The Expression Of Notch1 In Different Molecular Subtypes Of Glioblastoma And Its Study On The Regulation Mechanism Of NF-?B(p65)

Background: Glioblastomas(GBMs)are the most prevalent and devastating primary intracranial malignancies and have extensive heterogeneity.Notch1 signaling is a more complex process in the development of numerous cell and tissue types,including gliomagenesis and progression,and is upregulated in glioma-initiating cells.However,the contradictory expression of Notch1 among lower grade gliomas and GBMs confounds our understanding of GBM biology and has made identifying effective therapies difficult.The present study aimed to explore the specific mechanisms of Notch1 in the processes of glioma proliferation and apoptosis,the expression of Notch1 and NF-?B(p65)in the different GBM molecular subtypes and how Notch1 regulates the NF-?B(p65)signaling pathways in GBM.It is expected to be help drive the design of more effective therapies in Notch1-targeted clinical trials.Methods:Through MERAV and Oncomine database,we analyzed the expression of Notch1 in human normal tissues and tumor tissues,and then further analyzed the expression of Notch1 in GBM tissues and non-tumor brain tissues from the Murat Brain and Sun Brain datasets.Meanwhile,in order to study the activity of Notch1 pathway in different histological types,pathological grades and molecular typing of glioma,we also carry out bioinformatics analysis in TCGA and CGGA database.The levels of Notch1 in GBM tumor tissues,glioma stem cells(GSCs)and glioma cell lines were detected by western blotting,immunofluoresence and immunohistochemistry.The correlation between the Notch1 pathway and NF-?B(p65)in TCGA and CGGA datase was analyzed.Botch treatment of DAPT and sh RNA targeting Notch1 decreased NF-?B(p65)expression,suppressed cell proliferation,and induced apoptosis of U87,LN229 and U251 glioma cell lines in vitro.After DAPT or sh RNA targeting Notch1,the Notch1,NICD,Hes1,p65,Cylin D1,p21,Bcl-2,Pro-Caspase-3,Cleaved Caspase-3,Pro-Caspase-9 and Cleaved Caspase-9 expression levels were detected by Western blotting.DAPT treatment and sh Notch1-transduced glioma cells were subjected to the colony formation assay and flow cytometry.Immunofluorescence staining showed the distribution of NF-?B(p65)in U87,U251 and LN229 cells after sh RNA and DAPT treatment.we performed a co-IP analysis and observed that NICD can bind to NF-?B(p65).we investigated the effects of Notch1 on tumor growth in established intracranial glioma model.Results: Notch1 was overexpressed in GBM samples compared with normal brain controls.The results of the cluster analysis revealed that the Notch1 signaling pathway and NF-?B(p65)were significantly upregulated in classical and proneural subtypes of GBM.Targeting Notch1 induced apoptosis,suppressed the growth and proliferation of glioma cells.Intracranial model results confirmed that the knocdown of Notch1 could inhibit tumor growth in vivo.Conclusions:Notch1 was expressed at relatively higher levels in the classical and proneural subtypes from TCGA and CGGA databases.Patients with Notch1 overexpression had a significantly shorter overall survival in the classical subtype of GBM.We found that Notch1-expressing cells colocalized with CD133-expressing cells and Nestin-expressing cells in GSCs.Notch1 and stemness markers also surrounded the endothelial cells as indicated by immunohistochemical staining.Furthermore,based on the pearson correlation analysis of TCGA and CGGA,Notch1 expression was positively correlated with RELA(NF-?B(p65))expression in GBM.In addition,we showed that targeting Notch1 inhibited proliferation and induced apoptosis of GBM cells by regulating cell cycle-and apoptosis-related proteins in vitro and in vivo via suppression of the NF-?B(p65)pathway.Combination drug regimens designed to prevent activity of the Notch1 signaling and NF-?B(p65)pathways may be advantageous in treating the classical and proneural GBM.