Table 1.
Characteristics of 132 HPV-Positive Pap Smear Specimens.
Fig 1.
GO Biological Process Enrichment of Differentially Expressed Hub Genes Across Cervical Lesion Stages.
Bar plots illustrating Gene Ontology (GO) biological process (BP) enrichment of upregulated (Fig 1A), downregulated (Fig 1B) and hub genes across four cervical cytological stages: NILM (Negative for Intraepithelial Lesion or Malignancy), ASCUS (Atypical Squamous Cells of Undetermined Significance), LSIL (Low-grade Squamous Intraepithelial Lesion), and HSIL (High-grade Squamous Intraepithelial Lesion). Enrichment analyses were performed using WebGestalt (2023 release), applying a false discovery rate (FDR) threshold of <0.05. Cytological groups are color-coded as follows: blue (NILM), green (ASCUS), red (LSIL), and orange (HSIL). Bar length indicates the number of genes associated with each enriched term, and the x-axis shows –log10 transformed FDR values indicating statistical significance.
Fig 2.
Pathway Crosstalk Network Across Cervical Cytological Categories (Full Network).
This network diagram illustrates the inter-pathway crosstalk among statistically enriched biological pathways identified across four cervical cytological categories: NILM (blue), ASCUS (green), LSIL (red), and HSIL (orange). Each node represents a unique pathway enriched in one or more cytological stages (FDR < 0.05, ≥ 3 constituent genes) and edges represent gene overlap between pathways. The overlap between pathways is quantified using the Jaccard Coefficient (JC) and Overlap Coefficient (OC), with a minimum of two overlapping genes required for inclusion. Pathways exhibiting the highest centrality are visualized as hub nodes and reflect biologically critical signaling axes during neoplastic progression. Notably, NILM pathways clustered toward immune regulation and epithelial adhesion, ASCUS and LSIL retained significant overlap with NILM (OC > 0.9), while HSIL pathways formed a distinct cluster dominated by cell cycle, replication, and chromatin remodeling processes. Network topology was visualized in Cytoscape and pathway identities are annotated according to KEGG, Reactome, and WikiPathways databases.
Fig 3.
Pathway Crosstalk Network Across Cervical Cytological Categories (Node degree ≥5).
This network visualization depicts the subnetwork with nodal degree ≥ 5 representing crosstalk among significantly enriched molecular pathways (FDR < 0.05; ≥ 3 genes) across four cytological stages of HPV-positive cervical lesions: NILM (blue), ASCUS (green), LSIL (red), and HSIL (orange). Each node represents a unique biological pathway identified through enrichment analysis using KEGG, Reactome, and WikiPathways databases. Node color indicates the cytological stage(s) in which the pathway was enriched. Edges between nodes reflect shared gene content, with a minimum threshold of two overlapping genes for edge retention. The network illustrates how pathway architecture evolves with disease progression: early-stage NILM and ASCUS pathways cluster around immune signaling and homeostatic regulation, while LSIL pathways show transitional overlap with metabolic and growth factor signaling. In contrast, HSIL forms a distinct, tightly connected module centered on cell cycle regulation, DNA replication, mitotic spindle assembly, and chromatin remodeling. High-connectivity nodes such as “Cell Cycle,” “DNA Replication,” and “ATR Activation in Response to Replication Stress” emerge as central hubs in HSIL, reflecting oncogenic pathway consolidation. The increasing modularity and divergence of HSIL-associated pathways highlight a molecular shift from viral persistence to host genome destabilization and dysplastic transformation.
Fig 4.
Global Protein–Protein Interaction (PPI) Network of Hub Genes Across Cytological Categories.
This figure illustrates the comprehensive PPI network of all hub genes identified across the four cytological categories of HPV-positive cervical specimens: NILM, ASCUS, LSIL, and HSIL. The network was constructed using the STRING database (v11.5), with a high-confidence interaction score threshold of ≥ 0.7 to ensure biologically meaningful associations. Only nodes (genes) with a degree > 5 were retained for visualization, resulting in a network of 151 nodes and 2,459 edges. Each node represents a unique hub gene, and edges indicate protein–protein interactions based on experimental data, curated databases, and predictive algorithms. The spatial organization of the network was generated using force-directed layout in Cytoscape (v3.10.3), allowing clusters and highly interconnected regions to emerge visually without imposed functional categorization. This unlabeled network provides a global overview of hub gene connectivity and highlights the dense interaction patterns that underpin the transcriptomic reprogramming during HPV-driven cervical lesion progression. The visualization serves as a reference for understanding network topology prior to functional modular classification.
Fig 5.
Protein–Protein Interaction (PPI) Network of Hub Genes with Functional Cluster Labeling.
This figure displays the high-confidence PPI network constructed from all identified hub genes across the four cytological stages of HPV-positive cervical lesions. Network construction was performed using the STRING database (v11.5), with a minimum interaction confidence score threshold of 0.7 to retain high-confidence associations. The resulting network includes 151 nodes (hub genes) and 2,459 edges, visualized in Cytoscape (v3.10.3). Only genes with a node degree >5 were retained to minimize noise and highlight core interactors. Nodes represent individual hub genes, and edges denote experimentally validated or predicted protein–protein associations. Functional modules were identified through STRING functional enrichment clustering and labeled based on biological relevance. Three major functional clusters are highlighted: 1. Mitochondrial Translation Cluster: Encompassing genes involved in ribosomal biogenesis and protein synthesis, reflecting enhanced metabolic and translational activity characteristic of early lesion stages (e.g., NILM and ASCUS). 2. Chemokine Response Cluster: Including immune modulators and signaling components involved in cytokine–cytokine receptor interactions and inflammatory signaling pathways, largely downregulated in early lesions but indicative of host immune surveillance. 3. Mitotic and DNA Replication Cluster: Comprising cell cycle regulators, DNA replication licensing factors, and mitotic spindle components, predominantly enriched and upregulated in high-grade lesions (HSIL), representing proliferative dysregulation and genomic instability. This cluster-labeled network underscores the temporal transition from immune evasion and metabolic support (early lesions) to proliferative transformation (HSIL), providing insight into molecular reprogramming along the cervical neoplastic continuum.