摘要
目的检测骨形态发生蛋白7（BMP-7）对缺氧诱导的肝癌细胞上皮细胞间质转化（EMT）及细胞体外侵袭能力增强的调控作用，探讨逆转肝癌EMT，降低其转移潜能的途径。方法逆转录-聚合酶链反应（RT—PCR）和Westernblot分别检测正常和缺氧状态（O2浓度1％）下1种肝癌SMMC-721细胞的3种上皮／基质表型标志物黏附蛋白（E-cadherin）、Snail和波形蛋白（Vimentin）的mRNA和蛋白表达变化，凝胶成像系统MUVB-20对RT—PCR和Westem blot结果行半定量分析，Transwell侵袭小室测定细胞跨膜侵袭能力。同样方法观察BMP-7（50μg/L）对细胞上皮／基质表型标志物表达，以及侵袭能力的影响。结果SMMC-721细胞缺氧培养后E-cadhefinmRNA表达减少而Snail和Vimentin表达增加，呈时间依赖性（P〈0．05），24h后前者相对表达量由1．299±0．095降至0．542±0．092（尸〈0．05）；后两者则分别由0．189±0．021和0．182±0．046增加至0．715±0．053和0．773±0．064。相应的，E—cadhefin蛋白相对表达量由0．806±0．093降至0．456±0．074，Vimentin由0．471±0．091增至0．831±0．057。此外，缺氧培养后细胞侵袭能力明显增强，呈时间依赖关系（P〈0．05），24h后穿过Transwell小室滤膜的细胞数由32．33±3．22增加至76．67±7．09。BMP-7处理24h后，与对照组比较，E—cadbefinmRNA和蛋白表达均增高，而且，BMP-7和Vimentin的表达则均减少，两组间差异有统计学意义（P〈0．05）。而且，BMP-7处理后细胞跨膜细胞数明显减少，由75．43±7．76下降至33．34±5．86（P〈0．05）。结论BMP-7可逆转缺氧诱导的肝癌细胞EMT，降低其体外侵袭能力，可能作为抑制肝癌转移的新靶点。
Objective To investigate the impact of bone morphogenetie protein 7 （ BMP-7 ） on epithelial-mesenchymal transition （EMT） and promoted invasiveness induced by hypoxia in hepatocellular carcinoma cells （ HCCs）, and to explore a potential intervention for HCC metastasis by EMT reversion. Methods mRNA and protein expression levels of three epithelial or mesenchymal marker genes E-cadherin, Snail and Vimentin in SMMC-7721 cells were detected by using reverse transcription-polymerase chain reaction （RT-PCR） or Western blotting under normoxic and anaerobic （1%） culture. E-cadherin, Snail and Vimentin expression levels were semi-quantitatively determined by using MUVB-20 system, and invasion of cells through Matrigel was assayed by using a Transwell system. Meanwhile, the impact of BMP-7 （50μg/L） on the expression of epithelial or mesenchymal marker genes and the cell invasion was investigated as well using the methods mentioned above. Results mRNA expression of E-cadherin was reduced, and that of Snail and Vimentin increased in a time-dependent manner after hypoxia in HCC cells. The relative quantity of E-cadherin mRNA was decreased from 1. 299 ± 0. 095 to 0. 542 ± 0. 092, and that of Snail and Vimentin increased from 0. 189 ± 0. 021 to 715 ± 0. 053 and from 0. 182 ± 0. 046to 0. 773 ±0. 064, respectively （P 〈 0. 05）. Accordingly, protein expression of E-cadherin was reduced from 0. 806 ± 0. 093 to 0. 456 ±0. 074, and that of Vimentin increased from 0. 471±0. 091 to 0. 831 ±0. 057 （P 〈0. 05）. Cell invasive capacity was remarkably enhanced under hypoxic stress in a time-dependent fashion （ P 〈 0. 05 ）, and cell number through Transwell chamber was increased from 32. 33 ± 3.22 to 76.67 ±7.09 after 24-h treatment of hypoxia. Administration of BMP-7 up-regulated the expression of E-cadherin mRNA and down- regulated the expression of Snail and Vimentin as compared to control, and the differences between the groups were of statistical significance （P 〈 0.05）. Consequently, BMP-7 notably reduced the elevated invasive capacity induced by hypoxic stress, and cell number through Matrigel was decreased from 75.43 ± 7. 76 to 33.34 ± 5.86 （P 〈 0. 05）. Conclusion BMP-7 reverses EMT mediated by hypoxia and in turn diminishes invasion in HCC cells, and it can serve as a potential novel therapeutic agent for HCC metastasis.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2012年第10期1923-1926,共4页
Chinese Journal of Experimental Surgery
关键词
癌
肝细胞
骨形态发生蛋白7
缺氧
Carcinoma, hepatocellular
Bone morphogenetic protein 7
Hypoxia
