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

Clonorchis sinensis-driven Hepatocarcinogenesis via E2F1-CD24 Transcriptional Axis: Mechanistic and Therapeutic Implications | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Clonorchis sinensis-driven Hepatocarcinogenesis via E2F1-CD24 Transcriptional Axis: Mechanistic and Therapeutic Implications Wen-Min Lu, Jin Yan, Zhao-Ji Liu, Yong Wu, Qian-Ru Cui, Ji Feng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6378057/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Aug, 2025 Read the published version in Parasites & Vectors → Version 1 posted 7 You are reading this latest preprint version Abstract 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. Hepatocellular Carcinoma Clonorchis sinensis CD24 E2F1 Tumor Promotion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Globally, liver cancer constitutes the third most lethal malignancy and sixth most frequently diagnosed cancer according to 2022 International Agency for Research on Cancer statistics [1]. Chinese epidemiological surveillance demonstrates concordance, with liver cancer representing the second leading cause of cancer-related mortality and fourth most diagnosed malignancy nationally [2]. Among the various subtypes of liver cancer, hepatocellular carcinoma (HCC, referred to as liver hepatocellular carcinoma (LIHC) in database) is the most prevalent, accounting for approximately 85% of cases [3]. While nationwide HBV vaccination initiatives and aflatoxin control have driven progressive reductions in HCC incidence and mortality over two decades, significant regional disparities persist [4, 5]. National cancer registry data identify Guangxi province exhibits China's highest HCC mortality rates, with gender-specific rates of 69.0/100,000 (male) and 17.8/100,000 (female) - markedly exceeding national averages of 36.5/100,000 and 12.8/100,000 respectively [6]. Notably, Guangxi demonstrates paradoxical upward trends in HCC incidence (annual percentage change +5.38%) and mortality (+9.23%) during 2010-2016 [7], indicating distinct etiological factors beyond conventional HBV- or aflatoxin-associated hepatocarcinogenesis. Of particular epidemiological significance, this region demonstrates China's highest prevalence of endemic Clonorchis sinensis ( C. sinensis ) infection (6.68%) in China [8], strongly associated with local dietary practices of raw fish consumption. The persistent geographic overlap between exceptional HCC mortality burdens and helminthic infection prevalence implies potential proto-oncogenic interactions warranting mechanistic investigation. C. sinensis , commonly known as the liver fluke, is a food-borne parasite prevalent in China, Korea, Northern Vietnam, and the Russian Far East [9]. Human infection occurs through the consumption of raw or undercooked freshwater fish containing C. sinensis metacercariae. Chronic C. sinensis infection induces progressive hepatobiliary pathology including biliary hyperplasia, periductal fibrosis, and cholangiocarcinoma development through sustained inflammatory responses [10, 11]. With 15 million global cases (87% in China) [12], this IARC Group 1 carcinogen [13] demonstrates increasing oncogenic evidence in liver cancer pathogenesis. Recent clinical investigations revealed C. sinensis co-infection with HBV accelerates HCC progression by enhancing cancer stem cell properties, serving as an independent prognostic factor for reduced overall survival (5-year OS: 47.8% vs. 63.8% in HBV alone-infected cohorts; n=946) [14, 15]. Experimental models demonstrate infection promotes hepatic progenitor cell proliferation [16] and induces M1-like macrophage polarization via extracellular vesicle-derived Csi-let-7a-5p, mediating biliary injury through Socs1/Clec7a-dependent NF- κ B activation [17]. Other mechanistical studies revealed that the excretory-secretory protein CsGRN drives malignant transformation through EGFR-mediated RAS/MAPK/ERK and PI3K/AKT pathway activation in hepatocytes [18, 19], with complementary studies confirming its transformative effects on human intrahepatic cholangiocytes in vitro and biliary injury in vivo [20]. Building upon these findings, our preliminary single-cell sequencing data identify CD24 overexpression in C. sinensis -associated HCC specimens (unpublished observations). CD24, commonly referred to as the heat-stable antigen, is a surface protein anchored by glycosylphosphatidylinositol [21, 22]. It is overexpressed in various cancers and is implicated in regulating cancer cell proliferation, invasion, and immune evasion [23-25]. For instance, in ovarian and breast cancers, CD24 engages with Siglec-10 found on tumor-associated macrophages, thereby promoting immune evasion [21]. In liver, CD24 is absent in normally differentiated hepatocytes but detectable in hepatic progenitor cells (oval cells), suggesting a potential association with hepatic stem cell characteristics [26]. Notably, CD24 is overexpressed in HCC, where its expression correlates with increased tumor invasiveness, metastatic potential, enhanced proliferation, and activation of the Wnt/ β -catenin signaling pathway [27]. However, the role of CD24 in the pathogenesis of HCC following C. sinensis infection remains poorly understood. Given the potential link between C. sinensis infection and HCC progression, our objective was to explore the underlying mechanisms through which CsESPs influence CD24 expression and function in HCC cells and co-cultured immunocytes. Materials and methods Clinical sample collection Human primary HCC tissues and paired normal tissues were collected from September 2020 to December 2022 at the First Affiliated Hospital of Guangxi Medical University. All HCC patients underwent surgical operation. Informed consent was acquired from all participants, and the research received approval from the Ethics Committee at the First Affiliated Hospital of Guangxi Medical University (Ethics approval number: 2024-E580-01). Diagnosis of C. sinensis infection was established based on at least one diagnostic criterion: serological evidence from ELISA testing or microscopic identification of the parasite in stool, bile, or pathological specimens [28]. Bioinformatic analysis The study utilized GEPIA (http://gepia.cancer-pku.cn/index.html) to explore the expression of CD24 in HCC, UALCAN (http://ualcan.path.uab.edu/) to analyze the relationship between CD24 mRNA expression in HCC and various pathological parameters, and TIMER2.0 (http://timer.cistrome.org/) to assess the correlation between CD24 expression, immune cell infiltration, and immune checkpoints. Finally, we employed the Kaplan-Meier plotter database (http://kmplot.com) to evaluate the prognostic significance of CD24 regarding OS, progression-free survival (PFS), recurrence free survival (RFS) and disease specific survival (DSS). The analysis included hazard ratios (HR) with 95% confidence intervals (95% CI) and log-rank p-values. Potential transcription factors binding to the CD24 promoter region were predicted using the PROMO (https://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) and Harmonizome (https://maayanlab.cloud/Harmonizome/) databases. Preparation CsESPs Pseudorasbora parvas , recognized as the second intermediate host for C. sinensis , was sourced from Hengzhou, Guangxi, exhibiting natural infection with metacercariae. The fish specimens underwent digestion with an artificial gastric solution composed of 0.5% pepsin, 1% hydrochloric acid, and 0.9% sodium chloride NaCl, maintained at 37 °C overnight. Male Sprague-Dawley rats were procured from the Guangxi Medical University Laboratory Animal Center and were cared for in compliance with the National Institutes of Health's standards for animal welfare and ethical treatment (Ethics approval number: 202410011). Metacercariae were extracted using a stereomicroscope, and a total of 150 metacercariae were administered intragastrically to each rat. Following an 8-week infection period, the rats were euthanized post-anesthesia, allowing for the collection of adult worms from the bile ducts, which were subsequently cultured in phosphate-buffered saline (PBS), maintained at 37 °C in an atmosphere containing 5% CO₂. The culture medium was collected after a duration of 6 hours, followed by centrifugation at 12,000×g for 10 minutes at 4 °C. The resulting supernatant, designated as CsESPs, was sterilized through a 0.22 μm filter and preserved at -80 °C. The concentrations of CsESPs were quantified utilizing the bicinchoninic acid assay. Cell culture Human HCC cell lines Huh7 and Hep3B were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China) and validated through STR identification. These cell lines were maintained in Dulbecco’s Modified Eagle’s Medium enriched with 10% fetal bovine serum and 1% penicillin-streptomycin. Cell transfection Short interfering RNAs (siRNAs) targeting CD24 were synthesized by GenePharma and conducted as previously described [29]. For the siRNA sequences, please refer to Supplementary Table 1 for details. The silencing effect on mRNA expressions were examined by qPCR analysis. Detection of cell viability and colony formation Cell viability was evaluated utilizing the Cell Counting Kit-8 (CCK-8) assay. Huh7 and Hep3B cell lines were exposed to 100 µg/ml concentrations of CsESPs for a duration of 48 hours. Subsequently, CCK-8 was combined with the culture medium at a ratio of 1:10, applied to each well, and incubated for 1.5 hours, in accordance with previously established protocols [30]. The colony formation assay was conducted as previously outlined [31], with 1,000 cells being plated in 12-well plates and subjected to treatment with the specified drugs over a period of 10 days. After performing crystal violet staining, photographic documentation of the colonies was obtained. Apoptosis assay To evaluate cell apoptosis, we utilized the Annexin V-APC/PI Apoptosis Kit (Lianke). In summary, HCC cells were collected via accutase treatment. Following this, the cells were resuspended in binding buffer and subsequently stained with 5 µl of Annexin V-APC and propidium iodide for a duration of 5 minutes. The analysis of apoptosis was conducted using a CytoFLEX Flow Cytometer. Western blotting (WB) assay and immunofluorescence WB was performed as previously described [32]. Primary antibodies E2F1 (sc-251) were obtained from Santa Cruz Biotechnology (USA), CD24 (67627-1-Ig), Bcl-2-associated X protein (BAX) (50599-2-Ig), ACTIN (66009-1-Ig) and α-Tubulin (66031-1-Ig) from Proteintech (China). Immunofluorescence was performed as described previously [33]. Observational data and imaging were captured utilizing a confocal microscope (Zeiss LSM 800). Real-time quantitative polymerase chain reaction (qPCR) RNA extraction was performed utilizing TRIzol reagent in accordance with the guidelines provided by the manufacturer. To assess mRNA expression levels, qPCR was conducted employing the 2× Universal Blue SYBR Green qPCR Master Mix (Servicebio), adhering strictly to the manufacturer's protocol. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control for normalization. All primers utilized for qPCR were procured from Sangon Biotechnology Ltd., and the specific sequences of these primers are detailed in Supplementary Table 2. Dual-Luciferase reporter assay The wild-type and mutant luciferase plasmids of the CD24 promoter were constructed by Hunan Fenghui Biotechnology Co., Ltd. (China). Similarly, the vector and overexpression plasmids of E2F1 were constructed by Wuhan MiaoLing Biotechnology Co., Ltd. (China). The binding sites of E2F1 on the CD24 promoter were predicted using the JASPAR database (https://jaspar.elixir.no/). HEK-293T cells were initially resuspended and subsequently plated into 6-well culture plates. The CD24 promoter luciferase plasmids were co-transfected into the HEK-293T cells alongside either a control vector or plasmids designed for E2F1 overexpression. Following a 48-hour incubation period, cell lysates were harvested, and the activities of firefly and renilla luciferase were quantified utilizing the Dual-Luciferase Reporter Assay Kits (Promega). Chromatin immunoprecipitation (ChIP) assay ChIP was executed in accordance with the guidelines provided by the manufacturer (Beyotime Biotechnology). In summary, cells were harvested and subjected to crosslinking using 1% formaldehyde. Subsequently, the DNA underwent fragmentation through sonication. Pre-washing was conducted with agarose beads, and then the mixture was incubated with 5 μg of E2F1 (Santa Cruz, sc-251) in the culture medium. Ultimately, DNA fragments were isolated utilizing magnetic beads, followed by quantification through qPCR. Isolation of peripheral blood mononuclear cells (PBMCs) PBMCs were extracted from EDTA-anticoagulated peripheral blood obtained from healthy individuals. The whole blood sample was diluted with an equivalent volume of PBS and carefully layered over an equal volume of lymphocyte separation medium. PBMCs were then isolated by gradient centrifugation. Following centrifugation at 800g for 25 minutes, the mononuclear cell layer was harvested and washed three times with PBS. Co-culture of HCC cells with PBMCs PBMC and hepatocellular carcinoma cells were co-cultured using a non-contact co-culture transwell system (JETBIOFIL, China). HCC