*3.4. Exploration of DEGs in Protein–Protein Interaction (PPI) Network and Subnetworks (Modules)*

To find out the potential interactions at the protein level, DEGs were mapped in STRING, and the PPI network (Figure 4A) was constructed for top 300 DEGs (up/downregulated genes). The significant pairs of the network (*p*-value: 1.0 × <sup>10</sup>−164) in 2736 interactions between 180 nodes based on confidence score (0.007) were visualized by Cytoscape software and the network analyzer plug-in with a cut-off value for BC > 0 and K > 8. After analyzing PPI network, subnetworks (modules) with a MCODE plug-in of Cytoscape were extracted, and generally, 10 modules obtained. Three significant modules were identified with MCODE score ≥ 3 and nodes ≥ 3 (Figure 4B–D). CDK1, CCNA2, and BUB1B with the highest BC and K in the PPI network and with the highest score in the MCODE plug-in were significantly

identified in Module 1 (Figure 4D and Table 3). Accordingly, it is clearly obvious that three of the DEGs (CDK1, CCNA2, and BUB1B) in the PPI network and module 1 are the genes that interact the most with others in the PPI network.

**Figure 3.** Normalization of raw read counts and Differential Expression Analysis (DEA). The volcano plot (**A**) of non-significant DEGs (colored in dark gray), significant DEGs (padj < 0.01) (colored in light blue), and up/downregulated genes (Colored in red). The x-axis represents the log2 FC, while the y-axis represents the statistical significance for each gene based on −log10 (*p* value). Heatmap of top 22 up/downregulated genes associated with non-normalized counts (**B**). Heatmap of top 22 up/downregulated DEGs associated with normalized counts (**C**). From the heatmap (downregulated genes are colored in light blue and upregulated genes are colored in orange in both plots). NS: non-significant; FC: fold change.

**Table 2.** The list of top 10 up/downregulated Differentially Expressed Genes (DEGs) based on RNAseq data analysis. The DEGs (padj < 0.01) between control and treatment groups are shown with top 10 upregulated genes (padj < 0.01, log2 FC > 2), and top 10 downregulated genes (padj < 0.01, log2 FC < −2). Padj: adjusted *p* value.


**Figure 4.** Overview of PPI network and subnetworks (modules). The PPI network (**A**); the yellow colors represent key nodes (highest BC and K) in PPI networks. Generally, 10 modules were obtained from the main network and module 1 (**D**), module 2 (**B**) and module 3 (**C**) are significant modules. Module 1 with the score of 47.057 and 54 nodes is the more significant module, covering three genes CDK1, CCNA2, and BUB1B with the highest BC and K in the PPI network and with the highest score in the MCODE plug-in.

**Table 3.** The position of 5 CRC-related candidate genes in the PPI network and subnetworks (modules). As shown, CDK1, CCNA2 and BUB1B genes with the highest degree (K) and betweenness (BC) are clustered in subnetwork 1, demonstrating their significant influence in the network. In contrast, GADD45G and ATF3 genes are classified in clusters 5 and 8, perhaps indicating their independence to the networks in CRC.


#### *3.5. KEGG Pathway Analysis of DEGs*

For the characterization of DEGs, we performed pathway enrichment analysis in KEGG. In total, 300 upregulated (padj < 0.01, log2 FC > 2) and downregulated (padj < 0.01, log2 FC < −2) genes were mapped to 117 KEGG pathways. In order to identify which gene is associated with biological pathways, a gene-pathway annotated network was created using 300 DEGs (up/downregulated genes) alongside the most significant KEGG pathways using Cytoscape software.

Generally, the gene-pathway annotated network showed that DEGs were significantly enriched in the cell cycle, E2F-mediated regulation of DNA replication, G1/S-specific transcription, cell cycle checkpoints, G2/M checkpoints, FOXM1 transcription factor network, and G1 to S cell cycle control (Table 4). Surprisingly, a vital gene of our study, CDK1, was significantly enriched in all significant pathways that may act as a bridge between both the CRC-related genes (CCNA2, BUB1B, GADD45G and ATF3) and other gene-pathway annotated network genes (Figure 5).

**Figure 5.** Overview of the gene-pathway network (annotated network) created by cystoscope. The blue nodes represent the CRC candidate genes connected with the pathways and other nodes in the network. The orange nodes represent other pathways in connection with CRC genes. According to this network, it can be demonstrated that CDK1 is involved in all significant first-level pathways; it is associated with related genes in each of the pathways and with other most significant CRCrelated genes (CCNA2, BUB1B, GADD45G, ATF3). The dominant pathway is cell cycle with padj 1.82 <sup>×</sup> <sup>10</sup><sup>−</sup>34.


**Table 4.** Status of CRC-related genes—CDK1, CCNA2, BUB1B, GADD45G, and ATF3—in genepathway annotated networks. The table shows that CDK1 is prominently involved in different pathways. The gene pathways are significantly enriched in cell cycle-related networks. Padj: adjusted *p*-value.
