osteosarcoma ( os ) is a common histological form of bone cancer , which is most prevalent in children and adolescents .
it was reported that os was the eighth most common childhood cancer , accounting for about 20% of all primary bone cancers and 2.4% of all malignancies in pediatric patients .
symptoms include pain at night , overt localized swelling , and bones that fracture easily .
various treatments , such as wide - excision surgery of tumors , radiotherapy , chemotherapy , and neoadjuvant chemotherapy , are usually adopted in os . although significant improvements in the long - term outcome of os patients have been made , it is still not satisfactory .
the best reported 10-year survival rate was 92% , and the protocol was an aggressive intra - arterial regimen that individualizes therapy based on arteriographic response .
the 3-year survival rates ranged from 5075% , and 5-year survival rate ranged from 6085% .
in addition , 3040% children died of it , and 2550% patients subsequently developed metastatic disease , which was the major cause of death . recently , some biomarkers for os were selected , such as cd20 , ecm , itga , and pmp22 . hyperthermia therapy is a treatment approach in which a particular area or the whole body is heated above normal temperatures to achieve therapeutic effects .
hyperthermia has been used in cancer treatment since 1989 , and currently it is used either for ablation purposes as an alternative to surgery , or , less frequently , in combination with chemotherapy or radiation therapy . it was reported that hyperthermia can activate various systemic anti - tumor immune responses , and leads to methamphetamine - induced toxicity .
the principle may be as follows : heat alters membrane characteristics , leading to modification in cell morphology , intracellular sodium and calcium levels , and membrane potential [ 1517 ] .
some studies reported that hyperthermia can induce apoptosis and reduce migration of os . in this study
, we aimed to screen gene expression changes of os induced by hyperthermia via integrating the chromatin immunoprecipitation with generation sequencing ( chip - seq ) and targetscan analysis , and further explored the related potential biomarkers and mechanism .
the chip - seq dataset of gse60984   was downloaded from the gene expressed omnibus ( geo ) database ( http://www.ncbi.nlm.nih.gov/geo/ ) . in this dataset
, there were 3 samples of os cell : one was treated with both anti - hsf1 antibody and heat shocked for 10 min , another was treated with anti - hsf1 antibody and was heat shocked for 20 min , and the last was treated with neither antibody nor hyperthermia ( the control sample ) .
raw fastq read sequences were demultiplexed , and cutadapt was used to remove adapter sequences from dna high - throughput sequencing data .
afterwards , the expressed genes were aligned to a reference genome using bowtie , an ultrafast genome aligning tool that was used for the mapping of the raw reads to enesembl ( http://www.ensembl.org/index.html ) hg19 genome with maximum of 2 mismatches in every reads and those who had only 1 match were reserved .
model - based analysis of chip - seq ( macs ) was a computational algorithm that identified genome - wide locations of transcription / chromatin factor binding or histone modification from chip - seq data .
the hsf1-binding sites ( also known as peaks ) were respectively obtained via macs2 in the os cells heat shocked for 10 and 20 min compared with the control samples , and recorded as peek-10 and peek-20 , respectively .
based on the chipseeker package , their related genes , distance to the closest transcription start sites ( tss ) , and genome features were assigned to the peaks .
moreover , some genes of peek-10 and peek-20 were separately selected out when hsf1-binding sites were located in the promoter regions , and their overlaps were found .
the database for annotation , visualization and integrated discovery ( david )   ( https://david.ncifcrf.gov/ ) is a widely used web - based tool for functional and pathway enrichment analysis . in this study
it was used to perform gene ontology ( go ) and kyoto encyclopedia of genes and genomes ( kegg ) pathway enrichment analysis of the above overlaps . moreover ,
the go terms and kegg pathways were selected out with the criteria of p value < 0.05 .
targetscan is a widely used database that predicts biological targets of mirnas by searching for the presence of conserved 8mer , 7mer , and 6mer sites that match the seed region of each mirna .
the mirna , which might regulate the overlaps , as well as the mirna - gene pairs , were screened out with targetscan .
furthermore , the mirna - gene - regulated network was constructed based on the above mirna - gene pairs .
however , the selected mirna - gene pairs might be overwhelming , and the mirna - gene regulated network too complicated , so that it was not conducive to analyze and the accuracy was not high .
hence , module analysis of the network was conducted and the modules were visualized using cytoscape software .
the chip - seq dataset of gse60984   was downloaded from the gene expressed omnibus ( geo ) database ( http://www.ncbi.nlm.nih.gov/geo/ ) . in this dataset
, there were 3 samples of os cell : one was treated with both anti - hsf1 antibody and heat shocked for 10 min , another was treated with anti - hsf1 antibody and was heat shocked for 20 min , and the last was treated with neither antibody nor hyperthermia ( the control sample ) .