cells were maintained in the upper chamber, whereas PBMCs were grown in the lower chamber, using RPMI-1640 medium enriched with 10% fetal bovine serum and 1% penicillin-streptomycin. After 48 hours of co-incubation, PBMCs were harvested for further analyses. Statistical analysis Data analysis was conducted utilizing SPSS software (version 22.0), with results expressed as the mean ± standard error of the mean (SEM). Statistical visualizations were created employing GraphPad Prism 8 software. Group comparisons were carried out using either the independent samples t -test or the Mann-Whitney U-test, depending on the data distribution. Alternatively, one-way analysis of variance (ANOVA) was employed, followed by multiple comparisons using either the least significant difference (LSD) test or the Games-Howell correction. A p -value of less than 0.05 was considered statistically significant. Results CD24 expression is up-regulated in HCC Using our in-house cohort, we evaluated CD24 mRNA expression in HCC tumor and paired adjacent non-tumor tissues. qPCR analysis revealed significant upregulation of CD24 in C. sinensis -infected HCC tumors compared to adjacent tissues (Figure 1A-C). To validate these findings, we conducted comprehensive bioinformatic analyses using multiple independent datasets (GEPIA, UALCAN, and TIMER2.0). Consistent with our experimental results, database analyses confirmed that CD24 expression was significantly increased in HCC tissues when compared to those of normal liver tissues. (Figure 1D-E). Furthermore, pan-cancer analysis through TIMER2.0 demonstrated significantly higher CD24 expression in multiple malignancies relative to their corresponding normal tissues (Figure 1F). These consistent findings across experimental and bioinformatic approaches suggest that CD24 upregulation is particularly prominent in C. sinensis -associated HCC and could be crucial in the development of liver cancer and the advancement of tumors. CD24 expression and different HCC clinicopathological parameters Given the marked elevation of CD24 expression in HCC, we systematically evaluated its clinical relevance using the UALCAN database. Our analysis revealed several key findings. First, CD24 expression showed progressive upregulation across advancing tumor stages (Stages 1-3) compared to normal liver tissue (Figure 2A). This stage-dependent pattern was paralleled by Edmondson's pathological grading, with significantly higher CD24 levels in grades 1-3 HCC (Figure 2B). Notably, CD24 expression was particularly elevated in cases with nodal metastasis (Figure 2C), suggesting a potential role in disease dissemination. Further stratification analysis demonstrated that CD24 upregulation occurred independently of TP53 mutation status (Figure 2D), patient gender (Figure 2E) and aging (21-80 years) (Figure 2F). In all subgroups examined, CD24 expression remained consistently higher in tumor tissue than in normal liver tissues. These comprehensive analyses establish CD24 as a robust biomarker consistently associated with HCC progression across multiple clinical parameters. High CD24 expression is associated with poor prognosis in HCC To assess the prognostic value of CD24 in HCC, we performed survival analyses using the Kaplan-Meier plotter database. Patients were dichotomized into high- and low-expression groups based on the median CD24 expression level. The results demonstrated that the CD24-high HCC patients exhibited significantly worse OS, PFS, RFS, and DSS compared to those with low CD24 expression (Figure 3). Furthermore, we assessed the prognostic significance of CD24 in relation to diverse clinicopathological characteristics by utilizing the Kaplan-Meier database. Especially in those with vascular invasion, higher CD24 expression was substantially linked to worse OS and PFS (Supplementary Table 3). Collectively, these findings indicate that CD24 serves as a significant prognostic marker in HCC, highlighting its potential clinical relevance. Correlation analysis of CD24 expression with immune cell infiltration Given the complexity of the liver immune microenvironment, we systematically evaluated the correlation between CD24 expression and the tumor immune microenvironment characteristics using the TIMER2.0 database. Our comprehensive analysis revealed several key findings. CD24 expression exhibited notable positive correlations with the infiltration levels of six predominant immune cell types, namely B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells (Figure 4A). To further investigate the association between CD24 and immune responses, we analyzed the relationship between CD24 expression and a range of immune characteristics in HCC. These included evaluations of B cells, T cells, CD8 + T cells, monocytes, tumor-associated macrophages (TAMs), M1/M2 macrophages, neutrophils, natural killer cells, and dendritic cells. Following adjustments for tumor purity, our analysis confirmed a significant correlation between CD24 expression and most established markers representative of these immune cell types (Supplementary Table 4). Additionally, we identified a positive correlation between CD24 expression levels and various immune checkpoints, including CTLA-4, LAG3, PDCD1, HAVCR2, and BTLA (Figure 4B-F). Collectively, these results indicate that CD24 may serve a crucial function in the regulation of immune infiltration in HCC, thereby underscoring its potential as a target for therapeutic intervention. CsESPs promote proliferation and inhibit apoptosis in HCC cells To investigate the effects of CsESPs on HCC cells, we treated Hep3B and Huh7 cells with 100 μg/ml CsESPs. CsESPs treatment significantly enhanced the viability of HCC cells after 48 hours of exposure and clonogenic proliferation compared to the control group (Figure 5A-B). Furthermore, the flow cytometry analysis demonstrated that CsESPs markedly reduced basal label of apoptosis in HCC cells (Figure 5C-D). Collectively, these results indicate that CsESPs promote HCC cell proliferation and suppress apoptosis, suggesting a potential role in HCC progression. CsESPs may up-regulate CD24 expression through transcription factor E2F1 Consistent with the mRNA changes (Figure. 1B), CD24 was significantly upregulated in CsESPs-treated HCC cells (Figure 6A and 6B). To investigate the mechanism underlying CD24 upregulation by CsESPs, we predicted potential transcription factors binding to the CD24 promoter region using both PROMO and Harmonizome databases. By intersecting the results from both databases, we identified E2F1 and AR as candidate transcription factors (Figure 6C). Subsequent correlation analysis using the GEPIA database revealed that E2F1 exhibited a positive correlation with CD24, in contrast, AR showed a negative correlation with CD24 (Figure S1). Further WB and qPCR experiments confirmed that E2F1 was up-regulated in CsESPs-treated HCC cells (Figure 6B and 6D). Immunofluorescence assays also indicated that E2F1 translocated from the cytoplasm to the nucleus following CsESPs treatment (Figure 6E). To validate the binding of E2F1 to the CD24 promoter, we used the JASPAR database to predict potential binding sites. The region spanning 1325–1335 bp scored highly, suggesting strong binding affinity. We subsequently constructed E2F1 overexpression vectors and CD24 promoter vectors with either wild-type or mutated binding sites. Dual-luciferase reporter assays revealed that E2F1 enhanced luciferase activity conjugating wild-type CD24 promoter sequence, but not in those with mutant sequence (Figure 6F). More importantly, ChIP-qPCR confirmed that E2F1 binds to the CD24 promoter region (Figure 6G). Collectively, these findings demonstrate that CsESPs upregulate CD24 expression through transcriptional regulation by E2F1. CD24 silencing abolishes CsESPs-induced cell proliferation and apoptosis reduction To investigate the essential role of CD24 in CsESPs-treated HCC cells, we performed knockdown of CD24 using specific siRNA (Figure S2). As expected, CD24 silencing significantly reduced the viability of CsESPs-treated HCC cells (Figure 7A-B). Flow cytometry analysis further demonstrated that CD24 knockdown markedly increased apoptosis in these cells (Figure 7C-E). WB assays revealed elevated expression of the pro-apoptotic protein BAX in CD24-knockdown cells (Figure 7F). Together, these results confirm that CD24 promotes cell proliferation and inhibits apoptosis in CsESPs-treated HCC cells. Relationship between CD24 and immune checkpoints in CsESPs-treated HCC cells To further investigate whether CD24 up-regulation in CsESPs-treated HCC cells affect multiple immune checkpoint molecules, we established an in vitro co-culture system of HCC cells and PBMCs. qPCR analyses showed that CD24 knockdown significantly attenuated CsESPs-induced upregulation of CTLA-4 and LAG-3 (Figure 8A-B). However, CD24 knockdown failed to alter the levels of other genes associated with immune checkpoints, such as PDCD1, HAVCR2, and BTLA (Figure 8C-E). These results suggest that CD24 may selectively regulate specific immune checkpoint pathways in CsESPs -treated HCC cells. Discussion This study demonstrated that CD24 is significantly upregulated in HCC, particularly in cases associated with C. sinensis . Analysis from multiple databases indicates that the level of CD24 expression correlates with clinicopathological stages and prognosis in HCC patients. Mechanistically, CsESPs can promote proliferation and inhibit apoptosis of HCC cells through E2F1-mediated transcriptional regulation of CD24. Moreover, CsESPs up-regulated the immune checkpoint molecules CTLA-4 and LAG-3 in the co-cultured PBMCs, an effect that could be reversed by knockdown of CD24 (Figure 9). Consistent with the up-regulation of CD24 in multiple malignancies [34], our preliminary single-cell sequencing data revealed elevated CD24 expression in C. sinensis -associated HCC. We further validated its up-regulation in both in-house cohorts and online datasets. More importantly, the Kaplan–Meier and Cox regression analyses demonstrated that aberrant CD24 expression has the potential to act as a prognostic biomarker for HCC, consistent with prior research [27]. Furthermore, CD24 exhibits a strong association with immune checkpoint genes, suggesting its potential role in modulating tumor immune infiltration. Given its differential expression patterns and immunomodulatory implications, we hypothesize that CD24 contributes to tumorigenesis and progression in a context-dependent manner, possibly influencing immunotherapy responsiveness. Clonorchiasis, caused by the C. sinensis , is recognized as one of the most clinically overlooked tropical diseases in East Asia [35]. This persistent C. sinensis infection is recognized as a risk factor for cholangiocarcinoma development [12, 36]. Recent epidemiological evidence also associates C. sinensis infection with poorer clinical outcomes in HCC patients [8, 23, 24]. Notably, the parasite enhances malignant transformation through induction of cancer stem cell-like properties [8]. The molecular mechanisms underlying this oncogenic effect involve parasite-derived factors such as the calcium-binding protein Cs16, which drives hepatic inflammation by reprogramming metabolic pathways in innate immune cells [37]. Similarly, CsESPs contribute to infection-associated pathology. For instance, the CsESP component Csseverin exhibits potent anti-apoptotic effects in human HCC cell lines [38]. In alignment with these findings, our current study demonstrates that CsESPs promote HCC cell proliferation while suppressing apoptosis. Importantly, we observed that CD24-upregulation may play an essential role in CsESP-induced cell proliferation enhancement and apoptosis reduction in HCC cells. Our experimental findings corroborate CD24's critical role in CsESP-mediated HCC pathogenesis. Specifically, CD24 knockdown in CsESP-treated HCC cells significantly reduced viability while increasing apoptosis, accompanied by elevated BAX expression. These results support a model wherein CsESPs upregulate CD24 expression. Our mechanical study further identifies a direct transcriptional regulation mode of CD24 through E2F1. As a pivotal transcription factor regulating diverse cellular processes including proliferation, differentiation, migration and metabolism [39], E2F1 exhibits dual oncogenic and tumor-suppressive functions in cancer progression [40, 41]. Apart from E2F1, several other pathways have been reported to regulate CD24 expression. For example, ADAR, an RNA-editing enzyme, promotes HCC progression by enhancing CD24 expression through its interaction with SNRPD3 and RNPS1, thereby inhibiting STAU1-mediated mRNA degradation [42]. In addition, the Hippo-YAP1-SOX4 axis was identified as a key regulatory pathway responsible for YAP1-mediated CD24 overexpression in HCC cells. Notably, suppression of CD24 expression markedly inhibited YAP1-initiated HCC progression [43]. Our findings demonstrate that CD24 expression positively correlates with immune checkpoints, including LAG-3 and CTLA-4. Importantly, CsESPs-induced upregulation of these checkpoints was attenuated by CD24 knockdown, suggesting CD24’s pivotal role in mediating immune evasion in CsESP-treated HCC. As critical immune checkpoint molecules, CTLA-4 and LAG-3 suppress T cell activation