Multiomics analysis reveals putative neural invasion-associated transcriptional signatures of hepatocellular carcinoma

Perineural invasion (PNI) drives tumor progression, local recurrence, and metastasis in pancreatic, prostate, and head/neck cancers. In hepatocellular carcinoma (HCC), the most common primary liver cancer, classical PNI is rarely observed, and the molecular mechanisms governing HCC dissemination remain poorly understood. The tumor-neural microenvironment critically regulates cancer progression, yet whether HCC harbors transcriptional programs matching neural invasion-related gene expression patterns has never been systematically investigated. This knowledge gap limits accurate prognostic stratification and the development of targeted therapies. Accordingly, we aimed to identify a putative neural invasion-associated transcriptional signature in HCC and evaluate its clinical relevance. A neural invasion-related gene set was derived from GeneCards and scored in single-cell data using five algorithms (AUCell, UCell, singscore, ssGSEA, AddModuleScore). Endothelial cells, exhibiting the highest correlation, underwent differential analysis. A random survival forest (RSF) model with 10-fold cross-validation (importance > 0.65) identified hub genes, validated by RT-qPCR, siRNA knockdown, and HCMEC/D3-HCC co-culture transendothelial invasion assays. Multi-omics analyses included GSEA, GSVA, drug sensitivity prediction, RCTD spatial deconvolution, MISTy interaction analysis, and PROGENy pathway quantification. RSF identified ANXA5, STMN1, and YBX1 as core genes, overexpressed in Hep3B and Huh7 compared with THLE-2 cells. siRNA silencing suppressed proliferation, migration, invasion, and transendothelial migration. GSEA/GSVA revealed enrichment in cell cycle, DNA repair, Notch, and VEGF pathways. Spatial transcriptomics showed elevated TNF-α activity in endothelial and hepatocyte subtypes. Expression correlated with sorafenib and 5-fluorouracil sensitivity. High expression of each gene independently predicted poor overall survival in TCGA-HCC. ANXA5, STMN1, and YBX1 constitute a putative neural invasion-associated transcriptional signature in HCC, with in vitro evidence supporting oncogenic roles in proliferation, migration, and transendothelial invasion. However, this signature reflects transcriptomic similarity rather than histologically confirmed PNI, and causal links to authentic neural invasion remain unestablished. This exploratory study offers a preliminary reference for HCC prognostic stratification.