Bioinformatic analysis of the molecular mechanisms underlying the progression of bone defects

Abstract

Background: The pathophysiology of bone defects (BDs) is complex, and the treatment for bone defects, in particular massive bone defects, remains a major clinical challenge. Our study was conducted to explore the molecular events related to the progression of bone defects a common clinical condition.

Methods: First, microarray data of GSE20980 were obtained from the Gene Expression Omnibus (GEO) database, where 33 samples in total were used to analyze the molecular biological processes related to bone defects. Next, the original data were normalized and differentially expressed genes (DEGs) were identified. Additionally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted. Finally, a protein-protein interaction (PPI) network was constructed and the trends of the different genes were confirmed.

Results: Compared with the samples of non-critical size defects (NCSD), the samples of critical size defects (CSD) had 2057, 827, and 1,024 DEGs at 7, 14, and 21 days post injury, respectively. At day 7, the DEGs were significantly enriched in metabolic pathways, at day 14 the DEGs were predominantly enriched in G-protein coupled signaling pathways and the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, and at day 21 the DEGs were mainly enriched in circadian entrainment and synaptic-related functions. The PPI network showed similar results. Quantitative real-time PCR (qRT-PCR) and western blot (WB) were performed to validate the partial results of sequencing.

Conclusion: This study provides some clues about the molecular mechanism behind bone defects, which should contribute to scientific research and clinical treatment of this condition.

Keywords: Kyoto encyclopedia of genes and genomes (KEGG); bone defects; circadian rhythms; metabolic pathway; protein–protein interaction (PPI).

Figure 1

(A) Data normalization of differentially expressed genes (DEGs). Box plots of gene expression in the critical size defect (CSD) and non-critical size defect (NCSD) at the new bone site (left panel) before and (right panel) after normalization. (B) Principal component analysis and (C) Volcano plots at 1–3  weeks of the CSD vs. NCSD groups.

Figure 2

Heat maps of the genes at 1 (A), 2 (B), and 3 (C) weeks after bone defect in the CSD vs. NCSD groups. Horizontal axis represents each sample, and the vertical axis represents each gene. Blue and red colors represent low and high expression values, respectively.

Figure 3

Scatter plot of enriched KEGG pathways for DEGs at 7 (A), 14 (B), and 21 (C) days after bone defect. The color and size of the dots represent the range of −log10 (p value) and the number of DEGs mapped to the indicated pathways, respectively. KEGG, Kyoto Encyclopedia of Genes and Genomes. DEGs, differentially expressed genes.

Figure 4

PPI networks based on DEGs at 7 (A), 14 (B), and 21 (C) days after bone defect. Rectangular nodes indicate a biological process or a KEGG pathway, colored with gradient colors from yellow (smaller value of p) to blue (larger value of p). Circular nodes indicate genes/proteins. Star-shaped nodes indicate transcription factor. Light green-to-dark green colors indicate low-to-high log2 (fold change). Interactions are shown as solid lines between genes/proteins, and edges of KEGG pathways/Go terms are presented as dashed lines. DEGs, differentially expressed genes; PPI, protein–protein interaction; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology.

Figure 5

qRT-PCR and western blot results. (A–C) qRT-PCR analysis of the mRNA expression levels of Hox2a, Ccr8, and Abca13 at 7 days post injury, IL2, Kif5c, and Cib3 at 14 days post injury, and Atp2b2, Mef2a, and Nap1l5 at 21 days post injury. (D) Western blot analysis of the protein expression levels of JAK2, phospho-JAK2, STAT3, and phospho-STAT3 in bone callus from mice. (E–H) Relative protein expression level in the NCSD groups and CSD groups. Data are presented as means ± SEM (n = 3). *p < 0.05 and **p < 0.01, CSD, critical size defects, NCSD, non-CSD.