COL1A1-shRNA表达载体的构建及对胃癌细胞增殖迁移的影响

Construction of COL1A1 short hairpin RNA vector and its effect on cell proliferation and migration of gastric cancer cells

目的:构建COL1A1特异小干扰RNA(siRNA)表达载体,体外评价其对胃癌细胞BGC-823增殖迁移的影响。方法:根据文献获得3个COL1A1的siRNA靶序列,设计能转录短发夹状RNA(short hairpin RNAs,shRNA)的DNA序列,并与pSilencerTM4.1-CMV neo线性质粒载体连接,连接产物转化感受态大肠杆菌DH5α,筛选获得重组质粒;经DNA测序鉴定重组体DNA序列正确后转染胃癌BGC-823细胞,利用G418筛选稳定表达COL1A1-shRNA的BGC-823细胞株。通过实时荧光定量RT-PCR及Western blot检测转染后细胞COL1A1在mRNA及蛋白质水平的表达;MTT法及Transwell法检测COL1A1干扰后胃癌细胞增殖及迁移能力的变化。结果:序列测定表明成功构建3个COL1A1-shRNA表达载体。实时荧光定量RT-PCR及Western blot检测显示,COL1A1-shRNA转染胃癌BGC-823细胞的COL1A1 mRNA及蛋白表达水平明显降低(P<0.05);MTT试验及Transwell迁移试验显示,转染后细胞增殖及迁移能力明显降低(P<0.05)。结论:COL1A1-shRNA转染胃癌BGC-823细胞能有效抑制细胞COL1A1的表达及癌细胞的增殖迁移。

Objective： To construct COL1A1-targeted short hairpin RNA（shRNA） vector with pSilencerTM 4.1-CMV neo siRNA expression vector and to evaluate its effect on proliferation and migration of gastric cancer BGC-823 cells in vitro.Methods： Three COL1A1-shRNA plasmids（COL1A1-shRNA-1,COL1A1-shRNA-2,COL1A1-shRNA-3）,targeting different sites of COL1A1 gene,were constructed using pSilencerTM 4.1-CMV neo siRNA expression vector and transfected into gastric cancer BGC-823 cells.Real time quantitative RT-PCR and Western blot were performed to detect expression levels of COL1A1.MTT and Transwell migration assays were employed to evaluate the effects of COL1A1 gene silence on cell proliferation and migration.Results： Three recombinant plasmids targeting COL1A1 were constructed successfully.The expressions of COL1A1 in BGC-823 cells,including mRNA and protein levels,were significantly inhibited by the COL1A1-shRNA transfectants,which resulted in a clear reduction of cell proliferation and migration capacity.Conclusion： The COL1A1-shRNA can effectively knock down gene expression and inhibit proliferation and migration of gastric cancer BGC-823 cells.