Patient samples and cell lines
Tissues used in this research were acquired from BCa patients who underwent radical surgery at the first affiliated hospital of Nanjing Medical University. 48 pairs of BCa samples and adjacent normal tissues were included between 2014 to 2019. All human-related tissues for this study were approved by the Ethics Committee of The First Affiliated Hospital of Nanjing Medical University. All patients signed the informed consent before any clinical materials were collected. Liquid nitrogen was used to preserve samples.
Cell culture and Transfection
T24 and BIU87 BCa cells were acquired from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). T24 and BIU87 cells were respectively cultured in DMEM medium (Gibco, USA) and RPMI 1640 basic medium (Gibco, USA), containing 10% fetal bovine serum (BI, Israel) and 1% penicillin–streptomycin (Gibco, USA). Cells were maintained at 37°C in a humidified incubator containing 5% CO2.
CircZNF609 knockdown or overexpression lentiviral vectors were purchased from HANBIO (HANBIO, Shanghai, China). When T24 or BIU87 cells reached 50% or 60% confluence in six-wells plates, they were infected with circZNF609 overexpression lentivirus (termed as circZNF609), negative control lentivirus (termed as vector), circZNF609 knockdown lentivirus (termed as si circZNF609-1, si circZNF609-2) and scramble control lentivirus (termed as si NC), respectively. Puromycin were used for 2 times to select stable transfections.
MiR-1200 mimics and controls were achieved from GenePharma (Shanghai, China). The transfection was conducted using the Lipofectamine 3000 kit (Invitrogen, USA) according to the manufacturer’s guidelines.
RNA isolation, quantitative Real Time-PCR (qRT-PCR)
Total RNAs were isolated from tissues and cells using Trizol reagent (Invitrogen, USA) in accordance with the manufacturer’s protocol. cDNA was synthesized using HiScript II (Vazyme, China). The circRNA, miRNA and mRNA qRT-PCR assays were conducted on StepOne Plus Real-Time PCR system (Applied Biosystems, USA) or LightCycler 480 (Roche, USA). U6 or β-actin were used as controls. Data were analyzed by comparing CT values and each experiment was repeated for three times. All PCR primers were gained from TsingKe (TsingKe, Nanjing, China) and listed in Table S1.
Protein extraction and western blot
RIPA buffer (Sigma, USA) was used to lyse the tissues or cells. Protein extractions were absorbed and then quantified by bicinchoninic acid (BCA) assessment (Beyotime, China). Protein was isolated and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA) using 10% SDS-PAGE. After blocking with 5% skim milk, membranes were incubated with primary (Protech, USA) and secondary antibodies (Protech, USA). Chemiluminescence (Bio-Rad, USA) was used to identify signals , which were then evaluated by Image Lab Software.
RNase R treatment
T24 and BIU87 cells were incubated for 30 min at 37 °C with RNase R (0.2 μl/μg, Epicenter). Total RNA were then extracted, reverse transcribed and detected by qRT-PCR assay.
Actinomycin D treatment
When T24 or BIU87 cells reached 60%-70% confluence in six-wells plates, they were incubated for 2 h, 4 h, 6 h, 8 h and 10 h, respectively, with 1 μg/ml actinomycin D (Abcam, UK). The relative RNA expression of ZNF609 in the liner or circular form was then quantified by qRT-PCR.
Cell proliferation assay
To determine the proliferation ability of T24 or BIU87 cells, they were seeded in 96-well plates with starting number of 2000 or 5000 cells, respectively. Cell viability was determined every 24 h (24 h, 48 h, 72 h and 96 h) using the cell counting kit-8 (CCK-8) technique (Dojindo, Japan). The absorbance value at 450 nm was determined using a microplate reader (Tecan, Switzerland).
Cloning formation
T24 or BIU87 BCa cells were seeded at 1000 or 1500 cells per well in six-well plates. Two weeks later, cell colonies were fixed with 4% paraformaldehyde and stained by 0.1% crystal violet. Image J software was used to visualize and calculate cell colonies.
