| Glioblastoma multiforme(GBM)is a highly malignant and invasive CNS tumor.GBM has a high degree of heterogeneity.Targeted therapy for specific molecular subtypes will be important in GBM treatment.The significance.Mesenchymal(MES)has a higher degree of malignancy in GBM subtypes.Recent studies have suggested that TGF-β is over-expressed in the interstitial subtype of GBM,whereas The role of TGF-β signaling in the GBM stroma subtype has not yet been fully elucidated.The patient derived xenograft(PDX)model has become an important tool for biological research.It can better simulate the growth process of the tumor and the influence of the tumor’s microenvironment.It has been considered to be the closest in vivo animal model to the patient’s tumor.The PDX xenograft model plays an important role in the identification of GBM subtypes,particularly predictive treatment responses.In this study,the glioblastoma-derived PDX model and primary cultured cells were used as research subjects.TGFβR2 small molecule inhibitors were used to study in vivo and in vitro interventions.The in vivo inhibitory effect of TGFβR2 small molecule inhibitors was verified to elucidate the role of TGF-β signaling pathways in GBM interstitial subtypes.The first part: In this study,surgically resected fresh human brain glioblastoma tissues was subcutaneously tumorigenic and then passaged for the establishment of an intracranial in situ PDX model.This method increases the success rate of PDX.HE staining showed that the primary tumors and PDX tumors were similar in morphology.PDX tumors retained the morphological characteristics of primary tumor glioblastomas.PDX retains its biological properties with primary tumors and can serve as an ideal animal model for drug detection and functional analysis.The establishment of a PDX xenograft model provides an effective technique for the human glioma PDX model and provides an important approach for individualized treatment of gliomas.The second part: Based on the primary cultured cells of glioblastoma,the small molecule inhibitor of TGFβR inhibitor LY2109761 inhibited the primary cultured cells.The results suggested that the expression of interstitial GBM-related markers decreased.Further RNA-seq analysis revealed down-regulation of mesenchymal phenotypic genes such as MET,LIF,IL-6 and OSM,and TGFβR2 as a core marker of mesenchymal phenotypes.Genes play a key role in maintaining the GBM mesenchymal phenotype.To study the role of TGF-β signaling pathway in GBM,TGF-β signaling pathway was inhibited by LY2109761,a small molecule inhibitor of TGFβR,and multi-dimensional analysis of differentially expressed genes was performed using various bioinformatics methods.Through the KEGG pathway enrichment analysis of differentially expressed genes by the DAVID database,we found that the top biological processes involved the epithelial-mesenchymal transition(EMT),cell proliferation,and TGF-β signaling pathways,cytokine-cytokine rece ptor interactions,PI3K-Akt pathways,etc.The TGF-β signalingpathway is the core of the signal transduction network,regulates a variety of biological functions.It plays an important role in the proliferation and differentiation of gliobl astoma cells,inflammation,angiogenesis,and epithelial-mesenchymal transition(EMT).A protein-protein interaction network analysis was established via the STRING online software to clarify the protein-protein interactions of TGFβR2,RUNX1,PPARG,GIT2,ACSL1 and RAP1 B.TGFβR2,RUNX1,PPARG,GIT2,ACSL1 and RAP1 B interact with each other in a protein-protein network.T he interactions can be used as genotyping subtype markers ofglioblastoma cells and have important diagnostic value.The GBM-based PDX model was used to study the susceptibility of TGF-β2 R inhibitors.The inhibitory effect of LY2109761 was studied in vivo.It was found that TGF-β2 R inhibitors can inhibit tumor cell proliferation and tumor microangiogenesis.The effect of TGF-β signaling pathway can be a potential target for GBM mesenchymal subtype therapy.It is of great significance to evaluate the effect of targeted therapy based on individualized glioblastoma model.The third part: The relationship between human glioma TGF-β1、RUNX1 and EMT and the clinical significance.To investigate the relationship and clinical significance of TGF-beta 1,P-Smad3,RUNX1 and EMT,in this study we used the immunohistochemical method to detect the expression level and correlation of TGF-β1,P-Smad3,RUNX1,Vimentin,E-cadherin in different pathological grades of glioma and used Log Rank test for survival analysis.And draw the Kaplan-Meier survival curve.In high grade gliomas,the expression of TGF-β1,P-Smad3,RUNX1,Vimentin increased significantly(P< 0.01)compared to low grade gliomas.The expression of TGF-β1,P-Smad3,RUNX1 and Vimentin was positively correlated and negatively correlated with E-cadherin.TGF-β1,P-Smad3 and RUNX1 are related to EMT.In this study,we found that the expression of TGF-β1,P-Smad3,RUNX1,Vimentin,E-cadherin is related to the pathological grade and survival time of glioma,which can provide valuable indicators for the prognosis of glioma and provide a theoretical basis for the targeting therapy strategy for TGF-β1 signaling pathway.This study was based on the individualized study of the drug sensitivity of the small molecule inhibitor LY2109761 of TGF-βR2 kinase based on the PDX model of glioblastoma patient-derived xenograft tumors,which compensated for the lack of previous cell lines as the research object and confirmed the TGF-βR2 kinase.Small molecule inhibitors have anti-tumor effects and TGFβR2 may serve as a potential target for GBM clinical treatment.This study clarified that TGF-β signaling pathway plays an important role in the maintenance of GBM mesenchymal subtypes.Multi-dimensional perspectives and methods confirmed that TGFBR2,RUNX1,PPARG,ACSL1,GIT2,and RAP1 B can be used as markers of GBM mesenchymal subtypes.In addition,TGFBR2 is the most important core diagnostic factor for GBM interstitial subtypes. |