through distinct mechanisms. CTLA-4 competitively binds to B7 molecules with higher affinity than CD28, thereby inhibiting co-stimulatory signals, while LAG-3 negatively regulates T cell responses via MHC class II interactions. Co-expression of these checkpoints on exhausted T cells drives progressive functional impairment, characterized by diminished cytokine production and cytotoxic activity. Notably, recent evidence demonstrates that dual CTLA-4/LAG-3 blockade synergistically restores T cell function, highlighting their cooperative role in maintaining T cell exhaustion [44]. T cell exhaustion, a dysfunctional state marked by TOX-driven transcriptional reprogramming and sustained expression of inhibitory receptors (e.g., LAG-3, CTLA-4), is a hallmark of chronic viral infections and tumor microenvironments [45]. This exhaustion leads to CD8 + T cell proliferation failure and loss of effector capacity, ultimately compromising antitumor immunity. These results position CD24 as a potential regulator of T cell exhaustion and a therapeutic target for restoring antitumor immunity. While this study provides novel insights into CD24's role in C. sinensis -associated HCC pathogenesis, several limitations should be acknowledged. First, the study lacks in vivo validation, as findings derived from the in vitro experiments may not fully reflect the complexity of HCC progression. Clarification of HCC tumor microenvironment through single-cell RNA sequencing (scRNA-seq) and spatial transcriptomic sequencing (ST-seq) may shed light on this question. Second, the relatively small sample size in the in-house HCC cohort may limit the generalizability of the results. Particularly, it is yet unknown whether C. sinensis infection affect the treatment response and long-term prognosis of immunotherapy which is increasingly adopted as the first-line HCC treatment. Third, CsESPs may not fully represent the oncogenic ability of C. sinensis. The released non-coding RNAs and metabolites by extracellular vesicle by parasite are also important oncogenic factors. Addressing these limitations in future studies will be essential to dissect the oncogenic effects of C. sinensis -associated HCC and the potential application of CD24 as a predictive biomarker and potential therapeutic target. Conclusion This study comprehensively establishes CD24 as a clinically significant biomarker in HCC, with particularly pronounced overexpression in C. sinensis -associated cases. Integrated multi-database analyses revealed that CD24 expression levels not only correlate with advanced clinicopathological stages but also act as a standalone prognostic marker in patients with HCC. Mechanistically, CsESPs upregulates E2F1, which subsequently activates CD24 transcription, ultimately promoting tumor cell proliferation while suppressing apoptosis. Furthermore, CsESP treatment increases the levels of the immune checkpoint molecules CTLA-4 and LAG-3 in co-cultured PBMCs- an effect that was reversible by CD24 knockdown. These results reveal the essential roles of CD24 in both tumor-intrinsic growth pathways within HCC cells and immune evasion mechanisms in microenvironment in C. sinensis -associated HCC. Abbreviations CD24 cluster of differentiation 24 C. sinensis Clonorchis sinensis CsESP Clonorchis sinensis excretory-secretory products PBS phosphate-buffered saline Declarations Acknowledgements We gratefully acknowledge the financial support from the Natural Science Foundation of Shanghai (24ZR1414000), the Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor (Guangxi Medical University), Ministry of Education (GKE-ZZ202131), the National Natural Science Foundation of China (U22A20374 and 32160160), and the Innovation Project of Guangxi Graduate Education (YCBZ2024142). Authors’ contributions W.M.L., J.Z., and G.D.L. designed the experiments; W.M.L., J.Y., Y.W., Q.R.C. and Y.C. performed the experiments; Z.J.L., J.F. and G.Z.Z. analyzed the data; J.Z., and G.D.L. supervised the studies and contributed reagents/materials/ analysis tools; and W.M.L., J.Y., and Z.J.L. prepared the initial draft of the manuscript. All four authors approve the final version of the manuscript. Funding This study was partially supported by grants from the Natural Science Foundation of Shanghai (24ZR1414000), the Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor (Guangxi Medical University), Ministry of Education (GKE-ZZ202131), the National Natural Science Foundation of China (U22A20374 and 32160160), and The Innovation Project of Guangxi Graduate Education (YCBZ2024142). Availability of data and materials No datasets were generated or analyzed during the current study. Ethics approval and consent to participate All HCC patients underwent surgical operation. Informed consent was acquired from all participants, and the research received approval from the Ethics Committee at the First Affiliated Hospital of Guangxi Medical University (Ethics approval number: 2024-E580-01). All animal experiment procedures in this study were conducted in accordance with the Guide for the Guangxi Medical University Laboratory Animal Center (Ethics approval number: 202410011). Consent for publication Not applicable. Competing interests The authors declare no competing interests. Author details 1 Department of Toxicology, School of Public Health, Guangxi Medical University, Nanning, Guangxi, China. 2 School of Public Health, Fudan University, Shanghai, China. 3 Department of Physiology, School of Basic Medical Sciences, Guangxi Medical University, Nanning, Guangxi, China. 4 Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China. 5 Department of Pathology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital and Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Shenzhen, Guangdong, China. 6 Hengzhou Center for Disease Control and Prevention, Hengzhou, Guangxi, China. 7 Department of Hepatobiliary Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China. References Bray F, Laversanne M, Sung H, et al: Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229-263.http://dx.doi.org/10.3322/caac.21834 Han B, Zheng R, Zeng H, et al: Cancer incidence and mortality in China, 2022. J Natl Cancer Cent. 2024;4(1):47-53.http://dx.doi.org/10.1016/j.jncc.2024.01.006 Bray F, Ferlay J, Soerjomataram I, et al: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394-424.http://dx.doi.org/10.3322/caac.21492 Qiu H, Cao S, Xu R: Cancer incidence, mortality, and burden in China: a time-trend analysis and comparison with the United States and United Kingdom based on the global epidemiological data released in 2020. Cancer Commun (Lond). 2021;41(10):1037-1048.http://dx.doi.org/10.1002/cac2.12197 Qi J, Li M, Wang L, et al: National and subnational trends in cancer burden in China, 2005-20: an analysis of national mortality surveillance data. Lancet Public Health. 2023;8(12):e943-e955.http://dx.doi.org/10.1016/S2468-2667(23)00211-6 Zhou M, Wang H, Zhu J, et al: Cause-specific mortality for 240 causes in China during 1990-2013: a systematic subnational analysis for the Global Burden of Disease Study 2013. Lancet. 2016;387(10015):251-272.http://dx.doi.org/10.1016/S0140-6736(15)00551-6 Li Q, Cao J, Rong M, et al: Analysis of incidence and mortality of liver cancer in cancer registration areas in Guangxi, 2010—2016. J Chin J Oncol Prev Treat. 2021;13(2):138-143.http://dx.doi.org/10.3969/j.issn.1674-5671.2021.02.05 Jiang ZH, Wan XL, Lv GL, et al: High prevalence of Clonorchis sinensis infection in Guangxi, Southern China. Trop Med Health. 2021;49(1):6.http://dx.doi.org/10.1186/s41182-021-00297-0 Tang ZL, Huang Y, Yu XB: Current status and perspectives of Clonorchis sinensis and clonorchiasis: epidemiology, pathogenesis, omics, prevention and control. Infect Dis Poverty. 2016;5(1):71.http://dx.doi.org/10.1186/s40249-016-0166-1 Hatta MNA, Mohamad Hanif EA, Chin SF, et al: Pathogens and Carcinogenesis: A Review. Biology (Basel). 2021;10(6).http://dx.doi.org/10.3390/biology10060533 Pak JH, Lee JY, Jeon BY, et al: Cytokine Production in Cholangiocarcinoma Cells in Response to Clonorchis sinensis Excretory-Secretory Products and Their Putative Protein Components. Korean J Parasitol. 2019;57(4):379-387.http://dx.doi.org/10.3347/kjp.2019.57.4.379 Na BK, Pak JH, Hong SJ: Clonorchis sinensis and clonorchiasis. Acta Trop. 2020;203:105309.http://dx.doi.org/10.1016/j.actatropica.2019.105309 Bouvard V, Baan R, Straif K, et al: A review of human carcinogens--Part B: biological agents. Lancet Oncol. 2009;10(4):321-322.http://dx.doi.org/10.1016/s1470-2045(09)70096-8 Lin Q, Tang Z, Qin Y, et al: Clonorchis sinensis infection amplifies hepatocellular carcinoma stemness, predicting unfavorable prognosis. PLoS Negl Trop Dis. 2024;18(1):e0011906.http://dx.doi.org/10.1371/journal.pntd.0011906 Li YK, Zhao JF, Yang CL, et al: Effects of Clonorchis sinensis combined with Hepatitis B virus infection on the prognosis of patients with Hepatocellular Carcinoma following Hepatectomy. PLoS Negl Trop Dis. 2023;17(1):e0011012.http://dx.doi.org/10.1371/journal.pntd.0011012 Qi Y, Hu J, Liang J, et al: Clonorchis sinensis infection contributes to hepatocellular carcinoma progression in rat. Parasitol Res. 2022;121(12):3403-3415.http://dx.doi.org/10.1007/s00436-022-07699-x Yan C, Zhou QY, Wu J, et al: Csi-let-7a-5p delivered by extracellular vesicles from a liver fluke activates M1-like macrophages and exacerbates biliary injuries. Proc Natl Acad Sci U S A. 2021;118(46).http://dx.doi.org/10.1073/pnas.2102206118 Wang C, Lei H, Tian Y, et al: Clonorchis sinensis granulin: identification, immunolocalization, and function in promoting the metastasis of cholangiocarcinoma and hepatocellular carcinoma. Parasit Vectors. 2017;10(1):262.http://dx.doi.org/10.1186/s13071-017-2179-4 Wang C, He Q, Yin Y, et al: Clonorchis sinensis Granulin Promotes Malignant Transformation of Hepatocyte Through EGFR-Mediated RAS/MAPK/ERK and PI3K/Akt Signaling Pathways. Front Cell Infect Microbiol. 2021;11:734750.http://dx.doi.org/10.3389/fcimb.2021.734750 He Q, Pan X, Yin Y, et al: Clonorchis sinensis granulin promotes malignant transformation of human intrahepatic biliary epithelial cells through interaction with M2 macrophages via regulation of STAT3 phosphorylation and the MEK/ERK pathway. Parasit Vectors. 2023;16(1):139.http://dx.doi.org/10.1186/s13071-023-05765-6 Barkal AA, Brewer RE, Markovic M, et al: CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy. Nature. 2019;572(7769):392-396.http://dx.doi.org/10.1038/s41586-019-1456-0 Mensah FFK, Armstrong CW, Reddy V, et al: CD24 Expression and B Cell Maturation Shows a Novel Link With Energy Metabolism: Potential Implications for Patients With Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Front Immunol. 2018;9:2421.http://dx.doi.org/10.3389/fimmu.2018.02421 Kristiansen G, Sammar M, Altevogt P: Tumour biological aspects of CD24, a mucin-like adhesion molecule. J Mol Histol. 2004;35(3):255-262.http://dx.doi.org/10.1023/b:hijo.0000032357.16261.c5 Lim SC: CD24 and human carcinoma: tumor biological aspects. Biomed Pharmacother. 2005;59 Suppl 2:S351-354.http://dx.doi.org/10.1016/s0753-3322(05)80076-9 Tolkach Y, Zarbl R, Bauer S, et al: DNA Promoter Methylation and ERG Regulate the Expression of CD24 in Prostate Cancer. Am J Pathol. 2021;191(4):618-630.http://dx.doi.org/10.1016/j.ajpath.2020.12.014 Ochsner SA, Strick-Marchand H, Qiu Q, et al: Transcriptional profiling of bipotential embryonic liver cells to identify liver progenitor cell surface markers. Stem Cells. 2007;25(10):2476-2487.http://dx.doi.org/10.1634/stemcells.2007-0101 Yang XR, Xu Y, Yu B, et al: CD24 is a novel predictor for poor prognosis of hepatocellular carcinoma after surgery. Clin Cancer Res. 2009;15(17):5518-5527.http://dx.doi.org/10.1158/1078-0432.CCR-09-0151 Chang JI, Lee K, Kim D, et al: Clinical Characteristics of Clonorchis sinensis -Associated Cholangiocarcinoma: A Large-Scale, Single-Center Study. Front Med (Lausanne). 2021;8:675207.http://dx.doi.org/10.3389/fmed.2021.675207 Zhou J, Tan SH, Nicolas V, et al: Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion. Cell Res. 2013;23(4):508-523.http://dx.doi.org/10.1038/cr.2013.11 Wei H, Chen Y, Qin Z, et al: Artesunate demonstrates neuroprotective effect through activation of lysosomal function and inhibition of cGAS-STING pathway. Neuropharmacology. 2025:110426.http://dx.doi.org/10.1016/j.neuropharm.2025.110426 Li ZJ, Dai HQ, Huang XW, et al: Artesunate synergizes with sorafenib to induce ferroptosis in hepatocellular carcinoma. Acta Pharmacol Sin. 2021;42(2):301-310.http://dx.doi.org/10.1038/s41401-020-0478-3 Lu GD, Ang YH, Zhou J, et al: CCAAT/enhancer binding protein alpha predicts poorer prognosis and prevents energy starvation-induced cell death in hepatocellular carcinoma. Hepatology. 2015;61(3):965-978.http://dx.doi.org/10.1002/hep.27593 Li XY, Cui X, Xie CQ, et al: Andrographolide causes p53-independent HCC cell death through p62 accumulation and impaired DNA damage repair. Phytomedicine. 2023;121:155089.http://dx.doi.org/10.1016/j.phymed.2023.155089 Zhao C, Huang Y, Zhang H, et al: CD24 affects the immunosuppressive effect of tumor-infiltrating cells and tumor resistance in a variety of cancers. Discov Oncol. 