Clinical specimens and cell lines
BCa tumor tissues and healthy tissues were acquired through BCa patients who underwent an operation at the first affiliated hospital of Nanjing Medical University from 2013 to 2017. Tumor tissue is the tissue pathologically diagnosed as BCa, and adjacent normal tissue is the healthy tissue without tumor which near the tumor tissue more than 5cm in the same patient. Overall, 46 paired BCa tissues were frozen in liquid nitrogen and stowed at −80°C. Utilization of tissues was granted approval by the ethics board at the hospital. Participants were asked to sign informed consent before using clinical resources. Furthermore, a group of 46 BCa patients with clinical-pathological characteristics were monitored. The monitoring period varied between 1 to 62.5 months. Follow-up period was initiated on the day of the operation and lasted until time of disease advancement. The study methodologies conformed to the standards set by the Declaration of Helsinki. The BCa lines (SV-HUC, BIU87, TCC, T24, RT4, 5637, 253J, UMUC3 and J82) were acquired through the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). And we listed a brief introduction to the background of these cell lines in Additional table 1. Cells were maintained in DMEM with 10% FBS in a 37°C incubator with 5% CO2.
The stable transfection was used in circFAM114A2 relative transfection. Lentivirus constructs containing circFAM114A2 overexpression or knockdown was acquired through OBIO (Obio Technology Corp, China). T24 and 5637 BCa cells were placed in 6-well plates. Cells were then transfected with siRNA (si circFAM114A2-1, si circFAM114A2-2), knockdown negative control (si NC), overexpression lentivirus construct (circFAM114A2) and overexpression negative control (vector) at 50% confluency. The infected cells were treated with puromycin (4μg/ml) for two weeks. The transient transfection method was used in miRNAs relative transfection. The miRNA mimics and controls utilized for transfection were bought through Ribobio (Guangzhou, China). The procedure was conducted with the Lipofectamine 3000 kit (Invitrogen, USA) as per established guidelines (27).
RNA isolation and quantitative Real Time-PCR (qRT-PCR)
RNA was extracted through cells and tissues with Trizol as per established guidelines. Relative cDNA was produced by utilizing HiScript (Vazyme, China). LightCycler 480 (Roche, USA) was used for qRT-PCR of the circRNA and miRNAs. U6 and β-actin were selected as controls for circRNA, miRNA and mRNA identification. The CT values of target RNA were normalized by subtracting the CT value of β-actin. Every experiment was conducted three times and results were assessed through comparison of CT values. PCR primers were bought through Tsingke (Beijing, China) and listed in additional table 2.
Protein extraction and Western blotting
The cells were lysed using RIPA buffer that contained protease inhibitors (Sigma, USA). Protein was collected and measured using bicinchoninic acid (BCA) assessment. Protein was isolated using 10% SDS-PAGE and moved onto polyvinylidene fluoride (PVDF) membranes. Membrane was incubated with P27 (1:5000, Abcam, USA), P21 (1:5000, Abcam, USA) or β-actin (1:5000, Proteintech, USA) primary antibody. Next, membranes were treated with peroxidase (HRP)-conjugated anti-rabbit secondary antibody (1:1000, Cell Signaling Technology, USA). After conducting washes, proteins were identified utilizing chemiluminescence and assessed with Image Lab (Bio-Rad, USA).
Evaluation of cell proliferation and cloning formation
To evaluate cell growth, transfected T24 or 5637 BCa cells were placed in 96-well plates with 2000 or 4000 cells respectively per well. At 24, 48, 72 and 96h post-seeding, cell viability was calculated through the cell counting kit-8 (CCK-8) system as per established guidelines. In summary, 10 μl CCK-8 reagents were put into each well and the plate was placed at 37 °C for 1h in the dark. Absorbance was quantified (450 nm) using a microplate reader (Tecan, Switzerland).
To evaluate colony formation, cells were placed in 6-well plates at 500 or 1000 cells/well and cultured in DMEM with 10% FBS for two weeks. Post-fixation by methanol, cells were stained using 0.1% crystal violet for 30 min. Colonies were visualized and calculated.
The transfected T24 or 5637 BCa cells were gathered and placed in 96-well plate at 5000 cells/well. After an overnight incubation, a series of dilute concentrations of cisplatin (128, 64, 50, 40, 32, 16, 8, 4, 2, and 1 µM, Sigma, USA) were added to the transfected cells for 24h. Next, viability was quantified by CCK-8 technique as per established protocol. IC50 was generated with the probit regression model (28). Analyses were replicated three times.
Evaluation of cell cycle and apoptosis
To evaluate cell cycle, transfected BCa cells were stained with the cycle test and DNA reagent kit (BD Biosciences, USA) and quantified through flow cytometry (Becton Dickinson, USA). Cell proportion in G0/1, S and G2/M phases were analyzed by Modifit software (Version 5.0). To identify apoptosis, cells were stained by annexin V-APC/7AAD apoptosis kit (eBiosciences, USA) and assessed utilizing flow cytometry. To investigate the apoptotic rate of BCa cells treated with cisplatin, BCa cells at 70% confluence were treated with 4 µmol/ml cisplatin for 24h and then collected for follow-up apoptosis testing.
