Construction Of Human Bcl-2 ShRNA Eukaryotic Expressing Vectors And Screening The Most Effective Inhibitory Sequence

Objective:To construct eukaryotic vectors expressing short hairpin RNA (shRNA) sections targeting human Bcl-2 gene and screen the effective sequences,and to investigate its inhibitory effect on cell growth and Bcl-2 gene expression in human gastric carcinoma cell line SGC7901.Methods:4 pairs of specific Bcl-2 shRNA oligoes,shRNA1,shRNA2,shRNA3,shRNA4,and a non-coding sequence shNC were designed and synthesized,according to Bcl-2 cDNA sequence and the principle of shRNA designing.The complement form was obtained by annealing and cloned into plasmid vector pGPH1/GFP/Neo,then the recombinant plasmid were identified by restriction enzyme analysis and DNA sequencing.Finally the recombinant vectors were tansfected into SGC7901 by Lipofectamine 2000, then expression of Bcl-2 gene was detected by RT-PCR and cell growth of SGC7901 was measured by MTT.Results:The recombinant plasmid was cloned and the sequence was obtained by the results of restriction enzyme analysis and DNA sequencing. shRNA1,shRNA2,shRNA3,shRNA4 and shNC were respectively transfected into SGC7901 and the transfection rate was about 80%.RT-PCR showed Bcl-2 mRNA levels of 4 experimental groups had been reducted differently at 48,72hr after transfection,and the differences had statistical significance(P＜0.05) compared with shNC and lipofectamine group, especially shRNA2 led to a prominent reduction.MTT measurement showed the recombinant plasmids had a significant inhibition on cell proliferation of SGC7901,and it had statistical significance(P＜0.05) compared with shNC and lipofectamine group.Conclusion:Successful construction of human Bcl-2 shRNA eukaryotic expressing vectors shRNA1,shRNA2,shRNA3,shRNA4,which were certified to remarkably depress the expression of Bcl-2 mRNA and inhibit the cell proliferation of SGC7901 at 48,72hr after transfection,and established a favourable foundation for a further study on the function of Bcl-2.