Mmp-2重组真核表达载体的构建及MMP-2蛋白对肝癌细胞的作用机制探讨

Construction of Mmp-2 recombinant eukaryotic expression vector and study of the mechanism of action of MMP-2 on hepatocellular carcinoma cells

摘要:

目的：构建人基质金属蛋白酶2（matrix metalloproteinase 2，MMP-2）基因重组真核表达载体，探讨MMP-2蛋白对肝癌细胞迁移和侵袭的影响及其作用机制。方法：以肝癌SMMC-7721细胞为研究模型，以基因重组技术构建pEYFP-Mmp-2重组真核表达载体，pEYFP-Mmp-2一过性转染模型细胞过表达MMP-2-YFP，siRNA转染沉默模型细胞内源性MMP-2表达，设立空白对照、MMP-2沉默对照组、MMP-2沉默组、过表达MMP-2融合蛋白对照组、过表达MMP-2融合蛋白组，划痕实验检测细胞迁移能力，侵袭小室实验检测细胞侵袭能力，钙蛋白酶（calcium-activated neutral protease，Calpain）特异性抑制剂Calpeptin抑制Calpain活性，蛋白印记实验检测MMP-2、MMP-2-YFP、E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）、波形蛋白（Vi－mentin）表达变化。结果：成功构建人Mmp-2重组真核表达载体pEYFP-Mmp-2。pEYFP-Mmp-2转染过表达MMP-2融合蛋白组细胞高表达MMP-2-YFP。与MMP-2沉默对照组相比，MMP-2沉默组内源性MMP-2表达明显下调（P=0.000），细胞12 h、24 h及48 h迁移率明显降低（P=0.000或P=0.001），48 h侵袭率明显降低（P=0.004），E-cadherin表达明显上调，N-cadherin及Vimentin表达明显下调（P=0.000）。与过表达MMP-2融合蛋白对照组相比，过表达MMP-2融合蛋白组12 h、24 h及48 h迁移率明显增强（P=0.015或P=0.001），48 h侵袭率明显升高（P=0.011），细胞E-cadherin表达明显下调，N-cadherin及Vimentin表达明显上调（P=0.003或P=0.001）。Calpeptin预处理明显降低过表达MMP-2融合蛋白组细胞迁移率和侵袭率（P=0.000），上调E-cadherin，下调N-cadherin及Vimentin蛋白表达水平（P=0.000）。结论：本实验通过基因重组技术成功构建人Mmp-2重组真核表达载体pEYFP-Mmp-2，成功表达MMP-2-YFP 融合蛋白；并发现MMP-2通过调节Calpain介导肝癌SMMC-7721细胞迁移、侵袭及EMT。

Abstract:

Objective：To construct a recombinant eukaryotic expression vector of human matrix metalloproteinase 2（Mmp-2） and in－vestigate the effect and mechanism of action of MMP-2 on the migration and invasion of hepatocellular carcinoma cells. Methods：The human hepatocellular carcinoma SMMC-7721 cells were used as the research model，and the recombinant eukaryotic expression vector pEYFP-Mmp-2 was constructed by gene recombination technology. The cells of pEYFP-Mmp-2 transient transfection model overexpressed MMP-2-YFP，while the cells of siRNA transfection silencing model expressed endogenous MMP-2. Then the above cells were divided into five groups，namely control group，MMP-2 silencing control group（siCON），MMP-2 silencing group（siMMP-2），overexpressed MMP-2 fusion protein control group（ovNC），and overexpressed MMP-2 fusion protein group（ovMMP-2）. Wound healing assay was used to determine cell migration，and Transwell chamber assay was used to evaluate cell invasion. Calpeptin，a specific inhibitor of calcium-activated neutral pro－tease（calpain），inhibited the activity of calpain. The expression levels of MMP-2，MMP-2-YFP，E-cadherin，N-cadherin，and vimentin were measured by Western blotting. Results：The human Mmp-2 recombinant eukaryotic expression vector pEYFP-Mmp-2 was successfully constructed. High expression of MMP-2-YFP was observed in the ovMMP-2 group transfected with the pEYFP-Mmp-2. Compared with the siCON group，the siMMP-2 group showed a significant reduction in the expression level of endogenous MMP-2（P=0.000）. Meanwhile，the cell migration in the siMMP-2 group decreased significantly at 12 h，24 h，and 48 h（P=0.000 or P=0.001），and the cell invasion also decreased significantly at 48 h（P=0.004）. Furthermore，a significant up-regulation of E-cadherin and significant down-regulations of both N-cadherin and vimentin were manifested in siMMP-2 cells（P=0.000）. Compared with those in the ovNC group，the cell migration in the ovMMP-2 group increased significantly at 12 h，24 h，and 48 h（P=0.015 or P=0.001），and the cell invasion increased significantly at 48 h as well（P=0.011）. In addition to those，the ovMMP-2 group also had a significant down-regulation of E-cadherin along with significat up-regulations of N-cadherin and vimentin（P=0.003 or P=0.001）. Results also demonstrated that calpeptin pretreatment significantly reduced the cell migration and invasion，up-regulated the expression level of E-cadherin，and down-regulated the expression levels of N-cadherin and vimentin in the ovMMP-2 group（P=0.000）. Conclusion：In this study，the recombinant eukaryotic expression vector pEYFP-Mmp-2 is successfully constructed by gene recombination technology，which successfully expresses the MMP-2-YFP fusion protein. It finds that MMP-2 mediates the migration，invasion，and epithelial-mesenchymal transition of hepatocellular carcinoma SMMC-7721 cells by regulating calpain.