Lu Wen-Min, Yan Jin, Liu Zhao-Ji, Wu Yong, Cui Qian-Ru, Feng Ji, Chen Yu, Zhu Guang-Zhi, Peng Tao, Zhou Jing, Lu Guo-Dong
Department of Toxicology, School of Public Health, Guangxi Medical University, Nanning, Guangxi, China.
Department of Physiology, School of Basic Medical Sciences, Guangxi Medical University, Nanning, Guangxi, China.
Parasit Vectors. 2025 Aug 19;18(1):353. doi: 10.1186/s13071-025-06979-6.
Hepatocellular carcinoma (HCC) remains a global health burden, with disproportionately high mortality in China's Guangxi region, where endemic Clonorchis sinensis (C. sinensis) infection coincides with elevated HCC incidence. Preliminary single-cell sequencing revealed marked overexpression of cluster of differentiation 24 (CD24) in HCC tissues, suggesting its potential pathological role. This study aims to elucidate the oncogenic mechanisms of C. sinensis excretory-secretory products (CsESPs) and their link to CD24-mediated HCC progression.
We employed an integrated clinical and experimental approach. First, clinical cohort analysis assessed CD24 expression in C. sinensis-associated HCC cases. Multiplatform bioinformatics validation (GEPIA/UALCAN/TIMER) evaluated CD24's prognostic significance and immune microenvironment modulation. Functional studies (quantitative polymerase chain reaction (qPCR), Western blotting, CCK-8 assays, flow cytometry) examined CsESPs' effects on CD24 expression, cell proliferation, and apoptosis. Mechanistic investigations (chromatin immunoprecipitation, dual-luciferase reporter assays) identified E2F1-mediated transcriptional activation of CD24. siRNA-mediated CD24 knockdown validated its role in CsESPs-driven oncogenesis. Additionally, the expression of immune checkpoint (CTLA-4, LAG-3) was assessed in the co-cultures of peripheral blood mononuclear cells (PBMCs)-HCC cells.
Clinical cohort analysis confirmed significant CD24 upregulation in HCC, particularly in C. sinensis-infected cases. Bioinformatic analyses linked high CD24 expression to poor prognosis and immune microenvironment alterations. Functional assays demonstrated that CsESPs enhance CD24 expression, promoting proliferation and inhibiting apoptosis. Mechanistically, E2F1 directly binds to CD24 promoter, driving its transcription upon CsESPs exposure. CD24 silencing reversed CsESPs-induced oncogenic effects. Furthermore, CsESPs upregulated immune checkpoints (CTLA-4, LAG-3) in the co-cultures of PBMC-HCC cells, an effect reversed by CD24 knockdown.
Our findings establish a novel parasitic carcinogenesis paradigm wherein C. sinensis promotes HCC development through E2F1-mediated transcriptional activation of CD24, simultaneously identifying prognostic biomarkers and therapeutic targets while suggesting combinatory immunotherapy strategies for parasite-associated HCC.
肝细胞癌（HCC）仍然是一个全球性的健康负担，在中国广西地区死亡率极高，该地区华支睾吸虫（C. sinensis）的地方性感染与HCC发病率升高同时存在。初步的单细胞测序显示HCC组织中分化簇24（CD24）明显过表达，提示其潜在的病理作用。本研究旨在阐明华支睾吸虫排泄分泌产物（CsESPs）的致癌机制及其与CD24介导的HCC进展的联系。
我们采用了临床与实验相结合的方法。首先，临床队列分析评估了华支睾吸虫相关HCC病例中CD24的表达。多平台生物信息学验证（GEPIA/UALCAN/TIMER）评估了CD24的预后意义和免疫微环境调节。功能研究（定量聚合酶链反应（qPCR）、蛋白质免疫印迹法、CCK-8检测、流式细胞术）检测了CsESPs对CD24表达、细胞增殖和凋亡的影响。机制研究（染色质免疫沉淀、双荧光素酶报告基因检测）确定了E2F1介导的CD24转录激活。siRNA介导的CD24敲低验证了其在CsESPs驱动的肿瘤发生中的作用。此外，在人外周血单个核细胞（PBMC）-HCC细胞共培养中评估了免疫检查点（CTLA-4、LAG-3）的表达。
临床队列分析证实HCC中CD24显著上调，尤其是在华支睾吸虫感染的病例中。生物信息学分析将高CD24表达与不良预后和免疫微环境改变联系起来。功能试验表明，CsESPs增强CD24表达，促进增殖并抑制凋亡。机制上，E2F1直接结合到CD24启动子上，在暴露于CsESPs时驱动其转录。CD24沉默逆转了CsESPs诱导的致癌作用。此外，CsESPs上调了PBMC-HCC细胞共培养中的免疫检查点（CTLA-4、LAG-3），CD24敲低可逆转这一作用。
我们的研究结果建立了一种新的寄生虫致癌模式，即华支睾吸虫通过E2F1介导的CD24转录激活促进HCC发展，同时确定了预后生物标志物和治疗靶点，并提出了针对寄生虫相关HCC的联合免疫治疗策略。
