Background: The aim of this study was to explore crucial markers and uncover the regulatory mechanisms of fracture healing in the early stage.Methods: Gene expression proŞle of GSE45156 was downloaded, in which 3 fractured samples and 3unfractured samples were used in our present study. Based on the threshold value, differentiallyexpressed genes (DEGs) were selected between two kinds of samples using limma package in R.Enrichment analysis of these DEGs was performed by DAVID software. Furthermore, protein-proteininteraction (PPI) network was established integrating information in STRING database, and visualizedby Cytoscape software.Results: We identiŞed a set of 960 DEGs including 509 up-regulated and 451 downregulated genes.Biological processes involving RNA splicing and cell cycle were signiŞcantly enriched for the upregulated genes such as Snrpd2, Eftud2, Plk1 and Bub1b, whereas skeletal system development andbone development processes were predominant for down-regulated genes like Ubc. In the constructedPPI network, all theŞve genes were the predominant nodes, of which Snrpd2 was linked to Eftud2, whileBub1b was to interact with Plk1.Conclusion: Five candidate genes crucial for indicating the process of fracture in early stage wereidentiŞed. Eftud2, Snrpd2, Bub1b and Plk1 might function through the involvement of cell-cycle-relatedBP, while Ubc might inﬂuence the protein degradation during bone development. However, moreexperimental validations are needed to conŞrm these results.
1. Bender D, Jefferson-Keil T, Biglari B, Swing T, Schmidmaier G, Moghaddam A. Cigarette smoking and its impact on fracture healing. Trauma. 2014;16:18e22.
2. Giannoudis PV, Einhorn TA, Marsh D. Fracture healing: the diamond concept. Injury. 2007;38:S3eS6.
3. Claes L, Recknagel S, Ignatius A. Fracture healing under healthy and inflammatory conditions. Nat Rev Rheumatol. 2012;8:133e143.
4. Schindeler A, McDonald MM, Bokko P, Little DG, eds. Bone Remodeling during Fracture Repair: The Cellular Picture. Seminars in Cell & Developmental Biology. Elsevier; 2008.
5. Eghbali-Fatourechi GZ, Lamsam J, Fraser D, Nagel D, Riggs BL, Khosla S. Circulating osteoblast-lineage cells in humans. N Engl J Med. 2005;352:1959e1966.
6. Athanasopoulos AN, Schneider D, Keiper T, et al. Vascular endothelial growth factor (VEGF)-induced up-regulation of CCN1 in osteoblasts mediates proangiogenic activities in endothelial cells and promotes fracture healing. J Biol Chem. 2007;282:26746e26753.
7. Ai-Aql Z, Alagl AS, Graves DT, Gerstenfeld LC, Einhorn TA. Molecular mechanisms controlling bone formation during fracture healing and distraction osteogenesis. J Dent Res. 2008;87:107e118.
8. Chen Y, Whetstone HC, Lin AC, et al. Beta-catenin signaling plays a disparate role in different phases of fracture repair: implications for therapy to improve bone healing. PLoS Med. 2007;4:e249.
9. Liu R, Birke O, Morse A, et al. Myogenic progenitors contribute to open but not closed fracture repair. BMC Musculoskelet Disord. 2011;12:288.
10. Matthews BG, Grcevic D, Wang L, et al. Analysis of of o-labeled progenitor cell commitment identifies Notch signaling as an important pathway in fracture healing. J Bone Mineral Res. 2014;29:1283e1294.
11. Smyth GK. Limma: Linear Models for Microarray Data. Bioinformatics and Computational Biology Solutions Using R and Bioconductor. Springer; 2005: 397e420.
12. Consortium GO. The Gene Ontology (GO) database and informatics resource. Nucleic acids Res. 2004;32:D258eD261.
13. Zhang JD, Wiemann S. KEGGgraph: a graph approach to KEGG PATHWAY in R and bioconductor. Bioinformatics. 2009;25:1470e1471.
14. Dennis Jr G, Sherman BT, Hosack DA, et al. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 2003;4:P3.
15. Szklarczyk D, Franceschini A, Kuhn M, et al. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res. 2011;39:D561eD568.
16. Smoot ME, Ono K, Ruscheinski J, Wang P-L, Ideker T. Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics. 2011;27: 431e432.
17. Augat P, Simon U, Liedert A, Claes L. Mechanics and mechano-biology of fracture healing in normal and osteoporotic bone. Osteoporos Int. 2005;16: S36eS43.
18. Guo X, Chen Q-R, Song YK, Wei JS, Khan J. Exon array analysis reveals neuroblastoma tumors have distinct alternative splicing patterns according to stage and MYCN amplification status. BMC Med Genomics. 2011;4:35.
