Roles of the m(6)A Modification of RNA in the Glioblastoma Microenvironment as Revealed by Single-Cell Analyses

PURPOSE: Glioblastoma multiforme (GBM) is a common and aggressive form of brain tumor. The N(6)-methyladenosine (m(6)A) mRNA modification plays multiple roles in many biological processes and disease states. However, the relationship between m(6)A modifications and the tumor microenvironment in GBM remains unclear, especially at the single-cell level. EXPERIMENTAL DESIGN: Single-cell and bulk RNA-sequencing data were acquired from the GEO and TCGA databases, respectively. We used bioinformatics and statistical tools to analyze associations between m(6)A regulators and multiple factors. RESULTS: HNRNPA2B1 and HNRNPC were extensively expressed in the GBM microenvironment. m(6)A regulators promoted the stemness state in GBM cancer cells. Immune-related BP terms were enriched in modules of m(6)A-related genes. Cell communication analysis identified genes in the GALECTIN signaling network in GBM samples, and expression of these genes (LGALS9, CD44, CD45, and HAVCR2) correlated with that of m(6)A regulators. Validation experiments revealed that MDK in MK signaling network promoted migration and immunosuppressive polarization of macrophage. Expression of m(6)A regulators correlated with ICPs in GBM cancer cells, M2 macrophages and T/NK cells. Bulk RNA-seq analysis identified two expression patterns (low m(6)A/high ICP and high m(6)A/low ICP) with different predicted immune infiltration and responses to ICP inhibitors. A predictive nomogram model to distinguish these 2 clusters was constructed and validated with excellent performance. CONCLUSION: At the single-cell level, m(6)A modification facilitates the stemness state in GBM cancer cells and promotes an immunosuppressive microenvironment through ICPs and the GALECTIN signaling pathway network. And we also identified two m(6)A-ICP expression patterns. These findings could lead to novel treatment strategies for GBM patients.