Exploring miRNAs targeting the Wnt pathway in colorectal Cancer Patients
Understanding and targeting these miRNA-Wnt interactions hold promise for the development of innovative therapeutic strategies in CRC treatment(13).At the outset, Proteins targeted by miRNAs targeting the Wnt pathway were identified by miRDB database. We assembled a list of 1054 genes influenced by both up- and down-regulated miRNAs that impact the Wnt pathway. All selected genes had a score surpassing 95, as per the miRDB database. Subsequent analyses incorporated only those high-scoring genes regulated by miRNAs.
Constructing and Analyzing Protein-Protein Interaction Networks
To illustrate the complex protein network among these genes visually, we utilized the Database String and Cytoscape. Biological networks, which intricately represent diverse biological systems, often involve various entities. Cytoscape, a versatile software tool with support from various apps, facilitates the analysis and visualization of these networks (Fig. 1).
Protein-protein interaction networks (PPIN) occurring in defined binding regions, holding particular biological significance and serving specific functions. Researchers have increasingly emphasized protein substructures, utilizing interaction algorithms to predict interactions and comprehend functions, enabling the examination of key proteins crucial for optimal targeting (14). In Fig. 1, proteins directly linked to dysregulated miRNAs play pivotal roles in essential cellular pathways for growth and proliferation. In subsequent sections, our goal is to present a refined classification and understanding of these proteins, pinpointing those with the most interactions.
Using MCC, MNC, DMNC, and Degree algorithms we pinpointed 15 central proteins with extensive interactions, indicating their crucial roles in the network (Fig. 2). Table 2 offers in-depth information about these hub proteins, and the subnetwork they create is illustrated in Fig. 2. The identified hub proteins in your study includes: IGF1, TNF, FGF2, FGF18, FGF7, COL19A1, PLOD2, COL5A3, ADAMTS2, CACHD1, PTEN, ESR1, PIK3CA, SMAD3, and FOS, are associated with the pathogenesis and development of colorectal cancer.
IGF1 (Insulin-like Growth Factor 1): Implicated in cell proliferation and survival, potentially contributing to tumor growth (15). TNF (Tumor Necrosis Factor): Inflammatory cytokine linked to cancer development; its dysregulation may promote colorectal cancer progression (16). FGF2, FGF18, FGF7 (Fibroblast Growth Factors): Involved in cell growth, angiogenesis, and tissue repair, their aberrant expression may support cancer development (17). COL19A1, PLOD2, COL5A3 (Collagens): Structural components of the extracellular matrix, alterations in collagen expression can influence tumor invasion and metastasis (18). ADAMTS2 (ADAM Metallopeptidase with Thrombospondin Motifs 2): Associated with extracellular matrix remodeling, potentially affecting cancer progression (19). CACHD1: Its specific role in colorectal cancer may require further investigation as information may vary (20). PTEN (Phosphatase and Tensin Homolog): Acts as a tumor suppressor by regulating cell growth; mutations can lead to uncontrolled cell division (21). ESR1 (Estrogen Receptor 1): Hormone receptor associated with colorectal cancer, especially in estrogen-related pathways (22). PIK3CA (Phosphatidylinositol-4, 5-Bisphosphate 3-Kinase Catalytic Subunit Alpha): Frequently mutated in colorectal cancer, influencing cell survival and proliferation (23). SMAD3: Part of the TGF-beta signaling pathway, alterations may contribute to colorectal cancer progression. FOS: Proto-oncogene involved in cell proliferation; dysregulation can support tumor development. These proteins collectively play diverse roles in cellular processes, and their dysregulation or mutations can contribute to colorectal cancer pathogenesis, providing potential targets for further research and therapeutic interventions (24).
