Identification of differentially expressed genes in ulcerative colitis and verification in a colitis mouse model by bioinformatics analyses

Abstract

Background: Ulcerative colitis (UC) is an inflammatory bowel disease that is difficult to diagnose and treat. To date, the degree of inflammation in patients with UC has mainly been determined by measuring the levels of nonspecific indicators, such as C-reactive protein and the erythrocyte sedimentation rate, but these indicators have an unsatisfactory specificity. In this study, we performed bioinformatics analysis using data from the National Center for Biotechnology Information-Gene Expression Omnibus (NCBI-GEO) databases and verified the selected core genes in a mouse model of dextran sulfate sodium (DSS)-induced colitis.

Aim: To identify UC-related differentially expressed genes (DEGs) using a bioinformatics analysis and verify them in vivo and to identify novel biomarkers and the underlying mechanisms of UC.

Methods: Two microarray datasets from the NCBI-GEO database were used, and DEGs between patients with UC and healthy controls were analyzed using GEO2R and Venn diagrams. We annotated these genes based on their functions and signaling pathways, and then protein-protein interactions (PPIs) were identified using the Search Tool for the Retrieval of Interacting Genes. The data were further analyzed with Cytoscape software and the Molecular Complex Detection (MCODE) app. The core genes were selected and a Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed. Finally, colitis model mice were established by administering DSS, and the top three core genes were verified in colitis mice using real-time polymerase chain reaction (PCR).

Results: One hundred and seventy-seven DEGs, 118 upregulated and 59 downregulated, were initially identified from the GEO2R analysis and predominantly participated in inflammation-related pathways. Seven clusters with close interactions in UC formed: Seventeen core genes were upregulated [C-X-C motif chemokine ligand 13 (CXCL13), C-X-C motif chemokine receptor 2 (CXCR2), CXCL9, CXCL5, C-C motif chemokine ligand 18, interleukin 1 beta, matrix metallopeptidase 9, CXCL3, formyl peptide receptor 1, complement component 3, CXCL8, CXCL1, CXCL10, CXCL2, CXCL6, CXCL11 and hydroxycarboxylic acid receptor 3] and one was downregulated [neuropeptide Y receptor Y1 (NYP1R)] in the top cluster according to the PPI and MCODE analyses. These genes were substantially enriched in the cytokine-cytokine receptor interaction and chemokine signaling pathways. The top three core genes (CXCL13, NYP1R, and CXCR2) were selected and verified in a mouse model of colitis using real-time PCR Increased expression was observed compared with the control mice, but only CXCR2 expression was significantly different.

Conclusion: Core DEGs identified in UC are related to inflammation and immunity inflammation, indicating that these reactions are core features of the pathogenesis of UC. CXCR2 may reflect the degree of inflammation in patients with UC.

Keywords: Bioinformatics analysis; C-X-C motif chemokine ligand 13; C-X-C motif chemokine receptor 2; Colitis model mice; Neuropeptide Y receptor Y1; Ulcerative colitis.

Figure 1

Identification of differentially expressed genes in the two databases (GSE92415 and GSE87466) and Gene Ontology analysis of differentially expressed genes in ulcerative colitis. A: Upregulated differentially expressed genes (DEGs); B: Downregulated DEGs. DEGs were identified with a t-test, and statistically significant DEGs were defined by the GEO2R online tool with a |logFC| > 2 and adjusted P value < 0.05; C: The Gene Ontology analysis classified the DEGs into three groups: Molecular function, biological process and cellular component. Terms were selected with > 15 genes and arrayed in ascending order from top to bottom according to the count. GO: Gene Ontology.

Figure 2

Protein-protein interactions of differentially expressed genes and the most significant module cluster identified by Molecular Complex Detection in the protein-protein interaction network of ulcerative colitis. A: Protein-protein interaction network of differentially expressed genes determined using Cytoscape. One hundred and seventy-seven differentially expressed genes from the Search Tool for the Retrieval of Interacting Genes online database were screened using Cytoscape, including 130 nodes and 639 edges. Upregulated genes were shown in red, and downregulated genes were shown in blue; B: The most significant cluster was analyzed with the Molecular Complex Detection app in Cytoscape. Seventeen core genes were upregulated, and one gene was downregulated; a larger node indicated more interactions with a gene or a protein.

Figure 3

Hematoxylin-eosin staining and histological lesion score of colon tissues. Hematoxylin-eosin staining of colon tissues from the control and dextran sulfate sodium-induced colitis model mice. A: Control mice (× 100); B: Control mice (× 200); C: Colitis mice (× 100); D: Colitis mice (× 200); E: Histological lesion scores of colon tissues. Numerous neutrophils infiltrated and the crypts, goblet cells and normal four-layer structure of colon disappeared in the colitis model mice. Compared to the score of the control group (n = 4, 12 pieces), the score of the model group (n = 6, 18 pieces) increased significantly (P < 0.01).

Figure 4

Real-time polymerase chain reaction of the top three core genes from the first cluster in colon tissues from the colitis model and control mice. A, C, E, and G: Amplification curves for β-actin, C-X-C motif chemokine ligand 13, neuropeptide Y receptor Y1 and C-X-C motif chemokine receptor 2 (CXCR2); B, D, F and H: Melting peaks for β-actin, C-X-C motif chemokine ligand 13, neuropeptide Y receptor Y1 and CXCR2; I: Relative expression obtained using real-time polymerase chain reaction. C-X-C motif chemokine ligand 13, neuropeptide Y receptor Y1 and CXCR2 expression, particularly CXCR2 expression (P < 0.01), increased in the colitis model mice. The other two genes were expressed at higher levels in the colitis mice than in the control mice, but the difference was not significant. CXCL13: C-X-C motif chemokine ligand 13; NPY1R: Neuropeptide Y receptor Y1; CXCR2: C-X-C motif chemokine receptor 2.