过表达BMP9对人肺鳞状细胞癌细胞NCI－H520迁移和侵袭的影响

摘要

目的：探讨骨形态发生蛋白9（bone morphogenetic protein 9，BMP9）对人肺鳞状细胞癌细胞NCI－H520迁移和侵袭的影响及其机制。方法：采用RT－PCR和Western blot法分别检测NCI－H520细胞和正常人支气管上皮（ HBE）细胞中BMP9的mRNA和蛋白表达水平；用AdBMP9感染NCI－H520细胞，RT－PCR和Western blot法验证BMP9的变化；应用划痕愈合实验检测各组细胞的迁移能力， Transwell小室检测迁移和侵袭能力，并用RT－PCR和Western blot法检测迁移相关因子基质金属蛋白酶2（ MMP2）的mRNA和蛋白水平。 Western blot法检测BMP－Smad经典通路中Smad1／5的磷酸化水平（ p－Smad1／5）。用BMP特异性拮抗剂AdNoggin与AdBMP9共处理NCI－H520细胞，检测p－Smad1／5及细胞迁移能力的变化。结果：BMP9在NCI－H520细胞中的mRNA和蛋白水平均比在HBE细胞中低；AdBMP9感染的NCI－H520细胞中BMP9的mRNA和蛋白水平均明显升高，细胞迁移和侵袭能力均明显下降，MMP2的mRNA和蛋白水平下降，且p－Smad1／5的表达水平明显上调。在NCI－H520细胞中，Noggin能有效逆转BMP9导致的p－Smad1／5升高和细胞迁移抑制能力。结论：外源性BMP9转入肺鳞癌细胞NCI－H520可明显抑制其迁移侵袭的能力，该抑制作用可能与BMP－Smad信号通路的激活有关。%AIM:To investigate the effect of bone morphogenetic proteins 9 (BMP9) on the migration and in-vasion abilities of human lung squamous-cell carcinoma NCI-H520 cells and its mechanism.METHODS:The expression of BMP9 at mRNA and protein levels in the NCI-H520 cells and human bronchial epithelial ( HBE) cells was detected by RT-PCR and Western blot.The NCI-H520 cells were transfected with the recombinant adenovirus AdBMP9 and the expres-sion of BMP9 at mRNA and protein levels was validated by RT-PCR and Western blot.The migration and invasion abilities of the NCI-H520 cells were determined by wound-healing and Transwell assays.The mRNA and protein levels of the migra-tion-related factor matrix metalloproteinase 2 (MMP2) were detected by RT-PCR and Western blot.The level of phospho-rylated Smad1/5 (p-Smad1/5) was detected by Western blot.Meanwhile, NCI-H520 cells were treated with BMP specific antagonist AdNoggin and AdBMP9.The level of p-Smad1/5 and the cell migration ability were measured by Western blot, wound-healing and Transwell assays.RESULTS:The expression of BMP9 at mRNA and protein levels was lower in NCI-H520 cells than that in HBE cells.After AdBMP9 was stably transfected into the NCI-H520 cells, the expression of BMP9 at mRNA and protein levels was significantly up-regulated, cell migration and invasion abilities were significantly de-creased, and the mRNA and protein levels of MMP2 were decreased.Meanwhile, the level of p-Smad1/5 was increased. Noggin reversed BMP9-caused the increase in p-Smad1/5 and the decrease in cell migration ability.CONCLUSION:O-ver-expression of BMP9 inhibits the migration and invasion abilities of lung squamous-cell carcinoma NCI-H520 cells.The activation of BMP-Smad signaling pathway may be involved in this inhibitory process.