raw fastq read sequences were demultiplexed , and cutadapt was used to remove adapter sequences from dna high - throughput sequencing data .
afterwards , the expressed genes were aligned to a reference genome using bowtie , an ultrafast genome aligning tool that was used for the mapping of the raw reads to enesembl ( http://www.ensembl.org/index.html ) hg19 genome with maximum of 2 mismatches in every reads and those who had only 1 match were reserved .
model - based analysis of chip - seq ( macs ) was a computational algorithm that identified genome - wide locations of transcription / chromatin factor binding or histone modification from chip - seq data .
the hsf1-binding sites ( also known as peaks ) were respectively obtained via macs2 in the os cells heat shocked for 10 and 20 min compared with the control samples , and recorded as peek-10 and peek-20 , respectively .
based on the chipseeker package , their related genes , distance to the closest transcription start sites ( tss ) , and genome features were assigned to the peaks . moreover ,
some genes of peek-10 and peek-20 were separately selected out when hsf1-binding sites were located in the promoter regions , and their overlaps were found .
the database for annotation , visualization and integrated discovery ( david )   ( https://david.ncifcrf.gov/ ) is a widely used web - based tool for functional and pathway enrichment analysis . in this study
it was used to perform gene ontology ( go ) and kyoto encyclopedia of genes and genomes ( kegg ) pathway enrichment analysis of the above overlaps . moreover ,
the go terms and kegg pathways were selected out with the criteria of p value < 0.05 .
targetscan is a widely used database that predicts biological targets of mirnas by searching for the presence of conserved 8mer , 7mer , and 6mer sites that match the seed region of each mirna .
the mirna , which might regulate the overlaps , as well as the mirna - gene pairs , were screened out with targetscan .
furthermore , the mirna - gene - regulated network was constructed based on the above mirna - gene pairs .
however , the selected mirna - gene pairs might be overwhelming , and the mirna - gene regulated network too complicated , so that it was not conducive to analyze and the accuracy was not high .
hence , module analysis of the network was conducted and the modules were visualized using cytoscape software .
a total of 19 626 and 18 015 hsf1-binding sites were obtained in the os cells heat shocked for 10 and 20 min , respectively , compared with the control samples ( recorded as peek-10 and peek-20 ) .
their distributions in the genome - wide are shown in figure 1 , showing 10.017% peek-10 and 7.921% peek-20 located in the promoter regions .
in other words , 1880 genes of peek-10 and 1283 genes of peek-20 were located in the promoter regions . besides , 889 overlaps of them were found , and 40 of the overlaps are shown in table 1 .
a total of 122 go terms were found to be enriched in the overlapped genes , and the top 10 most significant go terms are exhibited in figure 2 .
the overlaps were enriched in 3 kegg pathways , namely p53 signaling pathway , methane metabolism and viral myocarditis .
the mirna - gene regulated network of the overlaps was constructed , including 13 657 mirna - gene - regulated pairs .
the network was divided into 4 modules , and they were recorded as module 1 , module 2 , module 3 , and module 4 .
a total of 19 626 and 18 015 hsf1-binding sites were obtained in the os cells heat shocked for 10 and 20 min , respectively , compared with the control samples ( recorded as peek-10 and peek-20 ) .
their distributions in the genome - wide are shown in figure 1 , showing 10.017% peek-10 and 7.921% peek-20 located in the promoter regions .
in other words , 1880 genes of peek-10 and 1283 genes of peek-20 were located in the promoter regions . besides , 889 overlaps of them were found , and 40 of the overlaps are shown in table 1 .
a total of 122 go terms were found to be enriched in the overlapped genes , and the top 10 most significant go terms are exhibited in figure 2 .
the overlaps were enriched in 3 kegg pathways , namely p53 signaling pathway , methane metabolism and viral myocarditis .
the mirna - gene regulated network of the overlaps was constructed , including 13 657 mirna - gene - regulated pairs .
the network was divided into 4 modules , and they were recorded as module 1 , module 2 , module 3 , and module 4 .
heat shock transcription factor 1 ( hsf1 ) is localized to the cytoplasm as an inactive monomer , and heat shock can induce the activation of hsf1 .
the active hsf1 leads to synthesis and cytoprotection of heat shock proteins ( hsps ) .
however , an increase in hsps might render hyperthermia less effective . in this study ,
the hsf1 binding sites in promoter regions were analyzed after 10 and 20 min of hyperthermia using the chip - seq approach .
after the functional enrichment analysis , more overlapped genes were enriched in processes of regulation of programmed cell death and regulation of cell death ( figure 2 ) . programmed cell death , especially the expressions of some receptors and ligands in the process , play critical roles in the progression and resistance to oncolytic adenovirus of os [ 3032 ] .
