Background Epithelial mesenchymal transition (EMT) plays an important role in pulmonary fibrosis. EMT of alveolar epithelial cells is a major source of myofibroblasts. Bioinformatic analysis used to screen hub genes in EMT process has attracted the attention of scholars as the highthroughput sequencing technology evolves.
Objective This study is conducted to screen potential hub genes and related key signaling pathways in the EMT process related to pulmonary fibrosis by analyzing the genes differentially expressed during the EMT of human alveolar type Ⅱ epithelial cells (A549 cells), aiming to provide new ideas for the study of EMT related to pulmonary fibrosis.
Methods An GSE17708 dataset was downloaded from the Gene Expression Omnibus of the National Center for Biotechnology Information, and used to identify differentially expressed genes by limma package in R language. DAVID 6.8 was used to perform Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis. An interaction gene database and Cytoscape software were used to draw protein-protein interaction (PPI) network. The hub genes were identified by the CytoHubba plugin of Cytoscape software. Finally, the hub genes in the process of EMT of A549 cells were verified by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR).
Results A total of 55 differentially expressed genes were screened out based on high-throughput chip screening. The GO enrichment results showed that 5 enriched GO terms fell in the biological process category, namely collagen catabolic process, positive regulation of cell migration, negative regulation of growth, negative regulation of endothelial cell apoptotic process, and extracellular matrix organization; 2 enriched terms in the cellular component category, mainly in extracellular region and extracellular space; and 5 enriched terms in the molecular function category, containing extracellular matrix structural constituent, heparin binding, phosphatidylserine binding, enzyme inhibitor activity, and fibronectin binding. The results of KEGG showed that differentially expressed genes were mainly involved in extracellular matrix-receptor interaction, amoebiasis, focal adhesion, PI3K-Akt signaling pathway, mineral absorption, platelet activation, complement and coagulation cascades, and small lung cell cancer. Subsequently, the top eight genes with the highest scores were determined as hub genes related to EMT through the maximal clique centrality (MCC) method: THBS1, COL4A1, COL5A1, COL4A2, FGG, SERPINE1, LAMC2, and IGFBP5. The qRT-PCR results showed that the abundance of IGFBP5 was lower and not significantly different in A549 cells after TGF-β stimulation for 24 h and 72 h compared with the control group. Except the expression of FGG, the expressions of the remaining 6 hub genes were consistent with the screening results of A549 cells after TGF-β stimulation for 24 h.
Conclusion Seven hub genes related to EMT are identified. The expression of FGG is down-regulated, and the expressions of THBS1, COL4A1, COL5A1, COL4A2, LAMC2 and SERPINE1 are up-regulated.
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