Identification And Mechanism Study Of Subtype Markers In Human Glioma

Objective: Based on the level of transcription,the currently recognized molecular classification of glioblastoma includes three types,Classical,Mes(Mesenchymal)and Proneural.These three types of patients present a significant difference in survival.This project plans to use bioinformatics analysis combined with experimental research to find two subtypes of Classical and Mes biomarkers with poor survival rates,and targeted therapy was explored.Methods: 1.EGFR is a taxonomic marker of the Classical subtype of glioblastoma.The CGGA m RNA and non-coding RNA sequencing results are analyzed to find protective mi RNAs of Classical glioblastoma.Developmental roles and mechanisms associated with EGFR,including phenotypic analysis: Transwell experiments,plate cloning experiments,CCK8 experiments,cell cycle experiments.The Ch IP assay verified the histone modification of the mi RNA promoter region under the activation of the EGFR pathway.q PCR,Western blots and dual luciferase reporter assays validate downstream target genes of mi RNA.We perform the Ch IP assay to verify the activation of the target gene pathway.2.Cell and tumor tissue typing were performed by RNA-sequencing.We used coimmunoprecipitation(co-IP)and immunofluorescence to identify members of the RUNX1 transcriptional protein complex.Bioinformatics analysis,chromatin immunoprecipitation(Ch IP)and luciferase reporter experiments were used to verify target genes.Analyses of The Cancer Genome Atlas(TCGA)and Chinese Glioma Genome Atlas(CGGA)verified the expression levels and prognoses associated with RUNX1 target genes.In vivo patient-derived xenograft(PDX)studies and in vitro functional studies verified the impact of RUNX1 on the occurrence and development of GBM.Results: 1.We found that mi R-524-3p and mi R-524-5p were suppressed in the classical molecular subtype of glioblastoma(GBM)from Chinese Glioma Genome Atlas(CGGA)data,and the suppression was associated with EGFR overexpression and EGFRv III mutation.These two mi RNAs improved overall survival time of patients with glioma,and their overexpression could restrain glioma cell migration,proliferation,and cell cycle,and control tumor formation in vivo.Interestingly,both of the mi RNAs had a synergistic inhibitory effect on glioma cells.Furthermore,we confirmed that EGFR amplification/EGFRv III mutation can repress the expression of Pri-mi R-524 by histone modification.Mi R-524-3p and mi R-524-5p inhibited TGF/β,Notch and the Hippo pathway by targeting Smad2,Hes1 and Tead1,respectively.These pathways repressed their common downstream transcription factor,C-myc.More interestingly,C-myc bound to the promoter region of EGFR/EGFRv III and activated its expression.2.RUNX1 is upregulated in Mes GBM cell lines,tissues and patients and promotes proliferation and invasion in GBM in a TGFβ pathway-dependent manner in vivo and in vitro.The RUNX1/p-Smad3/SUV39H1 transcription protein complex is tightly linked following the activation of the TGFβ pathway.We found and verified that BCL3,MGP and POSTN are transcriptionally activated by p-Smad3/RUNX1,while MXI1 is transcriptionally suppressed by the RUNX1/SUV39H1-H3K9me3 axis.Our study found that Mes GBM presented high levels of TGFβ pathway activation and RUNX1 protein expression and that the activated TGFβ pathway promotes the formation of the RUNX1/p-Smad3/SUV39H1 transcription protein complex and affects the expression of downstream genes(BCL3,MGP,POSTN and MXI1).Conclusions: Our experimental findings indicate that miR-524 mediates EGFR/EGFRv III activation.The transcriptional activation axis of RUNX1 formation maintains the glioblastoma interstitial subtype.They can be used as therapeutic drugs and specific biomarkers for Classical subtypes and Mes subtypes of glioblastoma,respectively.