to and regulate the deacetylation modification of H2AZK4/7AC.3.EGFR vⅢ down-regulates the transcription and translation of USP11 by the epigenetic approach via the PI3K/AKT-HDAC1/2 axis.4.EGFR vⅢ down-regulates the expression of USP11 by down-regulating the binding of H3K27 AC and H2AZK4/7AC in the USP11 promoter region,and FK228 treatment partially reverses the down-regulation of USP11 caused by EGFR vⅢ mutation.5.In vivo and in vitro experiments,FK228 can induce G1/S phase arrest in EGFR vⅢ mutant glioblastoma to exert therapeutic effects.