Integrated Single-Cell and Spatial Transcriptomics Identifies an RBM8A/HDAC9/p16 Axis Driving Early Recurrence in Hepatocellular Carcinoma

Background Surgical resection remains the primary curative therapy for early-stage hepatocellular carcinoma, yet high early recurrence driven by profound tumor heterogeneity severely limits long-term patient survival. Identifying specific malignant subpopulations and dissecting their core regulatory mechanisms represents a critical unmet clinical challenge. Methods We integrated single-cell and spatial transcriptomic profiling on primary tumor specimens from patients experiencing early postoperative recurrence. Core regulator functions were rigorously validated through comprehensive in vitro and in vivo experiments, including dominant-negative mutant modeling. Molecular interactions were precisely characterized using RNA immunoprecipitation, pull-down assays, chromatin immunoprecipitation, and complementary biochemical approaches. Results We identified a distinct malignant subpopulation, designated the C2 cluster, which was selectively enriched in early-recurrence tissues and exhibited intrinsic invasiveness alongside pronounced chemoresistance. Multi-omics analyses revealed extensive aberrant RNA-binding protein-mediated post-transcriptional regulation within this cluster, with RBM8A functioning as a central hub essential for sustaining aggressive phenotypes. Mechanistically, RBM8A directly binds and stabilizes HDAC9 messenger RNA, subsequently triggering histone H3K18 deacetylation and epigenetic silencing of p16. This establishes a novel RBM8A/HDAC9/p16 signaling axis that rapidly accelerates cell cycle progression. Importantly, the oncogenic activity of this pathway strictly depends on wild-type RBM8A functionality, as definitively confirmed by the potent inhibitory effects observed with the RBM8A-W73V dominant-negative mutant. Conclusions This study uncovers a unique hepatocellular carcinoma subpopulation harboring a novel signaling axis that drives early recurrence through intricate post-transcriptional and epigenetic crosstalk. RBM8A serves as a pivotal node governing the recurrence chemoresistance vicious cycle, providing promising prognostic biomarkers and precise therapeutic targets for future clinical applications.