Biotin-coupled miRNA capture
About 2×106 BCa cells at 50% confluence were transfected using 50 μM biotinylated miRNA mimics or nonsense control (NC) (Ribobio Guangzhou, China). After 24h, cells were collected and washed with phosphate-buffered saline (PBS) twice. Streptavidin magnetic beads (Thermo Fisher, USA) were placed in blocking buffer for 2h and put into every reaction to extract the biotin-coupled RNA compound. Tubes were placed in incubation while rotating at low speed (10rpm /min) for 4h. Beads were washed by lysis buffer five times. Then, Trizol reagent was utilized to retrieve the RNAs that interacted with miRNAs. Quantity of circFAM114A2 in bound portions was assessed by qRT-PCR and agarose gel electrophoresis.
Biotin-coupled probe pull down assay
Biotinylated probe was targeted to bind the junctional region of circFAM114A2. Oligo probe served as control. Approximately 1×107 cells were washed with pre-cooled PBS, and then lysed using lysis buffer. The lysed products were placed in incubation using 3 μg biotinylated probes at room temperature for 2h. Then, cell lysates were incubated with 50 μl streptavidin magnetic beads for an additional 4h. The beads were cleaned using lysis buffer five times and miRNAs in the pull-down material were isolated utilizing Trizol reagent and assessed with qRT-PCR assay.
Agarose gel electrophoresis
The agarose gel was made of agarose powder, 1×TAE solution and nucleic acid dye. About 2 µl DNA ladder was added to 8 µL of the PCR product, and loaded to agarose gels. The constant-pressure 200 V was set up for 20 min electrophoresis. When electrophoresis ends, the gel was viewed by ultraviolet lamp to identify the region of DNA.
Northern blot was conducted using northern blot kit. In short, approximately 30 μg of total RNA was denatured by formaldehyde and ran under electrophoresis in a 1% agarose-formaldehyde gel. Electrophoresed RNAs were moved onto a Hybond-N+nylon membrane. The biotinlabeled DNA probes were used for hybridization with relative RNAs. Biotin chromogenic detection was utilized to identify the bound RNAs. Lastly, membranes were assessed by Image Lab software (Bio Rad, USA).
Fluorescence in situ hybridization (FISH)
Cy3-labeled probes specific to circFAM114A2 and fam-labeled probes specific to miR-222-3p/miR-146a-5p were constructed and produced through Genepharma (Shanghai, China) and probe signals were identified through a Fluorescent In Situ Hybridization Kit (Genepharma, Shanghai, China) as per established protocol. Pictures were attained on a Zeiss LSM880 NLO confocal microscope system (Leica Microsystems, Germany).
Luciferase reporter assay
HEK293 cells were co-transfected with mutant or wild 3′-UTR fragments of P27 or P21 and miRNA mimics utilizing Lipofectamine3000 (Invitrogen, CA) as per established instructions. After 24h post-transfection, firefly and renilla luciferase activities were quantified consistently by utilizing dual luciferase reporter assay system (Promega, USA). Lastly, the proportion of luminescence through luciferase was measured and every experiment was conducted three times.
Paraffin-embedded tumors from mice were sliced to 4 μm slides. Tissue slides were rehydrated with different grade ethanol, and then placed in sodium citrate buffer (pH=6). Antigen was isolated utilizing heating through microwave. Slides were dipped in 3% H2O2 for 10 min and then treated with P27 or P21 antibody at 4°C overnight. After washed, the slides were incubated with HRP-conjugated antibody and standard avidin biotinylated peroxidase complex method. The images were viewed with a microscope. The degree of positivity was assessed by at least two pathologists based on the proportion of positive tumor cells.
Xenografts in mice
Approximately 1×107 cells underwent a stable transfection using si circFAM114A2, si NC, circFAM114A2 and vector were inoculated into axilla of the BALB/C nude mice (4-6 weeks old, 18-22 g, 4 mice/group) subcutaneously. We divided nude mice inoculated with si circFAM114A2 and si NC transfected cells into four groups and named them si circFAM114A2+cisplatin group, si NC+cisplatin group, si circFAM114A2+saline group and si NC+saline group. One week after inoculation, nude mice in the experimental groups were intraperitoneally injected with cisplatin (5mg/kg) every three days, while the control groups were injected with the same volume of saline. Tumor evolution was followed each week, and the width (W) and length (L) were quantified utilizing calipers. The volume (V) of tumor was measured utilizing the formula V= (W2×L)/2. After four weeks, the mice were sacrificed and tumor bulk was quantified. Animal experiments were conducted according to ethics guidelines for animal studies and granted approval through the animal ethics board of Nanjing Medical University.
Results were assessed utilizing SPSS version 22.0 and depicted as mean±standard deviation. The P value was statistically significant when less than 0.05. Two-tailed Student’s t-test and one-way ANOVA was conducted to assess variation among the groups. Correlation was assessed through Pearson’s correlation and Spearman’s rank correlation test. Survival curves were imaged utilizing the Kaplan-Meier method, and variation was assessed through log-rank test.