图1 HCC中糖酵解代谢途径及乳酸水平变化情况

Fig.1 Changes in pyruvate metabolic process and lactate levels in HCC. A-C: ScRNA-seq analysis of HCC tissues and paired adjacent normal liver tissues from 3 patients with primary HCC. D: Scatter plot of DEGs between normal and tumor samples. Red dots, upregulated genes in tumors; blue dots, downregulated genes; gray dots, genes without significant changes. Selection criteria: adjusted P value (Padj)<0.05 and |log2(fold change)|≥1. E: Schematic illustration of the glycolytic pathway. F: GSEA showing enrichment of pyruvate metabolism in HCC. G: GO enrichment analysis of upregulated DEGs in epithelial cells from tumor samples. H: Lactate levels in HCC and normal hepatic cell lines. Data are presented as Mean±SD (n=3). **P<0.01 vs L-O2 group.

图2 糖酵解驱动的组蛋白H3K18la修饰促进HCC进展

Fig.2 Glycolysis-driven H3K18la promotes HCC progression. A: Western blotting of Pan Kla and H3K18la levels in HCC and normal hepatic cell lines. B, C: Western blotting of H3K9la, H3K18la, and H4K8la levels in HepG2 cells under normoxia and hypoxia. D: Schematic of glycolytic pathway and its inhibitors. E, F: Western blotting and of H3K18la levels following treatment with graded concentrations of 2-DG and oxamate for 24 h in hypoxia (1% oxygen). G: CCK-8 assay for the effect of 2-DG and oxamate for 24 h in hypoxia (1% oxygen) on the proliferation of HepG2 cells. H-I: Colony formation assay of HepG2 cells in different treatment groups with hematoxylin staining. J-K: Transwell migration and invasion assays of HepG2 cells in different treatment groups with crystal violet staining (Scale bar=100 μm). Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs Normal, Normoxia or Ctrl group; ^P<0.05, ^^^P<0.001 vs 2-DG (1 mmol/L) or oxamate (1 mmol/L) group; &&P<0.01 vs 2-DG (2 mmol/L) or oxamate (2 mmol/L) group.

图3 H3K18la介导S100A2转录激活并与HCC不良预后相关

Fig.3 H3K18la-mediated transcriptional activation of S100A2 is associated with poor prognosis in HCC. A: ChIP-qPCR analysis of H3K18la enrichment at the S100A2 promoter. B, C: Western blotting of S100A2 expression in HepG2 cells under hypoxia at indicated time points. D, E: Western blotting of S100A2 expression in HepG2 cells treated with lactate at indicated time points. F-H: Western blotting of H3K18la and S100A2 expression in HepG2 cells treated with 2-DG, lactate or 2-DG with lactate. I: Analysis of S100A2 expression in LIHC using TCGA database. J: Kaplan-Meier analysis of the association between S100A2 expression and prognosis in LIHC patients. K, L: Western blotting of S100A2 expression in HCC and normal hepatic cell lines. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs IgG, Hypoxia (0 h), Lactate (0 mmol/L), Ctrl or L-O2 group; ^^P<0.01, ^^^P<0.001 vs Hypoxia (6 h), Lactate (5 mmol/L) group or Lactate group; &&P<0.01, &&&P<0.001 vs Hypoxia (12 h) or Lactate (10 mmol/L) group.

图4 S100A2促进HCC细胞的增殖、迁移和侵袭能力

Fig.4 S100A2 promotes proliferation, migration, and invasion of HCC cells. A-C: RT-qPCR and Western blotting of S100A2 mRNA and protein expression in 5 HCC cell lines. D-G: Western blotting and RT-qPCR analysis of S100A2 expression in HCC cells following overexpression or knockdown. H-I: CCK-8 assay for cell proliferation in different treatment groups. J, K: Colony formation assay of the cells with hematoxylin staining. L, M: Transwell migration and invasion assays with crystal violet staining (Scale bar=100 μm). Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs si-Ctrl or Ctrl group.

图5 H3K18la修饰通过调控S100A2表达促进HCC细胞的增殖、迁移和侵袭能力

Fig.5 H3K18la modification promotes proliferation, migration, and invasion of HCC cells by regulating S100A2 expression. A: Western blotting of H3K18la and S100A2 expression in HepG2 cells in different treatment groups. B, C: Colony formation assay with hematoxylin staining. D: Transwell migration and invasion assays with crystal violet staining(Scale bar=100 μm). E: Western blotting of H3K18la and S100A2 expression in Huh-7 cells in different treatment groups. F, G: Colony formation assay with hematoxylin staining. H: Transwell migration and invasion assays with crystal violet staining (Scale bar=100 μm). Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs si-Ctrl or Ctrl group; ^^P<0.01, ^^^P<0.001 vs si-S100A2 or S100A2 group.

图6 S100A2与KRT6A存在相互作用

Fig.6 Protein-protein interaction between S100A2 and KRT6A. A: Protein-protein interaction network of S100A2. B: Surface representation of protein docking model between S100A2 (pink) and KRT6A (blue) with docking residues. C, D: Co-IP assay for detecting the interaction between S100A2 and KRT6A.

图7 KRT6A调控Wnt/β-catenin信号通路与EMT促进HCC进展

Fig.7 KRT6A promotes HCC progression by regulating the Wnt/β-catenin signaling pathway and EMT. A,B: Western blotting of nuclear β‑catenin protein levels following KRT6A knockdown and overexpression; C: Western blotting of nuclear β-catenin in different treatment groups. D: Western blotting analysis of nuclear β-catenin in different treatment groups. E: Western blotting of vimentin and E-cadherin expression in different treatment groups. F-G: Western blotting analysis of Vimentin and E-cadherin expression following KRT6A knockdown and overexpression. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs si-Ctrl or Ctrl group; ^^^P<0.001 vs Lactate or 2-DG group.

图8 H3K18la激活 S100A2/KRT6A/β-catenin信号轴促进肝细胞癌上皮间质转化过程的机制示意图

Fig.8 Mechanism of H3K18la for activating the S100A2/KRT6A/β-catenin signaling axis to promote the EMT process in HCC.