Microarray analysis identification of key pathways and interaction network of differential gene expressions during osteogenic differentiation

Abstract

Background: Adult bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stem cells that can differentiate into three lineages. They are suitable sources for cell-based therapy and regenerative medicine applications. This study aims to evaluate the hub genes and key pathways of differentially expressed genes (DEGs) related to osteogenesis by bioinformatics analysis in three different days. The DEGs were derived from the three different days compared with day 0.

Results: Gene expression profiles of GSE37558 were obtained from the Gene Expression Omnibus (GEO) database. A total of 4076 DEGs were acquired on days 8, 12, and 25. Gene ontology (GO) enrichment analysis showed that the non-canonical Wnt signaling pathway and lipopolysaccharide (LPS)-mediated signaling pathway were commonly upregulated DEGs for all 3 days. KEGG pathway analysis indicated that the PI3K-Akt and focal adhesion were also commonly upregulated DEGs for all 3 days. Ten hub genes were identified by CytoHubba on days 8, 12, and 25. Then, we focused on the association of these hub genes with the Wnt pathways that had been enriched from the protein-protein interaction (PPI) by the Cytoscape plugin MCODE.

Conclusions: These findings suggested further insights into the roles of the PI3K/AKT and Wnt pathways and their association with osteogenesis. In addition, the stem cell microenvironment via growth factors, extracellular matrix (ECM), IGF1, IGF2, LPS, and Wnt most likely affect osteogenesis by PI3K/AKT.

Keywords: Bioinformatics analysis; Bone mesenchymal stem cells; Osteogenic differentiation; Protein-protein network.

Fig. 1

Venn diagram of biological process (BP) and KEGG pathway enrichment analysis on days 8, 12, and 25. a Upregulated differentially expressed genes (DEG). b Downregulated DEGs. Common biology terms between the biological process and KEGG pathway were shown in the same color

Fig. 2

Protein-protein interaction (PPI) network (STRING). a FRZB. b 14 genes involved in the PI3K/AKT pathway. The red nodes indicated upregulation DEGs, and the green nodes indicated downregulation DEGs. c Presence of β-catenin in the protein-protein interaction (PPI) network. Module 4 (day 8).

Fig. 3

Real-time PCR validation of candidate mRNA at days 8, 12, and 25. a–f the mRNA levels of MAPK3, TLR4, CTNNB1, CCNB1, ITGA5, and ITGAV were detected by real-time PCR (n = 3). All data are presented as mean ± SEM. **P < 0.01

Fig. 4

Model of PI3K/AKT regulation and Wnt/β-catenin in osteogenic differentiation. Important pathways in osteoblast that promote osteogenesis via PI3K/AKT and β-catenin. a PI3K/AKT and its relationship with growth factors, ECM attachment, IGF1, IGF2, LPS, and BMP2 are illustrated in the top portion of this figure. The question mark next to PI3K/AKT/NF-κB indicates whether PI3K/AKT plays a significant role during osteogenesis directly via BMP2 signaling or indirectly through the upregulation of NF-κB. b The interaction or connections of genes and pathways with β-catenin are shown. Wnt/β-catenin interacts or is affected by FGF, IGF-1/IGF-2, IGFBP7, VEGF, integrin-ILK, ITGA11/β1, ITGA5/β1, CDH11, ERK/MAPK, PI3K/AKT, IL-6, and adiponectin. The red star represents commonly upregulated genes on all 3 days. Only expression of MAPK3 (ERK1) at day 8, ITGA5 on days 8 and 25, and both IGFBP7 and ITGA11 at day 25 are represented. ECM, extracellular matrix; PI3K, phosphoinositide 3-kinase; MKK, MAP kinase kinases