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

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,p EYFP-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(Vimentin)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。

Objective:To construct a recombinant eukaryotic expression vector of human matrix metalloproteinase 2(Mmp-2)and investigate 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 protease(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 p EYFP-Mmp-2 was successfully constructed.High expression of MMP-2-YFP was observed in the ovMMP-2 group transfected with the pEYFPMmp-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 p EYFP-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 epithelialmesenchymal transition of hepatocellular carcinoma SMMC-7721 cells by regulating calpain.