CAJ | 학술논문

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Western blot分别检测正常和缺氧状态(O2浓度1％)下1种肝癌SMMC-7721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Western 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-7721细胞缺氧培养后E-cadherin mRNA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(P ＜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-cadherin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24 h后,与对照组比较,E-cadherin mRNA和蛋白表达均增高,而Snail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.
목적 검측골형태발생단백7(BMP-7)대결양유도적간암세포상피세포간질전화(EMT)급세포체외침습능력증강적조공작용,탐토역전간암EMT,강저기전이잠능적도경.방법 역전록-취합매련반응(RT-PCR)화Western blot분별검측정상화결양상태(O2농도1％)하1충간암SMMC-7721세포적3충상피/기질표형표지물점부단백(E-cadherin)、Snail화파형단백(Vimentin)적mRNA화단백표체변화,응효성상계통MUVB-20대RT-PCR화Western blot결과행반정량분석,Transwell침습소실측정세포과막침습능력.동양방법관찰BMP-7(50μg/L)대세포상피/기질표형표지물표체,이급침습능력적영향.결과 SMMC-7721세포결양배양후E-cadherin mRNA표체감소이Snail화Vimentin표체증가,정시간의뢰성(P＜0.05),24h후전자상대표체량유1.299±0.095강지0.542 ±0.092(P ＜0.05)；후량자칙분별유0.189 ±0.021화0.182 ±0.046증가지0.715±0.053화0.773±0.064.상응적,E-cadherin단백상대표체량유0.806±0.093강지0.456±0.074,Vimentin유0.471 ±0.091증지0.831 ±0.057.차외,결양배양후세포침습능력명현증강,정시간의뢰관계(P＜0.05),24h후천과Transwell소실려막적세포수유32.33±3.22증가지76.67±7.09.BMP-7처리24 h후,여대조조비교,E-cadherin mRNA화단백표체균증고,이Snail화Vimentin적표체칙균감소,량조간차이유통계학의의(P＜0.05).이차,BMP-7처리후세포과막세포수명현감소,유75.43±7.76하강지33.34±5.86(P ＜0.05).결론 BMP-7가역전결양유도적간암세포EMT,강저기체외침습능력,가능작위억제간암전이적신파점.
Objective To investigate the impact of bone morphogenetic protein 7 (BMP-7) on epitheIial-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 (50 μg/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 ±0.046 to 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 downregulated 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-7reverses 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.