Mice were housed in microisolator cages in a specific pathogen-free (SPF) condition with 12-hr light-dark cycles. Mice were subcutaneously implanted with 1 × 107 5637 cells. Once tumors reached approximately 60 μL in volume, the mice were allocated to receive either ASODN or MSODN treatment, with the concentration of 200 nmol/L and 0.2 ml/mice. The nude mice injected with ASODN were termed as treatment group and the nude mice injected with MSODN were termed as control group. Complexes of ASODN or MSODN plus 4 μL invivo-jetPEI™ (polyplus-transfection

Inc., U.S.A.) and also plus 160 μL 5% glucose were directly injected into the tumor once every other day with a total of 7 times. Tumor dimensions were measured once every three days and the tumor AZD2014 mw volumes calculated using the formula: 1/2 × a × b2, where a and b respectively represented the larger and smaller tumor diameter. At the end of the treatment, mice were killed by overdose of ketamine (400 mg/kg) and xylazine (50 mg/kg) and necropsy was performed. Tumor tissue samples were prepared for Immunohistochemistry or TUNEL cell apoptosis detection. Tumor growth inhibition (TGI) was calculated using the formula TGI (%) = (1-MT/MC) LY2835219 molecular weight × 100, where MT and MC are the mean tumor masses in the treatment group and control group respectively. TUNEL analyses for cell apoptosis detection For detection of apoptosis, TUNEL analyses were performed using the in

situ cell death detection kit (Roche Molecular Biochemicals, USA). Operations were carried very out according to kit instructions. 10 high-powerfields were selected for each case. Count the

number of apoptotic cells and total number of cells for each powerfield to calculate the percentage of apoptotic cells (number of apoptotic cells in each powerfield/total cell number in each powerfield) i.e., apoptosis index (AI). . Statistical analysis The results were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was used to determine the levels of difference between all groups. Comparisons for all pairs were made using Student-Newman-Keuls (SNK) test. p < 0.05 was considered statistically significant. Results Livin antisense oligonucleotide dose-dependently inhibit bladder cancer cell growth After transfected with different concentrations of Livin antisense oligonucleotides, cell growth of bladder cancer cell lines was determined by MTT and an obvious dose-dependently inhibitory effect was found (Fig 1). When the Livin antisense oligonucleotide concentration was 160 nmol/L, the cell growth inhibition rate reached 92.61 percent, although reagent concentration was continuously increasing, the inhibition rate will not increase significantly (P > 0.05). Accordingly, we chose 160 nmol/L oligonucleotide as the suitable concentration for further study. Figure 1 Inhibitory rate of 5637 cells transfected with Livin ASODN.