2024;15(1):399.http://dx.doi.org/10.1007/s12672-024-01284-7 Lai DH, Hong XK, Su BX, et al: Current status of Clonorchis sinensis and clonorchiasis in China. Trans R Soc Trop Med Hyg. 2016;110(1):21-27.http://dx.doi.org/10.1093/trstmh/trv100 Wei C, Chen J, Yu Q, et al: Clonorchis sinensis infection contributes to hepatocellular carcinoma progression via enhancing angiogenesis. PLoS Negl Trop Dis. 2024;18(11):e0012638.http://dx.doi.org/10.1371/journal.pntd.0012638 Li Q, Li X, Kan S, et al: Clonorchis sinensis calcium-binding protein Cs16 causes acute hepatic injury possibly by reprogramming the metabolic pathway of bone marrow-derived monocytes. Front Cell Infect Microbiol. 2023;13:1280358.http://dx.doi.org/10.3389/fcimb.2023.1280358 Chen X, Li S, He L, et al: Molecular characterization of severin from Clonorchis sinensis excretory/secretory products and its potential anti-apoptotic role in hepatocarcinoma PLC cells. PLoS Negl Trop Dis. 2013;7(12):e2606.http://dx.doi.org/10.1371/journal.pntd.0002606 Mandigo AC, Yuan W, Xu K, et al: RB/E2F1 as a Master Regulator of Cancer Cell Metabolism in Advanced Disease. Cancer Discov. 2021;11(9):2334-2353.http://dx.doi.org/10.1158/2159-8290.CD-20-1114 Kumari A, Iwasaki T, Pyndiah S, et al: Regulation of E2F1-induced apoptosis by poly(ADP-ribosyl)ation. Cell Death Differ. 2015;22(2):311-322.http://dx.doi.org/10.1038/cdd.2014.146 Cao LL, Wu YK, Lin TX, et al: CDK5 promotes apoptosis and attenuates chemoresistance in gastric cancer via E2F1 signaling. Cancer Cell Int. 2023;23(1):286.http://dx.doi.org/10.1186/s12935-023-03112-4 Sun L, Hu P, Yang H, et al: ADARp110 promotes hepatocellular carcinoma progression via stabilization of CD24 mRNA. Proc Natl Acad Sci U S A. 2025;122(3):e2409724122.http://dx.doi.org/10.1073/pnas.2409724122 He X, Zhang Y, Fang Q, et al: Unraveling the role of CD24 in Hepatocellular carcinoma: Involvement of inactivated Hippo signaling and SOX4-mediated regulation. Biochim Biophys Acta Mol Basis Dis. 2024;1870(4):167117.http://dx.doi.org/10.1016/j.bbadis.2024.167117 Andrews LP, Cillo AR, Karapetyan L, et al: Molecular Pathways and Mechanisms of LAG3 in Cancer Therapy. Clin Cancer Res. 2022;28(23):5030-5039.http://dx.doi.org/10.1158/1078-0432.CCR-21-2390 Khan O, Giles JR, McDonald S, et al: TOX transcriptionally and epigenetically programs CD8(+) T cell exhaustion. Nature. 2019;571(7764):211-218.http://dx.doi.org/10.1038/s41586-019-1325-x Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Graphicabstract.pdf Cite Share Download PDF Status: Published Journal Publication published 19 Aug, 2025 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Revision requested 11 Jun, 2025 Reviews received at journal 30 May, 2025 Reviewers agreed at journal 24 Apr, 2025 Reviewers invited by journal 24 Apr, 2025 Editor assigned by journal 09 Apr, 2025 Submission checks completed at journal 09 Apr, 2025 First submitted to journal 04 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6378057","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":447684905,"identity":"8e884d36-644b-4337-8896-269f185e4459","order_by":0,"name":"Wen-Min Lu","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wen-Min","middleName":"","lastName":"Lu","suffix":""},{"id":447684906,"identity":"2eca1aac-4f43-4b2d-872c-3f0e5ee56c01","order_by":1,"name":"Jin Yan","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Yan","suffix":""},{"id":447684907,"identity":"fae36927-b546-483a-8602-93fa802c724d","order_by":2,"name":"Zhao-Ji Liu","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhao-Ji","middleName":"","lastName":"Liu","suffix":""},{"id":447684908,"identity":"ad794010-4b47-4c73-8064-7bf825182b53","order_by":3,"name":"Yong Wu","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Wu","suffix":""},{"id":447684909,"identity":"a6b3a23b-189e-481e-99a6-1f850a541832","order_by":4,"name":"Qian-Ru Cui","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qian-Ru","middleName":"","lastName":"Cui","suffix":""},{"id":447684910,"identity":"62bb9b87-c356-4dfd-83bc-5744d13588d5","order_by":5,"name":"Ji Feng","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Ji","middleName":"","lastName":"Feng","suffix":""},{"id":447684911,"identity":"b664a0eb-5001-497f-810c-89ea27d83deb","order_by":6,"name":"Yu Chen","email":"","orcid":"","institution":"Hengzhou Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Chen","suffix":""},{"id":447684912,"identity":"df345d77-edfa-455c-a598-fe137456b459","order_by":7,"name":"Guang-Zhi Zhu","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guang-Zhi","middleName":"","lastName":"Zhu","suffix":""},{"id":447684913,"identity":"196b0eeb-a280-4471-b61c-5a3f701e1c57","order_by":8,"name":"Jing Zhou","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhou","suffix":""},{"id":447684917,"identity":"90e06e6d-f1a6-4334-9201-100f0bc8f264","order_by":9,"name":"Guo-Dong Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACPiCW+GDDwNgA4vEQo4UNiCVnpJGqRZqHNC0S6Rdv2yQclu2f3cD44G0bg7w5YS05xdY5CYeNZ9w5wGw4t43BcGcDYS1p0rk/Dic23Ehgk+ZtY0gwOECMFouEw4nzbySw/yZSS/oxaQaglg1AW5iJ08LzhtmyJyHdeOONxGbJOeckDDcQ0sLPnv7wxo8Ea9l5N5IPfnhTZiNP0BZgXBhAGeCokSCoHgjYHxCjahSMglEwCkYyAACytT5tjgrzdgAAAABJRU5ErkJggg==","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Guo-Dong","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2025-04-04 17:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6378057/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6378057/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-025-06979-6","type":"published","date":"2025-08-19T16:29:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82123319,"identity":"e78fc987-b5c1-4220-8983-6ee2f8099adf","added_by":"auto","created_at":"2025-05-07 03:32:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":692335,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of CD24 in \u003cem\u003eClonorchis sinensis\u003c/em\u003e associated LIHC. A. The expression of CD24 was detected in \u003cem\u003eClonorchis sinensis\u003c/em\u003e negative LIHC in 12 pairs of tumor tissues and paired adjacent tissues by q-PCR. B. The expression of CD24 was detected in \u003cem\u003eClonorchis sinensis\u003c/em\u003e associated LIHC in 24 pairs of tumor tissues and paired adjacent tissues by q-PCR. C. The fold change of tumor and normal tissues of CD24 in \u003cem\u003eClonorchis sinensis \u003c/em\u003enegative and positive tissues. D. Increased expression of CD24 in LIHC compared to normal tissues in the GEPIA database. E. Increased expression of CD24 in LIHC compared to normal tissues in the UALCAN database. F. CD24 expression in different types of cancers was examined by using the TIMER2.0 database. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt;\u003c/em\u003e0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.01, \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001.\u003c/p\u003e","description":"","filename":"Binder131.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6378057/v1/1e26907ca0e3ad9930735e14.jpg"},{"id":82125325,"identity":"c1a24e83-3b1f-45c0-95f6-0869e8cd89d1","added_by":"auto","created_at":"2025-05-07 03:48:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":574370,"visible":true,"origin":"","legend":"\u003cp\u003eBox plots evaluating CD24 expression among different groups of patients based on clinical parameters using the UALCAN database. A. individual cancer stages. B. tumor grade. C. nodal metastasis status. D. TP53 mutation status. E. gender. F. age. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt;\u003c/em\u003e0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.01, \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001.\u003c/p\u003e","description":"","filename":"Binder132.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6378057/v1/b2c8d5b46f3ad5045f1fd264.jpg"},{"id":82124749,"identity":"98a29787-31d5-4d71-b972-9b6137d50255","added_by":"auto","created_at":"2025-05-07 03:40:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":514612,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival curve evaluating the prognostic value of CD24. Survival curves using the Kaplan-Meier plotter are shown for A. OS; B. PFS; C. RFS; D.DSS. OS, overall survival; PFS, progression-free survival; RFS, recurrence free survival; DSS, disease specific survival.\u003c/p\u003e","description":"","filename":"Binder133.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6378057/v1/43dddf086fcedf7fc50bdbfe.jpg"},{"id":82123333,"identity":"9c1f74d5-7461-4556-adeb-a813431ce5f8","added_by":"auto","created_at":"2025-05-07 03:32:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":845213,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between CD24 and immune cells. A. CD24 significantly associated with tumor purity and positively correlated with the infiltration of\u003c/p\u003e\n\u003cp\u003edifferent immune cells using the TIMER 2.0 database. B–D. Scatterplots of the correlations between CD24 expression and PDCD1 (B), LAG3 (C), CTLA-4 (D), HAVCR2 (E) and BTLA (F) in LIHC using the TIMER2.0 database. PDCD1, programmed cell death protein-1; LAG-3, Lymphocyte-activation gene 3; CTLA-4, cytotoxic T lymphocyte-associated antigen-4; BTLA, B and T lymphocyte attenuator; HAVCR2, hepatitis a virus cell receptor 2.\u003c/p\u003e","description":"","filename":"Binder134.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6378057/v1/45cebcaf5b688c38c69d4300.jpg"},{"id":82124753,"identity":"4ac84fa2-b1f0-4a6a-b6a8-4037b1a7ae9c","added_by":"auto","created_at":"2025-05-07 03:40:17","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":661597,"visible":true,"origin":"","legend":"\u003cp\u003eExposure to CsESPs promotes cell proliferation and inhibits cell apoptosis in LIHC cells. A. The viability of cells was detected by CCK8 assay in LIHC cells after they are treated or not treated with CsESPs (100 μg/ml) for 48 h. B. The proliferation of cells was detected by clone formation assay in LIHC cells after they are treated or not treated with CsESPs (100 μg/ml). C-D. Cell apoptosis was detected in control and CsESPs-treated LIHC cells by flow cytometry. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001.\u003c/p\u003e","description":"","filename":"Binder135.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6378057/v1/4299612bdfccd0f9547810ae.jpg"},{"id":82125921,"identity":"df801250-efa6-46be-9c37-99e4aab09e41","added_by":"auto","created_at":"2025-05-07 03:56:17","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":515368,"visible":true,"origin":"","legend":"\u003cp\u003eCsESPs may up-regulate CD24 expression by up-regulating the expression of transcription factor E2F1. A. The expression of CD24 was detected in control and CsESPs-treated LIHC cells by q-PCR. B. Schematic illustration showing the overlapping transcription factors of CD24 predicted by PROMO and Harmonizome. C. The expression of E2F1 was detected in control and CsESPs-treated LIHC cells by q-PCR. D. The expression levels of CD24 and E2F1 protein were detected by western blot in LIHC cells after they are treated with or without CsESPs. E. Subcellular localization of E2F1 was determined by immunofluorescence, scale bar = 10 μm. F. The potential binding sites of E2F1 on the CD24 promoter sequence and the corresponding mutations, and CD24 promoter luciferase activity was detected by dual-luciferase assays. ns: no significance, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05 compared with pcDNA3.1. G. ChIP- qPCR showed that E2F1 binds to the promoter regions of CD24 in Huh7 cells. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.01, \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001 compared with IgG.\u003c/p\u003e","description":"","filename":"Binder136.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6378057/v1/2868809ba94c188f987d38cc.jpg"},{"id":82123326,"identity":"305b605b-f619-47bc-a0c5-43524e79ad3b","added_by":"auto","created_at":"2025-05-07 03:32:17","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":671548,"visible":true,"origin":"","legend":"\u003cp\u003eCD24 promotes cell viability and inhibits cell apoptosis in LIHC cells under CsESPs exposure. A-B. The cell viability was detected by CCK-8 assay in LIHC cells after they are transfected with si-CD24 or NC that were treated or not treated with CsESPs. C-E. The cell apoptosis was detected by flow cytometry in LIHC cells after they are transfected with si-CD24 or NC that were treated or not treated with CsESPs. F. The expression levels of CD24 and BAX protein were detected by western blot in LIHC cells after they are treated with or without CsESPs. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep \u0026lt;\u003c/em\u003e 0.001 compared with control NC; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u0026lt;\u003c/em\u003e0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.01\u003c/em\u003e,\u003csup\u003e ###\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001 compared with CsESPs NC. NC: negative control. CsESP: excretory–secretory products of Clonorchis sinensis.\u003c/p\u003e","description":"","filename":"Binder137.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6378057/v1/5409630efc43d77552c934ae.jpg"},{"id":82123331,"identity":"6c75f79e-b549-46e0-82bd-dad50ff6d440","added_by":"auto","created_at":"2025-05-07 03:32:17","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":447486,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between CD24 and different immune checkpoint in LIHC cells under CsESPs exposure.