The Mechanism Of Adiponectin Inhibiting The Development Of Osteosarcoma Through MTOR Signaling Pathway

Objective:To study the mechanism of adiponectin inhibiting the development of osteosarcoma through mTOR signaling pathway. To investigate the effects of recombinant adiponectin on the proliferation, migration and invasive ability of human osteosarcoma cells.Methods:1, Extract blood from10patients who are clinically diagnosed with osteosarcoma and without chemotherapy. The control group included normal person whose gender, age and BMI are matched with control group. Centrifuge the blood under3000RPM4℃for10minutes, take supernatant and detect the circulation level of Adiponectin by Western Blot method.2, Select the MG-63cells, SaOS-2cells, U2OS cells, hFOB1.19cells, which is in logarithmic growth phase, then extract the protein of the cells,and detect the levels of adiponectin receptor1and adiponectin receptor2by Western Blot method.3,To determine the optimized treatment time, then according to the optimized treatment time, determine the optimized concentration of action of adiponectin on a logarithmic growth of Osteosarcoma cells line MG-63. detect the phosphorylation of Akt, ERK, S6K and4EBP1after adiponectin acting on Osteosarcoma cells line MG-63by Western Blot method. 4, To investigate the influence of recombinant adiponectin on human Osteosarcoma cell line MG-63proliferation by CCK8assay.5,To investigate the effects of recombinant adiponectin on the migration ability of human Osteosarcoma cell line MG-63by scratch-wound assay.6, Osteosarcoma cell line MG-63were attacked with the optimized concentration of adiponectin for24hours after pretreated with50μmol/l LY294002for1h, detect the phosphorylation of Akt, ERK, S6K and4EBP1by Western Blot method. Investigate the ability of invasion of human Osteosarcoma cell line MG-63by tumor cell invasion assay.Result:The serum level of adiponectin in the patients with osteosarcoma is lower than normal people (P<0.01). The difference was statistically significant. Compared with the normal osteoblast hFOB1.19, the expression levels of AdipoR1in human Osteosarcoma cell line MG-63, SaOS-2and U2OS are significantly increased (P<0.01), AdipoR2in three kind cells has no obvious difference. Compared with control group, the best role of the time that adiponectin acts on human Osteosarcoma cell line MG-63is30min (P<0.01), the best concentration is1μg/ml (P<0.01). Adiponectin group can significantly promote phosphorylation of AMPK, ACC and inhibit phosphorylation of Akt, ERK, S6K and4EBP1in the MG-63cells. LY294002, one of PI3K inhibitors can inhibit phosphorylation of Akt, S6K and4EBP1in the MG-63cells. By CCK-8assay, we found that recombinant adiponectin can significantly reduce the osteosarcoma MG-63cells proliferation in vitro (P<0.01), in a concentration dependent manner. Scratch-wound assay confirmed that adiponectin can inhibit migration of MG-63cells. Adiponectin and the PI3K inhibitor LY294002at50μmol/1along with adiponectin can inhibit invasion of human osteosarcoma cells line MG-63.Conclusion:Adiponectin can inhibit cell proliferation, migration and invasion in human osteosarcoma cells. Adiponectin may suppress osteosarcoma development by interaction of AMPK/mTOR, PI3K/Akt/mTOR, ERK/mTOR signal pathway axis.