M6A modification-related genes characterize prognostic risk and immune regulation in lung adenocarcinoma

Lung adenocarcinoma (LUAD), a molecularly complex and aggressive malignancy, has a dismal 5-year survival rate. N6-methyladenosine (m6A) associated factors regulates cancer progression, but its role in LUAD prognosis and immune regulation remains unclear. By integrating LUAD gene expression data and clinical information from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), we analyzed the expression differences and genetic variations of m6A regulators. Clustering analysis identified distinct m6A modification patterns, while various regression algorithms were employed to construct a risk model based on m6A-related differentially expressed genes, and assessed its clinical relevance, genetic mutations and sensitivity to immunotherapy. PHLDA2 function was assessed by RT-qPCR, proliferation, migration and invasion assays. Its role in immune regulation was tested via MIF promoter luciferase reporter and MIF neutralization followed by STAT3/PD-L1 analysis. Single-cell RNA sequencing and tools including CellChat and NicheNet were used to map gene expression and cell–cell communication in the tumor microenvironment. Consensus clustering analysis based on 15 differentially expressed m6A regulatory factors identified two m6A clusters. 40 key genes closely associated with m6A modification were identified and a two-gene (PHLDA2 and RGS13) risk model correlated with the prognosis of LUAD patients was constructed. High-risk scores were linked to poor prognosis, advanced TNM(Tumor-Node-Metastasis) stage, and higher mutation frequency. Furthermore, PHLDA2 was found to be highly expressed in LUAD, predominantly localizing in epithelial and T cells, influencing late-stage T cell functions, and regulating LUAD cell proliferation, migration and invasion. Additionally, PHLDA2 was positively correlated with Programmed Death-Ligand 1 (PD-L1) and associated with the initiation or activation of immune cycle processes. Experimental validation shows that PHLDA2 promotes MIF transcription, and that MIF signaling is required for PHLDA2-induced PD-L1 upregulation via STAT3 activation. m6A modification patterns and the PHLDA2-RGS13 signature predict LUAD prognosis and immunotherapy response. PHLDA2 contributes to tumor progression and immune evasion through MIF-dependent signaling in mediating PHLDA2-driven PD-L1 expression, highlighting this axis as a potential therapeutic target.