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

Background: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-FITC/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 pRNAshSUR 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 cells.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.