Lentiviral vectors encoding the short hairpin rnas shrnas

Five HNSCC cell lines including SCC9, SCC25, Cal27, FaDu and a normal human oral keratinocytes (HOK) were obtained from American Type Culture Collection (ATCC,USA). And HN4 and HN6 were generously gifted from Dr. Wantao Chen (Shanghai Jiao Tong University). HNSCC cells were cultured in DMEM/F12 (Invitrogen, USA) with 10% fetal bovine serum (FBS, Gbico, USA) in a humidi ed atmosphere with 5% CO2 at 37 °C Small interfere RNAs, lentivirus production and cell transfection
The siRNAs against HOXB7 with siNC as a negative control were obtained from GenePharma (Shanghai, China). The and sequences of the HOXB7 siRNA including siRNA-1: 5'-GCUAUUGUAAGGUCUUUGUTT, 5'-ACAAAGACCUUACAAUAGCTT; siRNA-2: 5'-CCCUUUGAGCAGAACCUCUTT, 5'-AGAGGUUCUGCUCAAAGGGTT; The nal concentration of 100 nM siRNA or siNC pre-coated with Lipofectamine 3000 (Invitrogen, USA) were used for transfection.
Lentiviral vectors encoding the short hairpin RNAs (shRNAs) that target HOXB7 with the sequence of and a scramble shRNA were purchased from GenePharma (Shanghai, China). Transfection processes were conducted according to the instructions provided by the manufacturer. To generate the stable cell line, The transduced cells were then selected in culture medium containing puromycin (5.0 μg/ml).