Fig. 5.

Identification of survivin as a direct target of miR-454 in ovarian cancer cells. (A) An illustration of vector and miR-454 binding sequence in survivin 3′-untranslated region (3′-UTR). A mutation was generated in the 3′-UTR of survivin in the complementary site for miR-454 binding. (B) A2780 and A2780/DDP cells were co-transfected with vector/survivin-WT/survivin-MUT and miR-NC/miR-454 mimics. Luciferase activity was examined by dual luciferase reporter assay. Renilla luciferase activity was used to normalize the activity of firefly luciferase activity. (C) Down-regulation of survivin by miR-454. Survivin level was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting. (D) Cells were transfected with pcDNA3.1-survivin vector or negative control (vector) and the expression level of survivin was determined by qRT-PCR and Western blotting. (E) A2780 and A2780/DDP cells were transfected with transfected with miR-NC, miR-454 with or without pcDNA3.1-survivin under cisplatin condition (A2780 for 8 μM and A2780/DDP for 20 μM) for 24 hours. Apoptosis of cells were examined by flow cytometry. PI, propidium iodide. (F) Expression of Bcl-2, Bax, and survivin in transfected ovarian cancer cells without cisplatin were determined by Western blotting. Values are presented as the mean±standard deviation and performed in triplicate. ^*p < 0.05, ^**p < 0.01.