as the 2008 beijing olympics were successfully held in beijing , sports developed rapidly in china . more and more inhabitants , professional or amateur ,
take part in daily physical activities . however , improper movement may cause injury . the intense sports ( like pugilism , football , and basketball ) and hazardous sports ( like motorcycle race , drift motion , and bungee jumping ) are all high - risk sports .
collisions with the ground , objects , and other players are common , and unexpected dynamic force on limbs and joints can cause injury . in human ,
the femur is the only bone in the thigh with the formation of long , slender , and cylindrical bone and is capable of walking , running , or jumping .
the femoral fracture is involved in the femoral head , femoral neck , or the shaft of the femur , accounting for 1 - 2% of all fractures in children and adolescents [ 4 , 5 ] . for a long time
however , femur fracture is still difficult to manage because of the multifocal fractures of the femur [ 8 , 9 ] .
although numerous surgical operations have been described to manage this injury , evidence for which to choose is lacking and individual approach is strongly emphasized during the treatment of these injuries [ 9 , 10 ] .
it is necessary for us to study the differences of gene expression at different stages after femoral fracture , in the purpose of finding the indicator of fracture healing .
, the linear growth of femur has slowed due to mitosis and hypertrophy in the chondrocytes of the physis [ 11 , 12 ] .
based on rat model , changes in mrna gene expression of femoral heading have been reported [ 12 , 13 ] .
briefly , in 4-week - old female sprague - dawley ( sd ) rats , at 0 ( intact ) , 0.1 , 0.4 , 1 , 2 , 3 , 4 , and 6 weeks after fracture , mrna gene expression in the femoral heading after unilateral midshaft femoral fracture was identified , including 8,002 genes , about half increasing and half decreasing .
these upregulated genes were related to cartilage , blood vessels , osteoprotegerin , osteomodulin , and most ribosomal proteins . meanwhile ,
downregulated genes were related to bone , growth promoting cytokines , g proteins , gtpase - mediated signal transduction factors , cytokine receptors , mitosis , integrin - linked kinase , and the cytoskeleton .
the relevant microarray data were deposited in geo ( gene expression omnibus ) database ( i d : gse3298 ) [ 12 , 13 ] . in this present study , based on the microarray data of gse3298 , 2 weeks after femoral fracture was chosen as a split point , and thus the earlier stage and later stage were grouped .
we aimed to identify degs at different stages of femoral fracture healing by bioinformatics methods , in order to provide the basis for the treatment of sport - related fractures .
the mrna expression profiling data was obtained from the research of meyer et al . , which were displayed in geo ( http://www.ncbi.nlm.nih.gov/geo/ ) database ( i d : gse3298 ) .
briefly , female sd rats , aged 4 weeks at surgery , were subjected to a unilateral , simple , transverse , and middiaphyseal femoral fracture and stabilized with an intramedullary rod . at 0 ( intact ) , 0.1 , 0.4 , 1 , 2 , 3 , 4 , and 6 weeks after fracture ,
the rna was extracted , processed to biotin labeled crna , and hybridized to affymetrix rat 230 2.0 genechip microarrays .
the microarray data in cel files were downloaded from geo database , including 16 chips , converted into fluorescence intensity values and standardized via the robust multiarray average ( rma ) method . for genes corresponding to multiple probe sets that had a plurality of expression values ,
considering the different healing level in different periods after fracture , 2 weeks was set as the split point .
chips data were divided into 2 groups : earlier stage ( 0.1 , 0.4 , 1 and 2 weeks after fracture ) and later stage ( 2 , 3 , 4 , and 6 weeks after fracture ) . the limma package in r language was used to identify degs between earlier stage and later stage .
the p value < 0.05 and the |log2fc| > 0.5 were used as the cut - off criterion . for genes differentially expressed in two stages , the string ( search tool for the retrieval of interacting genes )   database
was used to analyze their interaction network . for genes with consistent expression in two stages , bisogenet   software
was performed to map these genes to string database or bond database for interaction network analysis .
the p value < 0.05 was chosen as cut - off criterion . for function analysis of degs ,
the degs of two stages were , respectively , inputted into david ( database for annotation , visualization , and integrated discovery ) [ 18 , 19 ] for kegg ( kyoto encyclopedia of genes and genomes )   pathway and go ( gene ontology )   enrichment analysis . the count number larger than 5 and p value less than 0.01 were chosen as cut - off criterion .
