Abstract
Lung adenocarcinoma (LUAD) is the most prevalent histological subtype of non-small cell lung cancer (NSCLC) and is characterized by high mortality and limited therapeutic efficacy in advanced stages. AVEN, an apoptosis inhibitor that interacts with Bcl-xL and Apaf-1 to suppress caspase activation, has been implicated in tumour progression and drug resistance in various cancers. However, its role in LUAD remains unclear. In this study, the prognostic importance, immune microenvironment association, and regulatory mechanisms of AVEN in LUAD were comprehensively investigated using bulk RNA sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), and experimental validation. Analysis of the TCGA and GEO datasets revealed that AVEN expression was significantly upregulated in LUAD tissues compared with normal tissues and that high AVEN expression correlated with advanced T/N stage and pathological stage and was associated with poor overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFS). Multivariate Cox regression identified AVEN expression as an independent prognostic factor, and a nomogram incorporating AVEN expression demonstrated high predictive accuracy for 1-, 3-, and 5-year OS. Functional enrichment analysis linked AVEN to keratinocyte differentiation, spliceosome activity, and cell cycle pathways, whereas the results of scRNA-seq highlighted its predominant expression in malignant epithelial cell subtypes (tS2), which is associated with aggressive proliferation and immune evasion. AVEN expression was positively correlated with Th2, NK CD56dim, and Tgd cell infiltration but negatively associated with TFH, eosinophil, and mast cell infiltration, suggesting its role in modulating the tumour immune microenvironment. Detection of clinical samples verified the high expression of AVEN in LUAD. In vitro, AVEN knockdown in A549 cells suppressed proliferation, migration, and invasion while promoting apoptosis. Furthermore, bioinformatics prediction and validation revealed that hsa-miR-30d-5p was an upstream regulator of AVEN, with its low expression in LUAD tissues inversely correlated with that of AVEN and predicting a favourable prognosis. Subsequent bioinformatics analysis further revealed that lncRNA-AC012236.1 functioned as an upstream regulator of hsa-miR-30d-5p. This lncRNA was found to be highly expressed in LUAD tissues, and its elevated expression was significantly associated with poor overall survival (OS) in LUAD patients. In conclusion, AVEN, as a promising diagnostic and prognostic biomarker in LUAD, affected tumour progression, immune infiltration and apoptosis resistance through the lncRNA-AC012236.1/hsa-miR-30d-5p-AVEN axis. These findings provided new insights into the pathogenesis of LUAD and highlighted potential therapeutic targets for improving patient prognosis.