Clinical sample acquisition

From January 2017 to August 2022, bladder cancer patients undergoing radical cystectomy or transurethral resection of bladder tumors in the Department of Urology at Xiangya Hospital, Central South University had bladder cancer tissues and adjacent noncancerous tissues collected intraoperatively. However, patients with history of other malignant tumors, severe systemic diseases, recent antitumor therapy, severe infections, or those unwilling or unable to sign informed consent forms were excluded. All specimens were promptly placed in liquid nitrogen for cryopreservation or fixed in formalin. The acquisition and utilization of tissues have obtained approval from the Ethics Committee of Xiangya Hospital, Central South University. Prior to sample collection, patients had signed relevant written informed consent forms.

Cell culture

Human normal urothelial cells (SVHUC1) and bladder cancer cell lines (T24, UMUC3, 5637) were cultured in a cell culture incubator at 37 °C with 5% CO2. The cell lines were obtained from the American Type Culture Collection (ATCC). SVHUC1 was cultured in Ham’s F-12K medium (Shanghai BasalMedia Technologies Co., Ltd., Shanghai, China), T24 and 5637 were cultured in RPMI-1640 medium (Shanghai BasalMedia Technologies Co., Ltd., Shanghai, China), and UMUC3 was cultured in MEM medium containing NEAA (Shanghai BasalMedia Technologies Co., Ltd., Shanghai, China). All culture media were supplemented with 10% fetal bovine serum (ExCell Bio, Shanghai, China) and 1% penicillin–streptomycin solution (NCM Biotech, Suzhou, China).

Cell lentiviral transfection

Lentiviruses overexpressing or knocking down hsa_circ_0000520 and Lin28a, as well as control lentiviruses, were purchased from Genechem in Shanghai, China. Initially, T24 or UMUC3 cells were seeded in six-well plates. When the cell density reached 20–30%, the supernatant was aspirated from each well. Then, 860 μl of complete culture medium, 40 μl of transfection enhancer reagent HiTransG A (Genechem, Shanghai, China), and 100 μl of virus solution at a concentration of 1 × 107 TU/ml were added. The mixture was thoroughly mixed and incubated further. After 16 h of incubation at 37 °C, the culture medium containing lentivirus was aspirated, and fresh complete medium was added for continued cultivation. After 72 h of transfection, stable cell lines were established by selecting with complete medium containing 2 μg/ml puromycin for 5 days.

Genomic DNA (gDNA) extraction

T24 or UMUC3 cells were seeded in a six-well plate, and when the cell confluence reached 90%, genomic DNA extraction was performed following the operational instructions of the SteadyPure Universal Genomic DNA Extraction Kit (Accurate Biology, Hunan, China).

Quantitative real-time polymerase chain reaction (qRT-PCR)

For cells and tissues, RNA extraction was performed according to the instructions of the RNA extraction kit, Evo M-MLV Reverse Transcription PreMix kit, and SYBR® Green Pro Taq HS PreMix qPCR kit (Accurate Biology, Hunan, China). The program was as follows: pre-denaturation at 95 °C for 30 s; PCR reaction: 95 °C for 5 s, 60 °C for 30 s. After the reaction, the amplification curve and melting curve were examined for quality assurance. The housekeeping gene β-actin was chosen as the reference, and the relative expression of the target gene was calculated using the 2−ΔΔCt method.

Western blot (WB)

Cells were seeded in a six-well plate and cultured until reaching 90% confluence. The cells were then lysed by adding a lysis buffer containing 1% protease inhibitor and phosphatase inhibitor (NCM Biotech, Suzhou, China). Moreover, 5× loading buffer (NCM Biotech, Suzhou, China) was added to the protein lysate, followed by boiling at 100 °C for 10 min. Subsequently, constant voltage electrophoresis, constant current transfer to a membrane, blocking, primary and secondary antibody incubation were performed, and the results were visualized using the Amersham ImageQuant 800 highly sensitive multifunctional imager.

RNase R experiment

T24 or UMUC3 cells were seeded in a six-well plate and cultured until reaching 90% confluence. Total RNA was extracted, and its concentration was measured. According to the Epicentre Cat#RNR07250 kit instructions, the total RNA was divided into two portions. One portion was treated with RNase R, and the other portion, serving as a control, underwent an identical reaction system but lacking RNase R. The reaction system included 5 μg of total RNA, 2 μl of 10 × Reaction Buffer, 1 μl of RNase R, and nuclease-free water to make up the total volume to 20 μl. After thorough mixing, the reactions were carried out at 37 °C for 20 min and 80 °C for 10 min. The obtained samples were then subjected to subsequent reverse transcription and qPCR.

Nucleocytoplasmic separation experiment

T24 or UMUC3 cells were seeded in T25 culture flasks and cultured until reaching 90% confluence. Following the instructions of the Norgen Biotek Cytoplasmic & Nuclear RNA Purification Kit, RNA samples were purified and stored at −80 °C for long-term preservation.

Cell wound experiment

To begin with, three horizontal lines were uniformly drawn on the back of a six-well plate using a black marker. T24 or UMUC3 cells were then seeded into the plate, and upon reaching approximately 70% confluence, the complete culture medium was replaced with serum-free medium. After 12 h of incubation, a 200 μl pipette tip was used to create a vertical scratch on the cell surface, perpendicular to the lines previously drawn on the back of the plate. The scratches were observed and photographed under a microscope at 0 h and 24 h. The distance between cells was measured using ImageJ.

