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. 2022 Jun 24:13:914382.
doi: 10.3389/fphys.2022.914382. eCollection 2022.

Identification of Hub Genes and Immune-Related Pathways for Membranous Nephropathy by Bioinformatics Analysis

Xiao-Yu Cai  1 , Zu-Feng Wang  1 , Shu-Wang Ge  1 , Gang Xu  1
Affiliations

Identification of Hub Genes and Immune-Related Pathways for Membranous Nephropathy by Bioinformatics Analysis

Xiao-Yu Cai et al. Front Physiol. .

Abstract

OBJECTIVE: We aim to explore the detailed molecular mechanisms of membrane nephropathy (MN) related genes by bioinformatics analysis. METHODS: Two microarray datasets (GSE108109 and GSE104948) with glomerular gene expression data from 65 MN patients and 9 healthy donors were obtained from the Gene Expression Omnibus (GEO) database. After processing the raw data, DEGs screening was conducted using the LIMMA (linear model for microarray data) package and Gene set enrichment analysis (GSEA) was performed with GSEA software (v. 3.0), followed by gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. The protein-protein interaction (PPI) network analysis was carried out to determine the hub genes, by applying the maximal clique centrality (MCC) method, which was visualized by Cytoscape. Finally, utilizing the Nephroseq v5 online platform, we analyzed subgroups associated with hub genes. The findings were further validated by immunohistochemistry (IHC) staining in renal tissues from MN or control patients. RESULTS: A sum of 370 DEGs (188 up-regulated genes, 182 down-regulated genes) and 20 hub genes were ascertained. GO and KEGG enrichment analysis demonstrated that DEGs of MN were preponderantly associated with cell damage and complement cascade-related immune responses. Combined with literature data and hub gene-related MN subset analysis, CTSS, ITGB2, and HCK may play important roles in the pathological process of MN. CONCLUSION: This study identified novel hub genes in MN using bioinformatics. We found that some hub genes such as CTSS, ITGB2, and HCK might contribute to MN immunopathological process, providing new insights for further study of the molecular mechanisms underlying glomerular injury of MN.

Keywords: GSEA; bioinformatics analysis; hub genes; immunology; membranous nephropathy.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Volcano plot and Heatmap analysis identifies DEGs. (A) Red dots represent upregulated genes and blue dots represent downregulated genes in renal glomerular tissue from MN patients compared with normal controls. (B) Red areas represent highly expressed genes and blue areas represent lowly expressed genes in renal glomerular tissue from MN patients compared with normal controls. DEG, differentially expressed gene; MN, membranous nephropathy.
FIGURE 2
FIGURE 2
GO and KEGG enrichment result of DEGs. The x-axis represents p value and y-axis represents GO terms. The size of circle represents gene count. Different color of circles represents gene ratio. (A) GO-BP enrichment result of DEGs. (B) GO-CC enrichment result of DEGs. (C) KEGG enrichment result of DEGs. DEG: differentially expressed gene; KEGG: Kyoto Encyclopedia of Genes and Genomes; GO: Gene Ontology; BP: biological process; CC: cell composition.
FIGURE 3
FIGURE 3
GSEA plot showing most enriched GO terms and KEGG pathways in the MN group. (A). The 7 most significant enriched GO term positively correlated with the MN group was apoptotic cell clearance, positive regulation of blood vessel endothelial cell migration, transforming growth factor beta receptor signaling pathway, positive regulation of endothelial cell migration, regulation of cell migration involved in sprouting angiogenesis, regulation of cellular response to transforming growth factor beta stimulus, vascular endothelial growth factor receptor signaling pathway. (B) The 7 most significant enriched KEGG pathway positively correlated with the MN group was chemokine signaling pathway, FcεRi signaling pathway, FcγR mediated phagocytosis, focal adhesion, natural killer cell mediated cytotoxicity, p53 signaling pathway, toll like receptor signaling pathway. MN, membranous nephropathy; GSEA, GO term enrichment analysis; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
FIGURE 4
FIGURE 4
PPI network of DEGs and top 10 hub genes. (A) PPI network of DEGs created by STRING. Circles represent genes and lines represent PPIs (B) The network of the top 10 hub genes identified by MCODE. DEG: differentially expressed gene; PPI: protein–protein interaction.
FIGURE 5
FIGURE 5
Association between mRNA expression of hub genes in MN patients and normal controls. (A) The expression of TYROBP in MN group was higher than that of normal control (B) The expression of ITGB2 in MN group was higher than that of normal control. (C) The expression of HCK in MN group was higher than that of normal control (D) The expression of CD53 in MN group was higher than that of normal control. (E) The expression of CTSS in MN group was higher than that of normal control (F) The expression of FCER1G in MN group was higher than that of normal control (G) The expression of NCF2 in MN group was higher than that of normal control. p < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001. MN: membranous nephropathy; mRNA: messenger RNA.
FIGURE 6
FIGURE 6
Immunohistochemistry (IHC) staining of CTSS, HCK, and ITGB2 in MN tissues and normal renal tissues. The expression of CTSS, HCK, and ITGB2 protein in MN tissues was higher than that in control group.
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