\u003c/p\u003e\n\u003cp\u003eA-E. The expression of LAG3, CTLA4, PDCD1, HAVCR2 and BTLA was detected by qPCR in PBMC cells co-culture with LIHC cells after they are transfected with si-CD24 or NC that were treated or not treated with CsESPs. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001 compared with control NC; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u0026lt;\u003c/em\u003e0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep \u0026lt;\u003c/em\u003e 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001 compared with CsESPs NC. PDCD1, programmed cell death protein-1; LAG-3, Lymphocyte-activation gene 3; CTLA-4, cytotoxic T lymphocyte-associated antigen-4; BTLA, B and T lymphocyte attenuator; HAVCR2, hepatitis a virus cell receptor 2.\u003c/p\u003e","description":"","filename":"Binder138.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6378057/v1/dc4ef050a935b3c62d79ba2b.jpg"},{"id":89847321,"identity":"e549e885-72e4-4e80-98c9-1814de87a583","added_by":"auto","created_at":"2025-08-25 16:43:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5921606,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6378057/v1/6be46750-f931-4ca8-868d-93574fd06dab.pdf"},{"id":82124750,"identity":"1f603b33-a312-4861-a226-2ff16263133b","added_by":"auto","created_at":"2025-05-07 03:40:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":206579,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6378057/v1/e826e30907bf602274ed68d2.docx"},{"id":82123324,"identity":"e2a8df0b-cc0a-4655-982a-b6f1baca897b","added_by":"auto","created_at":"2025-05-07 03:32:17","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":227388,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicabstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6378057/v1/5dfa8915933dd7c050dad1ad.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Clonorchis sinensis-driven Hepatocarcinogenesis via E2F1-CD24 Transcriptional Axis: Mechanistic and Therapeutic Implications","fulltext":[{"header":"Background","content":"\u003cp\u003eGlobally, liver cancer constitutes the third most lethal malignancy and sixth most frequently diagnosed cancer according to 2022 International Agency for Research on Cancer statistics [1]. Chinese epidemiological surveillance demonstrates concordance, with liver cancer representing the second leading cause of cancer-related mortality and fourth most diagnosed malignancy nationally [2]. Among the various subtypes of liver cancer, hepatocellular carcinoma (HCC,\u0026nbsp;referred to as liver hepatocellular carcinoma (LIHC) in database) is the most prevalent, accounting for approximately 85% of cases\u0026nbsp;[3].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile nationwide HBV vaccination initiatives and aflatoxin control have driven progressive reductions in HCC incidence and mortality over two decades, significant regional disparities persist [4, 5]. National cancer registry data identify Guangxi province exhibits China\u0026apos;s highest HCC mortality rates, with gender-specific rates of 69.0/100,000 (male) and 17.8/100,000 (female) - markedly exceeding national averages of 36.5/100,000 and 12.8/100,000 respectively [6]. Notably, Guangxi demonstrates paradoxical upward trends in HCC incidence (annual percentage change +5.38%) and mortality (+9.23%) during 2010-2016 [7], indicating distinct etiological factors beyond conventional HBV- or aflatoxin-associated hepatocarcinogenesis. Of particular epidemiological significance, this region demonstrates China\u0026apos;s highest prevalence of endemic \u003cem\u003eClonorchis sinensis\u003c/em\u003e (\u003cem\u003eC. sinensis\u003c/em\u003e) infection (6.68%) in China [8], strongly associated with local dietary practices of raw fish consumption. The persistent geographic overlap between exceptional HCC mortality burdens and helminthic infection prevalence implies potential proto-oncogenic interactions warranting mechanistic investigation.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. sinensis\u003c/em\u003e, commonly known as the liver fluke, is a food-borne parasite prevalent in China, Korea, Northern Vietnam, and the Russian Far East [9]. Human infection occurs through the consumption of raw or undercooked freshwater fish containing \u003cem\u003eC. sinensis\u003c/em\u003e metacercariae. Chronic \u003cem\u003eC. sinensis\u003c/em\u003e infection induces progressive hepatobiliary pathology including biliary hyperplasia, periductal fibrosis, and cholangiocarcinoma development through sustained inflammatory responses [10, 11]. With 15 million global cases (87% in China) [12], this IARC Group 1 carcinogen [13] demonstrates increasing oncogenic evidence in liver cancer pathogenesis. \u0026nbsp;Recent clinical investigations revealed \u003cem\u003eC. sinensis\u003c/em\u003e co-infection with HBV accelerates HCC progression by enhancing cancer stem cell properties, serving as an independent prognostic factor for reduced overall survival (5-year OS: 47.8% \u003cem\u003evs.\u0026nbsp;\u003c/em\u003e63.8% in HBV alone-infected cohorts; n=946) [14, 15]. Experimental models demonstrate infection promotes hepatic progenitor cell proliferation [16] and induces M1-like macrophage polarization via extracellular vesicle-derived Csi-let-7a-5p, mediating biliary injury through Socs1/Clec7a-dependent NF-\u003cem\u003e\u0026kappa;\u003c/em\u003eB activation\u003cem\u003e\u0026nbsp;\u003c/em\u003e[17]. Other mechanistical studies revealed that the excretory-secretory protein CsGRN drives malignant transformation through EGFR-mediated RAS/MAPK/ERK and PI3K/AKT pathway activation in hepatocytes [18, 19], with complementary studies confirming its transformative effects on human intrahepatic cholangiocytes \u003cem\u003ein vitro\u003c/em\u003e and biliary injury \u003cem\u003ein vivo\u003c/em\u003e [20].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBuilding upon these findings, our preliminary single-cell sequencing data identify CD24 overexpression in \u003cem\u003eC. sinensis\u003c/em\u003e-associated HCC specimens (unpublished observations). CD24, commonly referred to as the heat-stable antigen, is a surface protein anchored by glycosylphosphatidylinositol [21, 22]. It is overexpressed in various cancers and is implicated in regulating cancer cell proliferation, invasion, and immune evasion [23-25]. For instance, in ovarian and breast cancers, CD24 engages with Siglec-10 found on tumor-associated macrophages, thereby promoting immune evasion [21]. In liver, CD24 is absent in normally differentiated hepatocytes but detectable in hepatic progenitor cells (oval cells), suggesting a potential association with hepatic stem cell characteristics [26]. Notably, CD24 is overexpressed in HCC, where its expression correlates with increased tumor invasiveness, metastatic potential, enhanced proliferation, and activation of the Wnt/\u003cem\u003e\u0026beta;\u003c/em\u003e-catenin signaling pathway [27].\u0026nbsp;However, the role of CD24 in the pathogenesis of HCC following \u003cem\u003eC. sinensis\u003c/em\u003e infection remains poorly understood.\u003c/p\u003e\n\u003cp\u003eGiven the potential link between \u003cem\u003eC. sinensis\u003c/em\u003e infection and HCC progression, our objective was to explore the underlying mechanisms through which CsESPs influence CD24 expression and function in HCC cells and co-cultured immunocytes.\u0026nbsp;\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eClinical sample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman primary HCC tissues and paired normal tissues were collected from September 2020 to December 2022 at the First Affiliated Hospital of Guangxi Medical University. All HCC patients underwent surgical operation. Informed consent was acquired from all participants, and the research received approval from the Ethics Committee at the First Affiliated Hospital of Guangxi Medical University (Ethics approval number: 2024-E580-01). Diagnosis of \u003cem\u003eC. sinensis\u003c/em\u003e infection was established based on at least one diagnostic criterion: serological evidence from ELISA testing or microscopic identification of the parasite in stool, bile, or pathological specimens [28].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study utilized GEPIA (http://gepia.cancer-pku.cn/index.html) to explore the expression of CD24 in HCC, UALCAN (http://ualcan.path.uab.edu/) to analyze the relationship between CD24 mRNA expression in HCC and various pathological parameters, and TIMER2.0 (http://timer.cistrome.org/) to assess the correlation between CD24 expression, immune cell infiltration, and immune checkpoints. Finally, we employed the Kaplan-Meier plotter database (http://kmplot.com) to evaluate the prognostic significance of CD24 regarding OS, progression-free survival (PFS), recurrence free survival (RFS) and disease specific survival (DSS). The analysis included hazard ratios (HR) with 95% confidence intervals (95% CI) and log-rank p-values.\u0026nbsp;Potential transcription factors binding to the CD24 promoter region were predicted using the PROMO (https://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) and Harmonizome (https://maayanlab.cloud/Harmonizome/) databases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation CsESPs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePseudorasbora parvas\u003c/em\u003e, recognized as the second intermediate host for \u003cem\u003eC. sinensis\u003c/em\u003e, was sourced from Hengzhou, Guangxi, exhibiting natural infection with metacercariae. The fish specimens underwent digestion with an artificial gastric solution composed of 0.5% pepsin, 1% hydrochloric acid, and 0.9% sodium chloride NaCl, maintained at 37 \u0026deg;C overnight. Male Sprague-Dawley rats were procured from the Guangxi Medical University Laboratory Animal Center and were cared for in compliance with the National Institutes of Health\u0026apos;s standards for animal welfare and ethical treatment (Ethics approval number: 202410011). Metacercariae were extracted using a stereomicroscope, and a total of 150 metacercariae were administered intragastrically to each rat. Following an 8-week infection period, the rats were euthanized post-anesthesia, allowing for the collection of adult worms from the bile ducts, which were subsequently cultured in phosphate-buffered saline (PBS), maintained at 37 \u0026deg;C in an atmosphere containing 5% CO₂. The culture medium was collected after a duration of 6 hours, followed by centrifugation at 12,000\u0026times;g for 10 minutes at 4 \u0026deg;C. The resulting supernatant, designated as CsESPs, was sterilized through a 0.22 \u0026mu;m filter and preserved at -80 \u0026deg;C. The concentrations of CsESPs were quantified utilizing the bicinchoninic acid assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman HCC cell lines Huh7 and Hep3B were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China) and validated through STR identification. These cell lines were maintained in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium enriched with 10% fetal bovine serum and 1% penicillin-streptomycin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShort interfering RNAs (siRNAs) targeting CD24 were synthesized by GenePharma and conducted as previously described [29]. For the siRNA sequences, please refer to Supplementary Table 1 for details. The silencing effect on mRNA expressions were examined by qPCR analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of cell viability and colony formation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell viability was evaluated utilizing the Cell Counting Kit-8 (CCK-8) assay. Huh7 and Hep3B cell lines were exposed to 100 \u0026micro;g/ml concentrations of CsESPs for a duration of 48 hours. Subsequently, CCK-8 was combined with the culture medium at a ratio of 1:10, applied to each well, and incubated for 1.5 hours, in accordance with previously established protocols [30]. The colony formation assay was conducted as previously outlined [31], with 1,000 cells being plated in 12-well plates and subjected to treatment with the specified drugs over a period of 10 days. After performing crystal violet staining, photographic documentation of the colonies was obtained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate cell apoptosis, we utilized the Annexin V-APC/PI Apoptosis Kit (Lianke). In summary, HCC cells were collected via accutase treatment. Following this, the cells were resuspended in binding buffer and subsequently stained with 5 \u0026micro;l of Annexin V-APC and propidium iodide for a duration of 5 minutes. The analysis of apoptosis was conducted using a CytoFLEX Flow Cytometer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting (WB) assay and immunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWB was performed as previously described [32]. Primary antibodies E2F1 (sc-251) were obtained from Santa Cruz Biotechnology (USA), CD24 (67627-1-Ig), Bcl-2-associated X protein (BAX) (50599-2-Ig), ACTIN (66009-1-Ig) and \u0026alpha;-Tubulin (66031-1-Ig) from Proteintech (China). Immunofluorescence was performed as described previously [33]. Observational data and imaging were captured utilizing a confocal microscope (Zeiss LSM 800).