MOLECULAR MEDICINE REPORTS 15: 3969-3976, 2017 Identification of novel genes associated with fracture healing in osteoporosis induced by Krm2 overexpression or Lrp5 deficiency FENG GAO1, FENG XU2, DANKAI WU1, JIEPING CHENG1 and PENG XIA1 1Department of Orthopedics, The Second Hospital of Jilin University, Changchun, Jilin 130041; 2Department of Spine Surgery, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China Received January 20, 2016; Accepted January 30, 2017 DOI: 10.3892/mmr.2017.6544 Abstract. The aim of the present study was to screen potential of 841 DEGs (335 upregulated and 506 downregulated) were key genes associated with osteoporotic fracture healing. The identified in theCol1a1‑Krm2 vs. the WT group, and 50 DEGs microarray data from the Gene Expression Omnibus database (16 upregulated and 34 downregulated) were identified in the accession number GSE51686, were downloaded and used to Lrp5-/- vs. the WT group. The DEGs in Col1a1‑Krm2 mice identify differentially expressed genes (DEGs) in fracture callus were primarily associated with immunity and cell adhesion tissue samples obtained from the femora of type I collagen (GO: 0007155) functions. By contrast, the DEGs in Lrp5-/- (Col1a1)‑kringle containing transmembrane protein 2 (Krm2) mice were significantly associated with muscle system process mice and low density lipoprotein receptor-related protein 5-/- (GO: 0003012) and regulation of transcription (GO: 0006355). (Lrp5-/-) transgenic mice of osteoporosis compared with those In addition, a series of DEGs demonstrated a higher score in wild-type (WT) mice. Enrichment analysis was performed in the PPI network, and were observed to be coexpressed in to reveal the DEG function. In addition, protein-protein inter- the coexpression network, and included thrombospondin 2 actions (PPIs) of DEGs were analyzed using the Search Tool for (Thbs2), syndecan 2 (Sdc2), FK506 binding protein 10 the Retrieval of Interacting Genes database. The coexpression (Fkbp10), 2'-5'-oligoadneylate synthase-like protein 2 (Oasl2), associations between hub genes in the PPI network were inves- interferon induced protein with tetratricopeptide repeats tigated, and a coexpression network was constructed. A total (Ifit) 1 and Ifit2. Thbs2 and Sdc2 were significantly correlated with extracellular matrix-receptor interactions. The results suggest that Thbs2, Sdc2, Fkbp10, Oasl2, Ifit1 and Ifit2 may serve important roles during the fracture healing process in osteoporosis. In addition, this is the first study to demonstrate Correspondence to: Dr Feng Xu, Department of Spine Surgery, The First Hospital of Jilin University, 71 Xinmin Street, Chaoyang, that Sdc2, Fkbp10, Oasl2, Ifit1 and Ifit2 may be associated with Changchun, Jilin 130021, P.R. China osteoporotic fracture healing. E-mail: [email protected] Introduction Dr Dankai Wu, Department of Orthopedics, The Second Hospital of Jilin University, 218 Zi Qiang Street, Changchun, Jilin 130041, P. R. Ch i na Osteoporotic fracture is a common event in the elderly, E-mail: [email protected] resulting in substantial mortality, and the mortality rate of hip fracture for 6 months is ~10-20% (1). The prevalence Abbreviations: DEGs, differentially expressed genes; PPIs, of osteoporotic fractures, hip fractures in particular, is protein-protein interactions; BMP-2, bone morphogenetic protein-2; increasing in many regions of the world (2). Current thera- PTH, parathyroid hormone; LRP5, lipoprotein receptor-related pies focus on the prevention and treatment of osteoporotic protein 5; KREMEN2, kringle containing transmembrane protein 2; fractures; however, this may easily lead to complications, DKK1, dickkopf homolog 1; GEO, Gene Expression Omnibus thus it remains a worldwide public health concern. Therefore, database; RMA, robust microarray analysis; FDR, false discovery a greater understanding of the underlying molecular mecha- rate; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes nisms of fracture healing in the osteoporotic bone is required, and Genomes; DAVID, Database for Annotation, Visualization and Integrated Discovery; STRING, Search Tool for the Retrieval of as well as identifying candidate biomarkers for osteoporotic Interacting Genes; PCC, pearson correlation coefficient; CAMs, cell fracture therapies. adhesion molecules; Thbs2, thrombospondin 2; Sdc2, syndecan 2; Over the past few years, a number of remarkable Fkbp10, FK506 Binding Protein 10 achievements have been made in the genetic study of fracture healing in osteoporosis. One such study demon- Key words: fracture healing, osteoporosis, gene, network, strated that transgenesis of bone morphogenetic protein-2 coexpression promotes fracture healing in osteoporosis by inducing increased callus density and a larger