候选转移抑制基因通过调控肝细胞生长因子／c—Met信号通路影响膀胱癌BIU87细胞生物学功能的研究

Effect of metastasis suppressor 1 gene expression on migration and apoptosis of human gastric canc- er cell BIU87 by regulating the hepatocyte growth factor/c -Met signaling pathway

目的观察候选转移抑制基因（MTSS1）通过调控肝细胞生长因子（HGF）／c—Met信号通路对膀胱癌BIU87细胞生物学功能的影响。方法培养膀胱癌BIU87细胞，将传代的细胞分为以下3组：A组：PEFMTSSl和PSV2neo共转染组；B组：阴性对照小干扰RNA（shNC）和PSV2neo共转染组；C组：未转染组，A组为实验组，B、C两组为阳性对照组和阴性对照组。通过Westernblot和实时定量聚合酶链反应（Real-timePCR）检测MTSS1蛋白和mRNA在3组细胞中的表达水平，并检测3组BIU87细胞中c—Met、E一钙黏蛋白（E-cadherin）和N一钙黏蛋白（N—eadherin）蛋白和mRNA水平表达变化，并通过流式细胞法和划痕实验观察比较转染PEFMTSSl的BIU87细胞的的迁移能力和增殖凋亡情况。结果（1）Westernblot和Real-timePCR检测结果显示：与B、C组比较，PEFMTSS1组MTSS1蛋白及mRNA表达均显著下调（P〈0．01），c—Met蛋白及其mRNA的表达显著下调（P〈0．01）。（2）Westernblot和Real—timePCR检测结果显示：与B、C组比较，A组上皮标志物E—eadherin蛋白和及mRNA表达显著上调（P〈0．01），c-Met蛋白及其mRNA，间质细胞标志物N—cadherin蛋白及mRNA的表达显著下调（P〈0．01），B、C两组间E—eadherin和N—eadherin蛋白及mRNA表达的差异无统计学意义（P〉0．01）。（3）划痕实验检测细胞迁移：结果表明，A组划痕间距较宽，愈合被抑制，BIU87细胞迁移能力低于B、C组细胞（P〈0．01），B、C两组细胞的划痕间距差异无统计学意义（P〉0．叭）。（4）流式细胞仪分析细胞凋亡：A、B和C组细胞的凋亡率分别为（26．73±3．72）％、（4．28-t-O．68）％和（2．73±0．41）％，A组细胞的凋亡率较C组明显增加（P〈0．01），B、C两组细胞凋亡的差异无统计学意义（P〉0．05）。结论MTSSl基因转染对体外膀胱癌细胞株BIU87细胞具有促进增殖和抑制凋亡作用，其可能是通过调控HGF／c—Met信号转导通路来发挥作用。

Objective To investigate the effect of metastasis suppressor 1 （MTSS1） gene on the biological function of bladder cancer BIU87 cells by regulating the hepatocyte growth factor （HGF）/c - Met signaling pathway. Methods Cultured gastric cancer BIU87 cells and passaged cells were divided into three groups： group A （PEF MTSS1 and PSV2neo cotransfection group） ; group B： （shNC and PSV2neo co transfection group） ; group C （ non transfection group）. Western blotting and real - time quantitative polymerase chain reaction （Real - time PCR） were used to detect MTSS1, c - Met, E - cadherin and N - cadherin protein and mRNA expression levels. Flow cytometry and scratch test were performed to ex- amine the migration, proliferation and apoptosis of BIU87 ceils transfected with PEF MTSS1. Results （ 1 ） Western blotting and Real -time PCR results showed that as compared with groups B and C, MTSS1 and c - Met protein and mRNA expression levels were significantly down - regulated in group A （ P 〈 0. 01 ）. （2） Western blotting and Real - time PCR results showed as compared with groups B and C, the E - cadherin protein and mRNA expression was significantly increased （ P 〈 0. 01 ）, and N - cadherin mRNA and protein expression was significantly decreased in group A （P 〈 0. 01 ）. There was no significant difference in the expression of E - cadherin and N - cadherin mRNA and protein between groups B and C （P 〈 0. 05）. （3） Scratch assay showed that the scratch space was widened, healing was inhibited, and migration ability of BIU87 cells was reduced in group A as compared with groups B and C （P 〈 0. 01 ）, but there was no statistically significant difference in the scratch space between groups B and C （P 〉 0.05 ）.（4） Flow cytometry showed that cell apoptosis rate in groups A, B and C was respectively （26. 73 ± 3.72）%, （4. 28 ±0. 68）% and （2. 73±0. 41 ）%. The apoptosis rate in group A was significantly higher than in group C （P 〈0. 01 ）, and there was no significant difference between groups B and C. Conclusion The transfection of MTSS1 gene can promote the proliferation and inhibit the apoptosis of gastric cancer cell line BIU87 cells probably by regulating the HGF/c - Met signal transduetion pathway.