Human GC specimens

GC and the corresponding noncancerous tissue samples (21 pairs) were obtained from Southwest Hospital of the Army Medical University. All patients were diagnosed by histopathological examination and were without chemotherapy or radiotherapy prior to surgery. The specimens were collected immediately in EP tubes containing RNA Later (Thermo Scientific, Pittsburg, PA, USA) after surgical resection and stored at -80 °C. Clinical characteristics of GC patients were given in Supporting Information Table S1. Informed consent was obtained from patients for each sample and the study was approved by the Ethics Review Committee of Chongqing Medical University and Southwest Hospital of the Army Medical University.

Cell lines

The GC-derived cell lines SGC7901, BGC823, and HGC-27 cells or normal gastric epithelial cell GES-1 were obtained from the Army Medical University (Chongqing, China). AGS and MKN-74 cells were purchased from Meisen (Hangzhou, China). All cells were cultured at 37 °C in a 5% CO2 atmosphere in DMEM or RPMI 1640 medium (Basal Media, Shanghai, China) supplemented with 10% fetal bovine serum (PAN-Biotech GmbH, Adenbach, Germany).

Western blot

Total protein was extracted from cultured cells using RIPA lysis buffer containing protease inhibitors (Beyotime, Shanghai, China). Cell lysates were separated on SDS–polyacrylamide gels and electrotransferred onto polyvinylidene difluoride (PVDF) membranes that were incubated at 4 °C with primary antibodies in 5% nonfat milk in PBS containing Tween-20 (PBST). The antibodies used for this study were used at 1:1000 dilutions in primary antibody dilution buffer (Beyotime) and were as follows: GAPDH and β-actin (Beyotime), SLC7A5 (Proteintech, Chicago, USA), IGF2BP3 (Abcam, Cambridge, UK), mTOR, p-mTOR, AKT and p-AKT (Cell Signaling Technology, Beverly, MA, USA). Membranes were washed with PBST for three times and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The blots were visualized using an ECL chemiluminescent reagent (Bioground, Chongqing, China).

Gene knockdown and overexpression

SiRNAs targeting IGF2BP3, circARID1A, and SLC7A5 were synthesized by GenePharma (Shanghai, China) (Supporting Information Table S2). Briefly, cells that had been seeded into 6 well plates overnight were transfected with siRNAs using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and incubated for 6 h. The medium was then replaced and cells were collected at indicated time points (see below).

For stable expression of IGF2BP3, pCDH-IGF2BP3 plasmids were constructed and co-packaged using a commercial lentivirus system that was assisted using pMD2.G, pCMV-VSV-G, and pRSV-Rev plasmids in 293T cells. SGC7901 and BGC823 cells were then infected with lentivirus-IGF2BP3 overexpression and stable cell lines were selected by the addition of puromycin. Transfection efficiency was confirmed by Western blot using IGF2BP3 antibodies (see above). For IGF2BP3 transient overexpression, the overall CDS region of IGF2BP3 was inserted into pcDNA3.1( +) for IGF2BP3 overexpression. The vector was transfected into GC cells using a commercial DNA transfection reagent (Neofect, Beijing, China).

Cell Counting Kit-8 assay

Cells were seeded into 96-well plate at 5 × 104/mL using 100 μL/well and incubated for the indicated times (see below). 10 μL of Cell Counting Kit-8 (CCK-8) (Biosharp, Hefei, China) reagent was added to each well and the plate was incubated at 37 °C for 2 h in a 5% CO2 atmosphere, the absorbance was measured at 450 nm using a microplate reader (Thermo).

Plate colony formation assay

Cells were removed from monolayers using trypsin digestion and seeded into 12-well plates at 1 × 103 cells per well. Two weeks after seeding, tumor spheres were fixed using 4% paraformaldehyde for 10 min and then stained with 1% crystal violet solution for 10 min at room temperature. The colony spheres were washed 3 × with water and then dried at room temperature. Plate images were captured using an Epson scanner (Suwa, Nagano Prefecture, Japan).

EdU (5-ethynyl-2´-deoxyuridine) incorporation assay

GC cells were treated and seeded into 48-well plate at a density of 3 × 104 per well. EdU incorporation utilized a Cell-Light EdU Apollo 567 In Vitro Kit (RiboBio, Guangzhou, China). Cells were treated and exposed to EdU solution (50 μM) for 2 h at 37 °C and then processed according to the manufacturer’s instructions. Cells were observed and photographed using a fluorescent inverted microscope (Olympus, Tokyo, Japan).

RNA-binding protein immunoprecipitation (RIP) assay

RIP assays were performed according to the instructions of Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Burlington, MA, USA). Briefly, cells were lysed with RIP lysis buffer and incubated with antibodies specific for IGF2BP3 or rabbit IgG. Co-precipitated RNAs were purified for sequencing or cDNA synthesis and used as template for indicated gene expression detection by qRT-PCR with specific primers. Total RNA (Input) or antibody (Rabbit IgG) was used as references.

