Multi-Omics Analysis and Experimental Validation Uncover Neddylation-Associated Biomarkers and Cellular Landscape in Lung Adenocarcinoma

Background: Accumulating evidence suggests that neddylation contributes to tumor initiation and progression. Nonetheless, its biological functions and regulatory mechanisms in lung adenocarcinoma (LUAD) remain poorly understood. Methods: Bioinformatics analyses integrated single-cell RNA sequencing (scRNA-seq) datasets from the Gene Expression Omnibus (GEO) with bulk RNA-seq data from The Cancer Genome Atlas (TCGA). The Seurat package was used for scRNA-seq processing, including cell clustering, annotation, cell-cell interaction analysis, and pseudotime trajectory inference. The survival and pROC packages were used for Kaplan–Meier (KM) survival and receiver operating characteristic (ROC) analyses, respectively. Tissue microarray (TMA) and immunohistochemistry (IHC) analyses were performed to validate biomarker expression. Functional assays, including Cell Counting Kit-8 (CCK-8), colony formation, wound healing, and Transwell assays, were conducted in A549 and NCI-H1975 cells with DNAJB1 knockdown. Results: Eight major cell populations were identified at the single-cell level, among which T/NK cells, particularly CD8+ effector memory T cells (CD8+ Tem), exhibited strong associations with neddylation. T/NK cells displayed extensive ligand-receptor-mediated interactions with other cell types. A transcription factor (TF)–target regulatory network was constructed, revealing that Surfactant Protein C (SFTPC), Lipopolysaccharide-Induced TNF Factor (LITAF), Serine/Threonine Kinase 17B (STK17B), Enah/Vasp-Like Protein (EVL), FYN Proto-Oncogene, Src Family Tyrosine Kinase (FYN), and Lymphocyte-Specific Protein 1 (LSP1) were associated with favorable prognosis, whereas DNAJB1 correlated with poor survival in LUAD patients. Pseudotime trajectories further delineated dynamic differentiation pathways of T/NK subsets and differential TF target expression across states. IHC analysis showed elevated DNAJB1 expression in tumors compared with adjacent normal tissues. Moreover, silencing DNAJB1 significantly inhibited proliferation, migration, and invasion in A549 and NCI-H1975 cells. Conclusion: This study identifies neddylation-associated cellular phenotypes and clinically relevant biomarkers in LUAD, providing new mechanistic insights and highlighting potential therapeutic targets for LUAD treatment.