ISG20 Promotes Local Tumor Immunity And Contributes To Poor Survival In Human Glioma

Objective:Glioma is the most common primary brain tumor,accounting for about 30%of primary brain tumors^[7].Although comprehensive treatments such as surgery,radiotherapy and chemotherapy are currently used,the prognosis of patients is still poor,especially the most malignant glioblastoma multiforme?GBM?.The median survival time of patients is only 14 months¹.IDH mutations are currently thought to be early events in glioma gene alterations and widely affect glioma precursor cells^[3],leading to down-regulation of leukocyte chemotaxis,leading to inhibition of the tumor-associated immune system^[3,4].Therefore,we sought a new therapeutic target for glioma by analyzing the biological processes involved in the early development of IDH mutant tumors and affecting the prognosis of patients.Methods:1.Explore the impact of IDH on glioma immunity.Clinical information and RNA-seq gene expression data were collected for 932 glioma samples from the CGGA and TCGA databases,and differentially expressed genes in both lower-grade glioma?LGG?and glioblastoma?GBM?samples were identified according to IDH mutation status for rule out differences in the immune microenvironment.The immune genes in the IDH mutation group and the IDH wild group in each subgroup were analyzed?T test,corrected p value<0.01?,and the biological pathways involved in the differentially expressed genes in each group were explored.2.Explore the immune genes affected by IDH status in gliomas and analyze their effects on prognosis..The differentially expressed genes associated with IDH status in the GBM and LGG groups in the TCGA database and the CGGA database were used to determine the stability of the differences and to identify the immune microenvironment for early changes in glioma.The gene set was analyzed for survival?R language,COX analysis?to find genes that affect prognosis.3.Discover the gene affected by IDH,ISG20,and explore its specific role in the progression of glioma.Through the survival analysis of the target gene set,it was found that only interferon-stimulated gene product 20?ISG20?can significantly affect the prognosis of patients.The expression distribution of ISG20 in different subtypes of gliomas was analyzed,including age,sex,tumor grade,IDH status,and 1p19q combined deletion.To investigate the effect of ISG20 levels on the survival time of patients in each subgroup and to find out its role in the progression of glioma.4.Explore the immunological processes associated with ISG20.Analysis of genes associated with ISG20 expression levels in all transcripts for GO analysis?http://david.abcc.n-cifcrf.gov/,Database for Annotation,Visualization and Integrated Discovery DAVID v6.8;http://Www.kegg.jp/kegg/pathway.html,Kyoto Encyclopedia of Genes and Genomes,KEGG?to find ISG20-related immune pathways and biological processes,applying gene set enrichment analysis?GSEA?and gene set variation analysis?GSVA?Verify the reliability of the immune pathway.5.Explore ISG20-related enrichment of immune cells.Immune cells associated with ISG20 levels were analyzed using GSVA.During the period from January 2017 to March 2018,43 patients with glioma diagnosed by neurosurgery in the First Affiliated Hospital of China Medical University were enrolled,including 19 cases of WHO grade II glioma and 12 cases of WHO grade III glioma.There were 12 cases of WHO grade IV glioma.Immunohistochemical staining was performed on tumor specimens to verify the relationship between the level of ISG20 and the composition of tumor infiltrating immune cells.6.Explore the relationship between ISG20 and cytokines and explore the mechanisms that lead to their immune differences.Explore the relationship between cytokines and ISG20 levels in the TCGA and CGGA databases,and discuss the mechanisms that lead to differences in the microenvironment of immune cells.Results:1.More immune genes are overexpressed in IDH wild-type gliomas and are associated with more immune pathways.After analyzing the results of TCGA database and CGGA database,it was found that the expression of up-regulated immune genes in IDH wild-type glioma was more than that in IDH mutant glioma.In both GBM and LGG tumors,the number of up-regulated immune genes was significantly increased in IDH wild-type tumors?t test,p<0.05;SAM,FDR<0.01?.The CGGA database suggested that in LGG,43 genes were up-regulated in IDH mutant tumors,and 337 genes were up-regulated in IDH wild-type tumors.Of the GBM subpopulations,106 genes were up-regulated in IDH mutant gliomas and 406genes were up-regulated in IDH wild type.In the TCGA database,there were 150 and433 genes up-regulated by IDH mutants and wild-type in the LGG subpopulation,respectively.In GBM,there are 22 genes in IDHmut and 208 genes in IDHwt are up-regulated.According to GO analysis,IDH wild-type tumors are associated with more immunological processes.2.ISG20 is the only immune gene that is affected by IDH mutations in the early stages of glioma and affects patient survival.We found that IDHwt-type gliomas in both databases have 293 up-regulated genes,and IDHmut-type gliomas have 43 up-regulated genes;in GBM,IDHwt-type gliomas are simultaneously up-regulated.There were 82 of the up-regulated genes shared by IDHmut gliomas.Moreover,we found that among these up-regulated genes,12 immune genes were over-expressed in IDH mutant glioblastoma and lower-grade gliomas,in IDHwt glioblastoma and lower-grade gliomas.71 immune genes were stably overexpressed.Among these genes,only the ISG20 gene affects patient survival.3.The expression level of ISG20 is related to the degree of tumor malignancy,which shortens the survival time of patients.By analyzing the expression levels of ISG20 in different subgroups,it was found that the level of ISG20 increased with the increase of tumor grade,and was up-regulated in IDH wild-type glioma and 1p19q non-deletion glioma.At the same time,ISG20 high expression glioma showed poor prognosis in multiple subgroups of patients.Multivariate COX regression analysis suggested that ISG20 is a factor in the prognosis of patients with glioma independent of age,gender,tumor grade,adjuvant chemotherapy and radiotherapy,IDH,1p9q and MGMT promoter methylation status.4.ISG20 is associated with multiple immune pathways.ISG20 and innate immune response,inflammatory response,T cell activation,antigen processing and presentation,hematopoietic lineage,leukocyte migration,cytokine-cytokine receptor interaction,lysosomal activation,and type I and type II IFN signaling pathways Related.At the same time,the results of GSEA and GSVA analysis suggest that ISG20 and innate immune response regulation and activation,adaptive immune response,antigen processing and MHC class I peptide antigen extraction based on somatic recombination of immunoreceptors constructed based on immunoglobulin superfamily domain In contrast,T cell proliferation regulation is associated with viral defense responses.5.ISG20 is involved in the aggregation of innate immune cells and inhibition of T cells and their subpopulations.With the increase of ISG20 level,mononuclear-derived macrophages and neutrophils aggregated,and the levels of T cells and T cell subsets?central memory T cells,follicular helper T cells?decreased significantly.6.ISG20 is related to the levels of cytokines and immunosuppressive factors PD1,PD-L1and CTLA4.ISG20 highly expressed gliomas with elevated levels of CCL2,CCL5,CCL23,CCL26,CCR1,CCR2,CCR7,CCRL2,CXCL16,CXCR4 and CXCR6,and elevated levels of immunosuppressive factors PD1,PD-L1,and CTLA4.Conclusions:1.The local immune response of IDH wild type glioma is enhanced.The increase of ISG20 gene expression level is associated with poor prognosis of glioma patients.This may be related to the accumulation of immunosuppressive factors in tumors,regulation of various cytokines,and emergence of single nuclear sources.