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探讨在体外模拟的乳腺癌骨转移微环境中,采用RNAi技术下调骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)基因对人乳腺癌SK-BR-3细胞增殖、迁移的影响及可能机制。用Transwell小室将空白组SK-BR-3细胞、感染腺病毒AdRFP的对照组SK-BR-3/RFP细胞、感染腺病毒AdsiBMP9的实验组SK-BR-3/siBMP9细胞分别与人骨髓基质HS-5细胞间接共培养后,MTT增殖实验、划痕愈合实验、Transwell迁移实验分别检测下调BMP9对SK-BR-3细胞增殖、迁移的影响;RT-PCR和Western blot法检测相关因子的变化。结果显示,在共培养体系中,SK-BR-3/siBMP9组的BMP9显著低于对照组(P<0.05);下调BMP9可有效促进SK-BR-3/siBMP9细胞增殖(P<0.05),SK-BR-3/siBMP9细胞的划痕愈合率、穿膜细胞数也明显升高(P<0.05);SK-BR-3/siBMP9组血管内皮细胞生长因子(VEGF)、结缔组织生长因子(CTGF)mRNA和蛋白表达均显著上调(P<0.05),且Western blot结果显示p-AKT显著上调(P<0.05)。综上,在骨转移微环境中,干扰BMP9表达可促进人乳腺癌SK-BR-3细胞增殖及迁移,其作用机制可能与上调VEGF、CTGF表达有关,且这个过程可能涉及PI3K/AKT通路的激活。
To investigate the effect of bone morphogenetic protein 9 (BMP9) gene on the proliferation and migration of human breast cancer SK-BR-3 cells and its possible mechanism by using RNA interference technique in breast cancer bone metastasis microenvironment in vitro. SK-BR-3 cells in the blank group, SK-BR-3 / RFP cells infected with the AdRFP adenovirus, and SK-BR-3 / siBMP9 cells infected with the adenovirus AdsiBMP9 were transfected with human bone marrow stromal HS 5 cells, MTT proliferation assay, scratch healing assay and Transwell migration assay were used to detect the effects of BMP9 on the proliferation and migration of SK-BR-3 cells. The changes of related factors were detected by RT-PCR and Western blot. The results showed that the BMP9 of SK-BR-3 / siBMP9 group was significantly lower than that of the control group (P <0.05) in co-culture system; down-regulation of BMP9 could effectively promote the proliferation of SK-BR- The wound healing rate and the number of transmembrane cells in SK-BR-3 / siBMP9 cells were also significantly increased (P <0.05). The expressions of VEGF, connective tissue growth factor (P <0.05), and Western blot results showed that p-AKT was significantly up-regulated (P <0.05). In summary, BMP9 expression can promote the proliferation and migration of human breast cancer SK-BR-3 cells in the microenvironment of bone metastasis. The mechanism may be related to the up-regulation of VEGF and CTGF expression, and this process may involve the PI3K / AKT pathway activation.