Engrailed 1 promotes immune evasion and chemoresistance in glioma through single cell and CeRNA network analyses

Immune evasion is a major barrier to effective glioma treatment, yet the molecular drivers contributing to this process remain insufficiently understood. Engrailed-1 (EN1), a developmental transcription factor, has recently emerged as a potential oncogenic regulator, but its role in glioma immune modulation is unclear. This study aimed to comprehensively investigate the function of EN1 in glioma, with a particular focus on its involvement in immune evasion, by integrating bulk and single-cell transcriptomic analyses together with ceRNA regulatory network construction and experimental validation. EN1 expression, diagnostic value, and prognostic significance were assessed using TCGA, GTEx, and CGGA datasets. Immune infiltration and microenvironmental features were evaluated through CIBERSORT, ESTIMATE, and WGCNA. Single-cell RNA-seq data (GSE182109) were used to characterize the cell-type distribution and developmental trajectory of EN1. A NEAT1/miR-9-5p/miR-128-3p/EN1 ceRNA axis was constructed using multi-database predictions. Functional assays including Western blot, CCK-8, and Transwell experiments were performed to validate EN1's effects in glioblastoma cells. EN1 was markedly overexpressed in glioma and associated with poorer survival. Elevated EN1 expression correlated with increased infiltration of immunosuppressive cells, reduced tumor purity, and higher immune checkpoint expression. Single-cell analysis revealed progressive EN1 upregulation along malignant cell pseudotime. Drug sensitivity prediction suggested that the EN1-high group may have reduced sensitivity to temozolomide and additional chemotherapeutic agents. The identified NEAT1/miR-9-5p/miR-128-3p/EN1 ceRNA loop suggested a regulatory mechanism contributing to EN1 activation. EN1 knockdown significantly suppressed glioblastoma cell proliferation and invasion in vitro. EN1 is associated with glioma aggressiveness, immune microenvironmental features, and predicted chemotherapeutic response, and a putative NEAT1/miR-9-5p/miR-128-3p/EN1 axis may contribute to EN1 dysregulation. These findings identify EN1 as a promising biomarker and potential therapeutic target for improving glioma treatment.