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In order to investigate the effects of short hairpin RNA (shRNA) on the expression of Survivin, cell cycle and cell proliferation in MCF-7 cells, using a pEGFP vector which contained a U6 promoter shRNA plasmid targeted against survivin was constructed and transfected into MCF-7 cells. The change of the expression of Survivin and cell proliferation rates were detected by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and MTT methods respectively. The change of cell cycle after transfection was analyzed by flow cytometry. The results indicated that the recombinant plasmid containing Survivin shRNA was constructed successfully, which could suppress the expression of Survivin at mRNA and protein level. The growth of MCF-7 cells was arrested in G1 phase of the cell cycle and the proliferation activity was suppressed after transfection. It was concluded that Survivin shRNA plasmid could knock down the expression of Survivin in MCF-7 cells specifically. In addition, Survivin shRNA plasmid could lead to G1 arrest and inhibit the proliferation of MCF-7 cells, which suggested that Survivin shRNA might be used as a new therapeutic method for breast cancer.