APLP2 as a Molecular Link Between Immune Regulation and Bone Metabolism in Hepatocellular Carcinoma: Evidence from scRNA-Seq and Functional Validation

Background and Aims: Hepatocellular carcinoma (HCC) remains a significant health concern worldwide, characterized by elevated mortality rates that are often associated with diagnoses occurring in advanced stages and the restricted efficacy of treatment options currently available. Immune checkpoint inhibitors (ICIs) demonstrate promise in treating HCC; nonetheless, challenges related to therapeutic resistance and varied responses among patients underscore the necessity of identifying new biomarkers and comprehending the fundamental mechanisms involved. This research explores the molecular relationship between immune regulation and bone metabolism in HCC by employing integrated single-cell RNA sequencing (scRNA-seq), bulk transcriptomics, and functional validation. Methods: The analysis of publicly accessible scRNA-seq (GSE223204) and bulk RNA-seq (TCGA-LIHC) datasets was conducted to discover distinct cell subpopulations and signaling patterns. Clustering, ligand-receptor interaction analysis, and transcription factor mapping were performed using the Seurat, CellChat, and SCENIC pipelines. A random survival forest method helped identify important prognostic genes. The research examined how immune cells infiltrate and their relationship with components that regulate the immune response. Clinical HCC samples were obtained for validation using qPCR. The functional effects of the gene APLP2 were studied through small interfering RNA (siRNA) knockdown experiments in HCC cell lines and co-culturing with osteoblasts. Results: In the tissues of HCC, nine distinct cell types were recognized, where hepatocytes demonstrated significant involvement in pathways related to bone metabolism and immune functions. Seven key genes (APLP2, SERPINC1, CAT, PDIA6, SLC2A2, C1S, and CFB) were found to be prognostically significant and closely linked to immune cell infiltration, immunomodulatory checkpoints, and key metabolic signaling pathways, including WNT/β-catenin and PI3K-AKT-mTOR. Particularly, APLP2 showed increased expression specifically in cancerous tissues. Reduced APLP2 levels suppressed proliferation, invasion, migration, and promoted apoptosis in HCC cells. Moreover, the downregulation of APLP2 lessened the suppressive influence of tumor cells on osteoblast differentiation, indicating its potential regulatory function in bone metabolism. Conclusion: This research highlights APLP2 as a new molecular connector between immune evasion and dysregulation of bone metabolism in HCC. The combination of single-cell analysis along with experimental validation offers fresh perspectives on the underlying mechanisms of immunotherapy resistance and emphasizes APLP2 as a promising dual-function therapeutic target.