| Objective:1.To screen the serum differential proteins of prostate cancer patients with bone metastasis and non-metastasis,and validate the key differential proteins in prostate cancer patients.2.To establish co-culture systems of PC3 cells with human bone marrow mesenchymal stem cells(hBMSCs)and osteoblasts(hFOB1.19),respectively,and to study the interaction between the three cell lines and explore the mechanism of the expression changes of the differential protein(CD59).Methods:1.Serum samples of 30 prostate cancer patients with bone metastasis and 30 non-metastasis patients were collected,and iTRAQ proteomics was used to compare the serum differential proteins between the two groups.2.Each of 50 serum samples were collected from prostate cancer with bone metastasis,prostate cancer without metastasis and benign prostatic hyperplasia patients respectively,and each of 30 prostate tissue sections from the above three groups were collected respectively also.Another 5 bone metastasis sections of prostate cancer were collected.ELISA and immunohistochemistry were used to verify the three key differential proteins with significant fold changes.3.Western blot,flow cytometry and immunofluorescence were used to analyze the expression of three key differential proteins in PC3,LNCaP and DU145 prostate cancer cell lines.4.The co-culture systems of PC3 with hBMSCs and hFOB1.19 cells were established to study the effects of hBMSCs and hFOB1.19 on the cell proliferation,migration and invasion of PC3 cells and the effects of PC3 on the cell proliferation of hFOB1.19 cells.5.The expression of CD59,CyclinD1,RANK/RANKL/OPG,and NF-?B(P50 subunit)were analyzed by flow cytometry,Western blot,RT-PCR and cellular immunofluorescence,and the mechanism of CD59 expression was discussed.Results:1.Proteomic studies had screened 32 differentially expressed proteins related to bone metastasis of prostate cancer,of which 11 were up-regulated and 21 were down-regulated.CD59 and haptoglobin in up-regulated proteins and tetranectin in down-regulated proteins had significant fold changes.2.ELISA analysis showed that the mean concentrations of CD59,haptoglobin and tetranectin in serum of prostate cancer patients with or without bone metastasis were consistent with those of mass spectrometry analysis.The differences of the three proteins between the two groups were statistically significant(p<0.05).3.Immunohistochemical analysis showed that the expression intensity of CD59 in prostate cancer with bone metastasis was higher than that in non-metastasis prostate cancer(p<0.05);the expression of haptoglobin was not significantly different between the two groups(p>0.05);the expression intensity of tetranectin in prostate cancer epithelial cells that complicated with bone metastasis was higher than that in non-metastasis prostate cancer,but the expression intensity in the stroma is the opposite(p<0.05).4.The expression of CD59 was highest in PC3 and lowest in LNCaP cells.The expression of haptoglobin and tetranectin was highest in DU145 and lowest in PC3 cells.5.hBMSCs and hFOB1.19 can promote the proliferation,migration and invasion of PC3 cells,and PC3 can also promote the proliferation of hFOB1.19 cells.6.In the co-culture system,the expression of CD59,RANK,RANKL,OPG,CyclinD1 and nuclear NF-?B(P50)in PC3 cells were up-regulated,while the expression of cytoplasmic NF-?B(P50)did not change significantly.The expression of Cyclin D1,RANKL and nuclear NF-?B(P50)expression in hFOBl.19 cells were up-regulated,while CD59,RANK,OPG and cytoplasmic NF-?B(P50)did not change significantly.Conclusion:1.CD59,haptoglobin and tetranectin are bone metastasis related proteins of prostate cancer.2.The co-culture of PC3 with hBMSCs and hFOB1.19 can activate the RANK/RANKL/OPG signaling pathway,which can further activate the downstream NF-kappa B signaling pathway.On the one hand,the activation of NF-kappa B can promote the expression of cyclin D1 and promote cell proliferation;on the other hand,it can up-regulate the expression of CD59. |
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