Cell culture and lentivirus infection
EJ and T24 human bladder cancer cell lines were purchased from Genechem (Shanghai, China). J82 and RT4 were obtained from the BeNa Technology (Hangzhou, Zhejiang, China). EJ, RT4 and T24 cell lines were cultured in RPMI-1640 medium (Gibco, Rockville, MD, USA) containing 10% FBS (Gibco, Rockville, MD, USA) at 37°C with 5% CO2. J82 were cultured in 90% EME medium (Gibco, Rockville, MD, USA) with 10% FBS additive.
For lentivirus infection, the EJ, RT4, J82 and T24 cells in the logarithmic growth phase were washed with PBS, adjusted to a cell density of 2 × 105 cells/mL, and re-inoculated into a 6 well dish, lentivirus (1×107 TU/mL, 400 μL) were used to infect the cells for 72 h. Infection results were observed by microscope and successful expression cell lines were selected (EJ and T24) with a high (> 80%) cell infection efficiency.
Tissue microarray and immunohistochemistry analysis
Total of 105 formalin fixation bladder cancer and para-carcinoma tissues microarray was obtained from Shanghai Outdo Biotech Company (Shanghai, China). Tumor information of patients with bladder cancer and other related characteristics were collected as well. Written informed consents were collected from all participants. Our study was approved by the Ethics Committee of General Hospital of Northern Theater Command.
Formalin fixation bladder cancer and para-carcinoma tissues microarray was dewaxed and hydrated. After washing, antigen retrieval was accomplished by citric acid buffer heating at 110°C. The chip was washed three times with PBS and blocked with 3% hydrogen peroxide. Sections were incubated with ZNF280A primary antibody (1:400, Bioss, Beijing, China) overnight at 4°C and subsequently incubated with HRP-conjugated secondary antibody (Abcam, Cambridge, MA, USA) for 2 h at room temperature. 3,3-diaminobenzidine (DAB) were used for coloring and counterstained with hematoxylin, then dehydrated and sealed with cover slips. ZNF280A expression was observed with ImageScope and CaseViewer then quantified for analysis with IHC scores. Scoring standard for ZNF280A was graded as 0-4 (negative to +++positive). The staining extent was graded as 0 (0%), 1 (1-25%), 2 (26-50%), 3 (51-75%), or 4 (76-100%). The staining intensity varied from weak to strong. Specimens were classified based on the sum of the staining intensity and staining extent scores.
Plasmid construction and lentivirus infection
shRNAs targeting ZNF280A and its flanking control sequence were designed by Shanghai Bioscienceres (Shanghai, China) and cloned in BR-V-108 vector and transformed into E. coli competent cells (Tiangen, Beijing, China). PCR was used to confirm and sequence the plasmids and the target shRNA sequence was identified as 5’-CTGTCACTATGAAGTCTTCAT-3’. The plasmid was purified by EndoFree Plasmid Mega Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instructions and the qualified plasmids were packaged in lentivirus production. Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used for cell transfection. The transfected cells were screened under Puromycin (Takara Bio, Otsu, Japan) and verified through observing fluorescence of GFP by a fluorescence microscope (Olympus, Tokyo, Japan).
Western blotting assay
T24 and EJ cells were lysed in ice-cold RIPA buffer (Millipore, Temecula, CA, USA) and the lysates were cleared with centrifuging and the total protein concentration was detected by BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The proteins were separated by 10% SDS-PAGE (Invitrogen, Carlsbad, CA, USA) with 20 µg in each lane, and were transferred onto PVDF membranes. Then the membranes were incubated with anti-ZNF280A (1:1000, Abcam, Cambridge, MA, USA), anti-GAPDH (1:3000, Bioworld, St. Louis, MN, USA), anti-MAPK9 (1:1000, Abcam, Cambridge, MA, USA), anti-PIK3CA (1:1000, Abcam, Cambridge, MA, USA), and anti-Cyclin D1 (1:2000, CST, Danvers, MA, USA) overnight at 4°C on a rocker. Afterward, the membranes were subsequently inculated with corresponding secondary antibodies (Beyotime, Beijing, China) at room temperature for 30 min. The blots were visualized by enhanced chemiluminescence (ECL).
Total RNA was extracted with TRIzol reagent (Sigma, St. Louis, MO, USA) from cells and the quality of total RNA was evaluated by Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The reversely transcribe of cDNA was performed using the Hiscript QRT supermix kit (Vazyme, Nangjing, Jiangsu, China). The expression of mRNA was examined by qRT-PCR with SYBR Green mastermixs Kit (Vazyme, Nangjing, Jiangsu, China) and Applied Biosystems 7500 Sequence Detection system. GAPDH was used as inner control and the amplification results for qRT-PCR were calculated using the 2-ΔΔCt method. The PCR cycling conditions were 95°C for 6 min, 40 cycles of 95°C for 5 s, 60°C for 30 s, 95°C for 15 s, 70°C for 5 min. Primers used for PCR were shown as below:
ZNF280A forward, 5’-GATCTGATCTATGTTGGGGTGGA-3’;
ZNF280A reverse, 5’-CGTGAGCAGGATATTGACGGA-3’;
GAPDH forward 5’-TGACTTCAACAGCGACACCCA-3’;
GAPDH reverse 5’-CACCCTGTTGCTGTAGCCAAA-3’.