Actinomycin D assay

For circARID1A stability evaluation, SGC7901 and BGC823 cells were seeded into 12 well plates and treated with actinomycin D (5 μg/mL) (Genview, Beijing, China) for 0, 3, 6, and 9 h. Cells were lysed in RNAiso Plus (Takara, Kyoto, Japan) for RNA extraction and qRT-PCR measurements. For mRNA stability assay, SGC7901 and BGC823 cells were seeded into 12 well plates and treated with si-circARID1A or si-IGF2BP3 and then treated with actinomycin D (20 μg/mL) or vehicle for 0, 3, 6, and 9 h. Cells were lysed in RNAiso Plus (Takara) for RNA extraction and further qRT-PCR detection.

RNase R treatment

SGC7901 and BGC823 cells were seeded into 12 well plates and incubated overnight. Cells were used for total RNA extraction and a total of 5 µg RNA was incubated in the presence or absence of RNase R (6 U) (Lucigen, Middleton, WI, USA) at 37 °C for 10 min, followed by 85 °C for 5 s. After RNase R treatment, the expression of linear ARID1A and circARID1A were measured using qRT-PCR.

Nuclear-cytoplasmic fractionation

Nuclear and cytoplasmic RNA in SGC7901 and BGC823 cells were isolated using the Paris Kit (Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s instructions. For circARID1A, ARID1A, and GAPDH, cDNA was synthesized with a PrimeScript RT Master Mix (Takara) and for snoU6 the cDNA was synthesized by stem-loop methods (RiboBio). Quantitative real-time PCR (qRT-PCR) analysis was executed using a SYBR Green Master Mix (Bioground). The 2−ΔΔCt method was used to analyze the relative expression levels of genes in nuclear and cytoplasm [35].

RNA fluorescence in situ hybridization (RNA-FISH)

Biotin-labeled probes or FAM-labeled probes were synthesized by Genepharma and used to visualize circARID1A or SLC7A5 in situ. Briefly, SGC7901 and BGC823 cells were seeded into μ-Slide 8-well chamber slide (Ibidi, Martinsried, Germany). The medium was removed 24 h later and cells were washed 2 × with PBS. Then cells were fixed with 4% paraformaldehyde for 10 min and treated with Triton X-100 (0.5%) at room temperature for 15 min. Cells were washed 2 × with PBS and incubated with denatured probes targeting circARID1A, SLC7A5, 18S rRNA or negative control and the slides were incubated at 37 °C overnight. The next day, nuclei were counterstained with DAPI and images were taken using a laser confocal microscope (Leica SP8, Wetzlar, Germany). The probe sequences for RNA-FISH are listed in Supporting Information Table S3.

Total RNA extraction and quantitative real-time polymerase chain reaction

Total RNA was extracted with RNAiso Plus (Takara) following the manufacturer's instructions. RNA was quantified using a Nanophotometer (Implen, Munich, Germany) and cDNA was synthesized using a PrimeScript RT Master Mix (Takara). qRT-PCR was conducted using SYBR Green Master Mix (Bioground) with CFX connect Real-Time PCR System (BioRad, Hercules, CA, USA). Human GAPDH was used as an internal control for the relative expression of circRNAs and mRNAs. The 2−ΔΔCt method was used to calculate the relative expression of indicated genes and primers are listed in Supporting Information Table S4.

Pull-down assay with biotinylated circARID1A probe

RNA pull-down assay was performed using a Waals RNA Pull down Kit (Chongqing, China). Briefly, SGC7901 or BGC823 cells (1 × 107) were harvested and lysed in IP lysis buffer on ice for 30 min. Avidin-labeled magnetic beads were incubated with biotin-labeled circARID1A probes or control probes (GenePharma) at 25 °C for 2 h to generate probe-coated beads. The cell lysates were incubated with circARID1A probe or control probe to pull-down circARID1A. The RNA complexes bound to the beads were purification using phenol/chloroform/isoamyl alcohol (25: 24: 1) for real-time PCR measurements. The protein complexes bound to the beads were denatured by heating at 100 °C for 10 min for Western blot analysis. The probe sequences for RNA pull-down are listed in Supporting Information Table S5.

Immunofluorescence assay

GC cells grown on confocal dishes (Ibidi) were fixed using 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Cells were washed 2 × with PBS and blocked with 5% BSA for 30 min at 37 °C, and incubated with specific primary antibody at 4 °C overnight. The primary antibody was removed and cells were washed 3 × with PBS and incubated with corresponding secondary antibody for 30 min at 37 °C, followed by staining with DAPI. Fluorescent images were obtained using a Leica SP8 confocal microscope.