19. Sato N, Maeda M, Sugiyama M, et al. Inhibition of SNW1 association with spliceosomal proteins promotes apoptosis in breast cancer cells. Cancer Med. 2015;4:268e277.
20. Ellis P, Dawson M, Dixon M. Mutation testing in Treacher collins syndrome. J Orthod. 2002;29:293e297.
21. Zhang H, Zhuang Y, Gill J, Lin S. Proof that dinoflagellate spliced leader (DinoSL) is a useful hook for fishing dinoflagellate transcripts from mixed microbial samples: symbiodinium kawagutii as a case study. Protist. 2013;164:510e527.
22. Rambaldi D, Felice B, Praz V, Bucher P, Cittaro D, Guffanti A. Splicy: a webbased tool for the prediction of possible alternative splicing events from Affymetrix probeset data. BMC Bioinforma. 2007;8:S17.
23. Omenn GS, Guan Y, Menon R. A new class of protein cancer biomarker candidates: differentially expressed splice variants of ERBB2 (HER2/neu) and ERBB1 (EGFR) in breast cancer cell lines. J Proteomics. 2014;107:103e112.
24. Tian C, Liu D, Sun Q-L, et al. Comparative analysis of gene expression profiles between cortex and thalamus in Chinese fatal familial insomnia patients. Mol Neurobiol. 2013;48:36e48.
25. Pape H-C, Marcucio R, Humphrey C, Colnot C, Knobe M, Harvey EJ. Traumainduced inflammation and fracture healing. J Orthop Trauma. 2010;24: 522e525.
26. Chang J-K, Li C-J, Liao H-J, Wang C-K, Wang G-J, Ho M-L. Anti-inflammatory drugs suppress proliferation and induce apoptosis through altering expressions of cell cycle regulators and pro-apoptotic factors in cultured human osteoblasts. Toxicology. 2009;258:148e156.
27. Li X, Wang H, Touma E, et al. TP508 accelerates fracture repair by promoting cell growth over cell death. Biochem Biophysical Res Commun. 2007;364: 187e193.
28. Petronczki M, Len art P, Peters J-M. Polo on the rise dfrom mitotic entry to cytokinesis with Plk1. Dev Cell. 2008;14:646e659.
29. Stuermer A, Hoehn K, Faul T, et al. Mouse pre-replicative complex proteins colocalise and interact with the centrosome. Eur J Cell Biol. 2007;86:37e50.
30. Caballa-Serrano J, Schuldt Filho G, Bosshardt DD, Gargallo-Albiol J, Buser D, Gruber R. Conditioned medium from fresh and demineralized bone enhances osteoclastogenesis in murine bone marrow cultures. Clin Oral Implants Res. 2016;27:226e232.
31. Guan Y, Yang X, Yang W, Charbonneau C, Chen Q. Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development. FASEB J. 2014;28:4470e4481.
32. Hede K. Which came first? Studies clarify role of aneuploidy in cancer. J Natl Cancer Inst. 2005;97:87e89.
33. Whitfield ML, Sherlock G, Saldanha AJ, et al. Identification of genes periodically expressed in the human cell cycle and their expression in tumors. Mol Biol Cell. 2002;13:1977e2000.
34. Trost Z, Trebse R, Prezelj J, Komadina R, Logar DB, Marc J. A microarray based identification of osteoporosis-related genes in primary culture of human osteoblasts. Bone. 2010;46:72e80.
35. Czakai K, Müller K, Mosesso P, et al. Perturbation of mitosis through inhibition of histone acetyltransferases: the key to ochratoxin a toxicity and carcinogenicity? Toxicol Sci. 2011;122:317e329.
36. Ding Y, Hubert CG, Herman J, et al. Cancer-Specific requirement for BUB1B/ BUBR1 in human brain tumor isolates and genetically transformed cells. Cancer Discov. 2013;3:198e211.
37. Hecht J, Kuhl H, Haas SA, et al. Gene identification and analysis of transcripts differentially regulated in fracture healing by EST sequencing in the domestic sheep. BMC Genomics. 2006;7:172.
38. Foster LJ, Zeemann PA, Li C, Mann M, Jensen ON, Kassem M. Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation. Stem Cells. 2005;23:1367e1377.
39. Middleton AJ, Budhidarmo R, Day CL. Use of E2 $ ubiquitin conjugates for the characterization of ubiquitin transfer by RING E3 ligases such as the inhibitor of apoptosis proteins. Regul Cell Death Part B Necroptotic Autophagic other Nonapoptotic Mech. 2014;545:243. C