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal-time quantitative polymerase chain reaction (qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA extraction was performed utilizing TRIzol reagent in accordance with the guidelines provided by the manufacturer. To assess mRNA expression levels, qPCR was conducted employing the 2\u0026times; Universal Blue SYBR Green qPCR Master Mix (Servicebio), adhering strictly to the manufacturer\u0026apos;s protocol. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control for normalization. All primers utilized for qPCR were procured from Sangon Biotechnology Ltd., and the specific sequences of these primers are detailed in Supplementary Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual-Luciferase reporter assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wild-type and mutant luciferase plasmids of the CD24 promoter were constructed by Hunan Fenghui Biotechnology Co., Ltd. (China). Similarly, the vector and overexpression plasmids of E2F1 were constructed by Wuhan MiaoLing Biotechnology Co., Ltd. (China). The binding sites of E2F1 on the CD24 promoter were predicted using the JASPAR database (https://jaspar.elixir.no/). HEK-293T cells were initially resuspended and subsequently plated into 6-well culture plates. The CD24 promoter luciferase plasmids were co-transfected into the HEK-293T cells alongside either a control vector or plasmids designed for E2F1 overexpression. Following a 48-hour incubation period, cell lysates were harvested, and the activities of firefly and renilla luciferase were quantified utilizing the Dual-Luciferase Reporter Assay Kits (Promega).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatin immunoprecipitation (ChIP) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChIP was executed in accordance with the guidelines provided by the manufacturer (Beyotime Biotechnology). In summary, cells were harvested and subjected to crosslinking using 1% formaldehyde. Subsequently, the DNA underwent fragmentation through sonication. Pre-washing was conducted with agarose beads, and then the mixture was incubated with 5 \u0026mu;g of E2F1 (Santa Cruz, sc-251) in the culture medium. Ultimately, DNA fragments were isolated utilizing magnetic beads, followed by quantification through qPCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of peripheral blood mononuclear cells (PBMCs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePBMCs were extracted from EDTA-anticoagulated peripheral blood obtained from healthy individuals. The whole blood sample was diluted with an equivalent volume of PBS and carefully layered over an equal volume of lymphocyte separation medium. PBMCs were then isolated by gradient centrifugation. Following centrifugation at 800g for 25 minutes, the mononuclear cell layer was harvested and washed three times with PBS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-culture of HCC cells with PBMCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePBMC and hepatocellular carcinoma cells were co-cultured using a non-contact co-culture transwell system (JETBIOFIL, China). HCC cells were maintained in the upper chamber, whereas PBMCs were grown in the lower chamber, using RPMI-1640 medium enriched with 10% fetal bovine serum and 1% penicillin-streptomycin. After 48 hours of co-incubation, PBMCs were harvested for further analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData analysis was conducted utilizing SPSS software (version 22.0), with results expressed as the mean \u0026plusmn; standard error of the mean (SEM). Statistical visualizations were created employing GraphPad Prism 8 software. Group comparisons were carried out using either the independent samples \u003cem\u003et\u003c/em\u003e-test or the Mann-Whitney U-test, depending on the data distribution. Alternatively, one-way analysis of variance (ANOVA) was employed, followed by multiple comparisons using either the least significant difference (LSD) test or the Games-Howell correction. A \u003cem\u003ep\u003c/em\u003e-value of less than 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCD24 expression is up-regulated in HCC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing our in-house cohort, we evaluated CD24 mRNA expression in HCC tumor and paired adjacent non-tumor tissues. qPCR analysis revealed significant upregulation of CD24 in \u003cem\u003eC. sinensis\u003c/em\u003e-infected HCC tumors compared to adjacent tissues (Figure 1A-C).\u0026nbsp;To validate these findings, we conducted comprehensive bioinformatic analyses using multiple independent datasets (GEPIA, UALCAN, and TIMER2.0). Consistent with our experimental results, database analyses confirmed that CD24 expression was significantly increased in HCC tissues when compared to those of normal liver tissues. (Figure 1D-E). Furthermore, pan-cancer analysis through TIMER2.0 demonstrated significantly higher CD24 expression in multiple malignancies relative to their corresponding normal tissues (Figure 1F). These consistent findings across experimental and bioinformatic approaches suggest that CD24 upregulation is particularly prominent in \u003cem\u003eC. sinensis\u003c/em\u003e-associated HCC and could be crucial in the development of liver cancer and the advancement of tumors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD24 expression and different HCC clinicopathological parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the marked elevation of CD24 expression in HCC, we systematically evaluated its clinical relevance using the UALCAN database. Our analysis revealed several key findings. First, CD24 expression showed progressive upregulation across advancing tumor stages (Stages 1-3) compared to normal liver tissue (Figure 2A). This stage-dependent pattern was paralleled by Edmondson's pathological grading, with significantly higher CD24 levels in grades 1-3 HCC (Figure 2B). Notably, CD24 expression was particularly elevated in cases with nodal metastasis (Figure 2C), suggesting a potential role in disease dissemination.\u0026nbsp;Further stratification analysis demonstrated that CD24 upregulation occurred independently of TP53 mutation status (Figure 2D), patient gender (Figure 2E) and aging (21-80 years) (Figure 2F). In all subgroups examined, CD24 expression remained consistently higher in tumor tissue than in normal liver tissues. These comprehensive analyses establish CD24 as a robust biomarker consistently associated with HCC progression across multiple clinical parameters.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh CD24 expression is associated with poor prognosis in HCC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the prognostic value of CD24 in HCC, we performed survival analyses using the Kaplan-Meier plotter database. Patients were dichotomized into high- and low-expression groups based on the median CD24 expression level. The results demonstrated that the CD24-high HCC patients exhibited significantly worse OS, PFS, RFS, and DSS\u0026nbsp;compared to those with low CD24 expression (Figure 3).\u0026nbsp;Furthermore, we assessed the prognostic significance of CD24 in relation to diverse clinicopathological characteristics by utilizing the Kaplan-Meier database. Especially in those with vascular invasion, higher CD24 expression was substantially linked to worse OS and PFS (Supplementary Table 3). Collectively, these findings indicate that CD24 serves as a significant prognostic marker in HCC, highlighting its potential clinical relevance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation analysis of CD24 expression with immune cell infiltration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the complexity of the liver immune microenvironment, we systematically evaluated\u0026nbsp;the correlation between CD24 expression and the tumor immune microenvironment characteristics using the TIMER2.0 database. Our comprehensive analysis revealed several key findings. CD24 expression exhibited notable positive correlations with the infiltration levels of six predominant immune cell types, namely B cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, CD4\u003csup\u003e+\u003c/sup\u003e T cells, macrophages, neutrophils, and dendritic cells (Figure 4A). To further investigate the association between CD24 and immune responses, we analyzed the relationship between CD24 expression and a range of immune characteristics in HCC. These included evaluations of B cells, T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, monocytes, tumor-associated macrophages (TAMs), M1/M2 macrophages, neutrophils, natural killer cells, and dendritic cells. Following adjustments for tumor purity, our analysis confirmed a significant correlation between CD24 expression and most established markers representative of these immune cell types (Supplementary Table 4). Additionally, we identified a positive correlation between CD24 expression levels and various immune checkpoints, including CTLA-4, LAG3, PDCD1, HAVCR2, and BTLA (Figure 4B-F). Collectively, these results indicate that CD24 may serve a crucial function in the regulation of immune infiltration in HCC, thereby underscoring its potential as a target for therapeutic intervention.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCsESPs promote proliferation and inhibit apoptosis in HCC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effects of CsESPs on HCC cells, we treated Hep3B and Huh7 cells with 100 μg/ml CsESPs. CsESPs treatment significantly enhanced the viability of HCC cells after 48 hours of exposure and clonogenic proliferation compared to the control group (Figure 5A-B). Furthermore, the flow cytometry analysis demonstrated that CsESPs markedly reduced basal label of apoptosis in HCC cells (Figure 5C-D). Collectively, these results indicate that CsESPs promote HCC cell proliferation and suppress apoptosis, suggesting a potential role in HCC progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCsESPs may up-regulate CD24 expression through transcription factor E2F1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsistent with the mRNA changes (Figure. 1B), CD24 was significantly upregulated in CsESPs-treated HCC cells (Figure 6A and 6B). To investigate the mechanism underlying CD24 upregulation by CsESPs, we predicted potential transcription factors binding to the CD24 promoter region using both PROMO and Harmonizome databases. By intersecting the results from both databases, we identified E2F1 and AR as candidate transcription factors (Figure 6C). Subsequent correlation analysis using the GEPIA database revealed that E2F1 exhibited a positive correlation with CD24, in contrast, AR showed a negative correlation with CD24 (Figure S1). Further WB and qPCR experiments confirmed that E2F1 was up-regulated in CsESPs-treated HCC cells (Figure 6B and 6D). Immunofluorescence assays also indicated that E2F1 translocated from the cytoplasm to the nucleus following CsESPs treatment (Figure 6E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo validate the binding of E2F1 to the CD24 promoter, we used the JASPAR database to predict potential binding sites. The region spanning 1325–1335 bp scored highly, suggesting strong binding affinity. We subsequently constructed E2F1 overexpression vectors and CD24 promoter vectors with either wild-type or mutated binding sites. Dual-luciferase reporter assays revealed that E2F1 enhanced luciferase activity conjugating wild-type CD24 promoter sequence, but not in those with mutant sequence (Figure 6F). More importantly, ChIP-qPCR confirmed that E2F1 binds to the CD24 promoter region (Figure 6G). Collectively, these findings demonstrate that CsESPs upregulate CD24 expression through transcriptional regulation by E2F1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD24 silencing abolishes CsESPs-induced cell proliferation and apoptosis reduction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the essential role of CD24 in CsESPs-treated HCC cells, we performed knockdown of CD24 using specific siRNA (Figure S2). As expected, CD24 silencing significantly reduced the viability of CsESPs-treated HCC cells (Figure 7A-B). Flow cytometry analysis further demonstrated that CD24 knockdown markedly increased apoptosis in these cells (Figure 7C-E). WB assays revealed elevated expression of the pro-apoptotic protein BAX in CD24-knockdown cells (Figure 7F). Together, these results confirm that CD24 promotes cell proliferation and inhibits apoptosis in CsESPs-treated HCC cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRelationship between CD24 and immune checkpoints in CsESPs-treated HCC cells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate whether CD24 up-regulation in CsESPs-treated HCC cells affect multiple immune checkpoint molecules, we established an \u003cem\u003ein vitro\u003c/em\u003e co-culture system of HCC cells and PBMCs. qPCR analyses showed that CD24 knockdown significantly attenuated CsESPs-induced upregulation of CTLA-4 and LAG-3 (Figure 8A-B). However, CD24 knockdown failed to alter the levels of other genes associated with immune checkpoints, such as PDCD1, HAVCR2, and BTLA (Figure 8C-E). These results suggest that CD24 may selectively regulate specific immune checkpoint pathways in CsESPs -treated HCC cells.