Clonorchis sinensis-driven Hepatocarcinogenesis via E2F1-CD24 Transcriptional Axis: Mechanistic and Therapeutic Implications

Hepatocellular carcinoma (HCC) persists as a global health burden with disproportionately high mortality in China's Guangxi region, where endemic Clonorchis sinensis (C. sinensis) infection coincides with elevated HCC mortality. This study aims to elucidate the oncogenic mechanisms of C. sinensis excretory-secretory products (CsESPs) through integrated clinical and experimental approaches. Our preliminary single-cell sequencing initially revealed marked cluster of differentiation 24 (CD24) overexpression in HCC tissues, prompting systematic investigation of its pathological relevance. Analysis of the institutional clinical cohort demonstrated significant CD24 upregulation, particularly in C. sinensis-infected HCC cases, while multi-platform bioinformatics validation (GEPIA/UALCAN/TIMER) established its prognostic value for survival reduction and immune microenvironment modulation. Functional characterization using qPCR, immunoblotting, CCK-8 assays, and flow cytometry demonstrated that CsESPs upregulated CD24 expression, concomitant with accelerated cell proliferation and apoptosis suppression. Mechanistic studies employing chromatin immunoprecipitation and dual-luciferase reporter assays identified E2F1-mediated transcriptional activation through direct promoter binding as the principal regulator of CsESPs-induced CD24 expression. More importantly, siRNA-mediated CD24 silencing abrogated CsESPs-mediated HCC cell proliferation and apoptosis restoration. Furthermore, CsESPs upregulated immune checkpoints CTLA-4 and LAG-3 in PBMC that co-cultured with HCC cells, reversibly modulated through CD24 knockdown. Taken together, these findings establish a novel parasitic carcinogenesis paradigm wherein C. sinensis promotes HCC development through E2F1-CD24 transcriptional activation, simultaneously identifying prognostic biomarkers and therapeutic targets while suggesting combinatory immunotherapy strategies for parasite-associated HCC.