Integrated Transcriptome and Metabolome Analyses Uncover Cholesterol-Responsive Gene Networks

Figures

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  Figure 1. Differential expression analysis. (A,B) Volcano plots of DEGs in HeLa cells after 6 h (A: C_6h vs. V_6h) and 24 h (B: C_24h vs. V_24h) cholesterol exposure. DEGs are defined by |log2FC| > 0.38 (1.3-fold) and p-value < 0.05. Blue dots represent downregulated genes, red dots represent upregulated genes, and purple dots represent genes with no significant expression change. Numbers of downregulated, normal, and upregulated genes are indicated above each plot. (C,D) Venn diagram analysis of DEGs comparing 6 h and 24 h cholesterol treatments. (E,F) Heatmaps depicting the expression patterns of selected DEGs in HeLa cells. Panels illustrate gene expression changes between vehicle-treated and cholesterol-treated groups at 6 h and 24 h timepoints, respectively.

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  Figure 2. Gene enrichment analysis reveals functional pathway differences in HeLa cells responding to cholesterol stimulation. (A–D) GOBP and KEGG enrichment analyses of DEGs in HeLa cells after cholesterol stimulation. Panels show enrichment results for DEGs at different timepoints of cholesterol exposure, distinguishing biological processes and pathways.

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  Figure 3. WGCNA of cholesterol-responsive gene networks. (A) Cluster dendrogram of genes. Vertical color bars represent co-expression modules identified by WGCNA, showing hierarchical clustering of genes based on expression similarity across samples. “Height” represents the degree of difference of genes or modules during the clustering process. (B) Heatmap depicting the correlation between WGCNA modules (rows) and experimental groups/traits. Corresponding p-values are in parentheses. The numbers outside the parentheses represent the correlation coefficients between the modules and the traits. (C) Venn diagram showing the intersection of genes from the brown and yellow WGCNA modules with up/downregulated DEGs. (D) STRING-based PPI network of hub genes from the brown and yellow WGCNA modules. Nodes represent proteins, and edges represent interactions.

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  Figure 4. Lipidomic reprogramming in HeLa cells after cholesterol exposure. (A,B) Volcano plots of DAMs in the negative (A) and positive (B) ion modes. Each point represents a metabolite: blue = downregulated, red = upregulated, purple = no significant change. DAMs are defined by |log2FC| > 0.38 (1.3-fold) and p-value < 0.05. (C,D) Heatmaps of DAMs in the negative (C) and positive (D) ion modes. Rows = metabolites, columns = samples (cholesterol/vehicle groups). Color intensity reflects the expression ratio. (E,F) KEGG pathway enrichment of DAMs in the negative ion mode (E) and positive ion mode (F).

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  Figure 5. Broad spectrum metabolomic alterations in HeLa cells after cholesterol exposure. (A,B) Volcano plots of DAMs in the negative (A) and positive (B) ion modes. Each dot represents a metabolite: blue = downregulated, red = upregulated, and purple = no significant change. DAMs are defined by |log2FC| > 0.38 (1.3-fold) and p-value < 0.05. (C,D) Heatmaps of DAMs in the negative (C) and positive (D) ion modes. Rows = metabolites, columns = samples (cholesterol/vehicle groups). Color intensity reflects expression ratio. (E,F) KEGG pathway enrichment of DAMs in the negative ion mode (E) and positive ion mode (F).

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  Figure 6. Integrated transcriptomic and metabolomic analysis of cholesterol-mediated metabolic reprogramming in HeLa cells. (A) Enrichment analysis of key pathways altered by cholesterol treatment. Larger circles indicate greater pathway enrichment, while darker colorsdenote higher statistical significance. (B) Bubble plot visualizing pathway enrichment results. (C) A schematic diagram of UDP-GlcNAc generation dependent on GFPT1 and the protein O-GlcNAc modification signaling pathway. Black straight arrows indicate the direction of metabolic conversion (straight downward), the upregulation of a gene or metabolite are shown by green arrows adjacent to labels). The curved arrow represents the O-linked N-acetylglucosamine transferae (OGT) catalyzed transfer of O-GlcNAc from UDP-GlcNAc to target proteins.

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  Figure 7. Gene expression validation and clinical correlation analysis. (A) mRNA expression of cholesterol processing in HeLa cells at 6 h and 24 h. (B) HeLa cells were cultured with different concentrations of CHOL (50 to 100 μM) or vehicle (the same concentration of MβCD used to dissolve cholesterol) in 10% FBS-containing medium for 6 h. Then cells were harvested for Western blotting. (C) mRNA levels in the GSE126848 cohorts of NASH. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.