Tissue specimens/Tissue samples
All GC tissues and adjacent normal stomach mucosa tissues in this study were obtained from patients who had received radical gastrectomy at the Department of Gastrointestinal Surgery, the First Affiliated Hospital of Nanjing Medical University. All the specimens were collected and snap-frozen in liquid nitrogen immediately after surgical resection. TNM stage was based on the TNM classification system (American Joint Committee on Cancer classification, AJCC, 7th edition).
Cell culture and treatment
The human GC cell lines BGC-823, SGC-7901, MGC-803, and MKN-45 and normal GES-1 stomach mucosa epithelium cells were cultured in RPMI 1640 (Wisent, Shanghai, China) supplemented with 10% foetal bovine serum (FBS) (Wisent, Biocenter, China) and 1% pencillin-streptomycin. HGC-27 cells were cultured in RPMI 1640 (Wisent, Shanghai, China) supplemented with 20% FBS (Wisent, Biocenter, China) and 1% penicillin-streptomycin. All the cells were incubated in a humidified atmosphere of 5% CO2 at 37 °C.
RNA-seq analysis
Total RNA was isolated from GC tissues and cells using TRIzol reagent (Invitrogen). Next, complementary DNA (cDNA) was reverse transcribed using PrimeScript RT Reagent (RR036A; TaKaRa, Japan). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the SYBR™ GREEN PCR Master Mix kit (4913914001; Roche, Shanghai, China). The relative expression of RNA was normalized to the endogenous control glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and U6. RIBOBIO Biotech (Guangzhou, China) provided all the specific primers for circRNAs. The PCR primer sequences of miRNAs and mRNAs were synthesized by Realgene (Nanjing, China) and are listed in Additional file 1: Table S1.
RNase R treatment
The total RNA of GC cell lines was mixed with 3 U/mg of RNase R for 20 min at 37 °C. qRT-PCR was applied to detect the stable expression of circST3GAL6 and ST3GAL6 mRNA.
Actinomycin D assay
Cells were seeded at 5×104 cells per well in a 24-well plate overnight and then treated with 2 mg/L of actinomycin D (Sigma-Aldrich, USA). Total RNA was harvested at the indicated time points (4 h, 8 h, 12 h, 24 h), and qRT-PCR was performed to analyse the stability of the circRNA and mRNA.
Oligonucleotide transfection
The human gastric cell lines BGC-823 and SGC-7901 were seeded in a 6-well plate and incubated at 37 °C in a humidified 5% CO2 atmosphere overnight. siRNA, miRNA mimics and inhibitors (GenePharma, Shanghai, China) were transfected with Lipofectamine 3000 (Thermo Fisher, Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.
Plasmid construction and stable transfection
CircST3GAL6 cDNA was synthesized and cloned into the pcDNA3.1 vector (GenePharma, Shanghai, China). Cells were transfected with plasmids according to the manufacturer’s protocol.
Western blotting
Total protein from tissues and cells was extracted using RIPA lysis buffer (P0013B; Beyotime Biotechnology, China) containing PMSF. The protein concentration was determined using the Bradford method. Equal amounts of protein samples were resolved and separated by 10% SDS-PAGE using an electrophoresis apparatus (Bio-Rad, America) and transferred onto polyvinylidene difluoride (PVDF) membranes. Next, the membranes were blocked by incubating with Quick Block (P0252; Beyotime Biotechnology, China) for 20 minutes. Next, the membranes were treated with primary antibody, using GAPDH as an internal reference, at 4°C overnight. Finally, the membranes were washed and then incubated with secondary antibody for 2 h at room temperature. The blots were then visualized by enhanced chemiluminescence detection.
CCK-8 assay
We plated BGC-823 and SGC-7901 cells in 96-well plates at ten thousand cells per well, and then added 10 μl of CCK-8 solution (Dojindo Laboratories, Kumamoto, Japan) to each well every other day according to the manufacturer's protocols. After that, the cells were cultured for 2 hours at 37°C. Two hours later, we recorded the absorbance of the cells at 450 nm using a microplate reader (BioTek, Winooski, VT, USA).
Colony formation assay
BGC-832 and SGC-7901 cells were seeded in different six-well plates. Each well was inoculated with 1000 cells, and then the six-well plates were cultured in an incubator containing 5% CO2 for 2 weeks. Two weeks later, the cell proliferation state was observed after staining the cells.
Transwell assay
First, we inoculated specific cells on the upper side of the Transwell compartments (Millipore, Billerica, MA, USA) and added 200 μl serum-free RPMI-1640 at the same time. Next, we added 600μl of complete medium to the lower side of the Transwell compartments. After incubation for 24 hours in an incubator containing 5% CO2, we rinsed the cells with PBS and then removed the cells that did not pass through the membrane with a cotton swab. Finally, we fixed and stained the cells with 75% alcohol and crystal violet.
Flow cytometric analysis
First, we inoculated the transfected cells in six-well plates. Next, we collected all the cells and incubated them with the PE Annexin V Apoptosis Detection Kit reagents (BD Pharmingen, Franklin Lake, New Jersey, USA) for 2 days. After 15 minutes of incubation, we observed the cells using CELL Quest software (BD Biosciences, San Jose, CA, USA). The proportion of apoptosis is reflected by the corresponding quadrant on the apoptosis map.
