2.1 Ethical approval

This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of The First Affiliated Hospital, Zhejiang University School of Medicine for the utilization of clinical samples with patient consent as well as for the xenograft tumorigenesis model. All operations and experimental protocols were carried out as per NIH laboratory instructions. File S1 illustrates the comprehensive details regarding the surgical samples.

2.2 RNA immunoprecipitation (RIP)

An RIP kit from Geneseed (Guangzhou) was utilized for the RIP assay following the guidelines provided by the manufacturer. In summary, a volume of 100µL of cell lysates was reserved for the input control. To form the antibody-bead complex, magnetic Protein A + G beads, which had been pretreated, were incubated with specific antibodies and 5 µg of IgG at a temperature of 4 °C, with rotation occurring at 10 rpm for 120 min. Upon completion of the reaction, the supernatant was extracted utilizing a magnetic frame. The RNA complexes that adhered to the beads were subsequently rinsed, allowing for RNA extraction for further assessments.

2.3 CUT&tag

The CUT&Tag assay was carried out utilizing the Hyperactive In-Situ ChIP Library Prep Kit for Illumina, following the guidelines provided by the manufacturer. In summary, magnetic beads coated with concanavalin A (ConA beads) were introduced to the resuspended cells and allowed to incubate at room temperature for cell binding. The nonionic detergent digitonin facilitated the permeation of the cell membrane. The cells attached by the ConA beads were then incubated with a secondary antibody, Hyperactive pA-Tn5 Transposase, and the H3K18la antibody (PTM-1427RM, PTM BIO, Hangzhou, China). This specific interaction enabled the hyperactive pA-Tn5 transposase to effectively cleave the DNA fragments associated with the target protein. Additionally, the cleaved DNA fragments were joined with P5 and P7 adaptors by means of Tn5 transposase. Subsequently, the amplification of libraries was conducted by means of PCR with P5 and P7 primers. Agilent Technologies’ 2100 Bioanalyzer was used to examine the purified PCR products that were obtained. Lastly, these libraries were subjected to sequencing on the Illumina NovaSeq6000 platform, yielding paired-end reads of 150 bp for further examination. Files S4-5 illustrate the findings.

2.4 RNA fluorescence in situ hybridization (FISH)

The synthesis of the circBARD1 probe labeled with Cy3 was conducted, and a FISH Detection Kit from RiboBio (PR China) was utilized following the provided instructions. DAPI was utilized to stain the cell nuclei. Observations were recorded via a microscope (Olympus, Tokyo, Japan). Specifics regarding the probe utilized in this research can be found in Supplementary file S2.

2.5 Glycolytic process assessment

To assess the state of glycolysis, we utilized 2-NBDG along with Colorimetric Assay Kits for Glucose Uptake (Biovision, USA) to evaluate the capacity for glucose absorption, and lactate colorimetric assay kits (Biovision, USA) were utilized to quantify lactate production. All experiments were conducted following the instructions outlined in the manual.

2.6 Cell lines and culture

The Chinese Academy of Sciences provided the cell lines utilized in this study. The following cell lines were cultured: J82, 5637, T24, and EJ in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin Solution (P/S); SV-HUC-1 in F-12 K medium with 10% FBS and 1% P/S; and TCCSUP in MEM medium with 10% FBS and 1% P/S. A controlled environment with 5% CO2 was used to maintain all cultures at 37 °C.

2.7 RNA isolation and quantitative real-time PCR analysis

The RNA isolation reagent NucleoZOL® (MACHEREY-NAGEL, Germany) was employed to isolate total RNA. The PrimeScriptTM RT Reagent Kit (Perfect Real Time) was used for reverse transcription, and a QUANT5 Studio system from Applied Biosystems was used for polymerase chain reaction (PCR). The endogenous control, GAPDH, was utilized. A 10-s denaturation at 95 °C, a 20-s annealing at 72 °C, and a 20-s extension at 60 °C were the steps in the amplification technique, which was performed 40 times. To find the relative gene expression, the 2-ΔΔCt technique was used to examine the Ct data. File S2 provides the primers utilized in this research.

2.8 SiRNA, plasmid, and lentivirus

siRNAs were sourced from RiboBio (Guangzhou, China). The transfection of siRNAs into cells was performed using Lipofectamine RNAiMAX (Invitrogen). In order to enhance circBARD1 expression in BCa cells, full-length circBARD1 cDNA was designed and then inserted into the pcD-ciR vector. Subsequently, the plasmids were introduced into cells using Lipofectamine 3000 (Invitrogen). For experiments involving lentivirus, cells were subjected to puromycin for 7 days to confirm transfection efficiency. File S2 provides the sequences of the siRNAs utilized in this research.