cross-sectional callus area (3). During remodeling of fractured bone, parathyroid hormone (PTH) promotes the formation of osteoclasts 3970 GAO et al: NOVEL GENES ASSOCIATED WITH FRACTURE HEALING IN OSTEOPOROSIS to restore the mechanical strength and structure of bones, org.Hs.eg.db (version 3.4.0) (11) and illuminaHumanv3.db and polymorphisms in genes encoding PTH influence the (version 1.26.0) (12) packages of Bioconductor were used to genetic regulation of bone mineral density (4). Low density translate probe identifications (IDs) to gene symbols. If one lipoprotein receptor-related protein 5 (LRP5) serves a gene symbol was matched by multiple probe IDs, the mean significant functional role in skeletal homeostasis, and muta- expression value was selected as the expression level of this tions in LRP5 induce a variety of bone density-associated gene. diseases (5). Lrp5 deficiency results in decreased osteoblast proliferation and function, which induces a low bone mass Identification of DEGs. DEGs in Col1a1‑Krm2 mice and phenotype (6). Kringle containing transmembrane protein 2 Lrp5-/- mice compared with the WT controls were identi- (KREMEN2), also known as KRM2, is a high-affinity fied using the linear models for microarray data (LIMMA) transmembrane receptor of dickkopf homolog 1, and is package (version 3.30.3; http://www.bioconductor.org/pack- thought to be a regulator of bone remodeling (7). It has been ages/release/bioc/html/limma.html) (13), which is a commonly demonstrated that Krm2-/- mice develop a high bone mass used tool for the identification of DEGs. The P‑value for each phenotype and overexpression of Krm2 in type I collagen gene was calculated using the unpaired t-test in LIMMA, (Col1a1)‑Krm2 transgenic mice induces severe osteoporosis which was then adjusted for the false discovery rate (FDR) with decreased levels of osteoblasts and elevated osteoclast using the Benjamini-Hochberg method (14). Only the genes differentiation (8). Using a model of fracture healing in with FDR values <0.05 and log2 fold change values ≥0.5 were Col1a1‑Krm2 transgenic mice and Lrp5-/- mice, a previous selected as DEGs. study revealed that fracture healing is greatly damaged in The Venny online tool (version 2.0; http://bioinfogp.cnb. Col1a1‑Krm2 transgenic mice and Lrp5-/- mice; however, csic.es/tools/venny/index.html) (15) was utilized to construct the Col1a1‑Krm2 mice were more severely impaired than Venn diagrams for the upregulated and downregulated genes Lrp5-/- mice (9). In addition, this previous study identified identified between theCol1a1‑Krm2 vs. WT and Lrp5-/- vs. WT a set of differentially expressed genes (DEGs) in the two groups. mouse models using microarray analysis (9). However, DEG interactions and functions require further investigation in Enrichment analysis of DEGs. Functional Gene Ontology order to provide a more comprehensive understanding of the (GO) and Kyoto Encyclopedia of Genes and Genomes effect of osteoporosis on fracture healing. (KEGG) pathway enrichment analyses of upregulated and In order to investigate the interactions and functions of downregulated genes were performed using the Database DEGs in Col1a1‑Krm2 transgenic mice and Lrp5-/- mice for Annotation Visualization and Integrated Discovery further, the microarray data obtained by Liedert et al (9) (version 6.8; http://david.abcc.ncifcrf.gov/) database (16). were analyzed in the present study. Following identification The P‑value was calculated using the modified Fisher's exact of DEGs, enrichment analysis was performed. In addition, test, and P<0.05 was considered to indicate a statistically protein-protein interactions (PPIs) of DEGs and hub genes in significant difference. A gene count in each term ≥2 was set the PPI network were analyzed. Furthermore, coexpression as the cut-off criteria. Additional parameters were set to the associations between hub genes and additional DEGs were default values. examined. These results may contribute to a greater under- standing of the effect of osteoporosis on fracture healing, and Construction of PPI networks. PPIs of DEGs were obtained provide novel information that facilitates the development of from the Search Tool for the Retrieval of Interacting Genes future clinical therapies for osteoporotic fractures. database (version 10.0; http://string-db.org/), which integrates a variety of known and predicted protein associations (17). Materials and methods The combined score for each PPI was calculated, and a score of >0.4 was set as the cut-off criterion. Additional parameters Affymetrix microarray data. The raw gene expression were set to the default values. The PPI network was visualized profile dataset GSE51686 (9) was obtained from the Gene using the Cytoscape software (version