摘要
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 effects of siRNA-mediated down-regulation of human bone morphogenetic protein 9 （BMP9） on proliferation and migration of human breast cancer SK-BR-3 cells and its possible mechanism in simulated bone microenvironment in vitro, SK-BR-3 cells as blank group, SK-BR-3/RFP cells infected with ade- novirus RFP as control group, and SK-BR-3/siBMP9 cells infected with adenovirus siBMP9 as experimental group were indirectly co-cultured with human bone marrow stromal cells HS-5 with Transwell chamber. Effects of down- regulating BMP9 on SK-BR-3 cell proliferation and migration were investigated by MTT, wound-healing test and transwell migration test; The expression of related factors were screened by RT-PCR and Western blot. The result showed that in the co-culture system, the expression of BMP9 was decreased in SK-BR-3/siBMP9 group （P〈0.05）; Down-regulating BMP9 could promote the proliferation of SK-BR-3/siBMP9 cells （P〈0.05）, and the wound-heal- ing rate and the number of migratory cells were remarkably increased （P〈0.05）; Compared with the control group, the expressions of VEGF and CTGF were significantly up-regulated at mRNA and protein levels in SK-BR-3/ siBMP9 group （P〈0.05）, and Western blot showed p-Akt was higher than the control group （P〈0.05）. All together, down-regulating BMP9 can promote the proliferation and migration of breast cancer cells SK-BR-3 in microenvi- ronment of bone metastasis, which may be related to up-regulating the expressions of VEGF and CTGF, and PI3K/ Akt pathway may be involved in this process.
出处
《中国细胞生物学学报》
CAS
CSCD
北大核心
2013年第11期1626-1633,共8页
Chinese Journal of Cell Biology
基金
国家自然科学基金(批准号:81172017)
国家重点基础研究发展计划(973计划)(批准号:2011CB707906)资助的课题~~
关键词
骨形态发生蛋白9
乳腺肿瘤
骨微环境
共培养
bone morphogenetic protein 9
breast tumor
bone microenvironment
co-culture
