Multi-omics analysis of lactate metabolism gene regulation in Clonorchis sinensis-associated hepatocellular carcinoma

Abstract

Background: Although recent research has highlighted lactylation, a post-translational modification driven by elevated lactate levels, as a critical regulator of key cellular pathways in hepatocellular carcinoma (HCC), its contribution to the poor prognosis of Clonorchis sinensis (Cs)-infected HCC remains poorly understood.

Methods: We first identified the significant upregulation of the lactate metabolism enzyme LDH in Cs-infected HCC patients through clinical retrospective analysis. We then conducted a multi-omics analysis (RNA-Seq, ATAC-Seq, WGBS-Seq, oxWGBS-Seq, and ChIP-Seq) to examine the differences in 392 lactate metabolism-related genes (LMRGs) between Cs-infected and Cs-noninfected HCC tumors. Six key differentially expressed LMRGs were further validated using RT-qPCR assays to confirm their expression and potential role in HCC progression.

Results: The differential expression levels of 8 LMRGs, along with 71 accessible regions and 42 CpG sites in the promoters of LMRGs, were identified. Notably, we also demonstrated that histone modifications, including H3K9ac, H3K79me2, H3K4me2, H3K4me3, H3K27ac, and H3K4me1, were associated with chromatin accessibility in the promoters of LMRGs. Finally, the TCGA-LIHC cohort confirmed that the differential expression of LMRGs between Cs-infected and Cs-noninfected HCC tumors significantly affects the survival outcomes of HCC.

Conclusions: Our findings revealed that lactylation plays an important role in reshaping the characteristics of HCC during Cs infection, expanding our understanding of the unique features of Cs-infected HCC.

Keywords: Clonorchis sinensis; Hepatocellular carcinoma; Lactate metabolism; Multi-omics.

Fig. 1

Retrospective clinical analysis of the serum key enzyme LDH in lactate metabolism among Cs⁺ and Cs⁻ HCC patients. a Comparative analysis of serum LDH concentrations between Cs⁺ and Cs⁻ HCC patients. b Kaplan-Meier survival curves demonstrating the correlation between stratified serum LDH levels and overall survival outcomes

Fig. 2

Cs infection changes the expression profile of LMRGs in HCC tumors. a Volcano plot showing downregulated and upregulated LMRGs between Cs⁺ HCC tumors and Cs⁻ HCC tumors. b GO enrichment network of differentially expressed LMRGs between Cs⁺ HCC tumors and Cs⁻ HCC tumors. c RT-qPCR validation of differentially expressed LMRGs in Cs⁺ and Cs⁻ HCC tumor samples. Data were presented as means ± SD; n = 3. Student’s t-test was used. d Kaplan-Meier curves illustrating the association between the expression levels of differentially expressed LMRGs and survival outcomes in the TCGA-LIHC cohort. e Spearman correlation analysis assessing the relationships among differentially expressed LMRGs

Fig. 3

Cs infection changes the chromatin accessibility landscape of lactate metabolism-related genes in HCC tumors. a Different chromatin accessibility landscapes in the promoter regions of LMRGs between Cs⁺ HCC tumors and Cs⁻ HCC tumors. b The top de novo motifs enriched in differential chromatin accessibility regions in the promoter regions of LMRGs between Cs⁺ HCC tumors and Cs⁻ HCC tumors. c Heatmap representation of gene active scores in differential chromatin accessibility regions of LMRGs between Cs⁺ HCC tumors and Cs⁻ HCC tumors. d IGV shows representative difference peaks of LMRGs between Cs⁺ HCC tumors and tumor-adjacent tissues

Fig. 4

Joint analysis of ATAC-Seq and public ChIP-seq data between Cs⁺ HCC tumors and Cs⁻ HCC tumors. a Heatmap representation of histone modifications associated with differential chromatin accessibility regions of LMRGs between Cs⁺ HCC tumors and Cs⁻ HCC tumors. b IGV shows representative ATAC-seq and ChIP-seq signals between Cs⁺ HCC tumors and Cs⁻ HCC tumors

Fig. 5

Correlation between DNA methylation of LMRGs and survival outcomes in the TCGA-LIHC cohort. a Kaplan-Meier curves show survival outcomes of genes associated with differential methylation sites in LMRGs between Cs⁺ HCC tumors and Cs⁻ HCC tumors