| Objective: To observe the functions and mechanism of BMP9 on invasion and bone metastasis of breast cancer MDA-MB-231 cells.To further understand the mechanisam of BMP9 decreasing bone metastasis of breast cancer cells, the adenovirus vector expressing BMP9 and CTGF were used to infect MDA-MB-231. Experimental group: MDA-MB-231/BMP9/CTGF, control group: MDA-MB-231/BMP9/RFP. These two cells were separately injected into the proximal tibia of nude mice,and the lytic lesion was detected by X-ray and CTGF expression were detected by immunohistochemistry assay.Methods: The expression of BMP9 in breast cancer tissues and its non–tumor adjacent tissues, differnent breast cancer cells was detected by RT- PCR. High titer of adenovirus vector expressing BMP9 was used to infect MDA-MB-231 in oder to construct MDA-MB-231/BMP9 cells as experimental group, MDA-MB-231/GFP and MDA-MB-231 were as control groups. Cells colony-forming assay, cell wounding assay, transwell invasion assay were carried out to detect the changes of invasion and migration in the recombinant MDA-MB-231/BMP9 cells. The bone metastases-related genes CTGF, ID1, CXCR4 in MDA-MB-231/BMP9 were detected by Real time PCR. MDA-MB-231/BMP9 was stimulated with CTGF conditioned medium, and the changes of invasion and migration in MDA-MB-231/BMP9 cell were detected by wounding assay, transwell invasion assay.A xenograft model was constructed by injecting the cells into the proximal tibia of nude mice to observe the inhibitory effect of BMP9 on breast cancer bone metastasis. And the changes of CTGF expression was detected by immunohistochemistry in animal models.Results: BMP9 mRNA transcripts could be amplified in adjacent non-tumor tissues from five of the six breast cancer patients, while undetected in breast cancer tissues. Although it could be observed in the breast cancer tissue of one patient, the BMP9 expression was in a higher level in the adjacent non-tumor tissues, suggesting that BMP9 may represent a down-regulated tumor suppressor in breast cancer. BMP9 mRNA was undetectable in the metastatic breast cancer cell line MDA-MB-231 while it could be observed in non-metastatic breast cancer cell lines MCF-7 and HBL-100 respectively.Compared with MDA-MB-231/GFP cells and MDA-MB-231 cells, the mRNA level of BMP9 was increased in MDA-MB-231/BMP9 cells. the cell colony formation rate of experimental group was decreased from the control group (95.4±3.1)% and (92.5±2.4)% to (74.0±3.6)% (P <0.05), the healing rate of experimental group was decreased from control group (90.6±2.5) %and (85.6±3.5) %to (57.3±4.5)%(P<0.05) , the number of invading cells which moved across the matrix barrier for experimental group was decreased from control group (255.3±16.1) and (267.3±14.9) to(150.3±14.6)(P<0.05).MDA-MB-231/BMP9 was stimulated with CTGF conditioned medium, the healing rate of MDA-MB-231/BMP9+CTGF was increased from MDA-MB-231/BMP9+GFP (32.2±1.6) %to (85.3±3.1)%(P<0.05) , the number of invading cells which moved across the matrix barrier for MDA-MB-231/BMP9+CTGF was increased from MDA-MB-231/BMP9 +GFP (135.8±10.1) to(248.5±21.6)(P<0.05).Experimental group and control group were separately injected into the proximal tibia of BALB/c nude mice, BMP9 inhibitted the lytic lesions of MDA-MB-231 in animal models, the CTGF expression was down regulation by BMP9 with immunohistochemistry in animal models(P<0.05), the lytic lesions of MDA-MB-231/BMP9/CTGF was bigger than MDA-MB-231/BMP9/RFP after restored the expression of CTGF.Conclusions: Our data suggest that the decreased BMP9 expression in the human breast cancer is associated with elevated proliferation and migration of breast cancer. BMP9can inhibit invasion, and migration of breast cancer cells in vitro and in vivo. BMP9 inhibit the migration, invasion of breast cancer cell MDA-MB-231 through decreasing the expression of CTGF in vitro. Moreover, in breast cancer bone metastasis mouse models, BMP9 inhibit the tumor bone metastasis through decreasing tumor bone metastases-related genes CTGF in vivo.BMP9 is a putative tumor suppressor in breast cancer bone metastasis, and may present a novel molecular therapy for breast cancer bone metastasis. |
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