2.1 Cell line culture

The GC cell lines (HGC27, MGC803, MKN45, MKN28, SFC7901 and AGS) and gastric mucosal epithelial cells (GES-1) used in this study were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). These cells were incubated in 1640 medium (Hyclone, South Logan, USA) containing 10% fetal bovine serum (Gibico, Grand Island, USA) and 100 U/ml penicillin and streptomycin (Gibico, Grand Island, USA) in an atmosphere of 5% Carbon Dioxide and 37 °C.

2.2 Tissue specimens

All GC tissues involved in this study were obtained from patients who were diagnosed with GC and underwent surgical procedures at Department of Gastrointestinal Surgery of our institute. The approval of the Medical Ethics Committee of Yixing People's Hospital was obtained in advance and all methods were carried out in accordance with relevant guidelines and regulations. The gastric cancer and paracancerous tissues involved in this study were obtained from patients with T1-T3a gastric cancer who underwent surgical treatment at our gastrointestinal surgery department from 2017 to 2020. All patients had not received preoperative chemotherapy or immunotherapy and had signed an informed consent form before surgery.

2.3 Cell transfection

To explore the function and mechanism, we attempted to regulate the transcriptional level of nucleic acids. We commissioned Genepharma (Shanghai, China) to design and synthesize small interfering RNAs (siRNAs) and miRNA mimics. All cell transfection were performed using the Lipofectamine 3000 (Thermo, Waltham, USA).

2.4 Reverse transcription-quantitative PCR (RT-qPCR) assay

Total RNA of both GC tissues and cells was obtained with the help of Trizol reagent (Invitrogen, Carlsbad, USA) according to the protocol provided by the manufacturer. After that, cDNA was generated with miRNAFirst Strand cDNA Synthesis (Stem-loop Method, Sangon Biotech, Shanghai, China) or PrimeScript RT Reagent Kit (Takara, Tokyo, Japan). qRT-PCR was performed with QuantiNova SYBR Green PCR Kit (QIAGEN, Duesseldorf, Germany) in CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, USA). During this study, GAPDH and U6 were taken as internal reference. In addition, GAPDH and U6 were taken as internal references and their relative expression levels were compared using 2−ΔΔCt method, respectively.

2.5 RNA fluorescence in situ hybridization (FISH)

Cells were first fixed by 4% paraformaldehyde (Sigma-Aldrich, St. Louis, USA) and then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, USA). Subsequently, cells were subjected to an overnight hybridization reaction at 37 °C with the Fam-labeled circTSN and Cy3-labeled miR-1825 probes and the nuclei were stained with DAPI. The final images were obtained using a Leica system (Wetzlar, Germany).

2.6 Cell counting Kit-8 (CCK-8) assay

HGC27 and MKN45 cells were previously inoculated into 96-well plates overnight and incubated at 37 °C. After adding 10 μL CCK-8 (Dojindo, Kumamoto, Japan) to each well and incubating for another 2 h, the optical density (OD) values at 450 nm of the 96-well plates were measured using an enzyme labeler (Thermo, Waltham, USA).

2.7 Clone formation assay

Transfected cells were seeded in 12-well plates and cultured. Two weeks later, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, USA) for 15 min at room temperature and then stained with crystal violet (Sigma-Aldrich, St. Louis, USA) for 15 min.

2.8 5-Ethynyl-2′-deoxyuridine (EdU)

Cells in logarithmic growth phase were placed 4 × 103/well in a 96-well plate and cultured to normal growth phase. After that, we conducted the follow-up procedures based on an EdU Cell Proliferation Detection Kit (RiboBio, Guangzhou, China).

2.9 Transwell

Transwell inserts (Corning, Corning, USA) were placed in 24-well plates. 250 µl of serum-free medium was added to the upper layer and 750 µl of complete medium in the lower layer. 20,000 cells were seeded onto the upper layer. After 12–24 h of incubation, the medium was discarded and the cells were fixed with formaldehyde and stained with crystal violet.

2.10 Dual-luciferase reporter assay

The pmirGLO plasmid was constructed to contain the predicted binding sites between circTSN and miR1825 and a luciferase reporter gene, along with its corresponding mutant plasmid (GenePharma, Shanghai, China). Transfection was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, USA). Firefly luciferase activity was measured 48 h after co-transfection of pmirGLO plasmids and mimics using the Dual Luciferase Reporter Assay Kit (Vazyme, Nanjing, China).

2.11 Western blot assay

Total protein was extracted using RIPA buffer supplemented with protease inhibitors (CST, Danvers, USA) at 4 °C for 30 min. The supernatant was centrifuged at 12,000 g, mixed with loading buffer (CST, Danvers, USA) and denatured. Proteins were separated by electrophoresis on an SDS-PAGE membrane and transferred to a PVDF membrane (Merck Millipore, Billerica, USA). We cropped the PVDF membranes according to the size of the target proteins [27], then incubated them to label the proteins with primary and secondary antibodies, and finally detected the proteins using ECL (Merck Millipore, Billerica, USA).

2.12 Xenograft mouse model

Six-week-old BALB/c nude mice were purchased from GemPharmatech (Shanghai, China) and randomly clustered. Transfected gastric cancer cells were implanted into the axillary subcutaneous tissue of nude mice. The size of the subcutaneous neoplastic tumors was measured weekly (Statement: the diameters were all less than the maximum tumor size of 20 mm allowed by the Medical Ethics Committee of Yixing People’s Hospital). 4 weeks later, mice were anesthetized by intraperitoneal injection of sodium phenobarbital and then executed by cervical dislocation. The subcutaneous neoplastic tumors were weighed. All animal experiments were approved by the Animal Ethics Committee of our institution.

2.13 Statistical analysis

Each experiment in this study was independently replicated for three times. GraphPad Prism 7.0 was used for statistical analysis. P < 0.05 was considered significant.