Clinical Significance Of CXCR4 In Patients With Osteosarcoma And Related Experimental Study

Part one:The expression of CXCR4 and its effect to the pulmonary metastasis in osteosarcoma patientsObjective:To examine the expression of chemokine receptor CXCR4 in osteosarcoma patients who with lung metastasis or not,and explore the relationship between the expression of CXCR4 and lung metastasis of osteosarcoma.Aim to search a new method and strategy for the treatment of lung metastasis of osteosarcoma.Methods:Collect and examine the expression of CXCR4 in 18 cases with lung metastasis and 25 cases without lung metastasis of osteosarcoma by immunohistochemisrtry stain.Results:Overall 43 patients included in this study.The positive expression rate of CXCR4 is 44.19%(19/43)in all patients,24%(6/25)in patients without lung metastasis,while 72.2%(13/18)in patients with pulmonary metastasis.Chi-square test was used to statistic analysis,the positive expression rate was significantly higher in patients with pulmonary metastasis than patients without pulmonary metastasis(p<0.05).Conclusions:CXCR4 positive expression rate was significantly higher in patients with pulmonary metastasis than patients without pulmonary metastasis,suggested that CXCR4 might be associated with the process of the pulmonary metastasis in osteosarcoma patients.CXCR4 could be a valuable target in the treatment of pulmonary metastasis in osteosarcoma.Part two:Cells and animal research were used to examine the effect of CXCR4 to ability of pulmonary metastasis in osteosarcoma in vitro and in vivo,and explore the molecular mechanism of pulmonary metastasis ability changes.Objective:To study the effect of CXCR4 to pulmonary metastasis of osteosarcoma in vitro and in vivo,and the molecular mechanism of CXCR4 promoting pulmonary metastasis of osteosarcoma.Methods:Firstly,build CXCR4 gene expression carrier vector and transfection into osteosarcoma cell line MG-63 and U20S,detect the effect of overexpression of CXCR4 on the invasion and migration ability of MG-63 and U20S cell lines by transwell and scratch experiments in vitro.Secondly,explore the effects of knocking down CXCR4 on the proliferation ability of osteosarcoma cells by MTT assay.Then,western blot(WB)and qRT-PCR technique were used to explore the molecular mechanism of CXCR4 promoting invasion and migration of osteosarcoma cells in vitro.Last,establish a model of lung metastasis of osteosarcoma in situ,detect the effect of CXCR4 on lung metastasis of osteosarcoma in nude mice,and further explore the molecular mechanism by WB.Results:?The number of osteosarcoma cells MG-63 and U20S migrated to the lower compartment was significantly increased after overexpression of CXCR4 compared with the control group in vitro(p<0.05);? In vitro,the scratch repair ability of MG-63 and U20S cells was significantly enhanced after overexpression of CXCR4 compared with the control group;?The transient knockout of CXCR4 could significantly inhibit the proliferation of MG-63 in osteosarcoma cells.?In MG-63 cells,overexpression of CXCR4 protein could remarkably up-regulate the expression of VEGF,MMP-9 but down-regulate TIMP-1.On the contrary,the transient knockout of CXCR4 protein can significantly down-regulate VEGF,MMP-9,but up-regulation of TIMP-1 in MG-63 cells;?Compared with the control group,the fluorescence intensity of CXCR4 overexpression group was significantly increased in nude mice,suggesting that the number of osteosarcoma cells transferred to the lung increased significantly(p<0.01).At the same time,the number of pulmonary metastasis in the control group was significantly lower than that in the over-expressed CXCR4 group(p<0· 01).?The expression of VEGF and MMP-9 in the overexpression group of CXCR4 was higher than that of the control group in situ xenografts and lung tissues,while TIMP-1 was significantly lower than that in the control group.Conclusions:Overexpression of CXCR4 can obviously promote the migration of osteosarcoma cells and the pulmonary metastasis of osteosarcoma in vitro and in vivo experiments,and CXCR4 regulated the expression level of VEGF,MMP-9 and TIMP-1.It was revealed that CXCR4 promotes pulmonary metastasis in osteosarcoma maybe through regulating the expression of VEGF,MMP-9 and TIMP-1.