Screening Of Osteosarcoma Associated Proteins Based On Quantitative Proteomic Methods

Osteosarcoma is the most common malignant bone tumor.The rapid development of mass spectrometry technology has provided a powerful mean for tumor research in recent years.The aim of this study was to compare the differentially expressed total proteins and nuclear proteins between osteosarcoma cell lines(MMNG/HOS,MG63,U2OS)and osteoblast cell line(h FOB 1.19)by isobaric labeling for Relative and Absolute Quantitation(i TRAQ)and Squential Windowed Acquisition of Major Theoretical fragment ions(SWATHTM)techniques,and identify osteosarcoma-associated proteins or therapeutic targets.The expression of total protein between osteosarcoma cell lines(MNNG/HOS,MG63,U2OS)and osteoblast cell line(h FOB 1.19)was analyzed by i TRAQ technique.66 differentially expressed proteins were identified.Through clinical screening and si RNA-based functional evaluation,CNOT1(CCR4-NOT transcription complex subunit 1)and CSE1 L correlated to the growth of osteosarcoma cells.Immunoprecipitation,RNA-seq analysis,and rescue assay were used to identify the mechanism.The clinical analysis showed that CNOT1 and CSE1 L expression was associated with the prognosis in patients with osteosarcoma.The differentially expressed nuclear proteins between osteosarcoma cell lines(MNNG/HOS,U2OS)and osteoblast cell line h FOB 1.19 were analyzed by the unlabeled quantitative technique SWATHTM.149 differentially expressed nuclear proteins were identified.By si RNA-based functional screening,MCM2 and MCM3 were found to play an important role in osteosarcoma proliferation.Immunoprecipitation experiments and rescue assay showed that MCM2/3 functioned by interaction with DHX9 in osteosarcoma.The results of immunohistochemistry also showed that the expression of MCM2/3 was closely related to prognosis of osteosarcoma patients.Collectively,quantitative proteomic analysis was used to identify differentially expressed proteins between osteosarcoma cells and osteoblast cells.The function and mechanism of CNOT1,CSE1 L and MCM2/3 were identified,which may act as novel therapeutic targets for osteosarcoma patients.