| PART Ⅰ THE STUDY ON THE EFFECTS OF OVEREXPRESSION OF FBP1 GENE ON THE MALIGNANT BIOLOGICAL BEHAVIOR AND OSTEOGENIC DIFFERENTIATION OF OSTEOSARCOMA CELLS AT THE CELLULAR LEVELObjective:To investigate the regulatory effect of fructose-1,6-diphosphate-1(FBP1)gene on proliferation,apoptosis,migration and osteogenic differentiation of human osteosarcoma cells.Methods:(1)The m RNA expression levels of FBP1 gene in human osteosarcoma cell lines MG63,143 B and human osteoblast h FOB1.19 was detected by real-time fluorescence quantitative PCR;(2)The recombinant carrying FBP1 gene was transfected into human osteosarcoma MG63 and 143 B cells by liposome.The osteosarcoma MG63 and 143 B cells treated by liposome alone were used as negative control group,and the osteosarcoma cells without any treatment were used as a blank control group;(3)The expression levels of FBP1 m RNA and protein were detected by q RT-PCR and western blot to verify the overexpression of FBP1 gene.(4)Trypan blue counting method,crystal violet staining and CCK-8 assay were used to detect the cell proliferation ability of the osteosarcoma cells.(5)The apoptosis rates was detected by Annexin V-FITC double staining and DAPI staining and the apoptosis-associated proteins were detected by western blot.In addition,the cell cycle distribution was detected by FCM.(6)Wound-Healing assay and transwell assay were used to detect the the ability of cell migration.(7)ALP staining and Alizarin red S staining were used to detect the early and late osteogenic differentiation of the cells.The indexes of osteogenic differentiation,Runx2,OCN and OPN,were detected by western blot.Results:(1)The m RNA expression of FBP1 in human osteosarcoma cells MG63 and 143 B was significantly much lower than that in osteoblasts(p < 0.01);(2)The expression level of FBP1 in MG63 and 143 B cells was significantly higher than that in the control group,which indicated that the FBP1 gene was successfully up-regulated.(p < 0.01);(3)The increasing number of the cells in FBP1 over-expression group was significantly lower than that in the two control groups(p < 0.01);(4)The apoptosis rate increased significantly after FBP1 gene was over expressed.(p < 0.01);(5)There was no significant change in cell cycle among the three groups;(6)In the experiment group,the wound healing rate decreased significantly(p < 0.01),and the number of transmembrane cells in Transwell experiment also decreased dramatically.(p < 0.01);(7)After MG63 cells were induced,ALP staining and Alizarin red staining were positive in FBP1 over-expression group,and the number of calcium nodules was significantly increased;The expression level of osteogenesis related genes Runx2,OCN and OPN increased.Conclusion:The expression of FBP1 in osteosarcoma cells was significantly lower than that in normal osteoblasts;Overexpression of FBP1 gene can inhibit the proliferation of human osteosarcoma MG63 and 143 B cells,but its proliferation is not regulated by cell cycle;Overexpression of FBP1 gene can promote cell apoptosis,and inhibit the expression of anti apoptosis-related gene Bcl-2.Moreover,overexpression of FBP1 gene can inhibit the migration of osteosarcoma cells and promote the early and late osteogenic differentiation of osteosarcoma cells.PART Ⅱ THE STUDY ON THE EFFETS AND MECHANISM OF OVEREXPRESSION OF FBP1 GENE ON OSTEOSARCOMA AT ANIMAL LEVELObjective:To further confirm the effects of the FBP1 gene on the malignant biological behavior and osteogenic differentiation of osteosarcoma in vivo,and to explore the mechanism of FBP1 on osteosarcoma cells through AKT signaling pathway.Methods:(1)In vivo,the animal model of tibial tumorigenesis was established by tumor formation in situ.We also set up three groups,which are Blank group,NC group and FBP1 group.(2)The tumor size and distant metastasis were dynamically observed by In Vivo Imagine Technology.(3)In addition,the expression level of FBP1,apoptosis-related proteins and osteogenic-related protein were measured by Immunohistochemistry;(4)The glucose content and lactate production were tested by glucose oxidase test kit and lactate test kit;(5)The key protein expression of PI3 K / AKT signaling pathway was measured by western blot;Results:(1)After the tumor cells were inoculated on the left tibia of nude mice,the xenograft model was established successfully.(2)In vivo,the tumor size of the over-expressed FBP1 group significantly reduced and no distant metastasis was found.(3)The results of Immunohistochemistry showed that in overexpressed FBP1 group,the expression of FBP1 increased,the expression of anti-apoptosis protein Bcl-2 decreased and the expression of osteogenic differentiation protein OPN was higher than that in control groups;(4)Compared with the control groups,the glucose content rised and the lactate production reduced.(p < 0.01);(5)The protein expression level of p-Akt was decreased after FBP1 gene was overexpressed(p < 0.01).Conclusion: In vivo,compared with the control groups,the size of tumor in experimental group much reduced,which suggested that the up-regulation of FBP1 expression can decrease the tumorigenicity of nude mice.The results of immunohistochemistry suggested that overexpression of FBP1 promote the apoptosis and osteogenic differentiation ability of osteosarcoma cells.We also found after FBP1 was up-regulated in osteosarcoma cells,the glucose content increased and the production of lactate decreased,which suggested that FBP1 could change the glucose metabolism and reduce the anaerobic glycolysis of osteosarcoma cells.The overexpression of FBP1 gene decrease the expression of p-AKT protein in osteosarcoma cells,which indicated that FBP1 may play a role in osteosarcoma cells through PI3 K / AKT signaling pathway. |
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