Clonorchis sinensis promotes hepatocellular carcinoma progression via a potential PTTG1-β-catenin-c-MYC axis

Abstract

Clonorchis sinensis (C. sinensis) infection is a recognized risk factor for hepatocellular carcinoma (HCC) and is linked to poor overall survival. To explore the underlying mechanisms, RNA sequencing was conducted on HCC tissues from C. sinensis-positive and C. sinensis-negative patients, revealing significant upregulation of pituitary tumor-transforming gene 1 (PTTG1). Gene Set Enrichment Analysis indicated activation of the Wnt/β-catenin pathway. Functional assays demonstrated that PTTG1 overexpression promoted HCC cell proliferation, migration, invasion, and sphere formation, while PTTG1 knockdown suppressed these processes. Excretory-secretory products (ESP) from C. sinensis enhanced PTTG1 expression and partially restored malignant phenotypes in PTTG1-deficient cells. In vivo, PTTG1 overexpression accelerated tumor growth in subcutaneous models, and ESP treatment elevated the protein levels of PTTG1, β-catenin, c-MYC, and CD44. Immunohistochemistry confirmed higher expression of these markers in both human C. sinensis-positive HCC tissues and a rat model of C. sinensis-associated HCC. These findings suggest that C. sinensis infection promotes HCC malignancy and stemness via ESP-induced PTTG1 expression, potentially through Wnt/β-catenin signaling and its downstream targets, including c-MYC and CD44, particularly in the context of the specific carcinogen-driven models used in this study. Further exploration of the PTTG1 pathway may offer insights into potential therapeutic strategies for C. sinensis-associated HCC.

Graphical abstract

Graphical illustration of the C. sinensis infection enhanced HCC stemness. Preparation of ESP and RNA-seq for C. sinensis-infection HCC patients. ESP active PTTG1-beta-catenin-c-MYC axis to promote HCC stemness. Created with https://biorender.com

Highlights

• •Clonorchis sinensis infection upregulates PTTG1 in HCC via excretory-secretory products.
• •PTTG1 may activate Wnt/β-catenin signaling and increase c-MYC and CD44 expression.
• •ESP partially rescues malignant phenotypes in PTTG1-deficient HCC cells.
• •Targeting PTTG1 may offer therapeutic strategies for C. sinensis-associated HCC.

1. Introduction

Clonorchis sinensis (C. sinensis), a major foodborne pathogen endemic to East Asia, represents a significant public health threat (Qian et al., 2024). Human infection primarily occurs through the consumption of raw or undercooked freshwater fish harboring infectious metacercariae, with adult worms residing in the biliary tract (Apari and Földvári, 2025). Pathogenic mechanisms include mechanical damage induced by worm activity and biochemical modulation via excretory-secretory products (ESP) (Smout et al., 2024). ESP regulates host immune responses, promotes cell proliferation, and inhibits apoptosis, thereby accelerating hepatobiliary disease progression and facilitating tumorigenesis (Wu et al., 2017; Yi et al., 2024). C. sinensis is classified as a Group I carcinogen by the IARC, with a well-established link to cholangiocarcinoma (Brindley et al., 2015).

The mechanisms underlying C. sinensis-associated intrahepatic cholangiocarcinoma (ICC) primarily involve mechanical bile duct damage and chronic inflammation, leading to periductal fibrosis and subsequent carcinogenesis. While epidemiological studies have also suggested an association between C. sinensis infection and hepatocellular carcinoma (HCC), the specific molecular mechanisms by which C. sinensis promotes HCC progression remain poorly understood. Recent proteomic studies have highlighted elevated expression of stemness-associated markers in tumors from C. sinensis-positive patients with HCC(Chen et al., 2025; Lin et al., 2024; Ni et al., 2024; Tang et al., 2024; Wei et al., 2024). Clinical data further show that these patients have significantly shorter overall survival (OS), with a 5-year survival rate below 30% and a median survival of 6–10 months, indicating a strong correlation between C. sinensis infection and aggressive HCC progression (Toh et al., 2023). HCC accounts for 70–90% of primary liver cancers, with over 900,000 new cases annually worldwide, and China bears the highest global disease burden (Cao, 2024; Qian and Zhou, 2021). Previous studies have shown that C. sinensis ESP can induce epithelial-mesenchymal transition by activating the ERK/PI3K/AKT pathway or by mimicking growth factor functions to promote tumor proliferation, migration, and invasion (Chen et al., 2013; He et al., 2023; Wang et al., 2021; Xu et al., 2024). However, the role of ESP in enhancing HCC malignancy by modulating cancer stem cell (CSC) characteristics remains inadequately explored. While C. sinensis infection is known to upregulate CSC marker expression, the downstream signaling pathways and key drivers remain incompletely understood. CSCs are critical in chemoresistance and metastasis in HCC, with these processes heavily dependent on the aberrant activation of stemness-related pathways such as Wnt/β-catenin (Lee et al., 2022; Lin et al., 2024; Qi et al., 2022; Roskams, 2006). Notably, the proto-oncogene pituitary tumor-transforming gene 1 (PTTG1) has been implicated in regulating HCC stemness and activating the Wnt/β-catenin signaling pathway (Lin et al., 2019; Molina-Jiménez et al., 2010; Zhou et al., 2023). However, whether PTTG1 plays a pivotal role in C. sinensis ESP-mediated enhancement of HCC stemness remains unclear (Zhang et al., 2023).

This study aims to investigate whether C. sinensis infection enhances HCC progression and stemness through ESP-mediated regulation of PTTG1 and its downstream signaling network. The central hypothesis is that C. sinensis infection functions as a tumor-promoting stimulus, in line with the established paradigm that various infectious agents can promote cancer by altering signaling in tissue stem or progenitor cells (Sell, 2011). By elucidating the PTTG1-β-catenin-c-MYC axis, this study seeks to define the specific mechanisms underlying C. sinensis-driven HCC aggressiveness and identify potential targets for precise therapeutic intervention.

