The Proteomic Change Of Human Osteosarcoma And Validation Of FANCD2and TPI-1

Primary malignant Osteosarcoma (OS) is a kind of bone malignant tumorsoccurred early and transferred easily, and have earlier onset age and higher malignantdegree.It is serious harmful to teenagers on the type of disease in healthy.However,however pathogenesis of OS is still unclear. Five year survival rate of OS was only20%-50%with traditional treatments such as surgical resection,amputation plus andchemotherapy. Especially, high-risk types of OS were found frequently clinically,which have no relation with the time of tumorigenesis and measures of chemotherapy.As a result,the patient can not be effectively controlled in the short term and deathrapidly. These shows fully that the OS has complicated heterogeneity with multiplegenes involved in multi-signaling pathway. Therefore, the focus of research inoncology is the incidence of osteosarcoma, invasion, transfer mechanisms andtargeted intervention,and has important significance in research.The application of High-throughput proteomics,bioinformatics technology andclassical molecular biology techniques including immunohistochemistry, RT-PCR andwesten blotting, integrated protein expression spectrum of osteosarcoma andscreening the marker of OS, provides valuable clues for OS pathogenesis, earlydiagnosis and treatment.The content of study is divided into4parts:1. The proteomics research of the human osteosarcoma proteome.The tissue were divided into osteosarcoma group and the peritumoral tissue,prepare the specimen and extract total protein, and then conduct two-dimensional gelelectrophoresis, extract the total protein, thereby separate osteosarcoma protein andcarring silver staining, analysis maps by ImageMasLer2D Elite3.01analysissoftware, gel digestion, and identify the protein of significant differences bymatrix-assisted ionization resolution time-of-flight mass spectrometer.2. The bioinformatics research of differential proteomics of osteosarcoma.Searching the most relevant transcription factors of the susceptible genes ofosteosarcoma using SABiosciences’ Text Mining Application and the UCSC GenomeBrowser to establish osteosarcoma susceptible gene regulatory networks at the transcriptional level. Searching metabolic pathways of differencial proteins. Furtheranalyzing OS differential expressed proteins according to Gene Ontology (GO)annotation by cellular distribution，molecular function and biological process. Andanalysising the interaction networks of differencial proteins by STRING and EBI.wewill establish susceptible gene regulatory networks of osteosarcoma, analysis ofKEGG pathway,and GO annotation and the analysis of protein interaction.3. Verify and analyze the expression of FANCD2and TPI-1.After Culturing MG-63(human osteosarcoma cell line) and the people osteoblastcell line hFOB1.19and extractting the total RNA of MG-63and hFOB1.19, analysismRNA expression of FANCD2and TPI-1.Extract the total protein,and analysisFANCD2and TPI-1expression levels by immunohistochemistry and western blotanalysis.4. The comprehensive analysis of differential proteomics and thepathological mechanism of OS.Through protein mass spectrometry analysis and identification of differentialprotein, we can analysis of the occurrence and development of the OS，and furtherdiscuss the relationship among the pathogenesis of osteosarcoma and FCNCD2andTPI-1.Results:1. Eleven kinds of protein were identified initially.The expression of nine kindsof protein was Significantly higher than other protein in OS, such as transthyretin,glyceraldehyde phosphate isomerase (TPI-1), Fanconi anemia protein isoforms(FANCD2), slow contraction of skeletal troponin T, cardiac troponin, actin, myosin,annexin-5and the heat shock protein-1; The expression of two kinds of proteins wasSignificantly lower than others in OS, such as manganese superoxide dismutase andcarbonic acid.2. The Transcription factors involved in the transcription of osteosarcomadifferencial proteins through the study of osteosarcoma susceptible genes regulatorynetworks such as c-jun, NF-kalphaB, NF-kalphaB1, FOX01, AP-1,CREB, SP-1,FOX01a, AP-1and P53. Osteosarcoma differential proteins involved in purinemetabolism, glucose metabolism, Fanconi anemia pathway, calcium signalingpathway, apoptosis by the preliminary analysis of KEGG pathways. we found thatdifferencial proteins distributed in in the cell membrane, cytoplasm and nucleus, most proteins distributed in the cytoplasm by the Gene Ontology (GO) analysis ofCellular component of differencial proteins of osteosarcoma. Most differencialproteins play the important function by binding to other proteins molecules by theGene Ontology (GO) analysis of Molecular function. Differencial proteins areinvolved in transport, metabolism, signal transduction, DNA damage and repair,muscle contraction, kinase activity, oxidizing reaction, etc. by the Gene Ontology (GO)analysis of Biological process.3. The expression of FANCD2and TPI-1in OS was analysed by Realtime-PCR, Western blot and immunohistochemical staining. The result of experimentshow that the expression of FANCD2and TPI-1of MG63were higher compared tohFOB1.19.Conclusion:1. The differential protein plays an important role in pathological process of OS,and maybe involved in the malignant tumor growth, differentiation, and regulation ofother phenotype and biological characteristics. The protein of FANCD2with TPI-1may be an important related protein of OS which plays an important role in theprogress of the pathological process of OS.2. The osteosarcoma transcription factors may be important transcription factors,which play important roles in the expression of the differencial proteins andregulatory networks such as c-jun, NF-kalphaB, and NF-kalphaB1.3. Differencial proteins of Osteosarcoma involve in glucose metabolism, andFanconi anemia pathway, apoptosis and other life processes by the analysis of KEGGpathway. Osteosarcoma differencial protein plays important roles in the researching ofosteosarcoma mechanism,clinical diagnosis and treatment strategies.4. This experiment identified differential protein using two-dimensionalelectrophoresis and mass spectrometry analysis, and further identified the new thedifferencial protein of FANCD2and TPI-1using molecular biology technology.wefound that the change of related protein have important correlation with theformation of OS, progression and metastasis. We also confirmed that the validation ofthe expression of protein has objectivity by A variety of methods.Contributions and innovations：1. The osteosarcoma transcription factors including c-jun, NF-kalphaB,NF-kalphaB1FOX01, FOX01a,AP-1,CREB,SP-1,P53may be important transcription factors, which play important roles in the expression of the differencial proteins andregulatory networks. Differencial proteins of Osteosarcoma involve in glucosemetabolism, and Fanconi anemia pathway, apoptosis and other life processes by theanalysis of KEGG pathway. Differencial proteins of osteosarcoma play importantroles in the research of cellular component, molecular function, biological process inthe clinical diagnosis and the treatment strategy of osteosarcoma.2. The protein of FANCD2with TPI-1may be an important related protein ofOS which plays an important role in the progress of the pathological process ofOS,and provide scientific basis for further study of OS.