Table 2
Hob proteins in genes targeted by dysregulated miRNA
Hob proteins | Method | Rank | Gene description |
IGF1 | MCC | 1 | Insulin-like growth factor I; The insulin-like growth factors, isolated from plasma, are structurally and functionally related to insulin but have a much higher growth-promoting activity. |
TNF | MCC, MNC, Degree | 2,2,3 | Tumor necrosis factor, membrane form; Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. |
FGF2 | MCC | 3 | Fibroblast growth factor 2; Acts as a ligand for FGFR1, FGFR2, FGFR3 and FGFR4. Also acts as an integrin ligand which is required for FGF2 signaling. Binds to integrin ITGAV:ITGB3. Plays an important role in the regulation of cell survival, cell division, cell differentiation and cell migration. |
FGF18 | MCC | 4 | Fibroblast growth factor 18; Plays an important role in the regulation of cell proliferation, cell differentiation and cell migration. Required for normal ossification and bone development. Stimulates hepatic and intestinal proliferation. |
FGF7 | MCC | 5 | Fibroblast growth factor 7; Plays an important role in the regulation of embryonic development, cell proliferation and cell differentiation. Required for normal branching morphogenesis. Growth factor active on keratinocytes. |
COL19A1 | DMNC | 1 | Collagen alpha-1(XIX) chain; May act as a cross-bridge between fibrils and other extracellular matrix molecules. Involved in skeletal myogenesis in the developing esophagus. |
PLOD2 | DMNC | 2 | Procollagen-lysine,2-oxoglutarate 5-dioxygenase 2; Forms hydroxylysine residues in -Xaa-Lys-Gly- sequences in collagens. |
COL5A3 | DMNC | 3 | Collagen alpha-3(V) chain; Type V collagen is a member of group I collagen (fibrillar forming collagen). It is a minor connective tissue component of nearly ubiquitous distribution. Type V collagen binds to DNA, heparan sulfate, thrombospondin, heparin, and insulin. |
ADAMTS2 | DMNC | 4 | A disintegrin and metalloproteinase with thrombospondin motifs 2; Cleaves the propeptides of type I and II collagen prior to fibril assembly (By similarity). Does not act on type III collagen (By similarity). |
CACHD1 | DMNC | 5 | VWFA and cache domain-containing protein 1; May regulate voltage-dependent calcium channels. |
PTEN | MNC, Degree | 1,1 | Phosphatase and tensin homolog; Tumor suppressor. Acts as a dual-specificity protein phosphatase, dephosphorylating tyrosine-, serine- and threonine- phosphorylated proteins. |
ESR1 | MNC, Degree | 3,2 | Estrogen receptor; Nuclear hormone receptor. The steroid hormones and their receptors are involved in the regulation of eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues. |
PIK3CA | MNC | 4 | Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform; Phosphoinositide-3-kinase (PI3K) that phosphorylates PtdIns (Phosphatidylinositol), PtdIns4P (Phosphatidylinositol 4-phosphate) and PtdIns(4, 5)P2 (Phosphatidylinositol 4,5-bisphosphate) to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3). |
SMAD3 | MNC, Degree | 5,5 | Mothers against decapentaplegic homolog 3; Receptor-regulated SMAD (R-SMAD) that is an intracellular signal transducer and transcriptional modulator activated by TGF-beta (transforming growth factor) and activin type 1 receptor kinases. Binds the TRE element in the promoter region of many genes that are regulated by TGF-beta and, on formation of the SMAD3/SMAD4 complex, activates transcription |
FOS | Degree | 4 | Proto-oncogene c-Fos; Nuclear phosphoprotein which forms a tight but non-covalently linked complex with the JUN/AP-1 transcription factor. |
Gene ontology and KEGG enrichment analysis
Our investigation into miRNAs targeting the Wnt pathway unveiled a network of hub proteins with significant associations, as reflected in various Gene Ontology (GO) terms.
Biological Processes: The enrichment of terms related to positive and negative regulation of biological processes suggests that these hub proteins play pivotal roles in regulating cellular activities linked to CRC development. The involvement in developmental processes, organ development, and system development underscores their potential contributions to CRC initiation and progression (Fig. 3).
Cellular Components: The presence of terms such as intracellular organelle lumen, membrane-bounded organelle, and extracellular space emphasizes the spatial importance of hub proteins within CRC. Their involvement in collagen trimer and extracellular matrix structural constituents indicates potential interactions influencing the tumor microenvironment and CRC cell behavior (Fig. 3).
Molecular Functions: Subnetwork of hub proteins exhibit diverse molecular functions, including protein binding, transcriptional regulation, and signaling receptor binding. These molecular interactions align with their roles in CRC pathogenesis, offering insights into the intricate signaling networks involved in colorectal tumorigenesis (Fig. 3).
KEGG Pathways:
The identification of cancer-related pathways, such as PI3K-Akt signaling, MAPK signaling, and others, signifies the direct association of hub proteins with key cellular pathways implicated in CRC. These findings strengthen the understanding of the molecular mechanisms driving colorectal cancer progression (Fig. 3).