2. Materials and methods

2.1. Clinicopathological specimens

Paired tumor and para-cancerous tissues were collected from 20 patients with HCC who underwent curative hepatectomy at the Guangxi Medical University Cancer Hospital (Nanning, China). The cohort consisted of 10C. sinensis-positive and 10C. sinensis-negative patients, matched for age, tumor size, Barcelona Clinic Liver Cancer staging system stage, Edmondson grade, microvascular invasion status, tumor number, HBV or HCV infection status, and alpha-fetoprotein levels (Table 1). Importantly, no significant difference in the prevalence of HBV or HCV infection was observed between the two groups (p > 0.05), effectively eliminating viral hepatitis as a major confounding factor. C. sinensis infection status was confirmed by stool examination (microscopic detection of eggs), intraoperative examination, or pathological assessment. This study was approved by the Ethics Committee of Guangxi Medical University Cancer Hospital (KY2025048).

Table 1.

Characteristics of the Clonorchis sinensis-positive and the Clonorchis sinensis-negative group.

	Clonorchis sinensis positive	Clonorchis sinensis negative	p
	n = 10	n = 10
Age (years)	55.20 (11.07)	58.60 (5.93)	0.403
HBV
Present	6 (60.0)	8 (80.0)	0.62
Absent	4 (40.0)	2 (20.0)	6
HCV
Present	0 (0.0)	1 (10.0)
Absent	10 (100.0)	9 (90.0)	0.474
Tumor size
≤5 cm	2 (20.0)	1 (10.0)	1
>5 cm	8 (80.0)	9 (90.0)
Number
Solitary	6 (60.0)	3 (30.0)	0.369
Multiple	4 (40.0)	7 (70.0)
BCLC stage
A	8 (80.0)	8 (80.0)	1
B	2 (20.0)	2 (20.0)
Edmondson grade
I-II	8 (80.0)	6 (60.0)	0.626
III-IV	2 (20.0)	4 (40.0)
Microvascular invasion
Absence	6 (60.0)	4 (40.0)	0.655
Presence	4 (40.0)	6 (60.0)
Alpha-fetoprotein (ng/mL)
≤400	7 (70.0)	6 (60.0)	1
>400	3 (30.0)	4 (40.0)

Categorical data are n (%); Continuous data are reported as mean ± SD or as median (IQR). BCLC: Barcelona Clinic Liver Cancer staging system.

2.2. Animals

Four-week-old male BALB/c nude mice (n = 20) were purchased from the Guangxi Medical University Animal Center. Additionally, liver tissue samples from a C. sinensis-positive rat HCC model were included from a previously published study (Qi et al., 2022). The rat study involved four groups: normal saline control (NS), NS + C. sinensis infection, diethylnitrosamine (DEN)-induced HCC, and DEN + C. sinensis co-infection. Animals were randomly assigned to each group to ensure unbiased distribution of littermates and initial body weights across experimental conditions.

The DEN-induced HCC model was established following a previously validated protocol (Qi et al., 2022). Specifically, DEN was selected as the carcinogen due to its established efficacy in inducing HCC in rodent models, distinguishing it from dimethylnitrosamine (DMN), which is more commonly associated with cholangiocarcinoma (CCA) induction in C. sinensis infection models Briefly, hepatocarcinogenesis was initiated by a single intraperitoneal injection of DEN (50 mg/kg), providing a standardized and reproducible HCC background for investigating the tumor-promoting role of C. sinensis co-infection. This protocol was consistently applied to the DEN and DEN + C. sinensis co-infection groups to ensure comparability.

To ensure the specificity of our findings to HCC, liver tissues from the DEN and DEN + C. sinensis co-infection groups were meticulously screened and confirmed as HCC through hematoxylin-eosin (H&E) staining by two independent pathologists. Only samples exhibiting definitive HCC morphology were included for further molecular analysis. All animal procedures were approved by the Ethics Committee of Guangxi Medical University Cancer Hospital (LW2025012) and were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For all invasive procedures, animals were anesthetized with isoflurane. Euthanasia was performed by cervical dislocation under deep anesthesia for mice, and by overdose of pentobarbital sodium for rats, followed by confirmation of death by absence of pulse and respiration.

2.3. Cell culture and transfection

Human HCC cell lines MHCC-97H and MHCC-LM3 were obtained from the American Type Culture Collection. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, MCE, China) and 1% penicillin-streptomycin (Solarbio, China). Cultures were maintained in a humidified incubator at 37 °C with 5% CO₂. Lentiviral vectors for PTTG1 knockdown (sh-PTTG1) and overexpression (PTTG1-OE), along with corresponding negative controls (shNC and NC), were purchased from Obio Technology Co. (Shanghai, China) and Genechem Co. (Shanghai, China), respectively. Cells were infected with lentivirus according to the manufacturer's instructions, selected with 2 μg/mL puromycin for 48 h to establish stable cell lines, and the efficiency of PTTG1 overexpression/knockdown was verified by quantitative real-time PCR (qRT-PCR) and western blotting (WB). For functional rescue assays, transfected cells were treated with 20 μg/mL ESP or an equivalent volume of phosphate-buffered saline (PBS) as a control for 6 h, unless otherwise specified.

2.4. Preparation of excretory-secretory products secreted by Clonorchis sinensis

C. sinensis metacercariae were isolated from Pseudorasbora parva fish collected in Guangxi Province, China. Each rabbit was administered 800 metacercariae via gavage. Six weeks post-infection, rabbits were sacrificed, and adult C. sinensis worms were aseptically harvested from the bile ducts of euthanized rabbits. The worms were washed three times with PBS containing 1% penicillin-streptomycin and cultured in serum-free PBS with antibiotics at 37 °C under 5% CO₂ for 6 h. The supernatant was collected, centrifuged at 12,000 rpm for 10 min at 4 °C and filtered-sterilized through a 0.2-μm membrane.

The protein concentration of ESP was quantified using a Nanodrop spectrophotometer, adjusted to a stock concentration of 2.0 mg/mL, aliquoted, and stored at −80 °C until further use.