a pearson correlation coefficient was calculated between expression level of every expressed gene after fracture and fracture time via cor.test in r language .
then , david tool was used to identify function classification associated with these significant genes .
after standardization , there were 31,042 probes corresponding to 30,641 genes . in earlier stage ,
total 1,004 degs had been identified , including 301 upregulated genes and 703 downregulated genes . in later stage ,
the most significant degs from two stages were displayed in tables 1 and 2 . among degs from two stages , 87 degs occurred in both earlier stage and later stage , including 26 upregulated genes , 57 downregulated genes , and 4 differentially regulated genes . briefly , one deg ( genbankacc : bf402112 ) was upregulated in earlier stage and downregulated in later stage , and 3 degs ( genbankacc : af037203 ( rest ) , ai071395 , and nm019258 ( cst8 ) ) were downregulated in earlier stage and upregulated in later stage ( figure 1 ) .
the obtained 87 degs were mapped to string in order to construct the interaction network . among the 26 upregulated degs ,
only mobkl3 was the hub node that interacted with other genes ( figure 2(a ) ) . meanwhile , among the 57 downregulated degs , 5 degs showed interaction with other genes in rats ( figure 2(b ) ) , such as syt and stx families .
in addition , the protein - protein interaction ( ppi ) network of rest was constructed via string tool and displayed in figure 3 , suggesting that rest protein might interact with 11 proteins in rats .
the ppi network of cst8 was built as well , in which cst8 was the hub protein connected with 10 proteins ( figure 4 ) . for function analysis
, all degs were inputted into david for go function and kegg pathway enrichment analysis .
go function enrichment analysis of degs in earlier stage showed 170 significant go terms , which were divided into 20 clusters , including material transportation in cells , regulation of biological process , structure development , neurodevelopment , and the blood pressure regulation .
the most significant go term was go : 0051179 ( localization ) , of which the fold enrichment was 1.576 .
similarly , total 111 significant go terms were obtained from go analysis of degs in later stage and were divided into 13 clusters , including neurons and synapses development , ion transport , regulation of gene expression , and hormone secretion .
the most significant go term was go : 0045202 ( synapse ) , of which the fold enrichment was 2.53 .
the top 10 go terms of later stage were shown in table 3 ( lower ) .
additionally , kegg pathway enrichment analysis of degs in earlier stage showed 5 significant pathways ( table 4 , upper ) .
meanwhile , degs in later stage were enriched into 2 significant pathways , mainly focused on neurodevelopment ( table 4 , lower ) . among the expressed genes after fracture
, a pearson correlation coefficient was calculated between gene expression level and fracture time via cor.test in r language . with the strict cut - off of p < 0.05 ,
total 3,300 genes significantly associated with fracture time were collected , including negative correlation ( 1,714 genes ) and positive correlation ( 1,586 genes ) ( table 5 ) .
none of the 3,300 significant correlation genes was overlapped with degs identified using limma package .
the function annotation of these significant correlation genes showed relationship with illness , cancer , and immune system , indicating that surgical approach did not cause serious damage to health of animals .
besides , the correlation analysis of degs from two stages did not show significant correlation with fracture time .
in daily life , sport - related fractures are common in adolescents , particularly in males .
femoral fracture , a common sport injury , has great impact on human physical exercise ability and improper treatment can lead to nerve injury , infection , pain , or dyskinesia . for professional athletes ,
it is necessary for us to identify degs after femoral fracture and to explore the key gene of the bone healing , which will provide theoretical basis for future treatment of these sport - related fractures . in this study ,
the chip data were divided into earlier stage and later stage based on 7 time points after femoral fracture . in earlier and later stages , 1,004 and 986 degs were identified by comparing with control group , respectively .
for example , among degs in early stage , oprm1 was opioid receptor , the reduced expression of which in dorsal root ganglion neurons was found to be associated with bone cancer pain in mouse models .
meanwhile , among degs in later stage , bcl2l 1 encoded bcl-2-like 1 protein , a critical regulator of programmed cell death , belongs to bcl-2 protein family .
