体外模拟乳腺脂肪微环境中过表达BMP9对乳腺癌细胞增殖和迁移的影响及其机制探讨

Effects of BMP9 over-expression on proliferation and migration of human breast cancer MDA-MB-231 cells in simulated breast adipose microenvironment in vitro and its possible mechanism

目的 :采用体外共培养技术模拟乳腺脂肪微环境,研究人骨形态发生蛋白9(bone morphogenetic protein9,BMP9)对人乳腺癌MDA-MB-231细胞增殖和迁移的影响,并初步探讨其可能的机制。方法 :将重组腺病毒Ad-BMP9(以Ad-GFP作为对照)导入MDA-MB-231细胞中,构建MDA-MB-231/Ad-BMP9重组细胞,再与前体脂肪细胞3T3-L1在Transwell共培养体系中间接共培养3 d。荧光显微镜下观察共培养体系中重组腺病毒Ad-BMP9在MDA-MB-231细胞中的感染效率,并通过RT-PCR和蛋白质印迹法验证BMP9在共培养体系的MDA-MB-231细胞中成功过表达。然后,分别采用MTT法、FCM法、划痕愈合实验和Transwell迁移实验检测MDA-MB-231细胞增殖和迁移能力的变化;蛋白质印迹法检测共培养的2种细胞中增殖和转移相关因子瘦素(leptin)的表达水平变化;细胞免疫荧光法和蛋白质印迹法分别检测MDA-MB-231细胞中leptin受体OB-R及leptin信号通路关键分子的表达水平变化。结果 :荧光显微镜下观察到重组腺病毒Ad-BMP9在共培养体系的MDA-MB-231细胞中感染效率为70%左右,且BMP9在共培养体系的MDA-MB-231细胞中成功过表达(P<0.05)。在模拟乳腺脂肪微环境的共培养体系中过表达BMP9后,乳腺癌MDA-MB-231细胞的增殖和迁移受到明显抑制(P<0.05),并且细胞周期阻滞于G2/M期(P<0.001)。过表达BMP9后,前体脂肪细胞3T3-L1中leptin表达显著下调(P<0.05),而MDA-MB-231细胞中磷酸化的STAT3(phosphorylated STAT3,p-STAT3)和磷酸化的ERK1/2(phosphorylated ERK1/2,p-ERK1/2)水平均明显下调(均P<0.05)。结论 :模拟乳腺脂肪微环境的共培养体系中,BMP9过表达可以调节乳腺癌细胞与前体脂肪细胞的相互作用,抑制乳腺癌MDA-MB-231细胞的增殖和迁移,且该抑制作用可能与leptin经典信号通路有关。

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 simulated breast adipose microenvironment by co-culture technique, and to explore its possible mechanism. Methods： MDA- MB-231 cells were infected with recombinant adenovirus Ad-BMP9 or Ad-GFP （as the control）, and then they were indirectly co-cultured with adipose progenitor 3T3-L1 cells for 3 days. The infection efficiency of recombinant adenovirus Ad-BMP9 in MDA-MB-231 cells in the co-culture system was observed under a fluorescence microscope, and the expression level of BMP9 was detected by reverse transcription （RT）-PCR and Western blotting. The effects of BMP9 overexpression on proliferation and migration of MDA-MB-231 cells were investigated by MTT method, flow cytometry, wound-healing assay and Transwell migration assay, respectively. The expression of proliferation and metastasis-related factor leptin in the cells in co-culture system was detected by Western blotting. 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. Results： The infection efficiency of recombinant adenovirus Ad-BMP9 in MDA-MB-231 cells in co-culture system was about 70% under a fluorescence microscope. The over- expression of BMP9 was successfully detected in MDA-MB-231 cells in co-culture system （P 〈 0.05）. In co-culture system of simulated breast adipose microenvironment, BMP9 overexpression could significantly inhibit the proliferation （P 〈 0.05） and migration （P 〈 0.05） of MDA-MB-231 cells, and arrest the cell cycle at G2/M phase （P 〈 0.001）. Compared with the control group, BMP9 overexpression could decrease the expession level of leptin in 3T3-L1 cells （P 〈 0.05）, and down-regulate the expression levels of phosphorylated STAT3 （p-STAT3） （P 〈 0.05） and phosphorylated ERK1/2 （p-ERK1/2） （P 〈 0.05）. Conclusion： BMP9 can inhibit the proliferation and migration of breast cancer MDA-MB-231 cells in simulated breast microenvironment by regulating the cross-talk between MDA-MB-231 cells and adipose progenitor 3T3-L1 cells. Leptin signaling pathway may be involved in this process.