2.5. RNA sequencing and bioinformatic analysis

A total of 30 RNA samples were utilized for transcriptome sequencing library preparation, comprising 10 pairs (n = 20 samples) of tumor and matched para-cancerous tissues from C. sinensis-positive patients with HCC, and 10 tumor tissues from C. sinensis-negative patients with HCC. Total RNA was extracted using Trizol reagent. RNA integrity was assessed using an Agilent 2100 Bioanalyzer (RIN >7.0). Library preparation and sequencing were carried out by Wuxi NextCODE (Shanghai, China) on an Illumina NovaSeq 6000 platform. Approximately 40 million 150-bp paired-end reads were generated per sample. Raw reads were quality-checked with FastQC, and adapter sequences were removed using Trimmomatic. High-quality reads were aligned to the human reference genome (GRCh37/hg19) via STAR. The external transcriptomic dataset was obtained from the Fang.alt study. Differential gene expression analysis between C. sinensis-positive HCC and C. sinensis-negative HCC tissues was conducted with the limma R package (v3.62.2), using a significance threshold of |log2(Fold Change)| >1.5 and adjusted p-value <0.05. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, and Gene Set Enrichment Analysis (GSEA) were performed using the clusterProfiler R package (v4.17.0). Weighted Gene Co-expression Network Analysis (WGCNA) was applied to identify gene modules significantly associated with C. sinensis-positive status using the WGCNA package (v1.73). To ensure unbiased analysis, the sample groups were blinded during the initial data processing and quality control steps. Principal Component Analysis (PCA) was conducted in R using the prcomp function on the normalized gene expression matrix (variance-stabilizing transformation) to visualize overall data structure and sample relationships.

2.6. Imaging mass cytometry

Paraffin-embedded sections (5 μm thick) from C. sinensis-positive and C. sinensis-negative HCC specimens were incubated at 65 °C for 2 h. The sections were then deparaffinized with xylene, rehydrated through a graded ethanol series (absolute ethanol to 75% ethanol), and washed with double-distilled water. Antigen retrieval was performed by placing the sections in a 50 mL EP tube containing 40 mL Tris-EDTA buffer (pH 9.0, FC16FA0005, Sangon Biotech Co., Ltd., China) and heating at 96 °C for 30 min, followed by gradual cooling at room temperature for 20 min. After PBS washing, the sections were blocked with a 3% BSA DPBS solution at room temperature for 45 min and incubated overnight at 4 °C in a humid chamber with a metal-conjugated antibody mixture (diluted in DPBS containing 0.5% BSA). The following day, the sections were counterstained with Cell-ID™ Intercalator-Ir (Fluidigm) diluted 1:400 in DPBS at room temperature for 30 min, washed twice with DPBS containing 0.1% Triton-X (TC259563, Thermo Scientific, USA), and allowed to dry at room temperature for at least 20 min. Detection was performed using a Hyperion imaging mass cytometer at a resolution of 1 μm and a frequency of 200 Hz. A 1 × 1 mm² region of interest was selected, with energy parameters set to 4–6. Data were exported as MCD and .txt files, which were analyzed using the MCD Viewer software. The panel of metal-conjugated antibodies used for IMC is listed in Supplementary Table 1, with all antibodies showed with signals (Supplementary Fig. 1).

2.7. Histology and immunohistochemistry

Formalin-fixed paraffin-embedded (FFPE) tissue sections from human C. sinensis-positive (n = 10) vs C. sinensis-negative (n = 10) HCC specimens, rat HCC model livers (10 per group), and mouse xenograft tumors (3 per group) were utilized. Standard H&E staining was performed for morphological assessment. Immunohistochemistry (IHC) was carried out using the IHC kit (Maxin Biotechnology, Fuzhou, China) following the manufacturer's protocol. Primary antibodies were incubated overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at room temperature. Antigen-antibody complexes were visualized using DAB chromogen, and sections were counterstained with hematoxylin. To ensure consistency, all IHC assays for a given target were performed in a single batch under identical conditions, including the DAB incubation time.

For quantitative assessment, five non-overlapping and representative fields of view (at 40× magnification) were captured from each IHC-stained section. Field selection was systematically performed to cover the entire tumor area while excluding necrotic regions, tissue folds, and edges, ensuring that scoring was confined to viable tumor tissue.

To minimize observer bias, a strict blinding protocol was implemented throughout the evaluation process. All slides were coded by a research assistant not involved in the subsequent analysis, ensuring that the two independent pathologists who performed the scoring were unaware of the group assignments.

Semi-quantitative analysis was conducted using the H-score system. For each field of view, the staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong), and the percentage of positive cells was estimated. The H-score for each field was calculated using the formula: H-score = (% weakly stained cells × 1) + (% moderately stained cells × 2) + (% strongly stained cells × 3). The final H-score for each sample represents the mean value derived from the five assessed fields. This standardized scoring methodology was applied consistently across all compared groups to ensure unbiased and reproducible quantification. The antibodies used for IHC are listed in Supplementary Table 2.

2.8. Quantitative real-time PCR assay

Total RNA from cultured cells was extracted using TRIzol (Takara, Japan). cDNA was synthesized using the PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan). qRT-PCR was performed using TB Green Premix Ex Taq II (Takara, Japan) on a QuantStudio 6 Flex system (Applied Biosystems). GAPDH was used as the endogenous control. Relative gene expression was calculated using the 2^-ΔΔCt method. Primer sequences were as follows: PTTG1-F, 5’-GTGCAATTCCTGAGCTGACA-3′; PTTG1-R, 5’-CTTAAAGATGGCCAGCAAGC-3′; GAPDH-F, 5’-GCACCGTCAAGGCTGAGAAC-3′; GAPDH-R, 5’-TGGTGAAGACGCCAGTGGA-3′.

2.9. Western blotting assay

Cells were lysed with RIPA buffer supplemented with PMSF to extract total protein. Protein samples were separated on a 10% SDS-PAGE and transferred to a 0.2-μm PVDF membrane (Millipore, USA). Nonspecific binding was blocked with Quick Block Buffer (P0252, Beyotime, China). Membranes were incubated with primary antibodies overnight at 4 °C, followed by washed with TBST and incubation with corresponding secondary antibodies for 1 h at room temperature. Membranes were visualized using an Odyssey Dual-Color Infrared Imaging System. Band intensities were quantified using ImageJ software (NIH, USA) and normalized to β-actin or GAPDH as loading controls. The antibodies used in WB were listed in Supplementary Table 2.

2.10. Cell proliferation assays

For the cell counting kit-8 (CCK-8) assay, 1500 cells per well were seeded in 96-well plates. Cell viability assay was done by adding 10 μL of CCK-8 reagent into each well and by incubating at 37 °C for 2 h according to the manufacturer's instructions. Absorbance at 450 nm was measured using a microplate reader to determine cell viability.

In the colony formation assay, 800 cells per well were seeded in 6-well plates and cultured for 14 days. Colonies were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet, and colonies containing ≥50 cells were counted.