The association of these GO terms with colorectal cancer underscores the multifaceted nature of hub proteins in regulating biological processes, influencing cellular components, and participating in crucial molecular functions. The spatial implications within cellular components and their involvement in pathways directly linked to cancer emphasize the pivotal role of these hub proteins in CRC pathogenesis. By deciphering the intricate network of hub proteins and their functional annotations, this study provides a foundation for further exploration of therapeutic targets and intervention strategies tailored to the specific molecular landscape of colorectal cancer. The identified associations contribute valuable insights, guiding future research endeavors aimed at advancing our understanding and treatment of CRC.
Deciphering Functional Modules in Breast Cancer via Cluster Analysis.
Cytocluster analysis confers a valuable advantage in deciphering complex biological datasets by revealing intricate patterns of molecular interactions within biological systems. By identifying clusters of genes or proteins that exhibit coordinated behavior, this analytical approach unveils potential regulatory networks and functional modules. The benefit lies in its ability to elucidate the underlying structure of biological data, facilitating a more nuanced understanding of cellular processes, disease mechanisms, and the intricate relationships between molecular components. Cytocluster analysis thus empowers researchers to pinpoint key players in biological systems, aiding in the discovery of novel biomarkers, therapeutic targets, and pathways crucial for disease initiation, progression, and response to treatment.
Table 3 summarizes the outcomes of the CytoCluster analysis focused on dysregulated miRNA-targeted genes associated with colorectal cancer. 5 top clusters identified, each characterized by its rank, number of nodes, edges, and the results of KEGG enrichment analysis. Notably, Cluster 1 exhibits significant associations with pathways crucial in cancer, such as "Pathways in cancer" and "Breast cancer," suggesting potential involvement in colorectal cancer initiation. Similarly, other clusters reveal enrichments in pathways like "PI3K-Akt signaling pathway" and "MAPK signaling pathway," providing insights into the significant cross talk of Wnt, MAPK, and PI3K-Akt pathways as well as roles of dysregulated miRNAs in the development of colorectal cancer. These findings set the stage for a comprehensive exploration of the molecular mechanisms underlying colorectal cancer, paving the way for targeted therapeutic interventions and a deeper understanding of the disease progression (Table 3).
Table 3
Top 5 CytoCluster results for dysregulated miRNA targeted genes
Cluster | Rank | Nodes | Edges | KEGG enrichment analysis |
1 | 1 | 24 | 164 | Pathways in cancer Breast cancer Human T-cell leukemia virus 1 infection PI3K-Akt signaling pathway Human papillomavirus infection Melanoma Gastric cancer |
2 | 2 | 24 | 167 | Breast cancer Leishmaniasis Hepatitis B Melanoma Human papillomavirus infection Proteoglycans in cancer |
3 | 3 | 23 | 180 | Breast cancer Human papillomavirus infection PI3K-Akt signaling pathway Melanoma Focal adhesion Proteoglycans in cancer Gastric cancer |
4 | 4 | 23 | 173 | PI3K-Akt signaling pathway Pathways in cancer Breast cancer Human papillomavirus infection Focal adhesion Protein digestion and absorption |
5 | 5 | 23 | 163 | Pathways in cancer Proteoglycans in cancer PI3K-Akt signaling pathway Leishmaniasis Breast cancer MAPK signaling pathway |
Promoter Motif Analysis of Hub Proteins
Promoter analysis of target genes in colorectal cancer treatment holds significant therapeutic promise by unveiling crucial regulatory elements governing gene expression. Understanding the specific motifs and transcription factor binding sites in the promoters of key genes associated with colorectal cancer allows for the identification of potential targets for intervention. By deciphering these regulatory sequences, researchers can design targeted therapies aimed at modulating gene expression, disrupting oncogenic signaling, or promoting tumor suppressor activity. This approach enables the development of precision treatments tailored to the molecular characteristics of individual tumors, fostering more effective and personalized strategies in the pursuit of enhanced outcomes for colorectal cancer patients.
The identified motifs, represented by logos, provide insights into the potential regulatory elements associated with specific hub proteins. For instance, the motif associated with ENSG00000108821 is linked to the CC transcription factor complex and is implicated in biological processes (BP) such as negative regulation of signal transduction and inner ear morphogenesis. Similarly, motifs associated with ENSG00000130635 and ENSG00000139219 exhibit connections to transcription factor complexes and activities related to signal transduction and protein interactions. The analysis of ENSG00000197467 reveals motifs associated with protein heterodimerization activity and dendritic processes, shedding light on potential regulatory mechanisms. These findings enhance our understanding of the regulatory elements of hub genes at the motif level, providing a foundation for further investigations into the functional implications of these identified motifs and made them potential targets element candidates (Table 4).