Network Pharmacology and Transcriptomic Analysis Reveal the Mechanism by Which Ginsenoside CK Remodels the Tumor Immune Microenvironment to Inhibit Liver Cancer Progression.

The study aimed to investigate the mechanism by which ginsenoside CK downregulates EZH2 to activate the dendritic cell-NK cell axis, promoting antitumor immunity in hepatocellular carcinoma (HCC). Potential targets of ginsenoside CK for HCC treatment were identified utilizing network pharmacology, followed by protein-protein interaction (PPI) network analysis, and GO and KEGG functional enrichment analyses. Differential expression analysis of HCC-related transcriptomic data from the GEO database (GSE84005) was conducted to identify key genes. The expression and prognostic relevance of key genes were verified utilizing the GEPIA and Kaplan-Meier Plotter databases. Molecular docking technology was used to study the binding characteristics of ginsenoside CK to key targets. The liver cancer samples were further grouped according to EZH2 expression, and co-expressed genes were screened and functionally annotated. The correlation between key gene expression and immune cell infiltration was analyzed utilizing the ssGSEA algorithm. Human HCC cell lines MHCC97 and Hep3B were cultured, and cell proliferation and colony formation were assessed utilizing CCK-8 and colony formation assays. Transwell migration and invasion assays evaluated changes in cell migration and invasion. Flow cytometry was employed to analyze cell apoptosis and cell cycle distribution. A xenograft mouse model was established to monitor tumor volume and body weight changes. Immunohistochemistry was used to assess Ki67 expression in tumor tissues, and flow cytometry measured the proportions of NK cells and dendritic cells in tumor tissues. qRT-PCR and Western blotting were performed to evaluate the expression levels of related factors. Network pharmacology analysis identified 114 potential targets of ginsenoside CK and 3991 potential targets of HCC, with 83 intersecting targets. GO and KEGG analyses indicated these targets were involved in phosphatidylinositol-mediated signaling, epithelial cell proliferation and migration, and regulation of MAP kinase activity, suggesting regulation through multiple immune-related signaling pathways in HCC. Transcriptomic analysis revealed that the core target EZH2 was highly expressed in HCC, and high expression correlated with poorer overall survival and relapse-free survival. Molecular docking confirmed that ginsenoside CK was stably bound to the active site of EZH2 (binding energy: -9.1 kcal/mol). Co-expression analysis showed that EZH2 was closely related to the cell cycle, p53 pathway and transcription factor E2F8/MYBL2. Immune infiltration analysis indicated that EZH2 negatively regulated the dendritic cell-NK cell axis, contributing to the remodeling of the tumor immune microenvironment. In vitro experiments demonstrated that ginsenoside CK downregulated EZH2, inhibiting HCC cell proliferation, migration, and invasion, while EZH2 overexpression reversed these inhibitory effects. In vivo experiments confirmed that ginsenoside CK suppressed tumor formation by downregulating EZH2, activating the dendritic cell-NK cell axis, and remodeling the tumor immune microenvironment. Ginsenoside CK inhibits EZH2, activating the dendritic cell-NK cell axis and remodeling the tumor immune microenvironment, thereby suppressing HCC cell activity and tumorigenicity.