2.11. Cell cycle analysis

For the cell cycle assay, 2 × 10⁵ cells per well were seeded in 6-well plates and incubated for 24 h. After trypsinization, cells were fixed with 75% ethanol at −20 °C overnight, washed with PBS, and stained with propidium iodide containing RNase A using a Cell Cycle Staining Kit (Multi Sciences, China). Flow cytometry data were acquired and analyzed with FlowJo software (v.10.8.1) by fitting cycle curves.

2.12. Migration and invasion assays

For the wound healing migration assay, 8.5 × 10⁵ cells per well were seeded in 6-well plates. Once cells reached 95% confluence, a scratch was made using a 200-μL pipette tip, and cells were cultured in serum-free DMEM. Images of the scratch area were captured at 0, 24, and 48 h using an inverted microscope.

For the transwell invasion assay, 3 × 10⁴ cells per well were seeded into Matrigel-coated transwell inserts (8-μm pore size, Corning), with the lower chamber filled with DMEM containing 10% FBS. After 72 h of incubation, invaded cells on the lower surface of the membrane were fixed, stained with 0.1% crystal violet, and counted in five randomly selected fields under an inverted microscope.

2.13. Spheroid formation assay

To assess stemness properties, 1 × 10³ cells per well were seeded in ultra-low attachment 96-well plates in serum-free DMEM supplemented with 20 ng/mL recombinant human basic fibroblast growth factor, 10 ng/mL recombinant human epidermal growth factor, and 1:50 diluted B27 supplement (Thermo Fisher Scientific). After a 7-day incubation, tumor spheroids were captured using a microscope, and spheroids with a diameter ≥ 50 μm were counted.

2.14. Animal experiments

Male BALB/c nude mice (n = 5 per group) were randomly assigned to four groups, with randomization based on body weight to ensure statistically equivalent starting averages. Mice were subcutaneously injected with 3 × 10⁶ cells transfected with NC, PTTG1-OE, shNC, or sh-PTTG1 into the right flank. Tumor volume was measured every 3 days using the formula: volume = 1/2 × length × width². Mice were euthanized when the tumor diameter reached 15 mm. Tumors were then harvested, weighed, and processed for IHC analysis.

2.15. Statistical analysis

Data are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical analyses were conducted using GraphPad Prism 9 software and R version 4.4.2 (http://www.r-project.org/). Differences between two groups were assessed using a two-tailed Student's t-test, while comparisons among multiple groups were analyzed by one-way ANOVA or two-way ANOVA followed by Tukey's post-hoc test. A p-value <0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

3. Results

3.1. Principal component analysis of transcriptomic profiling highlights phenotypic differences by tissue types and Clonorchis sinensis infection status

PCA of the internal transcriptomic dataset showed that the first five principal components (PC1–PC5) accounted for 24.1%, 13.8%, 8.4%, 6.6%, and 5.7% of the total variance, respectively (Supplementary Fig. 2A). PCA indicated a trend of separation between liver C. sinensis-negative and C. sinensis-positive carcinoma tissues (Supplementary Fig. 2B). A more distinct separation was observed between C. sinensis-positive liver para-cancerous tissues and C. sinensis-positive cancer tissues (Supplementary Fig. 2C). In the external transcriptomic dataset, PC1 to PC5 explained 11.4%, 9.0%, 7.1%, 6.2%, and 5.4% of the variance, respectively (Supplementary Fig. 2D). PCA also showed a trend of separation between C. sinensis-negative and C. sinensis-positive liver carcinoma tissues (Supplementary Fig. 2E), and distinct molecular phenotypic differences between C. sinensis-positive para-cancerous tissues and C. sinensis-positive cancer tissues (Supplementary Fig. 2F). In summary, the internal dataset demonstrated higher variance explained by the leading principal components and more pronounced separation trends based on C. sinensis infection status and tissue type (cancer vs. para-cancerous tissues), while the external dataset exhibited more modest separation, likely due to its lower variance captured by PCA.

3.2. Wnt/β-catenin-c-MYC signaling pathway is activated in Clonorchis sinensis-positive HCC

Compared to C. sinensis-positive para-cancerous tissues, C. sinensis-positive tumors exhibited 946 genes upregulated and 941 downregulated genes (Fig. 1A). More importantly, when directly compared to C. sinensis-negative HCC tumors, C. sinensis-positive tumors displayed a distinct signature, with 233 upregulated and 105 downregulated genes (Fig. 1B), providing a clearer signature specific to C. sinensis infection in the context of HCC.

Fig. 1.

RNA-seq profiling of C. sinensis-positive HCC. (A) Volcano diagram of DEGs between C. sinensis-positive HCC tumors and para-cancerous tissues. (B) Volcano diagram of DEGs between Cs-positive and Cs-negative HCC tumors. (C—D) WGCNA diagram of gene modules correlated with Cs-infection status. (E) Venn diagram for two DEGs, gene modules correlated with Cs-positive status, and prognosis-related genes.

GO and KEGG analyses were performed on the differentially expressed gene (DEG) sets of C. sinensis-positive tumors vs. C. sinensis-positive para-cancerous tissues and C. sinensis-positive tumors vs. C. sinensis-negative tumors. Biological processes such as cellular response to copper ion and steroid metabolic process were enriched (Supplementary Fig. 3A and B), along with pathways including mineral absorption and retinol metabolism (Supplementary Fig. 3C and D). Additionally, GSEA based on KEGG annotations showed a positive correlation between C. sinensis-positive status and the Wnt signaling pathway, among other stemness-related pathways (Supplementary Fig. 3E and F). GSEA also revealed significant enrichment of oncogenic signatures, including E2F targets, G2M checkpoint, and MYC targets (Supplementary Fig. 4A and B).

To validate these findings at the protein level and in spatial context, imaging mass cytometry (IMC) was applied to human HCC tissues. Several cell clusters were found to be significantly enriched in C. sinensis-positive tumors. These clusters exhibited markedly high expression of both c-MYC and the stemness marker CD44 (Supplementary Fig. 4C), corroborating the RNA-seq results. This confirmed that protein levels of c-MYC and CD44 were significantly elevated in C. sinensis-positive compared to C. sinensis-negative HCC.

