survivin基因shRNA真核表达载体的构建及其对人胃癌细胞株中survivin表达的影响

背景:胃癌是我国最常见的恶性肿瘤之一,survivin是凋亡抑制蛋白家族的新成员,在胃癌组织中高表达。目的:构建survivin基因短发夹RNA(shRNA)真核表达载体并观察其对人胃癌细胞株BGC823和SGC7901中survivin表达的影响。方法:根据GenBank中survivin基因序列设计并合成能转录shRNA的双链DNA序列,插入含有绿色荧光蛋白(GFP)基因和U6启动子的真核表达载体pRNAT-U6.3中,构建重组载体pRNA-shSUR。重组载体经鉴定后转染胃癌细胞株BGC823和SGC7901,以转染pRNA-shControl作为阴性对照。荧光显微镜下观察转染情况,蛋白质印迹法检测survivin蛋白表达,Annexin V-FITC/PI双染法检测胃癌细胞凋亡情况。结果:成功构建了针对survivin基因的shRNA表达载体。转染胃癌BGC823和SGC7901细胞48 h后,与阴性对照组相比,pRNA-shSUR组GFP表达增强,survivin蛋白表达受到明显抑制(P<0.05),胃癌细胞早期凋亡率明显增加。结论:成功构建靶向survivin基因的特异性shRNA真核表达载体,转染胃癌细胞后可抑制survivin蛋白表达并促进细胞凋亡,为进一步研究survivin基因与胃癌生物学行为以及化疗耐药等的相关性奠定了基础。

Construction of shRNA Eukaryotic Expression Vector Targeting survivin Gene and its Effect on survivin Expression in Human Gastric Cancer Cell Lines

Gastric cancer is one of the most common malignant tumors in China. Survivin is a new family member of inhibitors of apoptosis, and is highly expressed in gastric cancer. Aims： To construct an eukaryotic expression vector of small hairpin RNA （shRNA） targeting survivin gene and evaluate its effect on survivin expression in human gastric cancer cell lines BGC823, SGC7901. Methods： In accordance with the sequence of survivin gene in GenBank, double strand DNA that could transcribe shRNA was designed and synthesized, and was inserted into pRNAT-U6.3 eukaryotic expression vector containing green fluorescent protein （GFP） gene and U6 promoter to construct the recombinant vector pRNA-shSUR. After identification, the recombinant vector was transfected into gastric cancer cell lines BGC823 and SGC7901; cells transfected with pRNA-shControl were served as negative controls. Fluorescence microscopy was used to assess transfection, the expression of survivin protein was detected by Western blotting and Annexin V-F1TC/PI double staining was used to determine the apoptosis of gastric cancer cells. Results： Recombinant vector pRNA-shSUR shRNA were successfully constructed. After transfection into gastric cancer BGC823 and SGC7901 cells for 48 hours, expression of GFP in pRNA- shSUR group was increased when compared with negative controls, expression of survivin was significantly suppressed （P 〈 0.05）, early apoptosis rates of BGC823 and SGC7901 cells were enhanced. Conclusions： shRNA eukaryotic expression vector targeting survivin gene is successfully constructed. Transfection with pRNA-shSUR can down-regulate the expression of survivin gene and enhance cell apoptosis in gastric cancer ceils. Thus it provides the foundation for further study on the correlation of survivin gene with biological behavior of gastric cancer and drug resistance of chemotherapy.