Oral cancer tissue samples

A total of 96 pairs of postoperative cancer tissue samples and corresponding adjacent noncancerous tissue samples were collected from oral cancer patients at Hunan Cancer Hospital between 2020 and 2023. These samples were preserved in fixative, embedded in paraffin, and sectioned to create oral cancer tissue microarrays. Detailed clinical parameters are provided in Supplementary Table 1. The collection, application, and utilization of these specimens were approved by the Ethics Committee of Hunan Cancer Hospital (Approval Nos. SBOLL-2023-039 and GZ2023-017).

Cell culture and transfection

Oral cancer cell lines CAL-27 and SCC-9 were obtained from Beina Bio and cultured at Hunan Cancer Hospital. CAL-27 was grown in DMEM high glucose medium, while SCC-9 was cultured in DMEM/F12 medium, both supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cultures were incubated at 37 °C with 5% CO2.

Cells were transfected using Lipofectamine 3000 (Invitrogen, USA) following the manufacturer’s instructions. The circRNF13 siRNA was designed and synthesized by Baiyi Genetics, with its sequence in Supplementary Table 2. GFP-IGF2BP1-GV739-Neomycin and ITGB1-GV657-puromycin plasmids were obtained from Genechem (Shanghai, China).

RT-qPCR

RNA was extracted with TRIzol (Invitrogen, USA), and first-strand cDNA was synthesized using a reverse transcription kit (Thermo Fisher, USA). RT-qPCR was conducted on a LightCycler 480 (Roche, USA) with SYBR Green Mix. Data analysis used the β-actin gene and U6 small nuclear RNA as internal references, calculating relative gene expression with 2^−ΔΔCT values. The primer sequences used are listed in Supplementary Table 2.

Cytoplasmic and nuclear RNA analysis

Nuclear and cytoplasmic RNA fractions were extracted with the PARIS Kit (Invitrogen, USA) per the manufacturer’s guidelines. RT-qPCR was used to measure circRNF13 expression in both compartments, with U6 and GAPDH serving as controls for the nuclear and cytoplasmic fractions, respectively.

RNase R resistance analysis

5 µg of RNA from Cal-27 cells was treated with 0.5 µL RNase R and 2 µL buffer, then diluted to 20 µL with enzyme-free water. The mixture was incubated at 37 °C for 5 min, followed by enzyme inactivation at 70 °C for 10 min. The control group received the same treatment without RNase R. Both treated and control samples were used for RT-qPCR with divergent or convergent primers.

RNA decay assay

Actinomycin D (ACTD) was introduced to the cell culture at 1 µg/mL. RNA was extracted at 0, 4, 8, and 12 h for RT-qPCR. Gene expression was normalized to circRNF13 or ITGB1 mRNA levels at 0 h.

Fluorescence in situ hybridization

The FISH assay followed the RNA-FISH kit protocol (GenePharma, Suzhou, China). Cells were seeded on glass slides in 24-well plates, fixed with 4% paraformaldehyde, and permeabilized with 0.3% Triton X-100. After 30 min of blocking at 37℃, they were hybridized overnight at 37℃ with the bio-circRNF13 probe and SA-Cy3 to produce a fluorescent signal for the target RNA. Nuclei were stained with DAPI and imaged using laser confocal microscopy. The probe sequences are provided in Supplementary Table 2.

Immunofluorescence

Cells were seeded on glass slides in 24-well plates, fixed with 4% paraformaldehyde, and permeabilized with 0.3% Triton X-100. They were blocked with 5% BSA for 1 h, incubated overnight with primary antibodies, washed with 0.5 M PBS, and then treated with fluorescent secondary antibodies for 1 h at room temperature. Nuclei were stained with DAPI for 10 min before confocal microscopy imaging (Zeiss). Antibody information is provided in Supplementary Table 3.

CCK-8 assay

Cal-27 or Scc-9 cells were initially seeded in 96-well plates, and OD450 values were recorded 2 h post CCK-8 reagent addition. Cell growth was tracked for 4 days.

Colony formation assays

Cal-27 or Scc-9 cells (1000–2000) were seeded in 6-well plates and cultured for 1–2 weeks. For cisplatin sensitivity assays, cells received the specified cisplatin dose 8–12 h post-seeding, then incubated for 1–2 weeks. Colonies were fixed with 4% paraformaldehyde, stained with crystal violet, scanned, photographed, and counted using ImageJ. Results were normalized to the control group.

Transwell assays

600 µL of medium with 10% FBS was added to the lower chamber, and 200 µL of Cal-27 or Scc-9 cell suspension (10⁵ cells) in serum-free medium was placed in the upper chamber of the Transwell system. After 12–48 h, cells on the upper membrane were removed with a cotton swab. Migrated cells on the lower membrane were fixed, stained, imaged, and counted using ImageJ.