Furthermore, differential expression analysis of an external cohort from Fang et al. revealed 1164 upregulated genes and 1446 downregulated genes in C. sinensis-positive tumors compared to C. sinensis-positive para-cancerous tissues (Supplementary Fig. 5A). GSEA of the external cohort also showed significant enrichment of oncogenic signatures, including E2F targets, G2M checkpoint, and MYC targets (Supplementary Fig. 5B). GSEA based on KEGG annotations indicated a positive correlation between C. sinensis-positive status and the Wnt signaling pathway, among other stemness-related pathways (Supplementary Fig. 5C), suggesting that C. sinensis infection promotes a stem-like phenotype, potentially through Wnt/β-catenin pathway activation.

3.3. PTTG1 is identified as a key regulatory molecule in Clonorchis sinensis-positive HCC

To identify key drivers of the C. sinensis-positive HCC phenotype, WGCNA was performed, revealing two gene modules (brown and yellow) strongly correlated with C. sinensis infection status (Fig. 1C-D). By intersecting the hub genes from these modules with DEGs and known prognosis-related genes from TCGA, PTTG1 emerged as a top candidate (Fig. 1E). Consistent with this, survival analysis confirmed that high PTTG1 expression was associated with poor OS and recurrence-free survival in patients with HCC (Supplementary Fig. 6), highlighting its clinical significance.

3.4. Clonorchis sinensis-infection influences PTTG1 expression in HCC

PTTG1 expression patterns were further investigated. IHC revealed a stepwise increase in PTTG1 protein levels, with the highest expression observed in C. sinensis-positive tumors, intermediate expression in C. sinensis-negative tumors, and the lowest in para-cancerous tissues (Fig. 2A and Supplementary Fig. 7A). This trend was replicated in a DEN-induced HCC rat model, where co-infection with C. sinensis significantly enhanced PTTG1 expression in HCC compared to DEN alone (Fig. 2B and Supplementary Fig. 7B). In vitro, treatment with C. sinensis ESP (20 μg/mL, 6 h) markedly upregulated PTTG1 mRNA expression in MHCC-97H and MHCC-LM3 cells (Fig. 2C). Notably, ESP treatment also increased the protein levels of β-catenin, c-MYC, and CD44 (Fig. 2D and Supplementary Fig. 7C), suggesting that PTTG1 may function within an ESP-driven oncogenic pathway.

Fig. 2.

Clonorchis sinensis-infection enhanced PTTG1 expression in HCC. (A) IHC analysis of C. sinensis-positive and C. sinensis-negative tumors, and para-cancerous tissues. (B) IHC analysis of C. sinensis-infected rat HCC model tumors. (C) Quantification of relative mRNA levels (PTTG1) in MHCC-97H and MHCC-LM3 after co-culturing with ESP. (D) WB analysis showed the upregulation of CD44, β-catenin, c-MYC, and PTTG1 in MHCC-97H and MHCC-LM3 after co-culturing with ESP.

3.5. PTTG1 promotes malignant phenotypes of HCC

To further assess the role of PTTG1 in hepatocellular carcinogenesis, stable overexpression of PTTG1 in MHCC-97H and MHCC-LM3 cells was confirmed by qRT-PCR and WB analysis (Fig. 3A-B and Supplementary Fig. 8A). Subsequent WB analysis of key downstream effectors revealed that PTTG1 overexpression increased the protein levels of CD44, β-catenin, and c-MYC in these cell models (Fig. 3B and Supplementary Fig. 8A), suggesting its involvement in activating Wnt/β-catenin signaling and promoting a stemness program. Findings from the transwell invasion assay (Fig. 3C and Supplementary Fig. 8B), spheroid formation assay (Fig. 3D and Supplementary Fig. 8C), and wound healing migration assay (Fig. 3E and Supplementary Fig. 8D) showed that PTTG1 overexpression significantly enhanced invasion, increased spheroid diameter, and promoted migration. Furthermore, CCK-8 (Fig. 3F), colony formation (Fig. 3G and Supplementary Fig. 8E), and cell cycle analysis (Fig. 3H and Supplementary Fig. 8F) indicated accelerated cell proliferation, elevated colony formation, and enhanced G1/S cell cycle transition in PTTG1-overexpressing cells. In contrast, PTTG1 knockdown using shRNA effectively suppressed all malignant phenotypes, including invasion, spheroid formation, migration, proliferation, colony formation, and induced G1 phase arrest (Fig. 4 and Supplementary Fig. 9).

Fig. 3.

PTTG1 overexpression promoted malignant phenotypes of HCC cells. (A) Quantification of relative PTTG1 mRNA levels in MHCC-97H and MHCC-LM3 cells following PTTG1 overexpression. (B) WB analysis demonstrated upregulation of CD44, β-catenin, c-MYC, and PTTG1 proteins in MHCC-97H and MHCC-LM3 cells following PTTG1 overexpression. (C) Transwell invasion assay of MHCC-97H and MHCC-LM3 cells after PTTG1 overexpression. (D) Spheroid formation assay of MHCC-97H and MHCC-LM3 cells after PTTG1 overexpression. (E) Wound healing migration assay of MHCC-97H and MHCC-LM3 cells after PTTG1 overexpression. (F) Quantification of proliferation rates in MHCC-97H and MHCC-LM3 cells after PTTG1 overexpression. (G) Colony formation assay of MHCC-97H and MHCC-LM3 cells following PTTG1 overexpression. (H) Flow cytometry analysis of cell cycle distribution in MHCC-97H and MHCC-LM3 cells following PTTG1 overexpression.

Fig. 4.

PTTG1 knockdown decreased malignant phenotypes of HCC cells. (A) Quantification of relative PTTG1 mRNA levels in MHCC-97H and MHCC-LM3 cells following PTTG1 knockdown. (B) WB analysis demonstrated downregulation of CD44, β-catenin, c-MYC, and PTTG1 proteins in MHCC-97H and MHCC-LM3 cells following PTTG1 knockdown. (C) Transwell invasion assay of MHCC-97H and MHCC-LM3 cells after PTTG1 knockdown. (D) Spheroid formation assay of MHCC-97H and MHCC-LM3 cells after PTTG1 knockdown. (E) Wound healing migration assay of MHCC-97H and MHCC-LM3 cells after PTTG1 knockdown. (F) Quantification of proliferation rates in MHCC-97H and MHCC-LM3 cells after PTTG1 knockdown. (G) Colony formation assay of MHCC-97H and MHCC-LM3 cells following PTTG1 knockdown. (H) Flow cytometry analysis of cell cycle distribution in MHCC-97H and MHCC-LM3 cells following PTTG1 knockdown.

