HO refers to a pathological process, involving a variety of etiology, location, mechanism and cell origin.(3) It is reported that early diagnosis of HO is difficult due to lack of evident signs and symptoms and limited treatment.(18, 19) How to manipulate the inflammatory cascades to control HO formation is just beginning to be understood.(3) Therefore, it is urgent to explore the mechanisms of the formation of HO at the molecular level, so as to find novel, adequate and effective treatment methods to alleviate, delay, or even reverse the development of HO.
The whole-genome microarray and bioinformatic analysis facilitate us to detect genetic differences in the development of HO, and provide a better way to explore the pathogenesis and identify novel candidates for early diagnosis and precise treatment. In current study, 275 DEGs were identified, including 122 downregulated genes and 153 upregulated genes.
After that, the functional enrichment analysis was performed, and the relationships and interactions among DEGs were predicted. The majority of DEGs, including KIT, FGF13, EFNB3, UNC5C, TMEFF2, WISP3 and PTH2 were intensively enriched in cell signal transmission items, including axon guidance, negative regulation of cell migration, peptidyl-tyrosine phosphorylation and cell-cell signaling. Moreover, KEGG analysis indicated that the majority of DEGs, including KIT, DDIT3, FGF13, EFNB3, UNC5C, MAPK10 and DKK2, primarily involved in axon guidance, MAPK signaling pathway, Ras signaling pathway, PI3K-Akt signaling pathway and Wnt signaling pathway. This is consistent with the recent research findings that muscle-derived mesenchymal stem cells in the soft tissue migrate to the area of trauma and inflammation, differentiate into osteoblasts and form heterotopic bone.(20)
The PPI network of the DEGs has been constructed. The results indicates that CX3CL1, CXCL1, ADAMTS3, ADAMTS16, ADAMTSL2, ADAMTSL3, ADAMTSL5, PENK, GPR18, CALB2 are hub genes.
Chemokines are able to mediate the migration and localization of immunocyte in the process of inflammation, which plays a vital role in manipulating the immune system. According to the conserved cysteine motifs, chemokines in mankind are divided into four families (C, CC, CXC, and CX3C). Most chemokines are involved in the differentiation of osteoblasts and/or osteoclasts to varying degrees. CX3CL1, belonging to the CX3C subgroup, is a combination of the properties of chemotactic agents and adhesion molecules, which has been shown to continuously control reshaping bone matrix by regulating bone remodeling at the cellular level.(21, 22) Current studies have reported that osteoblasts are able to express CX3CL1, and osteoclast progenitors are able to produce CX3C receptor 1 (CX3CR1).(23) Cytokines originated from inflammation are able to significantly induce the expression of CX3CL1 in osteoblasts. Meanwhile, CX3CR1 are identified as a candidate for screening osteoclasts with inflammatory reaction.(24–26) It was reported that the interaction between membrane-bound CX3CL1 on osteoblasts and CX3CR1 expressed by osteoclast progenitors is able to promote the progress of terminal differentiation of osteoclast progenitors.(23) CX3CR1-deficient mice showed moderate but significantly increased trabecular bone mass, which was mainly due to the decrease in the number of osteoclasts.(27) In vitro experiments suggested that this phenotype can be explained by the decreased ratio of receptor activator of nuclear factor-kappa B ligand/osteoprotegerin (RANKL/OPG), and the defect of spontaneous-formation of osteoclasts from CX3CR1-deficient bone marrow cells.(27) In general, it indicates that CX3CL1 may be involved in osteoclast-mediated bone loss. CXCL1, belonging to the CXC subgroup, is a kind of growth factors that could send signals through CXC receptor 2 (CXCR2).(28) CXCR2, a G protein-coupled receptor, is found remarkably expressed in osteoclast precursors, while it cannot be detected in the osteoblast lineage predominantly. Cell culture studies have confirmed that recombinant CXCL1 is able to stimulate the migration of osteoclast precursors in a dose-dependent manner.(29) Moreover, CXCL1 has been proven to promote osteoclast formation in vitro.(30)
The superfamily of ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS) comprises 19 distinct ADAMTS, which are consist of secreted enzymes and seven ADAMTS-like proteins (ADAMTSLs) without enzymatic activity.(31–33) Most of them are involved in the generation and degradation of extracellular matrix (ECM) molecules, and participate in the formation and remodeling of connective tissue and the occurrence and development of diseases.(32, 34) The ECM of normal cartilage maintains a dynamic balance between generation and degradation, which is in a state of equilibrium. There is a loss of balance between the proteases and their inhibitors that degrade the ECM in pathological cartilage. ADAMTS3 has been proven to be involved in the formation of type II fibrous collagen in articular cartilage.(35) ADAMTS16, there is no known specific function for articular cartilage yet. Current studies have shown that the expression of ADAMTS16 is increased in cartilage and synovium from osteoarthritis patients compared with the normal.(36, 37) The steady increase of expression of ADAMTS16 will cause the inhibition of cell proliferation, migration and adhesion, and a decreased expression of matrix metalloproteinase-13 (MMP13) in chondrosarcoma cells.(38) Studies have indicated that ADAMTSLs possess specific extracellular ligands and several of them are ECM-binding proteins that act at the cell-matrix interface.(39–41) It is well known that fibrillin microfibrils are able to bind to ADAMTSLs.(42–45) Therefore, ADAMTSLs can be regarded as matricellular proteins, which are a kind of non-structural proteins expressedin ECM dynamically and with regulatory effects.
PENK, a classically identified opioid gene, was initially shown to be expressed almost exclusively in the mature nervous and neuroendocrine systems. Current studies have revealed that the expression of PENK is selectively increased in mineralized cultures, and it is essential for the formation and remodeling of bone structure.(46) The expression of PENK in osteoblasts is regulated by bone-targeting hormones, which makes a valuable contribution toward bone development.(47)
The interaction among bone formation and hub genes GPR18 and CALB2 have not been reported yet. GPR18 is a receptor for endocannabinoid N-arachidonyl glycine (NAGly).(48, 49) GPR18 may be involved in the regulation of immune system, whose activity is mediated by G proteins that can inhibit adenylyl cyclase.(48) CALB2, a member of the troponin C superfamily, is an intracellular calcium-binding protein and is abundant in auditory neurons and functions as a modulator of neuronal excitability.