regulations the cell death processes of tumor cells and normal cells has become an important way to increase efficacy and reduce resistance to traditional treatments in os [ 3335 ] .
therefore , processes of programmed cell death and cell death might be involved in the mechanism of hyperthermia in os , and their rational regulation might contribute to a better curative effect .
the overlaps were found enriched in 3 kegg pathways , p53 signaling pathway , methane metabolism , and viral myocarditis .
the p53 signaling pathway was involved in many tumors , and os is no exception .
it was reported that the p53 signaling pathway participates in cell proliferation , metastasis , and angiogenesis of os [ 3840 ] .
furthermore , the p53 pathway is closely associated with cell cycle and apoptosis , which are involved in the enriched go terms used in this study [ 4143 ] .
matsumoto et al .   found that nitric oxide is an initiator of intercellular signal transduction after hyperthermia in mutant p53 cells of human glioblastoma .
our results indicate that the p53 signaling pathway , methane metabolism , and viral myocarditis might play critical roles in gene changes of os regulated by hsf1 .
however , further in - depth research is needed , especially on the relationships between os and pathways of methane metabolism and viral myocarditis .
after targetscan analysis of the overlaps , the mirna - gene - regulated network was constructed , including 13 657 mirna - gene regulated pairs that might be unreliable .
module 1 contained the most nodes and pairs , and sgms2 and cggbp1 were regulated by more mirnas than other nodes .
therefore , sgms2 and cggbp1 are important nodes in the mirna - gene - regulated network of the overlapped degs .
sgms2 encoded sphingomyelin synthase 2 ( sgms2 ) , which is a risk factor of liver steatosis and atherosclerosis .
moreover , nfb signaling pathways is closely related to the proliferation and progression mechanism of many cancers , and targeting it has been used in cancer prevention and therapy [ 4850 ] .
cggbp1 encoded cgg triplet repeat - binding protein 1 ( cggbp1 ) , which is a nuclear and midbody protein regulating abscission .
cell cycle directly affects cell proliferation and apoptosis , which is important in the progression and prognosis of os .
although few studies have verified that sgms2 and cggbp1 are associated with effects of hyperthermia on os , the present study indicates that they might be key genes of os treated with heat shock .
the gse60984 data set was provided by janus et al . , who mainly researched potential genes regulated by tnf- cytokine and heat shock based on chip - seq analysis , and explored functions of nf-b signaling pathways on os in the process of hyperthermia . in this article , some different methods were used and some novel findings were obtained . for example , functional and pathways enrichment analysis were performed , and some different biological processes ( e.g. , programmed cell death and cell death ) and more pathways ( e.g. , p53 signaling pathway , methane metabolism , and viral myocarditis ) were found to be involved in the effects of hyperthermia on os .
moreover , the mirna - gene network was constructed and analyzed with targetscan and cytoscape , which contained more interactions and thus might be more reliable .
sgms2 and cggbp1 might be biomarkers of heat shocked os , and programmed cell death and cell death might be involved in the mechanism of hyperthermia in os .
the p53 signaling pathway , methane metabolism , and viral myocarditis might play critical roles in gene changes in os regulated by hsf1 .
 backgroundthe purpose of this study was to research the effects of hyperthermia on osteosarcoma ( os ) by integrating the chromatin immunoprecipitation with the generation sequencing ( chip - seq ) and targetscan analysis of heat shock transcription factor 1 ( hsf1).material / methodsthe hsf1 chip - seq dataset of gse60984 was downloaded from the gene expressed omnibus ( geo ) database . 
 the hsf1-binding sites were screened by macs2 in os cells after 10 and 20 min of hyperthermia , and they were annotated using the chipseeker package . 
 the overlapped genes were selected out when hsf1-binding sites were located in the promoter region . 
 the database for annotation , visualization , and integrated discovery ( david ) was used to perform gene ontology ( go ) and kyoto encyclopedia of genes and genomes ( kegg ) pathway enrichment analysis of the overlaps . 
 the mirna - gene pairs of the overlaps were screened out via targetscan , and the mirna - gene - regulated network was constructed by cytoscape software.results1880 and 1283 genes of promoter regions were obtained in the osteosarcoma cells after 10 and 20 min of hyperthermia , respectively , and 889 of them were overlapped . 
 the overlapped genes were enriched in 122 go terms and 3 kegg pathways . 
 there were 13 657 pairs involved in the mirna - gene regulated network of the overlaps.conclusionssome biomarkers were identified for os treated with hyperthermia . moreover , some go terms ( regulation of programmed cell death and regulation of cell death ) and pathways ( p53 signaling pathway , methane metabolism , and viral myocarditis ) might be involved . 