Uncovering core stemness biomarkers in HBV-HCC through integrated multi-omics

Chronic hepatitis B virus (HBV) infection is a major etiology of hepatocellular carcinoma (HCC), with cancer stem cells (CSCs) playing a critical role in tumor progression and therapeutic resistance. However, the specific mechanisms linking HBV infection to stemness features in HBV-associated HCC (HBV-HCC) remain incompletely defined. To identify potential regulators, we conducted an integrated in silico multi-omics analysis using single-cell and bulk transcriptomic datasets. Comparative stemness scoring across major cell populations prioritized the endothelial compartment for further analysis. Subsequent pseudotime trajectory and CytoTRACE2 analysis of single-cell RNA sequencing (scRNA-seq) data identified Cluster 4 (C4) as a putative stemness-associated endothelial subpopulation, from which 21 differentially positive-HBV-expressed genes (DPEGs) were derived. In bulk RNA sequencing (bulk RNA-seq), weighted gene co-expression network analysis (WGCNA) identified genes strongly correlated with stemness in the blue module. Cross-referencing scRNA-seq and bulk RNA-seq yielded 52 shared genes. Random Forest feature selection prioritized two high-confidence biomarkers: SSR2 and UBE2D3. A combined diagnostic model based on these two genes showed favorable performance across independent cohorts with bootstrap-supported validation, and survival analyses indicated that high SSR2 expression and low UBE2D3 expression were associated with poor clinical outcomes. Mechanistically, pathway activity correlation analyses revealed that SSR2 and UBE2D3 are significantly associated with endoplasmic reticulum stress (ERS), and mTOR signaling, suggesting a potential coordinated role in regulating viral persistence and stemness maintenance. Overall, our findings propose SSR2 and UBE2D3 as candidate diagnostic and prognostic biomarkers, providing a hypothesis-generating framework for developing targeted therapeutic strategies against HBV-HCC.