Cells, plasmid and transfection procedures
The grade II human urinary bladder cancer cell line 5637 was selected for the UNC5B transfection and nude mouse tumor transplantation experiment because 5637 is a suitable transfection host and has tumorigenic ability. The cells were maintained in RPMI-1640 (Lonza, Verviers, Belgium) supplemented with 10 % fetal bovine serum (FBS) (EuroClone, West York, United Kingdom) at 37 °C in a 5 % CO2 humidified incubator. The human recombinant pcDNA-UNC5B-green fluorescent protein (GFP) construct was purchased from GenePharma (Shanghai, GenePharma Co., Ltd). 5637 cells stably expressing pcDNA-UNC5B-GFP (hereafter referred to as 5637-U cells) overexpressed UNC5B. For transfection, 2.5 μg of pcDNA-UNC5B-GFP and 12 μl of Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) were added to 2 ml of serum-free transfection medium and incubated for 24 h. The transfected 5637 cells were cultured in medium supplemented with G418 (500 μg/ml) (Invitrogen) to select cells stably transfected with pcDNA-UNC5B-GFP for approximately 14 days. Next, the 5637-U cells were cultured in RPMI-1640 medium supplemented with 10 % FBS containing G418 (500 μg/ml). Positive clones were determined by GFP immunofluorescence using a fluorescence microscope (Olympus, Tokyo, Japan). Non-transfected cells were used as controls.
Real-time RT-PCR analysis
TRIZOL reagent (Invitrogen, Carlsbad, CA) was used for RNA extraction according to the manufacturer’s instructions, and the RNA was quantified using a Thermo Scientific NanoDrop ND-100 (Wilmington, DE, USA). PCR reactions were conducted in a Roche quantitative PCR machine LC480 in a total reaction volume of 20 μl with SYBR Green PCR Master Mix (Takara, Kyoto, Japan). The PCR conditions were as follows: 50 °C for 2 min, 95 °C for 5 min, and 45 cycles of 95 °C for 40 s and 55 °C for 30 s. The primer sequences were as follows: β-actin sense: 5′-CTCCATCCTGGCCTCGCTGT-3′; β-actin anti-sense: 5′-GCTGTCACCTTCACCGTTCC-3′; UNC5B sense: 5′-CAGGGCAAGTTCTACGAGAT-3′; and UNC5B anti-sense: 5′-TGGTCCAGCAGGATGTGA-3′. The fold-change in UNC5B expression was normalized to β-actin and calculated using the ΔΔCT method. Experiments were performed in triplicate.
Western blot analysis
Cells were washed with pre-cooled PBS 3 times, lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with phenylmethanesulfonyl fluoride protease inhibitor cocktail and centrifuged at 12,000 rpm for 30 min. Total proteins in the supernatant were collected. The protein concentration was determined using the BCA assay (Beyotime, Shanghai, China), and the values were normalized using a standard BSA curve. Then, 60 μg of standardized protein per lane was separated by electrophoresis in an SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated at 4 °C overnight with primary antibodies against UNC5B (1:1000) (Sigma, USA), GAPDH (1:2000) (Sigma, USA), cyclin B1 (1:1000) (Abcam, Hong Kong), cyclin D1 (1:1000) (Abcam, Hong Kong) and cyclin E (1:1000) (Abcam, Hong Kong). The membranes were subsequently incubated with secondary IgG antibody (Santa Cruz Biotechnology) at 37 °C for 1 h with shaking, and the bound proteins were visualized using the EC3 Imaging System (UVP Inc., Cambridge, UK).
Immunofluorescence technique
Immunofluorescence analyses were conducted using 5637 and 5637-U cell lines cultured in 24-well plates. After the cells fusion, they were washed with PBS, permeabilized with 0.3 % Triton X-100 for 1 h, and 30 min at 37 °C of 5 % BSA. Then cells were incubated with UNC5B antibody (rabbit anti-human (1:1000)) overnight at 4 °C. After washing, the cells were incubated with TRITC-conjugated (labeled goat anti-rabbit IgG (1:200) secondary antibodies at 37 °C for 1 h in a dark place. Nuclear was stained with DAPI. Immunofluorescence images were observed utilizing an Inverted Flurescence Microscopy (Olympus, Tokyo, Japan).
Cell cycle analysis
We cultured 5637 and 5637-U cells in serum-free medium for 12 h and subsequently cultured them in RPMI-1640 with 5 % FBS for an additional 24 h. Next, the cells were harvested, washed once with PBS, slowly combined with 75 % ice-cold ethanol and incubated overnight at 4 °C. The cells were then centrifuged at 1200 g, resuspended in PI/RNase Staining Buffer (Becton Dickinson Biosciences, San Jose, CA) and incubated for 30 min at 4 °C. The data of flow cytometry were analyzed using CellQuest Pro and ModFit software (Becton Dickinson Biosciences, San Jose, CA).
Cell proliferation and wound healing assays
We used the Cell Counting Kit-8 (Beyotime, Shanghai, China) assay to compare the growth of 5637 and 5637-U cells. The two cell lines were plated at a density of 4.0 × 103 cells per well in 96-well plates, and OD values were measured on each one of the 7 days. The experiments were performed according to the manufacturer’s protocol. 5637 and 5637-U cells were plated at a density of 1 × 105 cells/well in 24-well plates and incubated in RPMI-1640 containing 10 % FBS for 24 h until they reached confluence. A wound was created in the adherent cells using a 200-μl pipette tip, followed by incubation with serum-free RPMI-1640 medium for 24 h. The changes in wound area were analyzed using an inverted microscope.
In vivo mouse models of bladder cancer
Animal experiments were formally approved by the China Medical University Ethics Committee. Four-week-old female SPF/VAF nude mice weighing approximately 13 g were purchased from Vitalriver China. Stably transfected cells (5637-U) or normal cells (5637) (1 × 105cells in 170 μl of RPMI1640 with 10 % FBS) were injected into the armpit or rear flank of nude mice to form implanted tumors. Tumor growth was monitored approximately every 2 days. At 47 days after injection, the mice were sacrificed, and the specimens (tumor or liver) were harvested for measurements and immunohistochemical analysis. The resected specimens were rinsed with PBS and fixed with 4 % formalin overnight.
Immunohistochemistry staining
Fresh tissues harvested from mice were fixed with 4 % formalin for a minimum of 24 h. Next, the tissues were embedded in paraffin, sectioned and transferred to glass slides. Whole-mount immunostaining assays were conducted by incubating the slides with antibodies against UNC5B (1:200) (Sigma, USA), followed by secondary antibodies. The samples were then stained with DAB and rinsed. Nucleus were stained with hematoxylin (Beyotime, Shanghai, China) for 5 min and rinsed with water for more than 3 h. Images were captured using an Upright Metallurgical Microscope (Olympus, Tokyo, Japan).
Statistical methods
We used the software SPSS for Windows 17.0 (SPSS Inc., Chicago, USA) for statistical analyses. Student’s t-test was used to analyze differences in UNC5B expression, cell migration and cell cycle arrest between 5637 and 5637-U cells. Analysis of variance of repeated measures was used to evaluate the growth curves of 5637 and 5637-U cells. P values <0.05 were considered statistically significant.