Transwell experimentCell migration assay

We added 600 μl of cell culture medium containing 10% FBS to a 24-well plate. Using tweezers, the Transwell chamber was transferred to the aforementioned 24-well plate with added medium. We added 200 μl of cells diluted to an appropriate concentration to the upper chamber of the Transwell and incubated for 24 h in a cell culture incubator. After fixation with 4% paraformaldehyde, crystal violet staining, and PBS washing, we observed and captured images under a microscope.

Cell invasion assay

We uniformly added 50 μl of diluted Matrigel to the bottom membrane of the Transwell chamber to form a thin film. We added 600 μl of cell culture medium containing 10% FBS to a 24-well plate. Using tweezers, the Transwell chamber was transferred to the 24-well plate with added medium. We added 200 μl of cells diluted to an appropriate concentration to the upper chamber of the Transwell and incubated for 24 h in a cell culture incubator. After fixation with 4% paraformaldehyde, crystal violet staining, and PBS washing, we observed and captured images under a microscope.

Vasculogenic mimicry formation experiment

We added 50 μl of Matrigel evenly and vertically to a 96-well plate and incubated at 37 °C for 1 h to allow gel formation. We slowly added 50 μl of diluted cell suspension to the 96-well plate with Matrigel and incubated in a 37 °C cell culture incubator for approximately 12 h, then observed and captured images under a microscope.

Fluorescence in situ hybridization (FISH)

We designed and synthesized specific probes based on the gene information of hsa_circ_0000520 and PTEN mRNA. Slides of T24 and UMUC3 cell smears or Xiangya bladder cancer tissue cores were prepared, fixed with 4% paraformaldehyde for 20 min, and digested with proteinase K (20 μg/ml), then hybridized overnight with hsa-circ-0000520 or PTEN mRNA probes at a concentration of 6 ng/μl. We incubated with anti-digoxin-HRP antibody on slides, stained with DAPI, and observed and captured images under a fluorescence microscope.

RNA pulldown experiment

The RNA pulldown experiment was conducted according to the Thermo Magnetic RNA–Protein Pulldown Kit instructions. We designed biotin-labeled 5′ oligonucleotide probes targeting the hsa_circ_0000520 binding site. We captured biotinylated hsa_circ_0000520 with streptavidin magnetic beads and incubated with cell lysates overnight at 4 °C. On the following day, washing and elution steps were performed. We analyzed the obtained products using mass spectrometry and validated with Western blot.

RNA binding protein immunoprecipitation (RIP)

RIP experiments were conducted following the instructions of the Millipore Magna RIP RNA Binding Protein Immunoprecipitation Kit. We prepared T24 or UMUC3 cell lysates and incubated with 5 µg of the target protein antibody overnight at 4 °C. After treatment with proteinase K buffer, we extracted RNA and used qRT-PCR to detect RNA bound to the target protein.

Cycloheximide (CHX) experiment

T24 or UMUC3 cells were seeded in a six-well plate until reaching 60% confluence. We added 10 μg/ml cycloheximide at 0 h, 4 h, 8 h, and 12 h to inhibit protein synthesis, extracted cell proteins, and performed Western blot to detect the expression of the target protein.

Actinomycin d (ACTD) experiment

T24 or UMUC3 cells were seeded in a six-well plate until reaching 60% confluence. We added 5 μg/ml actinomycin d at 0 h, 4 h, 8 h, and 12 h to inhibit RNA synthesis, extracted total RNA from cells, and used qRT-PCR to detect the relative residual amount (%) of the target gene.

Animal modelsBladder orthotopic tumor model

We introduced cells with luciferase using lentivirus into the desired cell line for stable expression of the luciferase gene. A bladder orthotopic tumor model was constructed using 24 female nude mice aged 6–8 weeks. We administered continuous isoflurane anesthesia and applied artificial tears to protect the eyes. We sterilized the experimental platform, fixed the anesthetized nude mice on the platform with medical tape, and disinfected. A midline incision was made in the lower abdomen to expose the bladder. A syringe was used to draw 30 μl of pretreated 50% Matrigel mixed with 50% serum-free medium containing 1 × 106 T24 cells. We gently lifted the bladder wall with the syringe and slowly injected the cell suspension into the bladder wall. Successful injection was confirmed by a clear boundary between urine in the bladder and the cell suspension in the bladder wall. The abdomen was closed and the mouse was observed until it recovered from anesthesia. We injected 150 mg/kg d-luciferin into the abdominal cavity weekly and monitored tumor progression using an In Vivo Imaging System (IVIS). The mice were sacrificed after 8 weeks, the number and location of metastatic foci were recorded, and bladder orthotopic tumors were collected for further experiments.

Tail vein lung metastasis model

We introduced cells with luciferase using lentivirus into the desired cell line for stable expression of the luciferase gene. A tail vein lung metastasis model was established using 24 female nude mice aged 6–8 weeks. We used a mouse restrainer to immobilize the nude mice, disinfected the tail with alcohol, and used a syringe to inject 1 × 106 cells/200 μl cell suspension prepared in PBS into the tail vein. We injected 150 mg/kg d-luciferin into the abdominal cavity weekly and monitored lung metastasis using an In Vivo Imaging System (IVIS). The mice were sacrificed after 8 weeks, and we recorded the number of tumors and collected lung tissues for further experiments.

Statistical methods

GraphPad Prism 8 and SPSS 26.0 were used for statistical analysis, and experiments were repeated at least three times. Count data were analyzed using the chi-square test or Fisher’s exact probability method. Measurement data are presented as mean ± standard deviation (x ± s). Independent-sample t-test was used for comparing differences between two groups, and one-way analysis of variance (ANOVA) was used for comparing differences among three groups or more. Survival data were analyzed using the log-rank test and Kaplan–Meier survival analysis. A two-tailed P-value < 0.05 indicated a statistically significant difference.