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

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.