In a subcutaneous tumorigenesis model, nude mice injected with PTTG1-overexpressing cells developed significantly larger and heavier tumors than controls, while tumors from PTTG1-knockdown cells grew more slowly and were smaller (Fig. 5A). IHC analysis of these tumors revealed that PTTG1 overexpression upregulated, while its knockdown downregulated, the expression of β-catenin, CD44, and c-MYC (B and Supplementary Fig. 10A). Consistent with the human data, liver tumors from C. sinensis + Fig. 5DEN HCC rats exhibited elevated expression of these markers compared to tumors from DEN-only HCC rats (Fig. 5C and Supplementary Fig. 10B), firmly establishing PTTG1 as a key driver in the activation of this pathway in C. sinensis-associated hepatocarcinogenesis.

Fig. 5.

PTTG1 aggravated hepatocellular carcinogenesis and promoted HCC stemness. (A) Representative images, growth curves, tumor weight quantifications, and tumor/body weight ratio quantifications of subcutaneous tumors following PTTG1 overexpression or knockdown. (B) IHC analysis for subcutaneous tumors. (C) HE and IHC analysis for C. sinensis-infected rat HCC model tumors.

3.6. Clonorchis sinensis promotes HCC stemness via the PTTG1-β-catenin-c-MYC axis

To investigate whether PTTG1 is essential for the aggressive phenotypes induced by C. sinensis ESP, rescue experiments were conducted in PTTG1-knockdown cells. ESP treatment partially restored the migratory capacity (Fig. 6A and Supplementary Fig. 11A), proliferative ability (Fig. 6B), invasive potential (Fig. 7A and Supplementary Fig. 11B), spheroid formation efficiency (Fig. 7B and Supplementary Fig. 11C), and G1/S cell cycle transition (Fig. 7C and Supplementary Fig. 11D) impaired by PTTG1 knockdown. Furthermore, the decreased protein expression levels of PTTG1, CD44, β-catenin, and c-MYC resulting from PTTG1 knockdown were partially reversed upon co-treatment with ESP (Fig. 7D and Supplementary Fig. 11E). These results suggest that PTTG1 acts as a crucial downstream mediator contributing to the tumor-promoting effects of C. sinensis ESP (Graphical Abstract).

Fig. 6.

ESP promotes HCC migration and proliferation in PTTG1-knockdown cells via co-culture. (A) Wound healing migration assay of PTTG1 knockdown cells after co-culturing with ESP. (B) Quantification of proliferation rates in PTTG1 knockdown cells after co-culturing with ESP.

Fig. 7.

ESP accelerates HCC progression by regulating cell invasion, stemness, cell cycle, and β-catenin/c-MYC signaling in PTTG1-knockdown cells. (A) Transwell invasion assay of PTTG1 knockdown cells after co-culturing with ESP. (B) Spheroid formation assay of PTTG1 knockdown cells after co-culturing with ESP. (C) Flow cytometry analysis of cell cycle distribution in PTTG1 knockdown cells after co-culturing with ESP. (D) WB analysis showed the upregulation of CD44, β-catenin, c-MYC, and PTTG1 in PTTG1 knockdown cells after co-culturing with ESP.

4. Discussion

Chronic and heavy infection with C. sinensis is a significant risk factor for the development of ICC and HCC (Cadamuro et al., 2022; Chu et al., 2023; Dong et al., 2024; Liu et al., 2023; Wang et al., 2023). Extensive research by Yongvanit et al. (2014), Thamavit et al. (Thamavit et al., 1993; Thamavit et al., 1994), and Lee et al. (1994) has firmly established the causal link between C. sinensis infection and cholangiocarcinoma (CCA), particularly when combined with nitrosamines like DMN. It is important to note the distinct pathological outcomes observed with different carcinogens in C. sinensis models. While DMN is a well-established inducer of CCA through its affinity for biliary epithelial cells (Prempracha et al., 1994), DEN exhibits a strong tropism for hepatocytes, typically favoring HCC development (Hassan et al., 2026; Xu et al., 2026; Zhao et al., 2025). Our observation of an 88.2% HCC induction rate is consistent with the ‘initiator-promoter’ framework where C. sinensis infection acts as a powerful promoter for hepatocyte-derived lesions initiated by DEN. However, we must acknowledge that the liver's response to infection is heterogeneous; thus, the potential for a spectrum of biliary and hepatic co-lesions remains.

While the causal link between C. sinensis infection and CCA, via mechanisms such as chronic biliary inflammation and periductal fibrosis is well-established (Pak et al., 2009; Sripa et al., 2009; Yoon et al., 2000; Zheng et al., 2013), its role in promoting HCC progression remains less clear. However, emerging evidence increasingly associates C. sinensis with HCC, with clinical studies indicating that patients with concomitant C. sinensis infection often experience poorer outcomes. This association is further supported by experimental models using DEN, a widely recognized agent for inducing HCC, in combination with C. sinensis infection. These studies demonstrate that C. sinensis infection promotes hepatic progenitor cell proliferation, thereby increasing the risk of HCC development. Building on this foundation, our study specifically aims to elucidate the mechanisms by which C. sinensis infection exacerbates HCC progression.

In this study, bulk RNA sequencing of HCC individuals with or without C. sinensis infection identified PTTG1 as a candidate prognostic marker associated with C. sinensis-positive HCC. PTTG1, a recognized oncogene, is implicated in HCC pathogenesis, with elevated expression correlating with unfavorable clinical outcomes (Gao et al., 2024; Tie et al., 2021; Zhang et al., 2025). This observation was further validated by data from an external sequencing cohort, suggesting a potential role for PTTG1 in C. sinensis-associated hepatocarcinogenesis. Proteomic analysis of liver tissues from C. sinensis-positive patients revealed increased expression of the CSC marker CD44 and the transcription factor c-MYC. This aligns with previous reports showing elevated CSC marker expression in tumor tissues from C. sinensis-positive patients with HCC and C. sinensis-infected HCC rat tumor tissues (Lin et al., 2024; Qi et al., 2022), indicating that C. sinensis infection may enhance stem-like properties in HCC (Lin et al., 2019; Yoon et al., 2012).