consistently , it is reported that bcl-2 plays an important role in regulating the apoptosis of osteoclast and osteocyte .
furthermore , wt1 ( wilms tumor 1 ) might act as a novel oncogene facilitating development of the lethal metastatic phenotype in prostate cancer . among degs between two stages
, there was no significant difference in the number of degs and total 87 degs were shared by two stages , indicating different expression profiles between two stages .
there were 4 degs oppositely regulated in earlier and later stages , which might act as indicators for femur healing . among the 4 degs ,
rest , similar to tmem200a , might inhibit overgrowth of myelocyte combined with myc gene .
rest gene is a transcriptional repressor of diverse neuronal genes , the downregulation of which contributed to the proper development of neurons .
similarly , in the current study , rest was downregulated in earlier stage but upregulated in later stage .
moreover , rest is involved in the differentiation from pluripotent cell to neural stem cell and from stem cell to mature neurons .
many members of the cystatin superfamily such as gelatin could protect matrix metalloproteinases without affecting their biological activities , which are critical for tissue modeling .
total 57 degs were downregulated in both two stages , of which interaction network showed that 5 genes were interacted with other reported genes in rats , such as syt and stx families .
consistently , this study showed that calcium signaling pathway was also enriched in early stage , suggesting the critical role of calcium signaling in bone healing after femoral fracture .
besides , syt1 might control neural signal transmission combined with snap-25 [ 37 , 38 ] and stx1a .
stx1a was involved in vesicle fusion process which is critical for calcium - dependent neurotransmitters release .
importantly , it has been reported that increase of syt-1 might play a role in impairment of learning and memories attributed to aging in mouse model .
pearson 's correlation coefficient analysis between gene expression and fracture time indicated that significant correlation genes between gene expression and fracture time were not overlapped with identified degs , which demonstrated that rats underwent surgical operation without other infections and injuries .
briefly , in earlier stage , abundant degs were related to material transportation and synthesis in cells , and a few genes were enriched in synapse growth , while , in later stage , in contrast , the majority of degs were related to synapse growth and a small number of genes were related to transporter activity .
besides , system development was enriched in both earlier and later stages , revealing its importance in the whole process of fracture healing .
kegg pathway analysis showed that neuroactive ligand - receptor interaction was needed in two stages , indicating its important role in fracture healing .
meanwhile , in earlier stage , degs were significantly enriched into calcium signaling pathway and neuroactive ligand - receptor interaction pathway . for later stage ,
neuroactive ligand - receptor interaction becomes the most important pathway , and axon guidance pathway was also enriched .
these findings indicated difference of physical growth between two stages . in earlier stage , more nutrients for vegetative growth were needed to repair fracture ; in later stage , nervous systems were repaired to restore movement ability , which were consistent with general understanding .
in conclusion , based on rat model , identification of degs after femoral fractures was useful for investigation of the proper treatment and providing foundation for exercise capacity recovery . however , further genetic studies were still needed to confirm our observation .
 we aimed to investigate differentially expressed genes ( degs ) in different stages after femoral fracture based on rat models , providing the basis for the treatment of sport - related fractures . 
 gene expression data gse3298 was downloaded from gene expression omnibus ( geo ) , including 16 chips . 
 all femoral fracture samples were classified into earlier fracture stage and later fracture stage . 
 total 87 degs simultaneously occurred in two stages , of which 4 genes showed opposite expression tendency . out of the 4 genes , 
 rest and cst8 were hub nodes in protein - protein interaction ( ppi ) network . 
 the go ( gene ontology ) function enrichment analysis verified that nutrition supply related genes were enriched in the earlier stage and neuron growth related genes were enriched in the later stage . 
 calcium signaling pathway was the most significant pathway in earlier stage ; in later stage , degs were enriched into 2 neurodevelopment - related pathways . 
 analysis of pearson 's correlation coefficient showed that a total of 3,300 genes were significantly associated with fracture time , none of which was overlapped with identified degs . 
 this study suggested that rest and cst8 might act as potential indicators for fracture healing . 
 calcium signaling pathway and neurodevelopment - related pathways might be deeply involved in bone healing after femoral fracture . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions