Construction of shRNA expression vector targetting survivin and research on its biologic effect

AIM: To construct an expression vector with short hairpin RNA of survivin,which paves the way for RNAi-mediated breast cancer therapy.METHODS: Eight strands of DNA fragment with the structure of survivin shRNA were designed and synthesized.After annealed,the strands were cloned into pShuttle vector to construct the recombinant plasmids.After the plasmids were transfected into MCF-7 cells,MTT assay was used to screen out the most powerful one and the effective concentration.Cell cycle and apoptosis were obversed by flow cytometry and Hoechst staining.RT-PCR and Western Blot were done to detect the expression of survivin.RESULTS: The recombinat plasmids were constructed successfully.MCF-7 cells were arrested in the period of G2/M,moreover,some cells began to die.Survivin expression was depressed.CONCLUSION: The constructed recombinants can knockout survivin expression in cell MCF-7,which offers a basis for the research on the RNAi-mediated gene therapy of human breast cancer.