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Glioblastoma (GBM) is one of the most common malignant and incurable brain tumors. The identification of a gene signature for GBM may be helpful for its diagnosis, treatment, prediction of prognosis and even the development of treatments. In this study, we used the GSE108474 database to perform GSEA and machine learning analysis, and identified a 33-gene signature of GBM by examining astrocytoma or non-GBM glioma differential gene expression. The 33 identified signature genes included the overexpressed genes <i>COL6A2</i>, <i>ABCC3</i>, <i>COL8A1</i>, <i>FAM20A</i>, <i>ADM</i>, <i>CTHRC1</i>, <i>PDPN</i>, <i>IBSP</i>, <i>MIR210HG</i>, <i>GPX8</i>, <i>MYL9</i> and <i>PDLIM4</i>, as well as the underexpressed genes <i>CHST9</i>, <i>CSDC2</i>, <i>ENHO</i>, <i>FERMT1</i>, <i>IGFN1</i>, <i>LINC00836</i>, <i>MGAT4C</i>, <i>SHANK2</i> and <i>VIPR2</i>. Protein functional analysis by CELLO2GO implied that these signature genes might be involved in regulating various aspects of biological function, including anatomical structure development, cell proliferation and adhesion, signaling transduction and many of the genes were annotated in response to stress. Of these 33 signature genes, 23 have previously been reported to be functionally correlated with GBM; the roles of the remaining 10 genes in glioma development remain unknown. Our results were the first to reveal that GBM exhibited the overexpressed <i>GPX8</i> gene and underexpressed signature genes including <i>CHST9</i>, <i>CSDC2</i>, <i>ENHO</i>, <i>FERMT1</i>, <i>IGFN1</i>, <i>LINC00836</i>, <i>MGAT4C</i> and <i>SHANK2</i>, which might play crucial roles in the tumorigenesis of different gliomas.