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目的:采用体外共培养技术模拟骨微环境,研究人骨形态发生蛋白9(bone morphogenetic protein9,BMP9)对人高骨转移性乳腺癌MDA-MB-231细胞迁移和凋亡的影响,并初步探讨其作用机制。方法:将重组腺病毒AdBMP9导入MDA-MB-231细胞中,与骨髓基质细胞HS-5在Transwell共培养体系中培养3d。然后,采用划痕愈合实验、Transwell小室和FCM法检测MDA-MB-231细胞迁移和凋亡的变化;RT-PCR法检测共培养的2种细胞中基质细胞衍生因子-1(stromal cell derived factor-1,SDF-1)、白细胞介素-6(interleukin6,IL-6)和单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)等转移相关因子的水平;细胞免疫荧光法检测MDA-MB-231细胞中趋化因子受体CXCR4的表达;蛋白质印迹法检测2种细胞中SDF-1/CXCR4-PI3K信号通路关键分子的表达水平变化。结果:在模拟骨微环境的共培养体系中过表达BMP9后,乳腺癌MDA-MB-231细胞的迁移受到明显抑制(P<0.05),而肿瘤细胞凋亡增加(P<0.001)。过表达BMP9后,骨髓基质HS-5细胞中转移相关因子SDF-1、IL-6和MCP-1表达显著下调(P<0.05),但MDA-MB-231细胞中SDF-1表达水平无明显变化,IL-6和MCP-1的表达也明显下调。另外,MDA-MB-231细胞中本身CXCR4阳性,BMP9过表达对MDA-MB-231细胞中CXCR4表达无明显影响,但可以下调其配体SDF-1在HS-5细胞中的表达水平,且MDA-MB-231细胞中磷酸化的Akt(phosphorylated Akt,p-Akt)水平也被下调(P<0.05)。结论:模拟骨微环境的共培养体系中,BMP9过表达可以调节乳腺癌细胞与骨髓基质细胞的相互作用,抑制乳腺癌MDA-MB-231细胞的迁移,并促进其凋亡,且该抑制作用可能与SDF-1/CXCR4-PI3K信号通路有关。
OBJECTIVE: To study the effects of bone morphogenetic protein 9 (BMP9) on the migration and apoptosis of human high metastatic breast cancer MDA-MB-231 cells by in vitro co-culture technique. Mechanism. Methods: Recombinant adenovirus AdBMP9 was transfected into MDA-MB-231 cells and cultured with bone marrow stromal cells HS-5 in Transwell co-culture system for 3 days. Then, the changes of migration and apoptosis of MDA-MB-231 cells were detected by scratch healing assay, Transwell chamber assay and FCM assay. The stromal cell derived factor-1 1, SDF-1), interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) Method was used to detect the expression of chemokine receptor CXCR4 in MDA-MB-231 cells. Western blotting was used to detect the expression of key molecules in SDF-1 / CXCR4-PI3K signaling pathway. Results: Migration of breast cancer MDA-MB-231 cells was significantly inhibited (P <0.05) and apoptosis of tumor cells was increased (P <0.001) after overexpression of BMP9 in a co-culture system simulating bone microenvironment. The expression of SDF-1, IL-6 and MCP-1 in bone marrow stromal HS-5 cells was significantly down-regulated after BMP9 overexpression (P <0.05), but the expression of SDF-1 in MDA-MB-231 cells was not significant Changes, IL-6 and MCP-1 expression was significantly down-regulated. In addition, the expression of CXCR4 was positive in MDA-MB-231 cells and BMP9 overexpression did not affect the expression of CXCR4 in MDA-MB-231 cells, but down-regulated the expression of its ligand SDF-1 in HS-5 cells The level of phosphorylated Akt (p-Akt) in MDA-MB-231 cells was also down-regulated (P <0.05). CONCLUSION: BMP9 overexpression can regulate the interaction between breast cancer cells and bone marrow stromal cells, inhibit the migration and promote the apoptosis of breast cancer MDA-MB-231 cells in the co-culture system simulating bone microenvironment May be related to the SDF-1 / CXCR4-PI3K signaling pathway.