Semi-quantitative IHC analysis revealed progressively higher PTTG1 expression in C. sinensis-positive tumor tissues compared to C. sinensis-negative counterparts. This pattern was replicated in a C. sinensis-positive rat model, suggesting a potential association between C. sinensis infection and PTTG1 upregulation. In vitro, treatment with C. sinensis ESP led to increased PTTG1 protein levels in HCC cell lines, indicating that C. sinensis ESP may contribute to PTTG1 induction. Functional characterization demonstrated that PTTG1 overexpression enhanced malignant behaviors, including migration, invasion, proliferation, and tumorigenicity, while PTTG1 knockdown suppressed these activities. These findings align with previous studies (Cho-Rok et al., 2006; Huang et al., 2018; Lin et al., 2019; Molina-Jiménez et al., 2010; Zhang et al., 2023; Zhou et al., 2023), and suggest that PTTG1 may modulate aggressive phenotypes in HCC cells. Similar pro-tumor effects were observed by Kang et al. (2020), Pan et al. (2024), and Shang et al. (2017) following ESP treatment of hepatobiliary cancer cells, supporting the possibility that C. sinensis ESP could influence malignant phenotypes through PTTG1 regulation.

To explore the mechanisms by which C. sinensis infection and PTTG1 might influence tumor stemness, the Wnt/β-catenin pathway, previously linked to both PTTG1 function (Zhang et al., 2023) and CSC maintenance, was investigated (Sharma and Pruitt, 2020). ESP treatment increased the protein levels of PTTG1, β-catenin, c-MYC, and CD44, suggesting activation of a Wnt/β-catenin-mediated program. Rescue experiments in PTTG1-knockdown cells indicated that ESP co-treatment partially restored some functional impairments, supporting PTTG1 as one mediator of ESP effects. The coordinated expression changes of these markers following ESP exposure support a model in which the ESP-PTTG1-Wnt/β-catenin axis contributes to stemness characteristics in HCC. In vivo observations from subcutaneous tumorigenesis models and C. sinensis-positive HCC rat tumor tissues showed comparable expression patterns, providing further evidence for the relevance of this pathway.

C. sinensis ESP may contribute to HCC progression by activating the PTTG1-β-catenin-c-MYC axis in hepatocyte-derived cells, thereby enhancing stemness and aggressive phenotypes. This model aligns with the conceptual framework proposed by Sell, which posits that various infectious agents—whether viral, bacterial, or parasitic—may converge on a common oncogenic mechanism: the dysregulation of tissue stem or progenitor cell signaling, leading to sustained proliferation and impaired differentiation (Sell, 2011). In the context of C. sinensis-associated HCC, it is hypothesized that ESP components act as functional mediators, dysregulating the Wnt/β-catenin pathway—a key axis for liver stem/progenitor cell maintenance—through PTTG1 upregulation. Collectively, these observations suggest that C. sinensis infection may function as a tumor-promoting stimulus, potentially reinforcing a stem-like, proliferative state in initiated hepatocytes.

Notably, some discrepancies between our results and previous reports merit consideration. First, while we observed ESP-induced proliferation at 6 h, Fang Min's group (Chen et al., 2025) reported no significant change at 12 h, a difference likely attributable to the stage-dependent complexity of ESP composition (Zhang et al., 2022). Second, the “DEN + C. sinensis” model presents inherent complexities. Although DEN is classically used to favor HCC induction—with our previous work achieving an 88.2% HCC rate compared to 46.7% in DEN-only groups (Qi et al., 2022)—the potential for concurrent CCA precursor lesions or micro-CCA components cannot be entirely dismissed. Such components might influence the HCC microenvironment through paracrine signaling. In this study, we addressed this by focusing exclusively on histologically confirmed HCC tissues; however, future research employing more refined lineage-tracing markers is necessary to disentangle the independent contributions of hepatic versus biliary lineages. Furthermore, while our clinical samples were carefully matched, the sample size remains limited. Larger multi-center cohorts are needed to validate the prognostic value of PTTG1. Additionally, future studies using immunocompetent models will be vital to understand how C. sinensis modulates the tumor immune microenvironment to promote stemness. Collectively, our findings suggest that the ESP-PTTG1-Wnt/β-catenin axis is a key mediator of C. sinensis-promoted HCC progression, offering a potential therapeutic target for this high-risk population.

5. Conclusion

In conclusion, our study identifies PTTG1 as a potential mediator of C. sinensis-associated HCC progression. While our data strongly link ESP-induced PTTG1 to enhanced stem-like traits in hepatocytes, the inherent complexity of the C. sinensis-associated tumor spectrum necessitates caution in generalizing these findings. These insights provide a foundation for further investigating the specialized roles of PTTG1 across different lineages in the fluke-infected liver. However, further validation is required to confirm these associations and elucidate the precise molecular mechanisms.

The following are the supplementary data related to this article.

Supplementary Table 1

Antibodies used for flow cytometry.

Supplementary Table 2

Antibodies used for immunohistochemistry and western blotting assay.

Supplementary Fig. S1.

The positive signal detection results in multiplex immunofluorescence.

Supplementary Fig. S2.

PCA of internal and external transcriptomic datasets by Clonorchis sinensis infection status and tissue type. (A) Variance explained by PC1–PC5 of internal transcriptomic datasets. (B) PCA plots of internal transcriptomic datasets showing separation trends between C. sinensis-negative and positive liver carcinoma tissues. (C) PCA plots of internal transcriptomic datasets showing separation and molecular phenotypes between C. sinensis-positive para-cancerous and carcinoma tissues. (D) Variance explained by PC1–PC5 of external transcriptomic datasets. (E) PCA plots of external transcriptomic datasets showing separation trends between C. sinensis-negative and positive liver carcinoma tissues. (F) PCA plots of external transcriptomic datasets showing separation and molecular phenotypes between C. sinensis-positive para-cancerous and carcinoma tissues.

Supplementary Fig. S3.

GO and KEGG enrichment analyses of DEGs. (A) GO enrichment analyses of DEGs for C. sinensis-positive HCC tumors vs. para-cancerous tissues. (B) GO enrichment analyses of DEGs for C. sinensis-positive vs. C. sinensis-negative HCC tumors. (C) KEGG enrichment analyses of DEGs for C. sinensis-positive HCC tumors vs. para-cancerous tissues. (D) KEGG enrichment analyses of DEGs for C. sinensis-positive vs. C. sinensis-negative HCC tumors. (E) GSEA revealed enrichment of KEGG in C. sinensis-positive HCC tumors vs. para-cancerous tissues. (F) GSEA revealed enrichment of KEGG in C. sinensis-positive vs. C. sinensis-negative HCC tumors.