2.9 Chromatin Immunoprecipitation (ChIP)

In our study, we utilized a SimpleChIP® Plus Enzymatic Chromatin IP Kit (CST, #9004, USA) following the manufacturer’s protocols to examine the relationship between H3K18la and the CCNA2 promoter region.

2.10 Protein Immunoprecipitation (IP)

The IP trial was conducted following the manufacturer’s guidelines via the Absin Co-IP kit (Absin, ab955, China). In summary, 1 × 107 cells were gathered and rinsed with PBS. Following a 15-minute lysis period, the supernatant was mixed with specific antibodies and incubated overnight at 4 °C while being rotated. The following day, 5µL each of Proteins A and G were introduced, and the mixture was rotated for 2 h at 4 °C before being spun at 12,000×g for 1 min. Lastly, the pellet was preserved for subsequent experiments.

2.11 CCK-8 assay

To assess cell viability, we utilized the Cell Counting Kit-8 (APEBIO, USA), following the guidance provided by the manufacturer. In brief, 1 × 104 pretreated cells were cultivated in 96-well plates, subsequent to enumeration, with each well receiving 100 µL of medium. On the following day, 10µL of CCK-8 solution was administered to every well, and the incubation of cells was conducted for 2 h at 37 °C. Ultimately, absorbance was recorded at 450 nm via a SpectraMax i3x reader. Prior to calculating the relative cell viability, the subtraction of absorbance readings from the blank control well was conducted.

2.12 Colony formation assay

Two thousand cells were cultivated in 12-well plates to facilitate colony formation. Crystal violet staining and fixation with 4% paraformaldehyde followed a week of incubation. The colonies were subsequently counted and photographed.

2.13 Transwell migratory assay

T24 cells (3 × 10⁴) were evenly cultivated into the top chamber and left to incubate overnight in order to evaluate cell migration. To encourage migration, 500-µL of complete medium containing 10% FBS was subsequently introduced to the bottom chamber of the Transwell insert. Using crystal violet staining, migrating cells were observed under a microscope at 100× magnification after 24 h.

2.14 Wound healing assay

We employed an Ibidi culture insert (Ibidi, Germany) to assess the migratory capabilities of various cell types. To begin, BCa cells were carefully seeded onto culture plates as per the manufacturer’s guidelines and allowed to incubate overnight. The subsequent day, the inserts were carefully eliminated to produce a gap for cell migration, after which serum-free medium was introduced to the plates. After a period of 36 h, bright field images of the cell cultures were taken using a microscope. ImageJ software was employed to analyze the resulting images to quantify the migratory rate of the cells.

2.15 Western blot

A BCA assay kit was used for protein quantification after total protein was extracted using RIPA buffer (Beyotime, Shanghai). Polyvinylidene fluoride (PVDF) membranes were used for sample transfer after SDS-PAGE separation. Before the secondary antibody incubation at room temperature for 2 h, the membranes were exposed to primary antibodies for one night at 4 °C, diluted in NCM Universal Antibody Diluent (WB500D, NCM Biotech, China). The levels of gene expression were measured using chemiluminescence. PTM BIO (PTM-1406RM, Hangzhou, China) was the source of the H3K18la antibody.

2.16 Immunofluorescence assay

Prior to permeabilization with 0.1% Triton-X, cells were fixed with 4% paraformaldehyde. The next step was to add a 2.5% BSA blocking solution. The samples were examined under a Nikon microscope (Japan) after being incubated with primary and secondary antibodies. Staining with DAPI allowed us to see the nuclei.

2.17 Subcutaneous tumorigenesis model

Nude mice that were four weeks old were subcutaneously injected with T24 cells (6 × 106) that had been suspended in PBS. Over the course of seven days, the subcutaneous tumors’ parameters were carefully monitored. It is noteworthy that tumor volume was measured by means of the subsequent formula: tumor volume = 0.5×length×width² (mm³). After a period of 35 days, mice were sacrificed, and the tumors were collected and weighed. We adhered to the care guidelines for animals set forth by the Laboratory Animal Center of Zhejiang University.

2.18 Statistical analysis

Using one-way or two-way ANOVA in SPSS, we analyzed the differences between the groups. With a sample size of 3, all data are represented as the mean ± standard deviation (SD). Significance comparative to the control group is denoted by *P < 0.05 and **P < 0.01.