The Investigation Of Differential Proteomics In Tissue Specimens Of Osteosarcoma Patients

Objective: Osteosarcoma(OS)is the most common,nonhematopoietic,primary malignant bone tumor.OS is derived from mesenchymal cells and is histologically characterized by the production of ‘tumor osteoid' or immature bone directly from a malignant spindle-shape cell stroma.The majority of OS cases arise in children and young adults between 10 and 20 years of age during the years of bone development.After the initial diagnosis,patients usually receive multiagent preoperative chemotherapy and surgical resection of the tumor,followed by postoperative chemotherapy.Then,Patients have a 5-year survival rate between 60 and 70%.However,OS has short process and quick progress.There's metastasis in OS at early time.OS preferentially metastasizes to the lungs resulting inrespiratory failure and patient death if treatments are ineffective.And those patients with metastases present at the time of diagnosis have poor outcome and their 5-year survival rate dramatically decrease to between 20-40%.Comparative proteomics explore the information of protein expression in cells or organism under different periods,organizations,states or different environmental conditions.And the information can clear different expression of proteomics in important physiological and pathological system,which can find the reasons for the exception.It's possible to high throughput screening for protein expression in different types of tumors or normal tissue with the development of technology.Analysis and identification of different proteins expression lay the foundation for the discovery of molecular markers of tumor and novel therapeutic targets.It also helps us understand the laws of life better.This research compares different expresseions of proteins based on whether there is lung metastasis in OS proteins at early time.Thus,we can explore development,drug resistance and metastasis of OS in protein level,which can provide the evidence for early diagnosis,prognosis and individualized treatment of OS.Methods: 6 cases of rescected OS specimens have been collected from January 2014 to January 2016 in Department of Orthopaedic Oncology of The Third Hospital of Hebei Medical University,which is divided into two group: there is lung metastasis in 3 cases at early time(?group)and there is no lung metastasis in 3 cases at early time(II Group).TCA-acetone precipitation method was employed to extract total protein of collection of I group and II group samples respectively.Mix the total protein based on different group and digest the total protein to more peptide by pancreatic enzyme.The peptides were marked by different iTRAQ reagents box based on different group.Mixed peptide paragraph was graded and separated with strong cation exchange column and C18 reverse column.Proteins were analyzed through iTRAQ-based quantltative differential LC/MS/MS.The data was retrieved by Mascot software in Uniprot_human database and got the results of relative total protein spectrum and quantitative qualitative protein of two groups.Based on the results,screen the data on the threshold(difference in multiples greater than the 1.5,p-value of less than 0.05),Analyze the identificated protein by pathway,and biological pathway information was obtained.Results: According to the selection criteria,we have found 202 differentially expressed protein between the two groups through Proteome Discoverer 1.3 software.We found that these differentially expressed protein were related to many cancer associated biological information pathway through the analysis of KEGG signal pathway database,such as viral carcinogenesis,proteoglycans in cancer,microRNAs in cancer,pathways in cancer.What's more,these differentially expressed proteins are also involved in cell migration related biological information pathway such as focal adhesion,tight junctions,adherens junction,leukocyte transendothelial migration,regulation of actin cytoskeleton etc.The 6 expressed differentially proteins with higher repetition rate in these signaling pathways are: ACTN1,ACTN4,MYL9,TNC,FN,LAMA4.TNC,FN and LAMA4 are highly expressed in I group.ACTN1,ACTN4 and MYL9 are highly expressed in II group.Conclusions:1 Using specimens of OS patients with or without lung metastasis-free at early time as the object of study,we screening 202 expressed differentially metastasis-associated protein in OS with i TARQ combined LC-MS/MS method for the first time.Analysis of KEGG pathway database shows the expressed differentially proteins are involved in some signaling pathways,which provides clues for the study of the mechanism of invasion and metastasis of OS.2 TNC,FN and LAMA4 are highly expressed in I group.ACTN1,ACTN4 and MYL9 are highly expressed in II group.These proteins maybe associated with invasion and metastasis of OS.3 We have preliminary explored the differentially expressed protein in OS patients with or without lung metastasis-free at early time and laid the foundation for the research of development,drug resistance and metastasis of OS,which can provide the evidence for early diagnosis,prognosis and novel therapeutic targets of OS.