Clonorchis sinensis infection remodels chromatin accessibility in hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is a major global health concern, accounting for a significant proportion of liver cancer cases and related deaths. Clonorchis sinensis (C. sinensis) infection, a recognized carcinogen, has been implicated in the progression of liver diseases, including HCC. However, the precise epigenetic mechanisms underlying C. sinensis-associated HCC remain to be elucidated.

Methods: To investigate the role of chromatin accessibility in C. sinensis-related HCC progression, we performed an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) analyses of C. sinensis-infected (C. sinensis⁺) and non-C. sinensis-infected (C. sinensis^-) HCC tumors. Integrated analyses were conducted to assess chromatin accessibility, transcription factor (TF) motifs, and histone modifications using ATAC-seq, RNA-seq, and classical chromatin immunoprecipitation-sequencing (ChIP-seq) datasets. A scratch wound assay was used to evaluate the effects of C. sinensis excretory/secretory products (CsESPs) on HCC cell migration.

Results: ATAC-seq analysis revealed 9,396 differentially accessible regions (DARs) in C. sinensis⁺ HCC tumors compared with C. sinensis^- HCC tumors. Additionally, several crucial TFs enriched in DARs were identified, including HNF4A, FOXO1, ELF4, and RELA. Combined ATAC-seq and RNA-seq analyses further revealed differentially expressed genes (DEGs) associated with metabolism, immune regulation, and cytoskeletal dynamics. Chromatin accessibility was closely associated with histone modifications such as H3K9ac, H3K4me2, H3K4me3, H3K27ac, H3K4me1, and CTCF binding. Notably, C. sinensis infection significantly increased the migratory capacity of HCC cells, as confirmed by molecular assays and clinical observations.

Conclusions: Our study demonstrates that C. sinensis infection remodels chromatin accessibility and may contribute to HCC progression. Our work offers valuable insights into the pathogenesis of HCC in the context of parasitic infection and lays the groundwork for future biomarker and therapeutic target discovery.

Keywords: Clonorchis sinensis; Chromatin accessibility; Hepatocellular carcinoma; Transcription factor; Tumor progression.

Fig. 1

Chromatin accessibility landscape of C. sinensis⁺ and C. sinensis⁻ HCC. a. Experimental strategy for genome-wide ATAC-seq assay and integration analysis of C. sinensis⁺ and C. sinensis⁻ HCC. b. Fragment size of ATAC data in each group of C. sinensis⁺ and C. sinensis⁻ HCC. c. Distribution of peaks relative to TSS and TES regions in each group. d. Distribution of reads from ATAC-seq data relative to TSS in each group. e. Genomic distribution of reads from ATAC-seq data in each group. f. Correlation analysis between all groups. Cs represents C. sinensis

Fig. 2

Chromatin accessibility landscape of C. sinensis⁺ HCC. a. Different chromatin accessibility landscape between C. sinensis⁺ HCC tumors and tumor-adjacent tissues. b. The top de novo motifs enriched in up and down accessibility peaks between C. sinensis⁺ HCC tumors and tumor-adjacent tissues. c, d. Enrichment analysis of GO terms for up (c) and down (d) accessibility peaks between C. sinensis⁺ HCC tumors and tumor-adjacent tissues. e, f. KEGG analysis of up (e) and down (f) accessibility peaks between C. sinensis⁺ HCC tumors and tumor-adjacent tissues. g. IGV shows representative difference peaks between C. sinensis⁺ HCC tumors and tumor-adjacent tissues. h. Venn diagram for the relationship between different peaks in C. sinensis⁺ and C. sinensis⁻ HCC. Cs represents C. sinensis

Fig. 3

C. sinensis infection changes the chromatin accessibility landscape of tumors in HCC. a. Different chromatin accessibility landscape between C. sinensis⁺ HCC tumors and C. sinensis⁻ HCC tumors. b. The top de novo motifs enriched in up and down accessibility peaks between C. sinensis⁺ HCC tumors and C. sinensis⁻ HCC tumors. c, d. Enrichment analysis of GO terms for up (c) and down (d) accessibility peaks between C. sinensis⁺ HCC tumors and C. sinensis⁻ HCC tumors. e. KEGG analysis of down accessibility peaks between C. sinensis⁺ HCC tumors and C. sinensis⁻ HCC tumors. f. IGV shows representative different accessibility peaks between C. sinensis⁺ HCC tumors and C. sinensis⁻ HCC tumors. Cs represents C. sinensis

Fig. 4

The potential core TFs in HCC after C. sinensis infection. a. TF motif enrichment in DARs. P values were calculated by HOMER. The color bar indicates Log10(p). b, c. Quantification of coverage of TF-binding motifs in increased (b) and decreased (c) DARs in C. sinensis⁺ HCC tumors compared with C. sinensis⁻ HCC tumors

Fig. 5

Joint analysis of RNA and ATAC from the same sample in the C. sinensis⁺ HCC tumors and C. sinensis⁻ HCC tumors. a. Heatmap representation of relative expression levels of genes that were differentially expressed between C. sinensis⁺ HCC tumors and C. sinensis⁻ HCC tumors. The relative changes of chromatin accessibility in the same sample along the same gene are shown to the right. b. Correlation of the RNA expression and promoter accessibility changes in the C. sinensis⁺ HCC tumors and C. sinensis⁻ HCC tumors. c. The RNA expression and promoter accessibility of four RNA expression and promoter accessibility genes. Cs represents C. sinensis

Fig. 6

Joint analysis of ATAC-seq and public ChIP-seq data. a. Heatmap representation of the aggregated ATAC-seq and paired tag histone modification signals around different accessibility regions between C. sinensis⁺ HCC tumors and C. sinensis⁻ HCC tumors. b. IGV shows representative ATAC-seq and ChIP-seq signals. Cs represents C. sinensis

Fig. 7

C. sinensis infection promotes the migratory behavior of HCC cells and is correlated with increased metastatic potential. a The scratch assay of HCCLM3 cells co-cultured with CsESPs or PBS. b Migration statistics of HCCLM3 cells co-cultured with CsESPs (N = 3) or PBS (N = 3). c Comparison of the metastasis percentage of C. sinensis⁺ (N = 92) and C. sinensis⁻ (N = 855) HCC patients. *P < 0.05, **P < 0.001. Cs represents C. sinensis