Comparative Proteomics Analysis Of The Inhibiting Effects That CADM1 Overexpression And The IGF-1R Inhibitor PPP On Human Osteosarcoma U-2OS Cells

[Objective]:To provide experimental basis for further experiments of making protein biochips,protein-protein interaction and research on molecular targets for designing new drugs,comparative proteomics analysis of the inhibiting effects that CADM1 overexpression and the IGF-1R inhibitor PPP on human osteosarcoma cells U-2OS were performed to identify proteins that are differentially expressed between two cells in each group.[Methods]:The experiment was divided overexpression CADM1 group and transfected with empty vector group and PPP experimental group and control group.1. overexpression CADM1 group and transfected with empty vector group: people take over-expression CADM1 osteosarcoma U-2OS-CADM1 cell line(TF-Open Patent201,510,924,952.7, China Type Culture Collection Accession Number: CCTCC NO: C201518)and turn transfected with empty vector U-2OS human osteosarcoma cell line for cell recovery,were cultured, total protein was extracted by using two-dimensional electrophoresis technique wherein the differentially expressed proteins were identified, and access to relevant databases and data were analyzed.2. PPP experimental group and control group: Take the U-2OS human osteosarcoma cell line for cell recovery, were cultured, PPP group join PPP 8.0?M(DMSO dissolved), the control group was added the same amount of DMSO, total cellular protein extract, using a two-way electrophoresis the differences identified proteins, and mass spectrometry and access to relevant databases and data were analyzed. PPP experimental group versus control group: Human osteosarcoma cell line U-2OS were thawed, expanded and cultivated. PPP( dissolved in DMSO)was added into Cells in PPP experimental group, and equal volume of DMSO was added into cells in control group. Total cellular proteins were extracted. Two-dimensional gel electrophoresis were performed to identify proteins that are differentially expressed between two cells, then followed by mass spectrometric analyses and database searching to analysis them.[Results]: 1. Two-dimensional gel electrophoresis were repeated three times with total proteins in CADM1 overexpression group and blank load transfection group. Total proteins were separated, backgrounds of gels were clear and protein spots were obvious. A total of 894±19protein spots were detected in CADM1 overexpression human osteosarcoma cell line U-2OS-CADM1 and blank load transfection human osteosarcoma cell line U-2OS. Compared to blank load transfection human osteosarcoma cell line U-2OS, expression of 23 proteins were significantly increased and 21 proteins were significantly decreased. Difference was statistically significant(P<0.05). 12 most differentially expressed protein spots were chosen to the mass spectrometric analyses, 6 of them were identified: L-lactate dehydrogenase B chain(LDHB),Alpha-enolase(ENOA), Galactokinase(GALK1), Keratin, type II cytoskeletal 1(K2C1), 14 k Da phosphohistidine phosphatase(PHP14) and Keratin, type I cytoskeletal 9(K1C9).2. Two-dimensional gel electrophoresis were repeated three times with total proteins in PPP experimental group and control group. Total proteins were separated, backgrounds of gels were clear and protein spots were obvious. A total of 795± 23 protein spots were detected in two groups. Compared to control group, expression of 25 proteins in PPP experimental group were significantly increased and 30 proteins were significantly decreased. Difference was statistically significant(P<0.05). 12 most differentially expressed protein spots were chosen to the mass spectrometric analyses, 6 of them were identified: Macrophage migration inhibitory factor(MIF),D-dopachrome decarboxylase(DDT), Keratin, type II cytoskeletal 1(K2C1), Sorcin(SORCN),Glutathione S-transferase P(GSTP1) and Flavin reductase(BLVRB).[Conclusion]:1.Two-dimensional gel electrophoresis and mass spectrometric analyses were successfully performed to separate and identify 6 differentially expressed protein spots in CADM1 overexpression group versus blank load transfection group: L-lactate dehydrogenase B chain(LDHB), Alpha-enolase(ENOA), Galactokinase(GALK1), Keratin, type II cytoskeletal1(K2C1), 14 k Da phosphohistidine phosphatase(PHP14) and Keratin, type I cytoskeletal9(K1C9). Then explored literature and analyze the above proteins.2. Two-dimensional gel electrophoresis and mass spectrometric analyses were successfully performed to separate and identify 6 differentially expressed protein spots in PPP experimental group versus control group: Macrophage migration inhibitory factor(MIF), D-dopachrome decarboxylase(DDT), Keratin, type II cytoskeletal 1(K2C1), Sorcin(SORCN), Glutathione S-transferase P(GSTP1) and Flavin reductase(BLVRB). Then explored literatures and analyze the above proteins.3. The results of experiments establish the experimental basis of the inhibiting effects that CADM1 overexpression and the IGF-1R inhibitor PPP on human osteosarcoma cells U-2OS and will help provide a better approach to study the mechanism of occurrence, development and treating on osteosarcoma.