| Objective : In this study, we investigated the stimulating effect of pre-adipocytes and adipocytes on the proliferation and migration of breast cancer cells MDA-MB-231 in vitro, then researched the inhibitory effect of BMP9 on growth and metastasis of breast cancer cells in adipose microenvironment in vitro and in vivo.Methods: Inducing 3T3-L1 pre-adipocytes to mature adipocytes.There were three groups: Control group(MDA-MB-231), pre-adipocytes group(MDA-MB-231 cells co-cultured with pre-adipocytes) and adipocytes group(MDA-MB-231 cells co-cultured with adipocytes). As for MDA-MB-231 cells in different groups, morphological changes were observed by microscope, lipids accumulation was stained by Oil red O, the ability of proliferation was detected by MTT assay, and the ability of migration was estimated by transwell assay and wound-healing assay.Western blot and ELISA assay were used to detect the expression of leptin in pre-adipocytes and adipocytes. Immunofluorescence staining was usedto identify the expression of leptin receptor(Ob-R) in MDA-MB-231 cells.The key molecules of leptin signaling pathway in MDA-MB-231 cells were detected by Western blot. The expression levels of key molecules in leptin signaling pathway after using leptin netralization were detected by western blot.MDA-MB-231 cells were infected with BMP9 overexpressing adenovirus(AdBMP9) or BMP9 interfering adenovirus(AdsiBMP9) to construct cells that highly expressed BMP9(MDA-MB-231/AdBMP9)and endogenetic decreased BMP9 expression(MDA-MB-231/AdsiBMP9).Adipocytes were co-cultured with MDA-MB-231 cells(Co-Blank group),MDA-MB-231/AdGFP cells(Co-GFP group), MDA-MB-231/AdRFP cells(Co-RFP group), MDA-MB-231/AdBMP9(Co-BMP9 group) and MDA-MB-231/AdsiBMP9 cells(Co-siBMP9 group), respectively.As for the cancer cells in different groups, MTT Assay was used to investigate the proliferation status, FCM( flow cytometry) was used to detect cell cycle, wound-healing assay and transwell assay were used to observe the ability of migration. Western blot was used to detect the expression of leptin and leptin signaling. The levels of cyclin D1, c-myc and MMP9 were detected by western blot, similarly. As for adipocytes,the level of leptin was detected by western blot and ELISA.The mixed breast cancer cells and adipocytes were subcutaneously injected into nude mice at the ratio of 1:5. The growth curve was observed.HE staining was used to observe cell morphology and immunohistochemistry was used to detect the expression of leptin and leptin sianaling.Results:(1) Lipid droplets in differentiated 3T3-L1 could be observed and stained by Oil red O.(1)Compared with control group,We could observe that: MDA-MB-231 cells in the two co-culture groups became more spindly, the proliferation and migration ability of them were significantly enhanced(P<0.05). The adipocytes had a stronger effect than pre-adipocytes. And lipids accumulation in MDA-MB-231 cells in adipocytes group could be observed.(2)The high levels of leptin could be detected in pre-adipocytes, adipocytes and culture medium, and adipocytes had a much higher expression level(P<0.05). Compared with control group, the expression of p-Akt, p-STAT3, cyclin D1 and MMP9 in MDA-MB-231 cells increased in the two co-cultre groups(P<0.05), and they were higher in adipocytes group than in pre-adipocytes group. But the expression of p-ERK in MDA-MB-231 cells only increased in pre-adipocytes group(P<0.05). Compared with co-culture groups, the expression of key molecules in leptin signaling decreased after treatment with leptin netralization.RT-PCR and western blot results showed that the BMP9 overexpressed cell MDA-MB-231/AdBMP9 and endogenetic decreased BMP9 expression cell MDA-MB-231/AdsiBMP9 were successfullyconstructed.(2) In Co-BMP9 group: BMP9 could inhibit the proliferation(P<0.05),arrest the cell cycle in G1 phase(P<0.05), and decrease the expression of cyclin D1 and c-myc(P<0.05) of breast cancer cells. It also decreased wound healing rate and the number of migrated tumor cells to reduce migration ability of MDA-MB-231(P<0.05). Western blot result showed that there’s no significant changes of leptin and Ob-R in breast cancer cells between different groups(P>0.05). But the levels of leptin in adipocytes was meaningfully decreased in Co-BMP9 group(P<0.05). The expression of leptin in medium detected by ELISA had a consistent result(P<0.05).Key molecules in leptin signaling were measured by western blot. The levels of p-Akt and p-STAT3 were reduced in Co-BMP9 group(P<0.05).Smaller xenografts were observed in Co-BMP9 group(P<0.05). HE staining showed that cells in Co-BMP9 group arranged loosely, and their nuclears were smaller than those in Co-GFP group. Less leptin, p-STAT3,p-Akt, cyclinD1, c-myc and MMP9 expression were detected by immunohistochemistry in Co-BMP9 group(P<0.05).(3) In Co-siBMP9 group: The proliferation ability of MDA-MB-231 was stronger(P<0.05), cell number in S phase was higher, and the expression of cyclin D1 and c-myc(P<0.05) of breast cancer cells were increased. Wound-healing assay and transwell assay showed that breast cancer cells in Co-siBMP9 group had a higher migration ability than thosein Co-RFP group(P<0.05). Western blot and ELISA showed that, the level of leptin in adipocytes was increased(P<0.05). The levels of p-Akt and p-STAT3 were increased in Co-siBMP9 group(P<0.05). Animal experiment showed that xenografts in Co-siBMP9 group were bigger than those in Co-RFP group(P<0.05). Cells in Co-siBMP9 group arranged closely and disorderly, they had bigger nuclear and deeply stained. Leptin,p-STAT3, p-Akt, cyclin D1, c-myc and MMP9 expression were higher in Co-siBMP9 group(P<0.05).Conclusion: 1.Either pre-adipocytes or adipocytes could promote the proliferation and migration of breast cancer cells MDA-MB-231, and adipocytes had a stronger effect. Leptin signaling pathway may be involved in this process. 2. BMP9 can inhibit the growth and metastasis of breast cancer cells by regulating cross-talking between adipocytes and MDA-MB-231 cells. Reducing the expression of leptin in adipocytes,decreasing its promoting effect on proliferation and migration of breast cancer cells may be involved. |
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