Ribosome biogenesis (RiboSis) and ribosomal stress are critical in tumor progression, positioning RiboSis as a promising therapeutic target for cancer treatment and for overcoming drug resistance. In this study, we examined the role of RiboSis in the progression from hepatitis B virus (HBV) infection to HBV-related hepatocellular carcinoma (HCC), focusing specifically on nucleolar protein 9 (NOL9) and its influence on HCC pathogenesis and therapeutic response. Our findings showed that NOL9 was significantly upregulated in HCC tissues, correlating with larger tumor sizes and more advanced pathological grades. High levels of NOL9 expression were associated with unfavorable prognosis in both the TCGA-LIHC and our HCC cohorts. Functional assays indicated that NOL9 regulated HCC cell proliferation and apoptosis
specifically, NOL9 knockdown inhibited cell proliferation and promoted apoptosis, while overexpression enhanced these processes. In vivo studies confirmed that NOL9 depletion reduced tumor growth. Mechanistically, NOL9 expression was regulated by DNA methylation and the transcription factor ZNF384. Our DNA methylation analysis revealed an inverse correlation between NOL9 expression and methylation at specific CpG sites, implicating DNMT1 in its epigenetic regulation. Additionally, NOL9-mediated cell proliferation was dependent on activation of the Wnt/β-catenin signaling pathway. This study highlights the multifaceted role of NOL9 in HCC pathogenesis, underscoring its potential as a diagnostic biomarker and therapeutic target.