5-Ethynyl-2′-deoxyuridine (EdU) assay
We inoculated the transfected cells in a 96-well plate at 10,000 per well and cultured them for 24 hours. The next day, we added EdU solution (RiboBio, China) to each well of the 96-well plate for incubation. After that, the cells were fixed and then were subjected to Apollo staining and DNA staining using Apollo reaction solution and Hoechst 33342, respectively. Finally, we observed the red and blue signals under the microscope to evaluate cell proliferation.
Fluorescent in situ hybridization (FISH)
Cells were first fixed with 4% paraformaldehyde for 10 minutes and incubated in 70% ethanol overnight at 4°C. Next, the cells were permeabilized with 0.5% Triton X-100 for 5 minutes and washed with saline sodium citrate (SSC) buffer. Next, the cells were hybridized with a labelled FISH probe of circRNA at 37°C in hybridization buffer overnight. The cells were then washed with 4× sodium citrate buffer containing 0.1% Tween-20 for 5 minutes and washed with 1× SSC for 5 minutes. Finally, the cells were stained with 4,6-diamidino-2-phenylindole (DAPI) for 10 minutes. Images were captured using a fluorescence microscope.
Pull-down assay
A biotin-labelled probe for circST3GAL6 was designed by RiboBio (Guangzhou, China). GC cells were harvested, lysed and incubated with the circST3GAL6 probe and oligo probe at 4°C overnight. The RNA mixture was bound to the magnetic beads with washing buffer several times. After that, the RNA was extracted using the RNeasy Mini Kit (QIAGEN, Germany) and detected by qRT-PCR. Biotinylated miR-300 and biotinylated miR-NC were produced by GenePharma (Shanghai, China), and the methods were the same as those described above.
Immunohistochemical (IHC) analysis of tissue samples
The GC tissues were fixed with 10% formalin and embedded in paraffin. Next, the tissues were cut into sections and incubated with specific primary antibody at 4°C overnight. After washing twice, the sections were incubated with HRP-polymer-conjugated secondary antibody(abcam, UK) at 37°C for one hour. These sections were then stained with 3,3-diaminobenzidine solution and haematoxylin. Finally, we observed the slices via microscopy.
Transmission electron microscopy (TEM)
We placed the cell pellet in a droplet of 2.5% glutaraldehyde in PBS buffer at pH 7.2 and fixed the cells overnight at 4 °C. The samples were then rinsed in PBS solution for 10 min three times and postfixed in 1% osmium tetroxide for 60 min at room temperature. Next, the samples were embedded in 10% gelatin, fixed in glutaraldehyde at 4 °C and cut into several blocks. Subsequently, the samples were dehydrated for 10 min in increasing concentrations of alcohol (30%, 50%, 70%, 90%, 95%, and 100% ×3). Next, we exchanged alcohol with propylene oxide and infiltrated samples with increasing concentrations (25%, 50%, 75%, and 100%) of Quetol-812 epoxy resin mixed with propylene oxide. Each step lasts at least 3 h. The samples were then embedded in pure Quetol-812 epoxy resin and polymerized at 35 °C for 12 h, 45 °C for 12 h, and 60 °C for 24 h. We cut samples into sections (100 nm) using an ultramicrotome and poststained them with uranyl acetate for 10 min and lead citrate for 5 min at room temperature. After that, we observed sections under a transmission electron microscope operated at 120 kV.
Nude mouse xenograft model
In total, 1×106 stably transfected GC cells or control cells were suspended in 100 µl of PBS and injected into each armpit of 4-week-old female BALB/c nude mice purchased from the Department of Laboratory Animal Center of Nanjing Medical University. After 4 weeks, the mice were sacrificed, and the tumours were separated to measure their weights and volume. The tumour volume was calculated as V = length × width2 × 0.5.
In vivo metastasis assay
Stably transfected GC cells (1 × 106) were injected into the tail veins of 4-week-old female BALB/c nude mice. After 28 days, the bioluminescent signals of lung metastasis were detected using an IVIS Imaging system (Caliper Life Sciences, Hopkinton, MA, USA). After the mice were sacrificed, the lung metastatic lesions were assessed using haematoxylin and eosin (HE)-stained sections.
Luciferase reporter assay
BGC-823 and SGC-7901 cells were seeded in 24-well plates and cotransfected with the corresponding plasmids and miRNA mimics. Luciferase reporter assays were conducted using a dual-luciferase reporter assay system (Promega, Madison, WI) according to the manufacturer’s instructions after transfection for 48 hours. The relative luciferase activity was normalized to Renilla luciferase activity.
Confocal microscopy
BGC-823 and SGC-7901 cells transfected with GFP-mRFP-LC3 lentivirus (GeneChem, China) were seeded into a 35-mm culture dish for confocal microscopy. The nucleus were stained with DAPI. Red and yellow puncta representing autolysosomes and autophagosomes, respectively, were detected by confocal microscopy (Carl Zeiss, Germany). Three random fields were selected for puncta quantification.
Chromatin immunoprecipitation assay
ChIP assays were performed using the Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (17–10085; Millipore Sigma, Burlington, Massachusetts, USA) according to the manufacturer’s protocol. Briefly, cells were fixed with 37% formaldehyde and collected in lysis buffer. Chromatin fragments were sonicated on ice, and then the lysates were immunoprecipitated with normal rabbit IgG and FOXP2 antibodies (5337; Cell Signaling Technology). Elution of the protein/DNA complexes was obtained after DNA purification using wash buffers and standard PCR. DNA was extracted for PCR amplification of specific DNA fragments.
Statistics
The data were presented as means ± SD in each experiment. A significant difference was considered at P < 0.05 in each experiment. One-way analysis of variance (ANOVA) and Student's t-test were applied.