Table 1.
The primer sequences used are listed.
Fig 1.
Flow chart.
Fig 2.
Identification of differentially expressed genes (DEGs).
(A-B) Volcano plot of DEGs between datasets GSE122060 and GSE100219. Red dots: upregulated, blue dots: downregulated. Parameter settings are |log2FC| > 1 and p-value (FDR correction) < 0.05. (C-D) Heatmap of DEGs in datasets GSE122060 and GSE100219. Rows represent genes; columns represent samples. Expression values were row-normalized (Z-score) based on log2(TPM + 1) data. Hierarchical clustering (Euclidean distance, average linkage) was applied to genes. (E) Venn diagram of upregulated and downregulated genes in two datasets.
Fig 3.
Functional enrichment analysis of DEGs, protein-protein interaction (PPI) network construction.
(A) Gene Ontology (GO) analysis of DEGs, including the major biological processes (BP) and molecular functions (MF) involved. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs. (C) The circular network diagram of functional enrichment analysis. (D) The PPI network constructed from DEGs, with 9 genes in the inner circle having a degree ≥ 15. The color intensity of the genes corresponds to their degree values. (E) Bar chart of the interaction number of each DEG.
Fig 4.
Hub gene identification, and GEO dataset validation.
(A) The top 10 hub genes identified in the PPI network through the Cytohubba plugin, with the color intensity of the genes corresponding to their score values. (B) The functional clustering module extracted from the PPI network through MCODE contain 12 genes, with the color intensity of the genes corresponding to their degree values. (C) The mRNA expression of 10 hub genes was determined from the GSE28242 validation dataset, six genes have significant expression differences. (D) Diagnostic value of 10 hub genes analyzed with ROC curves. AUC, area under the ROC curve. ns, no significant difference.
Fig 5.
The interaction network between transcription factors (TFs) and six hub genes, the co-regulatory network of TF-miRNA-hub genes, and miRNA target prediction.
(A) The TF-hub genes interaction network. The red circles represent hub genes, the light blue diamonds represent TFs interacting with hub genes, and the purple diamonds represent TFs interacting with no less than three hub genes. (B) The interaction network between TF-miRNA and six hub genes. The red circle represents the hub gene, the blue pentagon represents the miRNAs that interact with the hub gene, the yellow pentagon represents the miRNAs that interact with no less than three hub genes, the green diamond represents the TFs that interact with the hub gene, and the purple diamond represents the TFs that interact with no less than three hub genes. (C) The predictable miRNAs that interact with hub genes.
Fig 6.
qPCR validation of DEGs and functional assays.
(A) qPCR validation of DEGs identified through CytoHubba and MCODE analysis in the PPI network. Among the six hub genes, except for C3 which showed no statistical difference, CLEC4E, CSF3R, CXCR2, and FPR2 were upregulated in the disease group compared to the control group, while IDO1 was downregulated. (B) Assessment of FPR2 knockdown efficiency by qPCR. (C) CCK-8 assay for assessing the viability of SV-HUC-1 cells. (D, E) Transwell assay to determine the migration ability of SV-HUC-1 cells (Scale bar, 100 μm). *: p < 0.05, **: p < 0.01, ***: p < 0.001. ns: no significance.