Supplementary Fig. S4.

GSEA analyses of DEGs. (A) GSEA revealed enrichment of Hallmark in C. sinensis-positive HCC tumors vs. para-cancerous tissues. (B) GSEA revealed enrichment of Hallmark in C. sinensis-positive HCC tumors vs. C. sinensis-negative HCC tumors. (C) Imaging mass cytometry analysis was performed to compare proteomic profiles between C. sinensis-positive and C. sinensis-negative HCC tumors.

Supplementary Fig. S5.

RNA-seq profiling of C. sinensis-positive HCC in external cohort. (A) Volcano diagram of DEGs between C. sinensis-positive HCC tumors and para-cancerous tissues in external cohort. (B) GSEA revealed enrichment of Hallmark in Cs-positive HCC tumors vs. para-cancerous tissues in external cohort. (C) GSEA revealed enrichment of KEGG in C. sinensis-positive HCC tumors vs. para-cancerous tissues in external cohort.

Supplementary Fig. S6.

Survival analysis demonstrated that high PTTG1 expression was associated with poor OS in patients with HCC.

Supplementary Fig. S7.

Quantification relative protein expression. (A) Quantification of relative IHC scores for PTTG1 in tumor tissues. (B) Quantification of relative IHC scores for PTTG1 in rat HCC model tissues. (C) Quantification of relative protein expression in MHCC-97H and MHCC-LM3 after co-culturing with ESP.

Supplementary Fig. S8.

Functional Consequences of PTTG1 Overexpression. (A) Quantification of relative protein expression levels in MHCC-97H and MHCC-LM3 cells following PTTG1 overexpression. (B) Quantification of invasive cell numbers in MHCC-97H and MHCC-LM3 cells following PTTG1 overexpression. (C) Quantification of spheroid diameters in MHCC-97H and MHCC-LM3 cells following PTTG1 overexpression. (D) Quantification of migration rates in MHCC-97H and MHCC-LM3 cells following PTTG1 overexpression. (E) Quantification of colony numbers in MHCC-97H and MHCC-LM3 cells after PTTG1 overexpression. (F) Quantification of cell counts in each cell cycle phase in MHCC-97H and MHCC-LM3 cells after PTTG1 overexpression.

Supplementary Fig. S9.

Functional Consequences of PTTG1 knockdown. (A) Quantification of relative protein expression levels in MHCC-97H and MHCC-LM3 cells following PTTG1 knockdown. (B) Quantification of invasive cell numbers in MHCC-97H and MHCC-LM3 cells following PTTG1 knockdown. (C) Quantification of spheroid diameters in MHCC-97H and MHCC-LM3 cells following PTTG1 knockdown. (D) Quantification of migration rates in MHCC-97H and MHCC-LM3 cells following PTTG1 knockdown. (E) Quantification of colony numbers in MHCC-97H and MHCC-LM3 cells after PTTG1 knockdown. (F) Quantification of cell counts in each cell cycle phase in MHCC-97H and MHCC-LM3 cells after PTTG1 knockdown.

Supplementary Fig. S10.

Upregulation of β-catenin and its downstream targets CD44 and c-MYC in tumor tissues. (A) Quantification of relative IHC score for β-catenin, CD44, and c-MYC in subcutaneous tumors. (B) Quantification of relative IHC score for β-catenin, CD44, and c-MYC in Cs-infected rat HCC model tumors.

Supplementary Fig. S11.

Quantification of malignant phenotypes and protein expression in PTTG1 knockdown cells following ESP co-culture. (A) Quantification of migration rates in PTTG1 knockdown cells after co-culturing with ESP. (B) Quantification of invasive cell numbers in PTTG1 knockdown cells after co-culturing with ESP. (C) Quantification of spheroid diameters in PTTG1 knockdown cells after co-culturing with ESP. (D) Quantification of cell counts in each cell cycle phase in PTTG1 knockdown cells after co-culturing with ESP. (E) Quantification of relative protein expression levels in PTTG1 knockdown cells after co-culturing with ESP.

CRediT authorship contribution statement

Tai-Xin Yang: Writing – original draft, Validation, Formal analysis. Jie Zhang: Writing – review & editing, Data curation. Min-Jun Li: Writing – review & editing, Validation. Jin-Du Li: Writing – original draft, Visualization. Shuang Shen: Methodology, Investigation. Ting Lu: Resources. Guo-Dong Yang: Writing – original draft, Validation, Conceptualization. Qiu-Yan Wang: Software, Resources. Yu Wang: Investigation. Tai Luo: Visualization, Investigation. Yang-Song Lin: Validation, Data curation. Yue Deng: Investigation, Formal analysis. Ming-Jian Huang: Formal analysis, Data curation. Jun-Liang Nong: Supervision, Formal analysis. Bang-De Xiang: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. Wen-Feng Gong: Writing – review & editing, Supervision, Project administration, Conceptualization.

Ethics approval statement

This study was approved by the Ethics Committee of Guangxi Medical University Cancer Hospital (KY2025048). All animal experiments were approved by the Ethics Committee of Guangxi Medical University Cancer Hospital (LW2025012) and were performed following laboratory animal care guidelines.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work we used Generative Pre-trained Transformer in order to improve readability and language. After using this tool/service, we reviewed and edited the content as needed and take full responsibility for the content of the publication. All data analysis and interpretation were done by the authors.

Funding

This research was funded by the Science and Technology Plan of Qingxiu District, Nanning City (2022011 to Wen-feng Gong), the Guangxi Key Research and Development Program (AB24010055 to Wen-Feng Gong), the Self-financed Scientific Research Project of Health Commission of Guangxi Zhuang Autonomous Region (ZA20221273 to Jun-Liang Nong), the Chen xiao-ping foundation for the development of science and technology of Hubei Province (CXPJJH124009-076 to Jun-Liang Nong), the Technology Project for Disease Prevention in Guangxi Science (GXJKKJ2026ZD003 to Wen-Feng Gong), the Project for the Development and Promotion of Appropriate Medical and Health Technologies in Guangxi (S2022108 to Wen-Feng Gong), the National Natural Science Foundation of China (82260573 to Bang-De Xiang), and the Guangxi Science and Technology Program under Grant (AD25069077). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability statement

The datasets during the current study are available from the corresponding author on reasonable request.