Pathogenesis Study Of BACE2 Activation Of NF-κB Pathway To Promote Malignant Progression Of Glioma

Background:In the brain,the most common central nervous system malignancy is glioma,and about half of them are glioblastoma multiforme(GBM),with a five-year survival rate of 5%and a median survival of only about 14 months.Currently,surgical-based treatment,supplemented by postoperative temozolomide and radiotherapy,is not effective,mainly because of the malignant phenotype of glioma with high proliferative capacity,invasive growth,and tumor heterogeneity.Beta-site APP cleaving enzyme 2(BACE2)is belong to β-secratse family,and closely related to tumor metastasis and tumor microenvironment formation.Currently,studies on the bond of beta secretase 2 mRNA and proteinlevels in gliomas and prognosis are limited,and studies on the mechanisms that regulate the malignant progression of gliomas are even more lacking.Therefore,an in-depth exploration of itin the malignant progression of glioma will help improve treatment strategy and enhance the therapeutic effect.Objectives:(1)Explore the expression level and prognostic significance of BACE2 in glioma.(2)Clarify specific function of BACE2 in the malignant progression of glioma.(3)Elucidate upstream and downstream regulatory process of BACE2.Methods:(1)Download transcriptome data and corresponding patients’ clinical information from TCGA,CGGA and other databases,draw BACE2 expression profiles and survival curves,and analyze expression differences of BACE2 in different grades and various subtypes of glioma.(2)RT-qPCR and immunohistochemistry were used to detect BACE2 expression in 34 specimens of different grades of glioma and 6 cases of normal brain tissue.(3)Transcriptome sequencing analysis of control and BACE2 knockdown U87MG cells was performed,and differentially expressed genes were screened using the R language Deseq2 package,differential gene set enrichment analysis was used to predict the biological function of BACE2.(4)Assess the effect of BACE2 on glioma invasion,migration and proliferation using siRNA and lentiviral techniques in combination with in vitro assays(3D invasion assay,Transwell,EdU fluorescence,CCK-8,etc.).(5)Cells were treated with cytokines or specific pathway inhibitors,and relevant signaling pathway changes were detected by Western blot and immunofluorescence staining.(6)Control and knockdown BACE2 Luciferase-labeled U87MG cells were injected into the brain of nude mice by stereotactic injector,and tumor growth status was detected by animal live imaging system.Results:(1)BACE2 expression level was positively correlated with the malignancy of glioma and was highly expressed in Mesenchymal subtype(MES)glioma,and patients whose BACE2 expression higher than median value had significantly shorter median survival.(2)Bioinformatics analysis predicted that BACE2 was closely related to the malignant phenotype of glioma,such as invasion,migration and proliferation.(3)In vitro experiments showed that BACE2 promotes MES transformation and enhances the invasion and migration of glioma cells,and regulates the G1/S phase transition of the cell cycle to promote tumor cell proliferation;in vivo experiments confirmed that knockdown of BACE2 inhibited tumor growth and prolonged survival in nude mice with in situ tumors.(4)Knockdown of BACE2 inhibited TNF-α induced activation of NF-κB signaling pathway by reducing the phosphorylation level of PP1A and IKKβ protein,and reduced the invasive migration and proliferation ability of glioma.(5)In vitro treatment of TGFβ1 promoted the expression of BACE2 protein in glioma cells,and the application of specific inhibitor SB431542 or knockdown of Smad2 inhibited this effect.Conclusion:(1)BACE2 expression levels correlate with glioma grade,MES subtype,and prognosis of glioma patients.(2)TGFβ1-Smad pathway regulates BACE2 expression and activates the TNF-α induced NF-κB signaling pathway through the PP1A/IKKβ axis to promote invasive,migration and proliferation of glioma cells.(3)BACE2 could be a glioma candidate biomarker for diagnosis,assessment of patient prognosis and a new target for molecular therapy.