Study On The Role And Mechanism Of Osteoprotegerin Expression In Tumor Cell Regulating Breast Cancer Bone Metastasis

1. An in vivo model of breast carcinoma metastasis in boneObjective To establish animal models of human breast cancer bone metastasis. Methods Forty female nude mice aged 4～6 weeks were divided equally into 5 groups at random, with 8 in each group. MDA-MB-231 cells at concentrations of 1×104 ( group 1),1×105 ( group 2),5×105 ( group 3),1×106 ( group 4) and 5×106 ( group 5) were suspended in 0.1 ml of PBS and then injected into the left heart ventricles of the animals of the 5 groups respectively. Deaths after the injection were recorded. The surviving nude mice 49 days after the injection were subjected to pathological examination to determine bone metastasis. Results The rate of mortality caused by the injection of MDA-MB-231 cells in all animals was 32.50% (13/40). The mortality rate in 5 groups was 1/8,1/8,1/8,3/8, and 7/8 respectively. There was statistical significance in the mortality rate difference for groups comparison(P=0.004). The total bone metastasis rate was 48.15%(13/27). The bone metastasis rate in 5 groups was 0/7,3/7,5/7,4/5,and 1/1 respectively. There was statistical significance in the bone metastasis rate difference between the 5 groups(P=0.014). Conclusion We have successfully set up in vivo animal models of breast cancer bone metastasis by injecting MDA-MB-231 cells at different concentrations into the left heart ventricles of the nude mice, and found 5×105 MDA-MB-231 cells suspended in 0.1 ml of PBS was the suitable concentration for setting up the vivo animal models of breast cancer bone metastasis by left ventricle injection.2. A comparative study of four methods for establishing animal models of human breast cancer bone metastasisObjective To investigate four methods for establishing animal models of human breast cancer bone metastasis. Methods Thirty-two female nude mice aged 4～6 weeks were divided randomly into four groups (n=8 in each group). 5×105 MDA-MB-231 cells were injected into the body via the left second mammary fat pads (group A), the tail veins (group B), the left heart ventricles (group C) and the left tibia marrow cavities (group D), respectively. Tumor formations in situ were recorded in group A. Deaths after the injection were recorded. The surviving nude mice 49 days after the injection were subjected to pathological examination to determine bone metastasis. Results The rate of tumor formation in situ of group A was 87.5% (7/8). One mouse in group C died after the injection of MDA-MB-231 cells. The bone metastasis rate in groups A, B, C and D was zero (0/8), 12.5% (1/8), 71.4% (5/7) and 100% (8/8), respectively. There was statistically significant difference in the bone metastasis rate between group A and group C, group A and group D, group B and group C; and group B and group D. Conclusion Injections of tumor cells via the breast fat pads and tail veins were not suitable methods to establish animal models of human breast cancer bone metastasis. The bone metastasis model could be established efficiently by injecting tumor cells into the left heart ventricles or the bone marrow cavity of nude mice.3. Inhibitory effect of RNA interference on osteoprotegerin gene expression in MDA-MB-231 breast cancer cell lineObjective To construct a eukaryotic expression plasmid that expresses 3 shRNAs targeting osteoprotegerin gene and to investigate the inhibitory effect of RNAi on osteoprotegerin gene expression in MDA-MB-231 breast cancer cell line. Methods Three sites targeting osteoprotegerin gene were selected. Accordingly, three pairs of single stranded DNAs were designed, synthesized and annealed to double stranded DNAs. The double stranded DNAs were cloned into pGenesil-1.1, pGenesil-1.2, pGenesil-1.3, respectively. After repeated enzyme digestion and ligation, the construction of recombinant plasmid pGenesil-1.1-1.2-1.3-shRNA1-shRNA2-shRNA3 was completed. After identification of restriction endonuclease and sequencing, the recombinant plasmid was transfected into MDA-MB-231 cells. After G418 screening, the stably transfected cells were identified with RT-PCR and Western blot. Results The eukaryotic expression plasmid pGenesil-1.1-1.2-1.3-shRNA1-shRNA2-shRNA3 was constructed successfully. Compared with the control group, OPG mRNA and protein levels of MDA-MB-231 cells stably transfected with pGenesil-1.1-1.2-1.3-shRNA1-shRNA2-shRNA3 were lower, with a significant difference. The inhibition rates of OPG mRNA and protein were 91% and 73%, respectively. Conclusion The eukaryotic expression plasmid that expresses 3 shRNAs targeting osteoprotegerin gene was constructed successfully, and the osteoprotegerin gene expression in MDA-MB-231 cells was inhibited by RNA interference in our study. Our study result may provide a basis for related experimental studies further exploring the role of osteoprotegerin expression by tumor cell itself in the development and progression of breast cancer bone metastasis.4. The impact of inhibiting osteoprotegerin expression on breast cancer cell proliferation and TRAIL-induced apoptosisObjective To investigate the impact of inhibiting osteoprotegerin expression on breast cancer cell line MDA-MB-231 proliferation and TRAIL- induced apoptosis. Methods The proliferations of control MDA-MB-231 cell and MDA-MB-231i cell (osteoprotegerin expression inhibition) were detected by MTT. The apoptosis rate of the control MDA-MB-231 cell, and the apoptosis rates of MDA-MB-231 cell and MDA-MB-231i cell after 24 hours TRAIL induction were detected by flow cytometry. Results The doubling generation time in the first two groups was 43.5±2.9 and 45.8±3.6 hours, respectively, with no statistically significant difference between the two groups. The cell apoptosis rates in the last three groups were 6.4±1.3%,16.1±1.7% and 24.4±3.3%, respectively. There was statistically significant difference in the apoptosis rate between the control MDA-MB-231 cell and the MDA-MB-231cell, the control MDA-MB-231 cell and the MDA-MB-231i cell, and the MDA-MB-231 cell and the MDA-MB-231i cell, respectively. Conclusion To inhibit the osteoprotegerin expression does not change the proliferation capability of breast cancer cell. TRAIL can induce breast cancer cell apoptosis. To inhibit the osteoprotegerin expression enhances significantly TRAIL-induced apoptosis in breast cancer cells.5. The impact of inhibiting osteoprotegerin expression in tumor cell on breast cancer metastasis to boneObjective To investigate the impact of inhibiting osteoprotegerin expression in tumor cells on breast cancer cell line MDA-MB-231 induced bone metastasis. Methods Thirty-two female nude mice aged 4～6 weeks were divided randomly into four groups (n=8 in each group). MDA-MB-231 cells (5×105) were injected into the body via the left heart ventricles (group A) and via the left tibia marrow cavities (group C), respectively. MDA-MB-231i cells (5×105) (osteoprotegerin expression inhibition) were injected into the body via the left heart ventricles (group B) and via the left tibia marrow cavities (group D), respectively. Forty-two days after the injection, all the nude mice were subjected to pathological examination to determine bone metastasis. Results The bone metastasis rate of group A was higher than that of group B. But there was no statistically significant difference in the bone metastasis rate between group A and group B. The bone metastasis number in group A was greater than in group B. There was a statistically significant difference in the bone metastasis number between the two groups. The bone tumor volume of group C was larger than that of group D. There was a statistically significant difference in the bone tumor volume between group C and group D. Conclusion Breast cancer metastasis to bone is closely correlated with the osteoprotegerin expression level in tumor cells. To inhibit the osteoprotegerin expression can decrease the capability of breast cancer-induced bone metastasis.