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrated that CD24 is significantly upregulated in HCC, particularly in cases associated with \u003cem\u003eC. sinensis\u003c/em\u003e. Analysis from multiple databases indicates that the level of CD24 expression correlates with clinicopathological stages and prognosis in HCC patients. Mechanistically, CsESPs can promote proliferation and inhibit apoptosis of HCC cells through E2F1-mediated transcriptional regulation of CD24. Moreover, CsESPs up-regulated the immune checkpoint molecules CTLA-4 and LAG-3 in the co-cultured PBMCs, an effect that could be reversed by knockdown of CD24 (Figure 9).\u003c/p\u003e\n\u003cp\u003eConsistent with the up-regulation of CD24 in multiple malignancies [34], our preliminary single-cell sequencing data revealed elevated CD24 expression in \u003cem\u003eC. sinensis\u003c/em\u003e-associated HCC. We further validated its up-regulation in both in-house cohorts and online datasets. More importantly, the Kaplan\u0026ndash;Meier and Cox regression analyses demonstrated that aberrant CD24 expression has the potential to act as a prognostic biomarker for HCC, consistent with prior research [27]. Furthermore, CD24 exhibits a strong association with immune checkpoint genes, suggesting its potential role in modulating tumor immune infiltration. Given its differential expression patterns and immunomodulatory implications, we hypothesize that CD24 contributes to tumorigenesis and progression in a context-dependent manner, possibly influencing immunotherapy responsiveness.\u003c/p\u003e\n\u003cp\u003eClonorchiasis,\u0026nbsp;caused by the \u003cem\u003eC. sinensis\u003c/em\u003e, is recognized as one of the most clinically overlooked tropical diseases in East Asia [35]. This persistent \u003cem\u003eC. sinensis\u0026nbsp;\u003c/em\u003einfection is recognized as a risk factor for cholangiocarcinoma development [12, 36]. Recent epidemiological evidence also associates \u003cem\u003eC. sinensis\u003c/em\u003e infection with poorer clinical outcomes in HCC patients [8, 23, 24]. Notably, the parasite enhances malignant transformation through induction of cancer stem cell-like properties [8].\u0026nbsp;The molecular mechanisms underlying this oncogenic effect involve parasite-derived factors such as the calcium-binding protein Cs16, which drives hepatic inflammation by reprogramming metabolic pathways in innate immune cells [37]. Similarly, CsESPs contribute to infection-associated pathology. For instance, the CsESP component Csseverin exhibits potent anti-apoptotic effects in human HCC cell lines\u0026nbsp;[38]. In alignment with these findings, our current study demonstrates that CsESPs promote HCC cell proliferation while suppressing apoptosis. Importantly, we observed that CD24-upregulation may play an essential role in CsESP-induced cell proliferation enhancement and apoptosis reduction in HCC cells.\u003c/p\u003e\n\u003cp\u003eOur experimental findings corroborate CD24\u0026apos;s critical role in CsESP-mediated HCC pathogenesis. Specifically, CD24 knockdown in CsESP-treated HCC cells significantly reduced viability while increasing apoptosis, accompanied by elevated BAX expression. These results support a model wherein CsESPs upregulate CD24 expression. Our mechanical study further identifies a direct transcriptional regulation mode of CD24 through E2F1.\u0026nbsp;As a pivotal transcription factor regulating diverse cellular processes including proliferation, differentiation, migration and metabolism [39], E2F1 exhibits dual oncogenic and tumor-suppressive functions in cancer progression [40, 41].\u0026nbsp;Apart from E2F1, several other pathways have been reported to regulate CD24 expression. For example, ADAR, an RNA-editing enzyme, promotes HCC progression by enhancing CD24 expression through its interaction with SNRPD3 and RNPS1, thereby inhibiting STAU1-mediated mRNA degradation\u0026nbsp;[42]. In addition, the Hippo-YAP1-SOX4 axis was identified as a key regulatory pathway responsible for YAP1-mediated CD24 overexpression in HCC cells. Notably, suppression of CD24 expression markedly inhibited YAP1-initiated HCC progression\u0026nbsp;[43].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur findings demonstrate that CD24 expression positively correlates with immune checkpoints, including LAG-3 and CTLA-4. Importantly, CsESPs-induced upregulation of these checkpoints was attenuated by CD24 knockdown, suggesting CD24\u0026rsquo;s pivotal role in mediating immune evasion in CsESP-treated HCC. As critical immune checkpoint molecules, CTLA-4 and LAG-3 suppress T cell activation through distinct mechanisms. CTLA-4 competitively binds to B7 molecules with higher affinity than CD28, thereby inhibiting co-stimulatory signals, while LAG-3 negatively regulates T cell responses via MHC class II interactions. Co-expression of these checkpoints on exhausted T cells drives progressive functional impairment, characterized by diminished cytokine production and cytotoxic activity. Notably, recent evidence demonstrates that dual CTLA-4/LAG-3 blockade synergistically restores T cell function, highlighting their cooperative role in maintaining T cell exhaustion [44]. T cell exhaustion, a dysfunctional state marked by TOX-driven transcriptional reprogramming and sustained expression of inhibitory receptors (e.g., LAG-3, CTLA-4), is a hallmark of chronic viral infections and tumor microenvironments [45]. This exhaustion leads to CD8\u003csup\u003e+\u003c/sup\u003e T cell proliferation failure and loss of effector capacity, ultimately compromising antitumor immunity. These results position CD24 as a potential regulator of T cell exhaustion and a therapeutic target for restoring antitumor immunity.\u003c/p\u003e\n\u003cp\u003eWhile this study provides novel insights into CD24\u0026apos;s role in \u003cem\u003eC. sinensis\u003c/em\u003e-associated HCC pathogenesis, several limitations should be acknowledged. First, the study lacks \u003cem\u003ein vivo\u003c/em\u003e validation, as findings derived from the \u003cem\u003ein vitro\u003c/em\u003e experiments may not fully reflect the complexity of HCC progression. Clarification of HCC tumor microenvironment through\u0026nbsp;single-cell RNA sequencing (scRNA-seq) and spatial transcriptomic sequencing (ST-seq) may shed light on this question.\u0026nbsp;Second, the relatively small sample size in the in-house HCC cohort may limit the generalizability of the results. Particularly, it is yet unknown whether \u003cem\u003eC. sinensis\u0026nbsp;\u003c/em\u003einfection affect the treatment response and long-term prognosis of immunotherapy which is increasingly adopted as the first-line HCC treatment. Third, CsESPs may not fully represent the oncogenic ability of \u003cem\u003eC. sinensis.\u0026nbsp;\u003c/em\u003eThe released non-coding RNAs and metabolites by extracellular vesicle by parasite are also important oncogenic factors. Addressing these limitations in future studies will be essential to dissect the oncogenic effects of \u003cem\u003eC. sinensis\u003c/em\u003e-associated HCC and the potential application of CD24 as a predictive biomarker and potential therapeutic target.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study comprehensively establishes CD24 as a clinically significant biomarker in HCC, with particularly pronounced overexpression in \u003cem\u003eC. sinensis\u003c/em\u003e-associated cases. Integrated multi-database analyses revealed that CD24 expression levels not only correlate with advanced clinicopathological stages but also act as a standalone prognostic marker in patients with HCC. Mechanistically, CsESPs upregulates E2F1, which subsequently activates CD24 transcription, ultimately promoting tumor cell proliferation while suppressing apoptosis. Furthermore, CsESP treatment increases the levels of the immune checkpoint molecules CTLA-4 and LAG-3 in co-cultured PBMCs- an effect that was reversible by CD24 knockdown. These results reveal the essential roles of CD24 in both tumor-intrinsic growth pathways within HCC cells and immune evasion mechanisms in microenvironment in \u003cem\u003eC. sinensis\u003c/em\u003e-associated HCC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCD24\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecluster of differentiation 24\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eC. sinensis\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eClonorchis sinensis\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCsESP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eClonorchis sinensis\u003c/em\u003e excretory-secretory products\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge the financial support from the Natural Science Foundation of Shanghai (24ZR1414000), the Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor (Guangxi Medical University), Ministry of Education (GKE-ZZ202131), the National Natural Science Foundation of China (U22A20374 and 32160160), and the Innovation Project of Guangxi Graduate Education (YCBZ2024142).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW.M.L., J.Z., and G.D.L. designed the experiments; W.M.L., J.Y., Y.W., Q.R.C. and Y.C. performed the experiments; Z.J.L., J.F. and G.Z.Z. analyzed the data; J.Z., and G.D.L. supervised the studies and contributed reagents/materials/ analysis tools; and W.M.L., J.Y., and Z.J.L. prepared the initial draft of the manuscript. All four authors approve the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was partially supported by grants from the Natural Science Foundation of Shanghai (24ZR1414000), the Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor (Guangxi Medical University), Ministry of Education (GKE-ZZ202131), the National Natural Science Foundation of China (U22A20374 and 32160160), and The Innovation Project of Guangxi Graduate Education (YCBZ2024142).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analyzed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll HCC patients underwent surgical operation. Informed consent was acquired from all participants, and the research received approval from the Ethics Committee at the First Affiliated Hospital of Guangxi Medical University\u0026nbsp;(Ethics approval number: 2024-E580-01).\u003c/p\u003e\n\u003cp\u003eAll animal experiment procedures in this study were conducted\u003c/p\u003e\n\u003cp\u003ein accordance with the Guide for the Guangxi Medical University Laboratory Animal Center (Ethics approval number: 202410011).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Department of Toxicology, School of Public Health, Guangxi Medical University, Nanning, Guangxi, China. \u003csup\u003e2\u003c/sup\u003e School of Public Health, Fudan University, Shanghai, China. \u003csup\u003e3\u003c/sup\u003e Department of Physiology, School of Basic Medical Sciences, Guangxi Medical University, Nanning, Guangxi, China. \u003csup\u003e4\u003c/sup\u003e Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China. \u003csup\u003e5\u003c/sup\u003e Department of Pathology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital and Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Shenzhen, Guangdong, China. \u003csup\u003e6\u003c/sup\u003e Hengzhou Center for Disease Control and Prevention, Hengzhou, Guangxi, China. \u003csup\u003e7\u003c/sup\u003e Department of Hepatobiliary Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray F, Laversanne M, Sung H, et al: Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin.\u003cem\u003e \u003c/em\u003e2024;74(3):229-263.http://dx.doi.org/10.3322/caac.21834\u003c/li\u003e\n\u003cli\u003eHan B, Zheng R, Zeng H, et al: Cancer incidence and mortality in China, 2022. J Natl Cancer Cent.\u003cem\u003e \u003c/em\u003e2024;4(1):47-53.http://dx.doi.org/10.1016/j.jncc.2024.01.006\u003c/li\u003e\n\u003cli\u003eBray F, Ferlay J, Soerjomataram I, et al: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin.\u003cem\u003e \u003c/em\u003e2018;68(6):394-424.http://dx.doi.org/10.3322/caac.21492\u003c/li\u003e\n\u003cli\u003eQiu H, Cao S, Xu R: Cancer incidence, mortality, and burden in China: a time-trend analysis and comparison with the United States and United Kingdom based on the global epidemiological data released in 2020. Cancer Commun (Lond).\u003cem\u003e \u003c/em\u003e2021;41(10):1037-1048.http://dx.doi.org/10.1002/cac2.12197\u003c/li\u003e\n\u003cli\u003eQi J, Li M, Wang L, et al: National and subnational trends in cancer burden in China, 2005-20: an analysis of national mortality surveillance data. Lancet Public Health.\u003cem\u003e \u003c/em\u003e2023;8(12):e943-e955.http://dx.doi.org/10.1016/S2468-2667(23)00211-6\u003c/li\u003e\n\u003cli\u003eZhou M, Wang H, Zhu J, et al: Cause-specific mortality for 240 causes in China during 1990-2013: a systematic subnational analysis for the Global Burden of Disease Study 2013. Lancet.\u003cem\u003e \u003c/em\u003e2016;387(10015):251-272.http://dx.doi.org/10.1016/S0140-6736(15)00551-6\u003c/li\u003e\n\u003cli\u003eLi Q, Cao J, Rong M, et al: Analysis of incidence and mortality of liver cancer in cancer registration areas in Guangxi, 2010\u0026mdash;2016. J Chin J Oncol Prev Treat.\u003cem\u003e \u003c/em\u003e2021;13(2):138-143.http://dx.doi.org/10.3969/j.issn.1674-5671.2021.02.05\u003c/li\u003e\n\u003cli\u003eJiang ZH, Wan XL, Lv GL, et al: High prevalence of \u003cem\u003eClonorchis sinensis\u003c/em\u003e infection in Guangxi, Southern China. Trop Med Health.