siRNA沉默胃癌SGC-7901细胞HO1基因的细胞系的建立及其生物学行为

摘要

目的 构建HO1基因的真核干扰表达载体,评估其转染人胃癌细胞系SGC-7901细胞后对HO1基因的干扰效果及其功能.方法 将外源性重组真核干扰表达载体HO1基因(pS/HO1)转染到人胃癌细胞系SGC-7901内,经G418筛选并建立siRNA表达载体稳定沉默胃癌SGC-7901细胞HO1基因的细胞系,分为SGC-7901-pS/HO1组,转染空质粒细胞(SGC-7901-pS)组和未处理细胞(SGC-7901)组；用实时荧光定量PCR和蛋白印迹验证HO1基因在各组细胞中的表达,并通过CCK-8和克隆形成实验分别观察HO1基因被干扰后细胞的生物学行为.结果 与SGC-7901-pS组相比,SGC-7901-pS/HO1细胞中HO1基因蛋白表达明显减少,降低了5.58倍(0.321±0.051 vs 1.675±0.153,P＜0.05)；与对照组相比较,SGC-7901-pS/HO1实验组较SGC-7901-pS对照组细胞增殖数量明显减少(P＜0.001)；与转染pS空载体的SGC-7901-pS细胞对照组相比,SGC-7901-pS/HO1细胞的克隆形成明显减少,降低了3.45倍(8.32±1.142 vs 2.32 ±0.362,P＜0.05).结论 HO1基因真核siRNA表达载体筛选成功,为继续深入的研究HO1基因在胃癌中的功能提供了依据.%Objective To investigate the effect and function of HO1 gene in its human gastric cancer SGC-7901 cell line by constructing the eukaryotic siRNA expression vector of HO1 gene and to explore its potential role in gastric carcinogenesis. Methods A recombinant plasmid carrying siRNA specific for HO1 gene expression vector was constructed and transfected into SGC-7901 cells by lipofectin-mediated method. Cells(SGC-7901 -pS/HO1) stably expressing siRNA specific for HO1 gene were selected using G418. SGC-7901 cells untransfected and those transfected with empty pS plasmid ( SGC-7901-pS) were used as controls. The expression levels of HO1 gene were detected by real-time PCR and Western blot in stably transfected-SGC-7901 (SGC-7901 -pS/HO1) cells, biological function of HO1 gene was analyzed by cell growth activity and soft agar colony formation assay. Results Compared with SGC-7901-pS cells, the expression of HO1 protein of SGC-7901-pS/HO1 cells was an average 5. 58-fold decrease　(0.321 ±0.051 vs 1.675 ±0. 153, P <0. 05). The colony formation rate of SGC-7901-pS/HO1 cells was significantly reduced as compared to that of control group ( P < 0. 05 ). The cell growth activity showed same tendency in SGC-7901-pS/HO1 group, SGC-7901-pS cells group and SGC-7901 cells group. Conclusion The eukaryotic expression vector of siRNA specific for HO1 gene was constructed successfully, which might contribute to probe the functions of HO1 in human gastric cancer in depth.