Bioinformatics analysis of gene expression network regulation in osteogenic differentiation of human bone marrow mesenchymal stem cells RunningTitle: Bioinformatics analysis of osteogenic differentiation

Abstract Background: Among mesenchymal stem cells (MSCs), bone marrow mesenchymal stem cells (BMSCs) are considered the gold standard treatment for bone tissue regeneration. BMSCs have become an important cell source in bone and cartilage tissue engineering and regenerative medicine. However, the mechanism of osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) remains incompletely understood. In this study, we aimed to analyze the function of key gene in osteogenic differentiation of hBMSCs. Methods: From the Gene Expression Omnibus (GEO) database, we downloaded three microarray data sets: GSE12266, GSE18043 and GSE80614. Differentially expressed genes (DEGs) were screened using the ‘limma’ package, and enrichment analysis was performed. Protein–protein interaction network analysis and visualization analysis were performed in the String website and in Cytoscape software. We construct core gene regulatory network, and a reactome-based Gene Set Enrichment Analysis (GSEA) was performed on the differential genes. Experimental verification of target gene expression, osteogenic differentiation and the related markers was assessed by alkaline phosphatase (ALP) activity detection, calcium content detection, alizarin red (ARS) staining and RT-qPCR, respectively. Results: A total of 11,077 DEGs were identified, including 5296 upregulated genes and 5781 downregulated genes. Functional enrichment pathways of the identified DEGs are related to cell cycle, nuclear division, apoptosis, organelle fission, chromosomal region, centromeric region, ATPase activity, tubulin binding, microtubule blinding, shear stress, TNF signaling, etc. Further functional and correlation analyses of four core genes BRCA1, BIRC5, GMNN and TSC22D3, as well as the followed verification experiments including qPCR, ARS and ALP staining all showed that TSC22D3 and BIRC5 were significantly related to the osteogenic differentiation of hBMSCs. Conclusions：This study provides a novel insight into the biological process of osteogenic differentiation of hBMSCs, and identifies novel genes for osteogenic differentiation of MSCs. Meanwhile, the findings in this study also provide potential therapeutic targets for bone tissue engineering.