Study On The Metabolism Of Cancer Stem Cells And Proteins Related With Metastasis In Osteosarcoma Using Omics

?Objective?1.To verify the characteristics of osteosarcoma CD133+ cells,and to explore the relationship between them and the sphere forming cells.So as to find a model for the study of cancer stem cells in osteosarcoma.2.To identify the main metabolic pathways of osteosarcoma stem cells by the method of metabonomics.3.To investigate the mitochondrial function related with metabolism of osteosarcoma stem cells in order to provide new ideas for treatment of osteosarcoma;4.To screen the major proteins of osteosarcoma stem cells by proteomic method and identify the key proteins for the sake of prevention and treatment of osteosarcoma.5.To investigate the role of protein ACTN4 in osteosarcoma.?Method?1.Using CCK-8 test,clone formation test,invasion experimengt,drug resistance test,serum free suspension culture into a ball and verify CD133+ osteosarcoma cell proliferation,survival,invasion,resistance,ball,and PCR was used to detect the stem cell related gene expression;2.Using the method of LC/MS to identify the metabolic spectrum of osteosarcoma stem cells,and to explore the main signal pathway by bioinformatics;3.The mitochondrial content and morphology of osteosarcoma stem cells was determined by flow cytometry and electron microscopy,and then the mitochondrial metabolic function was measured by the cell metabolism and respiratory analyzer.Then the level of ATP in the cells was also determined;4.Differentially expressed proteins of osteosarcoma stem cells were identified by using the method of label free chromatography tandem mass spectrometry;5.The expression of the key protein ACTN4 in different subsets of osteosarcoma cells and osteosarcoma tissues was detected by Western blot and immunohistochemistry.?Result?1.Compared with CD133-cells,CD133+ cells had stronger proliferated ability,clone formation ability,invasion ability,resistance to adriamycin,and relatively high expression of Sox2 and Nanog,stem cell related genes expression,as well as very good sphere forming ability in serum-free suspension culture.2.By metabonomics identification,154 different compounds were found in osteosarcoma stem cells,and further bioinformatics analysis showed that the main metabolic pathway changes around the mitochondrial expansion including alanine,aspartic acid and glutamic acid metabolic pathway,arginine and proline metabolism,glutathione metabolism,cysteine and methionine metabolism pathway.The citric acid cycle.3.Osteosarcoma stem cells had more mitochondrial mass since that their relative fluorescence intensity of mitochondrial probe were larger than normal cells.And their mitochondrias were more mature,with a relatively large diameter,regular shape,elongated or curly style,and more mitochondrias sulcus under electron microscope.Respiratory metabolism analyzer found their parameters related with energy phenotype located in the axis at the top right,and the ability of mitochondrial stress was higher as well as the level of intracellular ATP content.4.Using label free proteomics,more than 6600 proteins were detected.And 124 proteins had more than 1.5 times change with statistically significant difference.Msot proteins are enriched in tumor metastasis pathway,ACTN4 protein located in the key nodes of corresponding pathway.5.Higher expression of ACTN4 in osteosarcoma stem cells,consistent with the proteomics results.And higher expression of ACTN4 in 43 cases of osteosarcoma specimens in distant metastasis occurred in high proportion.But no significant correlation with age,gender,location,type of organization.?Conclusion?1.Osteosarcoma CD133+ cells can be used as a model of osteosarcoma stem cells in osteosarcoma cell lines.2.There are differences between the cancer stem cells and normal cells with respect to metabolism in osteosarcoma,and main changed metabolic pathways around the mitochondria including alanine,aspartic acid and glutamic acid metabolic pathway,arginine and proline metabolism,glutathione metabolism,cysteine and methionine metabolism pathway,citric acid cycle.3.The content of mitochondria and ATP in osteosarcoma stem cells is more than that of ordinary tumor cells as well as the more mature structure.So the energy base of osteosarcoma stem cells' proliferation and invasion was revealed.4.Differentially expressed proteins between osteosarcoma stem cells and ordinary tumor cells are mainly concentrated in the signaling pathway related with tumor metastasis.Thus the molecular basis of the higher ability of invasion and metastasis in osteosarcoma stem cells is explained.5.The expression of ACTN4 in osteosarcoma stem cells are higher,and the patients with higher expression of ACTN4 are prone to have a higher proportion of distant metastasis.