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mples was pretty much around the very same line and close to 0 (Figure 1), indicating superior high quality of standardization. At the 1-day time-point, within the BIT group, 222 genes were upregulated in comparison with all the BI group, in which 148 genes were downregulated. In the 2-day time-point, inside the BIT group, 328 genes were upregulated in comparison together with the BI group, in which 375 genes have been downregulated. In the 3-day time-point, the corresponding numbers were 533 upregulated and 515 downregulated, and at the 7-day time-point, the corresponding numbers had been 786 upregulated and 754 downregulated. The DEGs from the four time-points had been combined, as well as the overlap of theFrontiers in Genetics | frontiersin.orgNovember 2021 | Volume 12 | ArticleDu et al.Crucial Genes of Osteogenic and Adipogenic DifferentiationFIGURE 2 | Venn diagrams showing (A) the 98 upregulated genes and (B) the 66 downregulated genes. A differentially expressed gene (DEG) volcano plot (C) as well as a heatmap (D) are shown. Red represents upregulated genes, and green represents downregulated genes (p 0.05, logFC 1 or logFC -1).pathways. Though the p-value of “sa05200: Pathways in cancer” was 0.05, it contained a sizable number of enriched genes.Protein rotein Interaction PKCĪ¹ Formulation networks with the Differentially Expressed Genes and Identification of Hub GenesTo systematically analyze the PPIs of DEGs, PPI networks from the upregulated and downregulated genes had been constructed utilizing Cytoscape software program (Figures 4A,B). Inside the PPI networks of your upregulated genes, the DEGs with all the highest connectivity degrees were BMP2, CTGF, IGF1, TGFB3, MMP13, MMP3, SERPINE1, COMP, ASPN, and IL11. Similarly, in the PPI networks of upregulated genes, the DEGs with the highest connectivity degrees were PPARG, TIMP3, ANXA1, ADAMTS5, TIMP4, AGTR1, NQO1, CXCL12, CEBPA, and CFD. The PPI networks from the DEGs from theSTRING database have been deposited into Cytoscape v3.7.two, then the cytoHubba plugin from Cytoscape was utilised to identify hub genes with the PPI networks, and hub genes overlapped by MCC, MNC, and Degree. The top seven upregulated hub genes were CTGF, IGF1, BMP2, MMP13, TGFB3, MMP3, and SERPINE1; and also the top seven downregulated hub genes had been PPARG, TIMP3, ANXA1, ADAMTS5, AGTR1, CXCL12, and CEBPA (Figures 4A,B).Hub Gene mRNA Expression Levels and ValidationmRNA expression levels of upregulated hub genes involved in osteogenic differentiation were substantially greater in the BIT group than in the BI group. Nonetheless, the mRNA expression levels of downregulated hub genes involved in adipogenic differentiation were significantly reduce within the BIT group thanFrontiers in Genetics | frontiersin.orgNovember 2021 | Volume 12 | ArticleDu et al.Important Genes of Osteogenic and Adipogenic DifferentiationFIGURE 3 | Gene Ontology (GO) functional enrichment of differentially expressed genes (DEGs) in osteogenic and adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). These genes were enriched in several biological procedure (BP), cellular element (CC), and molecular function (MF) terms. The ordinate is ROCK Purity & Documentation indicated on a -log10 (p-value) scale. (A) The principle enrichment results of your upregulated genes. (B) The main enrichment benefits of the downregulated genes.TABLE two | KEGG pathways enrichment analyses of upregulated DEGs. Category KEGG_PATHWAY KEGG_PATHWAY KEGG_PATHWAY Term hsa04550: Signaling pathways regulating pluripotency of stem cells hsa04390: Hippo signaling pathway hsa04960: Aldosterone-regulated sodium reabsorpti