| Background:Malignant glioma is a disease with a dismal prognosis.In this study,we investigate the functions of Galectin-9/TIM-3/IL-6 signaling in glioma and its role in the regulation of the local disrupted immune microenvironment in glioblastoma.In addition,in order to find the key gene of interleukin-6(IL-6)associated cytokine receptor activation molecule family in gliomas,we investigate the role of the family and get the molecule,oncostatin M receptor(OSMR).Then we research the role of OSMR in gliomas and its function in regulating the glioblastoma(GBM)microenvironment.Methods:we first collected the expression data of inhibitory immune checkpoint genes including T cell immunoglobulin domain and mucin domain protein 3(TIM-3),cytotoxic T lymphocyte-associated protein 4(CTLA-4),programmed cell death protein 1(PD-1),programmed cell death protein ligand 1(PD-L1),programmed cell death protein ligand 2(PD-L2),lymphocyte activating 3(LAG-3),T cell immunoreceptor with Ig and ITIM domains(TIGIT),and indoleamine-2,3-dioxygenase(IDO1)in glioma from four datasets,Chinese Glioma Genome Atlas(CGGA),The Cancer Genome Atlas(TCGA),Gene Expression Omnibus(GEO)dataset(GSE16011)and the Repository for Molecular Brain Neoplasia Data(Rembrandt).Examination of these four datasets demonstrated which genes of the immune checkpoint genes is the highest expression in WHO grade II,grade III,and GBM,respectively.Then,the expression of these immune checkpoint genes was verified with western blot of glioma samples.Tissue immunofluorescence double labeling revealed the expression location of TIM-3 in glioma specimens.The expression of TIM-3 and its ligand galectin-9(Gal-9)was examined by RT-qPCR,immunostaining,and western blotting.In glioma cells,the role of TIM-3 in the regulation of in vitro proliferation,colony formation,migration and invasion capabilities and underlying mechanisms were investigated by in vitro proliferation and colony formation assays,wound healing tests,and Transwell assays,respectively.Furthermore,the effect of TIM-3 on in vivo tumorigenicity of glioma cells was examined using subcutaneous and intracranial tumor xenograft model.In addition,bioinformatics methods were applied to investigate the relation between TIM-3 and Gal-9.To further confirm this result,we performed immunofluorescence on clinical glioma samples to detect the expression of TIM-3 and Gal-9 protein.Moreover,we confirmed the direct combination of Gal-9 with TIM-3 by immunoprecipitation.In order to understand the molecular mechanism by which TIM-3 regulates the proliferation,migration and invasive capabilities of glioma cells,we further investigated the role of TIM-3/Gal-9 interaction with a TIM-3 overexpression vector and an anti-Gal-9 blocking antibody.The functions and pathways of TIM-3 in GBM was investigated by Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),Gene set enrichment analysis(GSEA)and gene set variation analysis(GSVA)et al.To further confirm these above observations,we performed western blotting and enzyme-linked immunosorbent assay(ELISA)to investigate the effect of TIM-3 overexpression and inhibition on the protein expression and secretory of IL-6 and its related NF?B-IL-6 signaling pathway.Simultaneously,we investigated the function of secreted IL-6 on regulating tumor immune microenvironment.We next evaluated the correlation between TIM-3 and two non-tumor cell populations by the MCP-counter method and further analyzed the correlation between TIM-3 and immune cell subsets through CIBERSORT.Finally,TIM-3 signaling in glioma cells,regulating the differentiation and migration of THP1-derived macrophages was determined by Trans well assays and qPCR,respectivelyThree gene expression profiles,CGGA,TCGA,and GSE16011,were enrolled in our study and employed for investigating the key gene of cytokine receptor activity related genes,which was OSMR.We then analyzed the expression and survival of OSMR.The expression of OSMR was further verified with RT-qPCR,immunohistochemistry and western blot in glioma tissues.GISTIC2.0 was applied to evaluate the frequency of copy number alteration(CNA)of OSMR in TCGA.We also investigate the correlation between OSMR expression and CNA frequency of OSMR and EGFR,and confirmed the mutant frequency of OSMR.Microenvironment cell populations-counter(MCP-counter)was applied for analyzing the relationship between OSMR expression and non-tumor cells.We conduced survival analyses to examine whether OSMR could serve as a marker for the prediction of the response to radiation and chemo-therapy.The functions of OSMR in GBM was investigated by Gene Ontology(GO),Gene set enrichment analysis(GSEA)and gene set variation analysis(GSVA)et al.In order to find the key gene which participated in OSMR regulating immune microenvironment,we further investigated the correlation between OSMR expression and immune checkpoint genes in CGGA,TCGA and GSE16011 datasets.And we confirmed the correlation between OSMR and them in glioma tissues with western blot.Results:Examination of these four datasets and western blot demonstrated that expression of TIM-3 was the highest amongst immune checkpoint genes in WHO grade II,grade III,and glioblastoma(GBM),respectively.Tissue immunofluorescence double labeling reveal that the expression of TIM-3 was located both in glioma cells and non-glioma cells in glioma specimens.The results of immunohistochemistry and western blot analysis confirmed that the expression of TIM-3 protein were elevated in glioma,especially in GBM,in comparison with non-tumor samples.And TIM-3 expression is correlated with the unfavorable prognosis in GBM.After TIM-3 combining with the ligand Gal-9,glioma cell intrinsic Gal-9/TIM-3 signaling facilitates the growth,migration,and invasion of glioma cells.Blocking Gal-9 attenuates the enhanced proliferation,migration and invasion of glioma cells induced by TIM-3 overexpression.Gal-9/TIM-3 contributes the expression and secretory of IL-6 by acting NF-kB pathway.The results of MCP-counter and CIBERSORT showed that the population of cells with monocytic lineage most significantly correlated with TIM3 expression,especially M2 phenotype of tumor associated macrophages.Gal-9/TIM-3 signaling not only participates in the regulation of proliferation,colony formation,migration and invasion of glioma cells,but also induces M2 phenotype of tumor associated macrophages and increases the migration capabilities of THP1-derived macrophages.OSMR was one of most obvious cytokine receptor genes differentially expressed between low grade glioma(LGG)and GBM in the three datasets.GBM patient had a higher OSMR expression than grade II or grade III glioma patient.Furthermore,we found that isocitrate dehydrogenase 1(IDH1)wild-type patients had a higher level of OSMR expression than IDH1 mutant patient.Additionally,in GBM,mesenchymal subtype exhibited a higher expression level of OSMR than the other two subtypes(proneural and neural).OSMR worked as a gene with an independent predictive factor for progressive malignancy in GBM.OSMR high-expression-group had a high amplification and low deletion frequency compared with OSMR low-expression-group in GBM.There was a close correlation between OSMR expression and CNA frequency of OSMR and EGFR.Besides,we found that the mutant frequency of OSMR was lower than 1%.OSMR could serve as a marker for the prediction of the response to radiation and chemo-therapy and OSMR participating in chemotherapy resistance in GBM was related to MGMT promoter methylatedConclusions:Our findings demonstrate that glioma intrinsic TIM-3 signaling facilitates tumor growth and induces M2 phenotype of TAMs,which may further promote tumor progression.IL-6 plays an important role in the progress of TIM-3 regulating glioma microenvironment.Moreover,OSMR is the most important molecule of cytokine receptor activation molecule family in glioma.Our findings define an important role of OSMR in the regulation of local immune response in GBM,which may suggest OSMR as a possible biomarker in developing new therapeutic immune strategies in GBM. |
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