\u003cem\u003e \u003c/em\u003e2021;49(1):6.http://dx.doi.org/10.1186/s41182-021-00297-0\u003c/li\u003e\n\u003cli\u003eTang ZL, Huang Y, Yu XB: Current status and perspectives of \u003cem\u003eClonorchis sinensis \u003c/em\u003eand clonorchiasis: epidemiology, pathogenesis, omics, prevention and control. Infect Dis Poverty.\u003cem\u003e \u003c/em\u003e2016;5(1):71.http://dx.doi.org/10.1186/s40249-016-0166-1\u003c/li\u003e\n\u003cli\u003eHatta MNA, Mohamad Hanif EA, Chin SF, et al: Pathogens and Carcinogenesis: A Review. Biology (Basel).\u003cem\u003e \u003c/em\u003e2021;10(6).http://dx.doi.org/10.3390/biology10060533\u003c/li\u003e\n\u003cli\u003ePak JH, Lee JY, Jeon BY, et al: Cytokine Production in Cholangiocarcinoma Cells in Response to \u003cem\u003eClonorchis sinensis\u003c/em\u003e Excretory-Secretory Products and Their Putative Protein Components. Korean J Parasitol.\u003cem\u003e \u003c/em\u003e2019;57(4):379-387.http://dx.doi.org/10.3347/kjp.2019.57.4.379\u003c/li\u003e\n\u003cli\u003eNa BK, Pak JH, Hong SJ: \u003cem\u003eClonorchis sinensis\u003c/em\u003e and clonorchiasis. Acta Trop.\u003cem\u003e \u003c/em\u003e2020;203:105309.http://dx.doi.org/10.1016/j.actatropica.2019.105309\u003c/li\u003e\n\u003cli\u003eBouvard V, Baan R, Straif K, et al: A review of human carcinogens--Part B: biological agents. Lancet Oncol.\u003cem\u003e \u003c/em\u003e2009;10(4):321-322.http://dx.doi.org/10.1016/s1470-2045(09)70096-8\u003c/li\u003e\n\u003cli\u003eLin Q, Tang Z, Qin Y, et al: \u003cem\u003eClonorchis sinensis\u003c/em\u003e infection amplifies hepatocellular carcinoma stemness, predicting unfavorable prognosis. PLoS Negl Trop Dis.\u003cem\u003e \u003c/em\u003e2024;18(1):e0011906.http://dx.doi.org/10.1371/journal.pntd.0011906\u003c/li\u003e\n\u003cli\u003eLi YK, Zhao JF, Yang CL, et al: Effects of \u003cem\u003eClonorchis sinensis \u003c/em\u003ecombined with Hepatitis B virus infection on the prognosis of patients with Hepatocellular Carcinoma following Hepatectomy. PLoS Negl Trop Dis.\u003cem\u003e \u003c/em\u003e2023;17(1):e0011012.http://dx.doi.org/10.1371/journal.pntd.0011012\u003c/li\u003e\n\u003cli\u003eQi Y, Hu J, Liang J, et al: \u003cem\u003eClonorchis sinensis\u003c/em\u003e infection contributes to hepatocellular carcinoma progression in rat. Parasitol Res.\u003cem\u003e \u003c/em\u003e2022;121(12):3403-3415.http://dx.doi.org/10.1007/s00436-022-07699-x\u003c/li\u003e\n\u003cli\u003eYan C, Zhou QY, Wu J, et al: Csi-let-7a-5p delivered by extracellular vesicles from a liver fluke activates M1-like macrophages and exacerbates biliary injuries. Proc Natl Acad Sci U S A.\u003cem\u003e \u003c/em\u003e2021;118(46).http://dx.doi.org/10.1073/pnas.2102206118\u003c/li\u003e\n\u003cli\u003eWang C, Lei H, Tian Y, et al: \u003cem\u003eClonorchis sinensis\u003c/em\u003e granulin: identification, immunolocalization, and function in promoting the metastasis of cholangiocarcinoma and hepatocellular carcinoma. Parasit Vectors.\u003cem\u003e \u003c/em\u003e2017;10(1):262.http://dx.doi.org/10.1186/s13071-017-2179-4\u003c/li\u003e\n\u003cli\u003eWang C, He Q, Yin Y, et al: \u003cem\u003eClonorchis sinensis\u003c/em\u003e Granulin Promotes Malignant Transformation of Hepatocyte Through EGFR-Mediated RAS/MAPK/ERK and PI3K/Akt Signaling Pathways. Front Cell Infect Microbiol.\u003cem\u003e \u003c/em\u003e2021;11:734750.http://dx.doi.org/10.3389/fcimb.2021.734750\u003c/li\u003e\n\u003cli\u003eHe Q, Pan X, Yin Y, et al: \u003cem\u003eClonorchis sinensis\u003c/em\u003e granulin promotes malignant transformation of human intrahepatic biliary epithelial cells through interaction with M2 macrophages via regulation of STAT3 phosphorylation and the MEK/ERK pathway. Parasit Vectors.\u003cem\u003e \u003c/em\u003e2023;16(1):139.http://dx.doi.org/10.1186/s13071-023-05765-6\u003c/li\u003e\n\u003cli\u003eBarkal AA, Brewer RE, Markovic M, et al: CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy. Nature.\u003cem\u003e \u003c/em\u003e2019;572(7769):392-396.http://dx.doi.org/10.1038/s41586-019-1456-0\u003c/li\u003e\n\u003cli\u003eMensah FFK, Armstrong CW, Reddy V, et al: CD24 Expression and B Cell Maturation Shows a Novel Link With Energy Metabolism: Potential Implications for Patients With Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Front Immunol.\u003cem\u003e \u003c/em\u003e2018;9:2421.http://dx.doi.org/10.3389/fimmu.2018.02421\u003c/li\u003e\n\u003cli\u003eKristiansen G, Sammar M, Altevogt P: Tumour biological aspects of CD24, a mucin-like adhesion molecule. J Mol Histol.\u003cem\u003e \u003c/em\u003e2004;35(3):255-262.http://dx.doi.org/10.1023/b:hijo.0000032357.16261.c5\u003c/li\u003e\n\u003cli\u003eLim SC: CD24 and human carcinoma: tumor biological aspects. Biomed Pharmacother.\u003cem\u003e \u003c/em\u003e2005;59 Suppl 2:S351-354.http://dx.doi.org/10.1016/s0753-3322(05)80076-9\u003c/li\u003e\n\u003cli\u003eTolkach Y, Zarbl R, Bauer S, et al: DNA Promoter Methylation and ERG Regulate the Expression of CD24 in Prostate Cancer. Am J Pathol.\u003cem\u003e \u003c/em\u003e2021;191(4):618-630.http://dx.doi.org/10.1016/j.ajpath.2020.12.014\u003c/li\u003e\n\u003cli\u003eOchsner SA, Strick-Marchand H, Qiu Q, et al: Transcriptional profiling of bipotential embryonic liver cells to identify liver progenitor cell surface markers. Stem Cells.\u003cem\u003e \u003c/em\u003e2007;25(10):2476-2487.http://dx.doi.org/10.1634/stemcells.2007-0101\u003c/li\u003e\n\u003cli\u003eYang XR, Xu Y, Yu B, et al: CD24 is a novel predictor for poor prognosis of hepatocellular carcinoma after surgery. Clin Cancer Res.\u003cem\u003e \u003c/em\u003e2009;15(17):5518-5527.http://dx.doi.org/10.1158/1078-0432.CCR-09-0151\u003c/li\u003e\n\u003cli\u003eChang JI, Lee K, Kim D, et al: Clinical Characteristics of \u003cem\u003eClonorchis sinensis\u003c/em\u003e-Associated Cholangiocarcinoma: A Large-Scale, Single-Center Study. Front Med (Lausanne).\u003cem\u003e \u003c/em\u003e2021;8:675207.http://dx.doi.org/10.3389/fmed.2021.675207\u003c/li\u003e\n\u003cli\u003eZhou J, Tan SH, Nicolas V, et al: Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion. Cell Res.\u003cem\u003e \u003c/em\u003e2013;23(4):508-523.http://dx.doi.org/10.1038/cr.2013.11\u003c/li\u003e\n\u003cli\u003eWei H, Chen Y, Qin Z, et al: Artesunate demonstrates neuroprotective effect through activation of lysosomal function and inhibition of cGAS-STING pathway. Neuropharmacology.\u003cem\u003e \u003c/em\u003e2025:110426.http://dx.doi.org/10.1016/j.neuropharm.2025.110426\u003c/li\u003e\n\u003cli\u003eLi ZJ, Dai HQ, Huang XW, et al: Artesunate synergizes with sorafenib to induce ferroptosis in hepatocellular carcinoma. Acta Pharmacol Sin.\u003cem\u003e \u003c/em\u003e2021;42(2):301-310.http://dx.doi.org/10.1038/s41401-020-0478-3\u003c/li\u003e\n\u003cli\u003eLu GD, Ang YH, Zhou J, et al: CCAAT/enhancer binding protein alpha predicts poorer prognosis and prevents energy starvation-induced cell death in hepatocellular carcinoma. Hepatology.\u003cem\u003e \u003c/em\u003e2015;61(3):965-978.http://dx.doi.org/10.1002/hep.27593\u003c/li\u003e\n\u003cli\u003eLi XY, Cui X, Xie CQ, et al: Andrographolide causes p53-independent HCC cell death through p62 accumulation and impaired DNA damage repair. Phytomedicine.\u003cem\u003e \u003c/em\u003e2023;121:155089.http://dx.doi.org/10.1016/j.phymed.2023.155089\u003c/li\u003e\n\u003cli\u003eZhao C, Huang Y, Zhang H, et al: CD24 affects the immunosuppressive effect of tumor-infiltrating cells and tumor resistance in a variety of cancers. Discov Oncol.\u003cem\u003e \u003c/em\u003e2024;15(1):399.http://dx.doi.org/10.1007/s12672-024-01284-7\u003c/li\u003e\n\u003cli\u003eLai DH, Hong XK, Su BX, et al: Current status of \u003cem\u003eClonorchis sinensis\u003c/em\u003e and clonorchiasis in China. Trans R Soc Trop Med Hyg.\u003cem\u003e \u003c/em\u003e2016;110(1):21-27.http://dx.doi.org/10.1093/trstmh/trv100\u003c/li\u003e\n\u003cli\u003eWei C, Chen J, Yu Q, et al: \u003cem\u003eClonorchis sinensis\u003c/em\u003e infection contributes to hepatocellular carcinoma progression via enhancing angiogenesis. PLoS Negl Trop Dis.\u003cem\u003e \u003c/em\u003e2024;18(11):e0012638.http://dx.doi.org/10.1371/journal.pntd.0012638\u003c/li\u003e\n\u003cli\u003eLi Q, Li X, Kan S, et al: \u003cem\u003eClonorchis sinensis\u003c/em\u003e calcium-binding protein Cs16 causes acute hepatic injury possibly by reprogramming the metabolic pathway of bone marrow-derived monocytes. Front Cell Infect Microbiol.\u003cem\u003e \u003c/em\u003e2023;13:1280358.http://dx.doi.org/10.3389/fcimb.2023.1280358\u003c/li\u003e\n\u003cli\u003eChen X, Li S, He L, et al: Molecular characterization of severin from \u003cem\u003eClonorchis sinensis\u003c/em\u003e excretory/secretory products and its potential anti-apoptotic role in hepatocarcinoma PLC cells. PLoS Negl Trop Dis.\u003cem\u003e \u003c/em\u003e2013;7(12):e2606.http://dx.doi.org/10.1371/journal.pntd.0002606\u003c/li\u003e\n\u003cli\u003eMandigo AC, Yuan W, Xu K, et al: RB/E2F1 as a Master Regulator of Cancer Cell Metabolism in Advanced Disease. Cancer Discov.\u003cem\u003e \u003c/em\u003e2021;11(9):2334-2353.http://dx.doi.org/10.1158/2159-8290.CD-20-1114\u003c/li\u003e\n\u003cli\u003eKumari A, Iwasaki T, Pyndiah S, et al: Regulation of E2F1-induced apoptosis by poly(ADP-ribosyl)ation. Cell Death Differ.\u003cem\u003e \u003c/em\u003e2015;22(2):311-322.http://dx.doi.org/10.1038/cdd.2014.146\u003c/li\u003e\n\u003cli\u003eCao LL, Wu YK, Lin TX, et al: CDK5 promotes apoptosis and attenuates chemoresistance in gastric cancer via E2F1 signaling. Cancer Cell Int.\u003cem\u003e \u003c/em\u003e2023;23(1):286.http://dx.doi.org/10.1186/s12935-023-03112-4\u003c/li\u003e\n\u003cli\u003eSun L, Hu P, Yang H, et al: ADARp110 promotes hepatocellular carcinoma progression via stabilization of CD24 mRNA. Proc Natl Acad Sci U S A.\u003cem\u003e \u003c/em\u003e2025;122(3):e2409724122.http://dx.doi.org/10.1073/pnas.2409724122\u003c/li\u003e\n\u003cli\u003eHe X, Zhang Y, Fang Q, et al: Unraveling the role of CD24 in Hepatocellular carcinoma: Involvement of inactivated Hippo signaling and SOX4-mediated regulation. Biochim Biophys Acta Mol Basis Dis.\u003cem\u003e \u003c/em\u003e2024;1870(4):167117.http://dx.doi.org/10.1016/j.bbadis.2024.167117\u003c/li\u003e\n\u003cli\u003eAndrews LP, Cillo AR, Karapetyan L, et al: Molecular Pathways and Mechanisms of LAG3 in Cancer Therapy. Clin Cancer Res.\u003cem\u003e \u003c/em\u003e2022;28(23):5030-5039.http://dx.doi.org/10.1158/1078-0432.CCR-21-2390\u003c/li\u003e\n\u003cli\u003eKhan O, Giles JR, McDonald S, et al: TOX transcriptionally and epigenetically programs CD8(+) T cell exhaustion. Nature.\u003cem\u003e \u003c/em\u003e2019;571(7764):211-218.http://dx.doi.org/10.1038/s41586-019-1325-x\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hepatocellular Carcinoma, Clonorchis sinensis, CD24, E2F1, Tumor Promotion","lastPublishedDoi":"10.21203/rs.3.rs-6378057/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6378057/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHepatocellular carcinoma (HCC) persists as a global health burden with disproportionately high mortality in China's Guangxi region, where endemic \u003cem\u003eClonorchis sinensis\u003c/em\u003e (\u003cem\u003eC. sinensis\u003c/em\u003e) infection coincides with elevated HCC mortality. This study aims to elucidate the oncogenic mechanisms of \u003cem\u003eC. sinensis\u003c/em\u003e 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 \u003cem\u003eC. sinensis\u003c/em\u003e-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 \u003cem\u003eC. sinensis\u003c/em\u003e promotes HCC development through E2F1-CD24 transcriptional activation, simultaneously identifying prognostic biomarkers and therapeutic targets while suggesting combinatory immunotherapy strategies for parasite-associated HCC.\u003c/p\u003e","manuscriptTitle":"Clonorchis sinensis-driven Hepatocarcinogenesis via E2F1-CD24 Transcriptional Axis: Mechanistic and Therapeutic Implications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 03:32:12","doi":"10.21203/rs.3.rs-6378057/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-11T10:14:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-30T13:55:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"96134405940272127074501138028506018479","date":"2025-04-24T22:08:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-24T19:30:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-09T12:56:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-09T09:16:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-04-04T17:30:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"afacba5f-fe07-47d0-8ca4-d13a48e0d0a8","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-25T16:35:46+00:00","versionOfRecord":{"articleIdentity":"rs-6378057","link":"https://doi.org/10.1186/s13071-025-06979-6","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2025-08-19 16:29:28","publishedOnDateReadable":"August 19th, 2025"},"versionCreatedAt":"2025-05-07 03:32:12","video":"","vorDoi":"10.1186/s13071-025-06979-6","vorDoiUrl":"https://doi.org/10.1186/s13071-025-06979-6","workflowStages":[]},"version":"v1","identity":"rs-6378057","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6378057","identity":"rs-6378057","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}