Prognostic Signature of NETs-Related Genes in Hepatocellular Carcinoma Based on Bulk and Single-Cell Transcriptomics.

Abstract

Background: Inflammation drives tumor development, with neutrophil extracellular traps (NETs) promoting progression through metastasis, immune suppression, and microenvironment modulation. However, the role of NETs-related genes in hepatocellular carcinoma (HCC) immunity response is still unclear.

Methods: We integrated single-cell RNA sequencing (GSE202642, seven tumor samples and four normal liver samples) and The Cancer Genome Atlas (TCGA, n=312) transcriptomic data to identify NETs-related gene signatures. Weighted gene co-expression network analysis (WGCNA) identified NETs-correlated gene modules, and LASSO-COX regression selected prognostic genes for risk stratification. A nomogram was developed to predict survival, while functional, mutation, immune, and drug sensitivity analyses highlighted intergroup differences. EdU and CCK-8 cell proliferation assays confirmed the role of NETs-related genes in HCC cell proliferation.

Results: The analysis revealed significant differences in survival time between high- and low-NETs groups. GAS2L3 and RTN3 were identified and validated as independent prognostic factors. ROC and decision curve analysis (DCA) demonstrated that the nomogram model combining NETs risk scores with clinical parameters exhibited robust prognostic performance. The high-risk subgroup was enriched in glycosphingolipid biosynthesis pathways and showed higher mutati843on rates, especially in TP53, CTNNB1, and MUC16, along with overexpression of immunosuppressive genes (VTCN1, LAIR1). In vitro experiments confirmed that GAS2L3 knockdown inhibited HepG2 and Huh7 cell proliferation.

Conclusion: Integrated multi-omics analysis revealed NETs-associated prognostic signatures in HCC, with GAS2L3 identified as a key gene linking NETs to tumor progression and therapeutic potential.