Scratch wound healing assay
When T24 or BIU87 cells reached 90%-100% confluence in six-wells plates, the monolayer cells were scraped off with the 200 μl pipette tip and cultured in serum-free medium. Cells were imaged at 24 h and 48 h, respectively, using microscope (Olympus, Japan). As a control, images of BCa cells at 0 h were also obtained. The migration rate was determined using a formula based on the distance traveled by BCa cells divided by the initial width.
Transwell assays
To examine the migration ability, 200 µl serum-free medium with 4×104 T24 or 6×104 BIU87 cells was seeded in upper chamber (Corning, USA), while 600 µl culture medium with 20% FBS was added into the lower chamber. After 24 h, the top chambers were fixed with 4% paraformaldehyde and then stained for 15 min with 0.5% crystal violet. Finally, the transwell chambers were photographed using Olympus microscope.
Evaluation of cell cycle
To detect distinct phases of cell cycle, BCa cells were fixed in 75% ethanol and dyed with the cycle test and DNA reagent kit (BD Biosciences, USA). Then using flow cytometry (Beckman Coulter, USA), cell proportion in various phases were enumerated and analyzed via ModFit software (Version 5.0).
IC50 determination
After an overnight incubation of 5000 BCa cells per well in a 96-well plate, various concentration gradients of cisplatin (128, 64, 50, 40, 32, 16, 8, 4, 2, and 1 µM, TCA, Japan) were added. After 24 h incubation, the cell viability was determined by CCK-8 method and IC50 value was obtained by the probit regression model.
RNA-fluorescence in situ hybridization (FISH) assay
Cy3-labeled circZNF609 and FAM-labeled miR-1200 probes were achieved from Genepharma (Shanghai, China). The T24 cells were stained by relative probes utilizing Fluorescent in Situ Hybridization kit (Genepharma, Shanghai, China). All images were captured from Zeiss LSM880 NLO confocal microscope system (Leica Microsystems, Germany).
Biotin-coupled probe pull-down assay
Biotinylated circZNF609 and miR-1200 (GenePharma, China) pull down assays were conducted. Oligo probe was used as control. A total of 107 cells were collected and lysed using lysis buffer. All lysed products were incubated at room temperature for 2 h with 3 μg biotinylated probes. Following that, streptavidin magnetic beads (50 μl) were incubated with the products generated in the previous step for 4 h. The RNA complexes attached to the beads were evaluated by qRT-PCR assay, after being washed 5 times with lysis buffer and isolated using Trizol reagent.
Luciferase reporter assay
293T cells were cultured in 96-well plates and co-transfected using Lipofectamine3000 (Invitrogen, Foster city, CA) with miR-1200 mimics and plasmids expressing wild-type or mutant fragments. After 24 h incubation, firefly and renilla luciferase activities were measured utilizing dual luciferase reporter assay system (Promega, USA).
Xenograft in nude mice
Approximately 1×107 T24 cells transfected with sh circZNF609, circZNF609 over expression or their negative control were subcutaneously injected into axilla of the athymic BALB/C nude mice (4-6 weeks old, 18-22 g, five mice/group). Mice in experimental groups received intraperitoneal injections of cisplatin (2.5 mg/kg) every three days for one week following inoculation, while mice in control groups received saline injections. The volume (V) was determined every week by measuring the width (W) and length (L) using calipers, and the formula V= (W2 × L)/2 was used. After 4 weeks, we euthanized the mice and quantified the tumor bulk. All animal experiments were performed according to the ethics guidelines for animal experiments, which were authorized by the animal management committee of Nanjing Medical University.
Immunohistochemistry (IHC)
Mouse tumor was fixed in paraffin and mounted on 4 μm slide each. Tumor slides were rehydrated by different concentrations of ethanol. Antigen was separated by microwave heating. The slides were soaked in 3% H2O2 for 10 min and then dealt with CDC25B antibody (1: 500, Proteintech, USA) in a humidified incubator at 4°C overnight. Then slides were incubated with HRP-conjugated antibody at room temperature for 30 min. The data were collected by a microscope.
Statistical analysis
Data were analyzed using SPSS version 22.0 (IBM Corp., Armonk, NY, USA) and were presented as mean ± standard deviation (means ± SD). Student’s t-test and one-way ANOVA were performed to analyze differences among the groups. Survival curves were plotted using the Kaplan-Meier method, and differences were analyzed by the log-rank test. P values <0.05 were considered statistically significant.