Cell viability was assessed using MTT assay. T24 and EJ cells (2×103 cells/well) were seeded in a 96-well plate and incubated at 37°C for 120 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT, 20 µL) was added into each well and incubated for another 4 h at 37°C. Afterwards, the culture medium was removed and 100 µL DMSO was added and the cells were incubated at room temperature in the dark for 20 min. The absorbance was measured using a microplate spectrophotometer (Bio-Tek instruments, Winooski, VT, USA) at 490/570 nm and the cell viability was calculated.
Flow cytometry for cell apoptosis and cycle
EJ and T24 cells groups were inoculated in 6-well plates at a seeding density of 1 × 103 cells/mL (2 mL/well) in triplicate and further cultured for 5 days. The cells were harvested with centrifuge (1000 × g), washed with 4°C cold D-Hanks and resuspended with binding buffer, then 5 μL Annexin V-APC (eBioscience, San Diego, CA, USA) was added for staining without light. The percentage of apoptotic cells was measured using FACScan (Millipore, Schwalbach, Germany) to assess the apoptotic rate.
For cell cycle detection, EJ and T24 cells were seeded in a 6-well plate (5 mL/well). After 5 days of culture, cells were stained by PI (Sigma, St Louis, MO, USA) and cell cycle distribution was detected by flow cytometry and observed using micropublisher.
Wound healing assay
Lentivirus infected EJ and T24 cells (5×104 cells/well) were plated onto a 96-well dish and grew until 90% confluence. Scratch was made by a pipette tip across the cell layer, the floating cells were washed and cultured. Photograph were taken by fluorescence micrograph at the time point (0 h, 4 h and 8 h). Cell migration rate of each group were calculated based on the randomly selected areas in each well.
Cell transwell assay were operated by Corning Transwell Kit (Corning, NT, USA) and was done in triplicate and repeated three times. EJ and T24 exponentially growing cells were trypsinized, counted and incubated in the upper chamber with 100 μL of medium (5×104 cells/well) in 24-well plate. 600 μL medium supplemented with 30% FBS was added in the lower chamber. Cells were incubated for 24 h at 37°C with 5% CO2 and non-metastatic cell were removed with a cotton swab. Cells were fixed by 4% formaldehyde and 400 µL Giemsa were added for staining and the migration ability of cells were analyzed. Experiment result was repeated three times.
Human Apoptosis Antibody Array
Human Apoptosis Antibody Array (R&D Systems, Minneapolis, MN, USA) was performed in T24 cells transfected with ZNF280A or NC following the manufacturer’s instructions. Briefly, lentivirus infected EJ and T24 cells were collected and washed and then lysed by lysis buffer. Total protein was extracted and diluted with Array Diluent Buffer. Each array antibody membrane was blocked and then incubated with protein samples (0.5 mg/mL) overnight at 4°C and then HRP linked Streptavidin was added to the membranes. The spots were visualized by chemiluminescence and the signal densities were analyzed with ImageJ software (National Institute of Health, Bethesda, MD, USA).
In vivo tumorigenicity assay
Our animal study was reviewed and approved by the Institutional Animal Care and Use Committee of General Hospital of Northern Theater Command. 4-week-old BALB/c female nude mice were purchased from Shanghai Lingchang Experimental Animals Co., Ltd (Shanghai, China) and randomly divided into shZNF280A group and shCtrl group, with 5 mice in each group. 0.2 mL T24 cells (2.5 × 107 cells/mL) suspension was injected into the mice for the construction of tumor model. The tumor size was observed and recorded every 2 days (volume of tumor = π/6×L×W×W, L represent longest dimension and W means dimension perpendicular to length). Mice were sacrificed 25 days post injection and tumor weight was measured. The removed tissues were stored at -80°C for subsequent measurement.
Tumor tissues were fixed with formalin and embedded using paraffin, then 2 μm sections were immersed in xylene and 100% ethanol for deparaffinization and rehydration, then blocked with PBS-H2O2 and washed. Then Ki-67 primary antibody (1/200) was added and incubated with all slides at 4°C overnight, following incubated with 1:400 HRP-conjugated secondary antibody. Slides were stained by Hematoxylin and Eosin. Stained slides were examined with a microscopic.
The statistical analysis was performed using SPSS 17 (IBM, SPSS, Chicago, IL, USA) and GraphPad Prism 5.0 (Graphpad Software, La Jolla, CA). Continuous variables were shown as the mean ± SD from and Student’s T-Test was used to analyze the statistical significance. All experiments were repeated at least 3 times. P<0.05 was considered statistically significant. Chi-square analysis was utilized to analyze the expression difference of ZNF280A gene in bladder cancer tissues and para-carcinoma tissues. Mann-Whitney U analysis and Spearman Rank correlation analysis were used while analyzing the relationship between ZNF280A expression and tumor characteristics in patients with bladder cancer.