Identification of key pathways and hub genes in the myogenic differentiation of pluripotent stem cell: a bioinformatics and experimental study

Abstract

Background: The regeneration of muscle cells from stem cells is an intricate process, and various genes are included in the process such as myoD, mf5, mf6, etc. The key genes and pathways in the differentiating stages are various. Therefore, the differential expression of key genes after 4 weeks of differentiation were investigated in our study. Method: Three published gene expression profiles, GSE131125, GSE148994, and GSE149055, about the comparisons of pluripotent stem cells to differentiated cells after 4 weeks were obtained from the Gene Expression Omnibus (GEO) database. Common differentially expressed genes (DEGs) were obtained for further analysis such as protein-protein interaction (PPI) network, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and GSEA analysis. After hub genes and key pathways were obtained, we manipulated in vitro cell research for substantiation such as immunohistochemical staining and semi-quantitative analysis and quantitative real-time PCR. Results: A total of 824 DEGs including 350 upregulated genes and 474 downregulated genes were identified in the three GSEs. Nineteen hub genes were identified from the PPI network. The GO and KEGG pathway analyses confirmed that myogenic differentiation at 4 weeks was strongly associated with pathway in cancer, PI3K pathway, actin cytoskeleton regulation and metabolic pathway, biosynthesis of antibodies, and cell cycle. GSEA analysis indicated the differentiated cells were enriched in muscle cell development and myogenesis. Meanwhile, the core genes in each pathway were identified from the GSEA analysis. The in vitro cell research revealed that actin cytoskeleton and myoD were upregulated after 4-week differentiation. Conclusions: The research revealed the potential hub genes and key pathways after 4-week differentiation of stem cells which contribute to further study about the molecular mechanism of myogenesis regeneration, paving a way for more accurate treatment for muscle dysfunction.