Nonsense sirna

Cell line and cell culture. The MCF-7 human breast cancer cell line was obtained from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 50 µg/ml streptomycin and 50 units/ml penicillin, in a humidified atmosphere incubator of 5% CO 2 at 37˚C. The selective EGFR blocker AG1478, PI3K phosphorylation inhibitor LY294002, and the MEK inhibitor PD0325901 were purchased from Sigma-Aldrich. Cells from AG1478, LY294002 and PD0325901 group were cultured with 20 µM AG1478, LY294002 and PD0325901 for 48 h before migration and proliferation assay, real-time PCR and western blot analysis, respectively.
Transfection of MCF-7 cells with ADAM17-siRNA. ADAM17small interference RNA (ADAM17-siRNA): 5'-TGAGGCAG TCTCTCCTATTCCTGACCAGC-3' and nonsense siRNA: 5'-TGACCACCCTGACCTACGGCGTGCAGTGC-3' were from Shanghai GenePharma, Co., Ltd. (Shanghai, China). MCF-7 cells of the ADAM17-siRNA groups and the nonsense siRNA groups were transfected with Lipofectamine 2000 (Invitrogen) following the manufacturer's instruction. The same amount of PBS was added to the control group cells.