| Objective:Glioma is one of the most common invasive primary brain tumors,which is more common in adults.According to pathological and clinical criteria,the World Health Organization(WHO)classifies gliomas as Ⅰ,Ⅱ,Ⅲ,Ⅳ gliomas.Among which,the grade IV gliomas,that is,glioblastoma multiforme(GBM),account for nearly 80%of all primary malignant brain tumors in adults.A molecular pathology diagnosis was added into WHO 2016,in which IDH wild/mutant differentiation has also become the basis for the molecular pathological classification of glioma.For the treatment of glioma,progress has been made in surgery,radiation and chemotherapy,benefiting many patients.However,the median survival time of GBM patients is only 14.6 months.Immunotherapy,although effective in multiple other tumor treatments,is still not ideal for gliomas,especially GBM.According to recent research,the specific tumor microenvironment of glioma may lead to its immune escape.However,the related mechanisms and molecular markers are still insufficient.This study attempts to mine existing transcriptomics databases to expand the genes that might affect the phenotype of glioma.We used a variety of bioinformatics tools to analyze transcriptome data from various public databases,such as TCGA and GEO,to explore new markers associated with glioma.We found that multiple members of the SOX gene family were closely associated with glioma,and SOX9 highly expressed in tumor cells.SOX9 was positively related to the poor clinical prognosis of the patients.Studies have shown that SOX9 is a member of the superfamily of sex-determinant genes SRY related high mobility group,a highly conserved HMG domain,which can regulate stem cell behavior,participate in embryonic development and sex determination,or oncogenes of multiple malignancies.In pancreatic cancer,gastric cancer,colon cancer,skin.cancer,and breast cancer,all of which have SOX9 abnormal high expression.Abnormal high expression of SOX9 in gliomas has also been noted.At present,research focuses on the role of SOX9 in glioma resistance,stem cell maintenance and tumor proliferation.Methodology is mostly limited to in vitro cell culture and immunohistochemical analysis of pathological tissue,and mechanistic study is still lacking.For the further study of SOX9 in glioma,after preliminary analysis of glioma transcriptome data,LASSO regression was used to screen variables,and a SOX9-based glioma risk prediction model was established.Through analyses of gene differential expression and enrichment,clustering,pseudo-time differentiation trajectory and cell communication,the malignant cells with high expression of SOX9 were mainly divided into AQP1 positive cells and TNFRSF12A positive cells,which can promote tumor cell proliferation,invasion,and recruitment of myeloid immunosuppressive cells(e.g.BMM),which was involved in the formation of the glioma immunosuppressive microenvironment and enhances immune escape.We also used glioma tissue microarray chips for immunohistochemical experiments.From the level of protein expression,the relationship between SOX9 and clinical prognosis of glioma patients was analyzed and verified.This study showed that high expression of SOX9 can lead to poor prognosis in glioma patients and analyzed the underlying molecular mechanism,which is important for the development of new glioma tumor markers for early detection and prognostic assessment,for improving treatment strategies.Methods:1.The expression of SOX family genes in various common tumors including GBM and LGG tissues,was analyzed by using Oncomine online analysis tools.2.Compared with those in the cancer genome map(TCGA)and genotype-tissue expression database(GTEx),the expression levels of SOX family genes in glioma were verified.3.Use of GEO single cell sequencing transcriptome data to further verify the expression differences of SOX family genes in glioma.4.Use of LASSO regression to screen variables,and to establish a SOX risk prediction model.Use of single factor and multivariate COX regression analysis to determine the correlation between SOX expression and clinicopathological characteristics of glioma.5.Gene differential expression,functional annotation and enrichment analysis are used to analyze the response of different genes and functional signals in high-risk gliomas.6.Use of high-risk and low-risk group transcriptome data to score stromal and immune cells in tumor samples to evaluate the immune microenvironment of glioma at bulk-seq level.7.Single-cell transcriptome data were analyzed using immunocyte deconvolution,pseudo-sequence trajectory reconstruction,and Cellchat to evaluate SOX9 expression in specific cell clusters and their relationship to cell proliferation and immune activity,as well as intercellular interactions.8.Immunohistochemical staining of glioma tissue microarray chip to determine the expression of SOX9 protein,combined with the clinical data of the samples,to clarify the relationship between SOX9 gene expression and tumor proliferation and patient prognosis.Results:1.The expression of several SOX family genes is upregulated in gliomas,and SOX9 is a glioma risk factor.2.SOX3/6/8/9 genes were selected by LASSO regression to construct a glioma risk prognosis model.3.According to the risk model,the patients were grouped and the differential expression gene function annotation and pathway enrichment were found in the high and low risk groups.It was found that the activities of glioma proliferation,immunity,inflammation and hypoxia microenvironment in the high-risk group were enhanced.4.High risk factors were significantly associated with tumor immunosuppressive microenvironment.5.SOX risk models predict benign and malignant subclusters in glioma single cell sequencing data.6.TNFRSF12A and AQP1 positive cells increased cell proliferation,invasion and immunosuppressive microenvironment.7.Patients with upregulated SOX9 expression had a poor prognosis. |
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