In vivo tumor formation assay

Female BALB/c nude mice, aged 4–6 weeks and weighing about 20 g, were obtained from Silaikejingda in Changsha, China. They were kept in the SPF Animal Room at Hunan Cancer Hospital’s Animal Experiment Center and acclimated for 7 days before use. The mice were randomly grouped and injected subcutaneously with 5 × 10⁶ Cal-27 cells, either control or experimental. Tumor growth was checked every four days, and volume was calculated using the formula V = 1/2 × length × width². The experiment ended when the subcutaneous tumors grew to the desired size. The nude mice were euthanized with CO2 anesthesia, and their tumors were removed, weighed, and photographed. The tumor tissue was then paraffin-embedded, sectioned, and analyzed for gene expression through in situ hybridization, immunohistochemistry, and other methods. In the cisplatin-treated subcutaneous xenograft tumor model, mice were randomly split into two subgroups post-tumor establishment and received intraperitoneal injections of either saline or 2 mg/kg cisplatin every four days for five sessions. The Ethics Committee of Hunan Cancer Hospital (GZ2023-017) approved all animal experiments.

In situhybridization (ISH)

Using the Bosterbio sensitivity-enhanced in situ hybridization kit, paraffin-embedded tissue sections were deparaffinized, rehydrated, treated with 3% H₂O₂ for 10 min, pepsin for 20 min, and prehybridized for 2 h. They were then hybridized overnight at 37 °C with the Dig-circRNF13 probe. A scoring system evaluated expression levels by combining the percentage of positively labeled cells (0 to 4) and staining intensity (0 to 3). The H-score, calculated as their product, ranged from 0 (no expression) to 12, with 1–4 indicating low expression and 6–12 indicating high expression.

Immunohistochemistry (IHC)

Tissue sections were deparaffinized, rehydrated, and boiled in citric acid solution for antigen retrieval. They were then incubated with a peroxidase blocker at room temperature for 10 min. Primary antibodies were applied and incubated overnight at 4 °C, followed by a 20-minute incubation with an enzyme-labeled secondary antibody at 37 °C. Finally, DAB and hematoxylin staining were performed. DAB chromogenic development (ZSGB-Bio, Beijing, China) and hematoxylin counterstaining were then performed. Detailed information on the antibodies is provided in Supplementary Table S3.

Western blotting

Proteins were lysed with RIPA buffer and protease inhibitors, separated by 10% SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked with 5% skimmed milk for 2 h at room temperature, then incubated with the primary antibody overnight at 4 °C. After three TBST washes, it was incubated with an HRP-labeled secondary antibody for 2 h at room temperature and washed again. Protein bands were detected using ECL reagent. Detailed information on the antibodies is provided in Supplementary Table S3.

RNA pull-down

Following cell lysis with RIP lysis buffer, the lysate was incubated overnight at 4 °C with streptavidin magnetic beads and either the biotinylated bio-circRNF13 probe or the control bio-LacZ probe. After multiple washes, the enriched proteins were prepared for Western blot analysis.

RNA immunoprecipitation (RIP)

The Magna RIP kit (Millipore, USA) was utilized following the manufacturer’s instructions. Cells were lysed with RIP lysis buffer, and the lysates were incubated overnight at 4 °C with magnetic beads and specific or IgG antibodies. The RNA obtained from this process was purified for RT-qPCR analysis.

Methylated RNA immunoprecipitation (MeRIP)

MeRIP was performed with the MeRIP m6A Transcriptome Profiling Kit (Ribobio, Guangzhou, China) following the manufacturer’s protocol. Total RNA was fragmented and incubated with anti-m6A magnetic beads at 4 °C for 2 h. After washing, m6A-enriched RNA was eluted and recovered using a microRNA extraction kit (Absin, Shanghai, China) for RT-qPCR analysis.

Droplet formation assay

HEK293 cells transfected with GFP-IGF2BP1 were cultured in confocal dishes for 48 h, washed with PBS, and fixed with 4% paraformaldehyde. After DAPI staining for 5 min, the cells were washed again and imaged using a Zeiss confocal microscope. Green fluorescent puncta indicated IGF2BP1 phase separation droplets.

Fluorescence recovery after photobleaching assay (FRAP)

FRAP experiments were performed with a Zeiss confocal microscope using a 63× oil immersion objective in a live-cell chamber. A 488 nm laser pulse b.

leached droplets in a chosen region of interest (ROI), and time-lapse images were taken to monitor fluorescence recovery. Recovery curves were plotted for analysis, and the experiment was repeated three times independently.

Statistical analysis

Statistical analysis and graphing were performed with GraphPad Prism 9.5. The independent samples t-test and Mann-Whitney U test analyzed data between two independent samples. Paired samples t-test assessed gene expression differences between oral cancer tissues and adjacent tissues. The chi-square or Fisher’s exact test evaluated the correlation between circRNF13 expression and clinical parameters in oral cancer patients. Data are presented as mean ± standard deviation, with p < 0.05 indicating significant differences between groups.