Tissue microarray (TMA) and in situ hybridization (ISH)

TMA were produced from 180 paraffin-embedded GC or adjacent tissues by Outdo Biotech (Shanghai, China). The clinical pathological characteristics including gender, age, survival time, pathological grading, tumor volume, TNM stage, AJCC stage, the expression of PDL1 or CD8 were public data and provided by Outdo Biotech (Shanghai, China). ISH was employed to detect the expression of circARID1A in GC tissues. The probes targeting the junction of circARID1A were synthesized by Boster (Wuhan, China) and the ISH Kit was purchased from Boster. Briefly, TMA was dewaxed in xylene and rehydrated with 100, 95, 85 and 75% ethanol and digested with trypsin. The TMA were hybridized with specific digoxin-labeled circARID1A probes overnight. The specimens were incubated with biotin-conjugated anti-digoxin antibody (Roche, Basel, Switzerland), and incubated with avidin-conjugated peroxidase and stained with NBT/BCIP (Roche) and photographed. circARID1A expression was quantified and analyzed. The positive staining intensity (0, no expression; 1, mildly positive; 2, moderately positive; and 3, markedly positive) was multiplied by the percentage of positive staining cells (0, < 5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; 4, > 75%) to calculate the score for ISH staining. The probe sequence is listed in Supporting Information Table S6.

Tumor-bearing nude mice model construction

Lentivirus constructs containing an shRNA targeting circARID1A was packaged by Hanbio (Shanghai, China) with a virus titer of 108/UI. SGC7901 cells were infected with lentivirus at a MOI = 50 using polybrene. The transfection efficiency was measured and proliferation of the SGC7901 stable circARID1A knockdown cells was detected. Briefly, 10 male BABL/C nude mice (Gempharmatech, Jiangsu, China) aged at 3–4 weeks were randomly divided into two groups. SGC7901 cells transfected with sh-NC or sh-circARID1A were cultured and digested with 0.25% trypsin. Cells were washed twice with cold PBS and diluted with PBS at a concentration of 3 × 107/mL. 100 µL of cell suspension was subcutaneously implanted in the upper-right flank of nude mice. One week later, the length and width of the tumor tissues was measured every two days. The volume of the tumor size was calculated by the formula: volume (mm3) = length × width2 / 2. The experiment was terminated one month after xenograft mouse model construction and the animals were sacrificed and tumor weight was measured. The tumor tissues were collected for indicated gene expression evaluation and IHC staining.

For in vivo rescue assay of circARID1A and SLC7A5, HGC-27 cells were employed to construct xenograft tumor model in nude mice. Briefly, 3-week-old male BALB/C nude mice (Gempharmatech) were subcutaneously injected in the right flank with HGC-27 cells (1.5 × 106) in 0.1 mL phosphate-buffered saline (PBS). When the tumor had grown to an appropriate volume, the tumor-bearing mice were randomly divided into three groups (n = 4). In vivo treatment with siRNA in the mouse models was performed as previously described [36]. Then, cholesterol-modified si-circARID1A or si-RNA control (GenePharma, 5 nmol/kg) with 5 μL in vivo transfection reagent (Entran-ster™-in vivo, Engreen, Beijing, China) was locally injected into the tumor mass once every 2 days for 2 weeks. Moreover, lentivirus containing SLC7A5 were also injected into the tumor mass at day 1 and day 3. The study was approved by the Ethics Review Committee of Chongqing Medical University.

Immunohistochemistry (IHC) staining

IHC was performed on formalin-fixed, paraffin-embedded tissue sections as described previously [37]. The primary antibody used was Ki-67 (1:500, Servicebio, Wuhan, China). Tissue sections were incubated with primary antibody at 4 °C overnight and then incubated with secondary antibody. DAB complex was used as the chromogen and the nuclei were counterstained with hematoxylin. Three different visual fields were randomly selected for each slice and all images were scored as follows: proportion score: 0–4 (0, < 5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; 4, > 75%); intensity score: 0, negative; 1, weak; 2, intermediate; 3, strong; total score = proportion score × intensity score.

RNA-sequencing

SGC7901 cells were seeded to 6-well plate at a density of 5 × 105 cells per well. Twenty-four hours later, cells were transfected with siRNAs targeting to circARID1A with the help of lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Six hours later, the supernatant was replaced with fresh DMEM containing 10% fetal bovine serum. Cells were collected after 48 h for further RNA sequencing by Sinotech Genomics (Shanghai, China).

Statistical analysis

Student’s t test or one-way analysis of variance (ANOVA) was used to compare the differences of continuous variable between two groups or among multiple groups. Kaplan–Meier analysis was employed for survival analysis, and the differences in the survival probabilities were estimated using the log-rank test. ISH score of circARID1A was categorical variable, and was analyzed by Kruskal–Wallis Test. The statistical analyses were performed using SPSS version 22.0 (IBM, Chicago, Ill, USA) and Prism 8.0 (GraphPad, San Diego, CA, USA). P < 0.05 was considered to indicate statistical significance.