The Inhibiting Effects Of BMP9 On Breast Cancer By Regulating The Interaction Between Breast Cancer Cells And Preadipocytes

Objective To investigate the effects of human bone morphogenetic protein 9 (BMP9) on proliferation and migration of human breast cancer MDA-MB-231 cells in vitro in vivo by coculturing with preadipocytes 3T3-L1 and its possible molecular mechanism.Methods The highly malignant breast cancer cell line MDA-MB-231 was used as the study model.MDA-MB-231 cells were infected with recombinant adenovirus AdBMP9 (AdGFP as the control) and AdsiBMP9 (AdRFP as the control).Then the BMP9 overexpressed cell lines MDA-MB-231/AdBMP9 and the BMP9 down-regulated cell lines MDA-MB-231/AdsiBMP9 were constructed. After that,the recombinant cells were indirectly cocultured with preadipocytes 3T3-L1 respectively for 3 days.The experimental groups were set up as follows:①MDA-MB-231 cells were cocultured with 3T3-L1 cells as the blank group,which was described as the Co-Blank group.② MDA-MB-231 cells/AdGFP were cocultured with 3T3-L1 cells as the overexpressed control group,which was described as the Co-AdGFP group. ③MDA-MB-231 cells/AdRFP were cocultured with 3T3-L1 cells as the down-regulated control group,which was described as the Co-AdRFP group.④MDA-MB-231 cells/AdBMP9 were cocultured with 3T3-L1 cells as the overexpressed exprimental group,which was described as the Co-AdBMP9 group. ⑤ MDA-MB-231cells/AdsiBMP9 were cocultured with 3T3-L1 cells as the down-regulated exprimental group,which was described as the Co-AdsiBMP9 group.As for the different breast cancer cells in the coculture system:The overexpression of BMP9 was detected by RT-PCR and western blotting.The effects of BMP9 overexpression on proliferation and migration of MDA-MB-231 cells were investigated by MTT,flow cytometry(FCM),wound-healing assay and Transwell migration assay, respectively.The expressions of leptin receptor (OB-R) and the key molecules of leptin signaling pathway in MDA-MB-231 cells were detected by immunofluorescence staining and western blotting,respectively.Western blotting was used to test the leptin expression in 3T3-L1 cells, and the secretory leptin proteins in the medium were detected by ELISA.The mixed cell suspension of MDA-MB-231 cells(5×106 per mouse) from each recombinant group and 3T3-L1 cells(2.5×107 per mouse) was injected subcutaneously (sc) into nude mice.The growth of xenograft was detected and tumor volume was measured with calipers.Mice were sacrificed after 21 days and the tumor tissue sections were removed to do HE staining for the tumor differentiation and immunohistochemical staining for he expression of leptin, c-Myc, CyclinD1, MMP9, p-ERK1/2 and p-STAT3.Results ① RT-PCR and western blotting showed that the BMP9 overexpressed breast cancer cell lines MDA-MB-231/AdBM9 and the endogenous BMP9 down-regulated cell lines MDA-MB-231/AdsiBM9 was successfully constructed.②The results for the breast cancer cells MDA-MB-231 in the cocultured system:The MTT results showed that the cell proliferation of BMP9 overexpressed group (Co-AdBMP9) was significantly inhibited,which was compared with the control group (Co-AdGFP) (P <0.05).But the cell proliferation activity of Co-AdsiBMP9 group was significantly higher than the control group (Co-AdRFP)((P<0.05).Flow cytometry (FCM) showed that the cell cycle of Co-AdBMP9 group was arrested at G2/M phase (P<0.05).Wound-healing assay found that the scratch healing rate of Co-AdBMP9 group was significantly lower than the control group (P<0.05) after 48h, while the scratch healing rate of Co-AdsiBMP9 group was significantly higher than the control group (P <0.05).Transwell migration assay showed that the breast cancer cells of Co-AdBMP9 group getting through a few films after 24h was significantly lower than the control group (P<0.05), while the migrated number of breast cancer cells in Co-AdsiBMP9 group was significantly higher(P<0.05). Immunofluorescence showed that the expression of leptin receptor OB-R in MDA-MB-231 breast cancer cells was positive.Western blotting showed that the expression levels of leptin,OB-Rb and OB-Rt in Co-AdBMP9, Co-AdsiBMP9 group compared with the control group had no significant change(P>0.05).Results of the molecules expression in leptin signaling pathway showed that the expression of p-ERK1/2 and p-STAT3 in Co-AdBMP9 group was significantly lower(P<0.05),while their expression in Co-AdsiBMP9 group compared with the control group was significantly higher (P<0.05).But the expression of p-Akt in Co-AdBMP9 group and Co-AdsiBMP9 group had no significant change (P>0.05).The target factors of leptin signaling pathways such as c-Myc,CyclinD1,MMP9, and VEGF in Co-AdBMP9 group were significantly lower(P<0.05), but they were significantly higher in Co-AdsiBMP9 group compared with the control group(P<0.05).③ Western blotting showed that the expression of leptin in Co-AdBMP9 group were significantly reduced compared to the control group (P<0.05) in preadipocytes 3T3-Ll,while its expression in Co-AdsiBMP9 group was significantly higher than the control group(P <0.05).The results of ELISA releaved that Co-AdBMP9 group compared with the control group had less released leptin(P<0.05) in the medium, while the Co-AdsiBMP9 group compared with the control group had more released leptin(P<0.05).④The results of animal experiments showed that the subcutaneous tumor volume in Co-AdBMP9 group was significantly smaller than that in the control group (P<0.05),while the subcutaneous tumor volume in Co-AdsiBMP9 group compared with the control group was significantly bigger(P<0.05).HE staining showed that the degree of differentiated tumor cells in Co-AdBMP9 group compared with the control group was higher, whereas the degree of differentiation tumor cells in Co-AdsiBMP9 group was lower than that in the control group.The results of immunohistochemistry realved that the staining extent of leptin, c-Myc, CyclinD1, MMP9,p-ERK1/2 and p-STAT3 was significantly lower in Co-AdBMP9 group than that in the control group, while the staining extent of that in Co-AdsiBMP9 group was significantly higher.Conclusion BMP9 could inhibit the proliferation and migration of highly malignant breast cancer cells MDA-MB-231 by modulating the interaction between breast cancer cells and preadipocytes 3T3-L1 in vitro and in vivo.The possible mechanism was that BMP9 might inhibit the leptin secreting from preadipocytes 3T3-L1 in the coculture system,thereby decreasing the binding of leptin with OB-R in breast cancer cells, which then could reduce the expression of related factors in leptin signaling pathway.