Tissues samples collection
Fifteen paired gastric cancer and adjacent normal tissues were collected from The Affiliated Suqian First People’s Hospital of Nanjing Medical University from February to June 2017. Characteristics of all participants are provided in Additional file 1. Collected samples were stored at −80°C immediately after surgery. All participants signed a consent agreement and the project was approved by the Ethics Committee of The Affiliated Suqian First People’s Hospital of Nanjing Medical University.
Cell culture and transfection
Human gastric epithelial (GES-1) and gastric cancer (AGS, NCI-N87, MKN-45, HGC-27 and SNU-1) cell lines were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cells were cultured in DMEM or RPMI 1640 medium (Gibco, USA) with 10% foetal bovine serum (FBS, Gibco, USA) at 37°C with 5% CO2. Small interfering RNA circ_0035277 (si-circ_0035277, 5′-GCCTCATTCATGTGTGGTGTT-3′), NC mimic (5′-UUUGUACUACACAAAAGUACUG-3′), miR-576-3p mimic (5′-AAGAUGUGGAAAAAUUGGAAUC-3′), NC inhibitor (5′-CAGUCCUUUUGUGUAGUACAA-3′) and miR-576-3p inhibitor (5′-GAUUCCAAUUUUUCCACAUCUU-3′) were synthesised by GenePharma (Shanghai, China). cDNA of circ_0035277 and LIN28B was cloned into pcDNA3.1 vector for overexpression of circ_0035277 and LIN28B. Cells were transfected with 100 nM si-circ_0035277, miR-576-3p mimic, miR-576-3p inhibitor, pcDNA-3.1-circ_0035277 or pcDNA-3.1-LIN28B using Lipofectamine 2000 (Invitrogen, USA) following the manufacturer’s instructions.
Real-time quantitative PCR assay
The total RNA of tissues and cells was isolated using Trizol reagent (TransGen, China) and reverse-transcribed to cDNA using PrimeScript®RT (Takara, Japan). Reactions were then performed with the ABI Prism 7500 PCR system (Applied Biosystems, USA) utilising SYBR Green Master Mix and TaqMan MicroRNA Assay Kit (Takara, Japan), as recommended by the supplier. U6 and GAPDH were used as internal control. The expression of all genes was calculated using 2 − ΔΔCT. Primer sequences of mRNA and miRNAs are shown in Additional file 2.
HGC-27 and AGS cells were placed into 96-well plates with 1×104 cells/well. CCK-8 solution (10 µL, Beyotime, China) was then added to cells after 96 hours (h). Cell optical density was detected with a microplate reader (Bio-Rad, China) at 450 nm after incubation for 1 h at 37°C.
Colony formation assay
HGC-27 and AGS cells were seeded into 6-well plates with 500 cells/well and incubated with 5% CO2 at 37 ℃ for 14 days. Cells were then fixed with methanol and stained with Giemsa dyeing for 15 minutes. The number of clones was obtained using a light microscope (Olympus, Japan).
HGC-27 and AGS cells were seeded into the upper Transwell chamber with 5×104 cells/well and incubated with FBS-free medium at 37 ℃. Cells were then seeded into the upper chamber with precoated Matrigel for invasion. The bottom Transwell chamber was supplied with 500 µL medium with 20% FBS. Cells migrated, invaded and became fixed on the small outdoor surface after 24 h and then stained with crystal violet for 15 minutes. Migrated and invaded cells were counted with a light microscope (Olympus, Japan).
Fluorescence in situ hybridisation assay
HGC-27 cells were placed into 48-well plates with 1×104 cells/well, fixed in 4% paraformaldehyde and permeabilised with 0.5% Triton X-100 for 5 minutes. HGC-27 cells were then incubated with circ_0035277-labeled fluorescence in situ hybridisation (FISH) probes (RiboBio, China) at 37 ℃ for 12 h. Lastly, DAPI staining (Beyotime, China) was used to reverse-stain nuclei. Images were observed with a fluorescence microscope (Nikon, Japan).
The online database Circular RNA Interactome was utilised to assess miRNAs binding with circRNA. Three online databases, namely, Targetscan, miRDB and miRTarbase, were used to find potential target genes of miRNAs.
Dual-luciferase reporter assay
Wild-type or mutant 3’UTR of circ_0035277 and LIN28B were cloned into pGL3-Firefly-Renilla vector (Promega, USA) separately. Cells were placed into 24-well plates, cotransfected with pGL3-Firefly-Renilla vector and mimicked or inhibited with Lipofectamine 2000. The luciferase activity was measured using dual-luciferase reporter assay (Promega, USA) according to the manufacturer’s instruction. Lastly, the luciferase activity was normalised to Renilla signals.
Western blot assay
Tissues and cells were fully lysed with 500 µL protein lysate of RIPA:PMSF=100:1 (Beyotime, China) and then iced for 15 minutes. The protein concentration of samples was determined using the BCA kit (Beyotime, China). Protein samples (20 µg) were added to the loading buffer at 95 ℃ for 5 minutes, placed in SDS-PAGE gel and shifted into PVDF membranes. Specific primary antibodies anti-LIN28B (ab229629, 1:1000, abcam, USA) and anti-GAPDH (ab9485, 1:1500, abcam, USA) were incubated at 4 ℃ overnight. Membranes were incubated with the secondary antibody (ab150082, 1:10,000, abcam, USA) for 2 h. Lastly, images were collected via ECL luminescence (Beyotime, China) and Western blot imaging system.
Tumour xenograft model
Six to eight week-old BALB/c nude mice were obtained from Jiangsu Medical Laboratory Animal Centre (Nanjing, China). Mice were raised in a specific pathogen-free room with free sterile food and water. Lentivirus with firefly luciferase-infected HGC-27 cell line was applied to establish a stable luciferase-positive HGC-27 cell line. The HGC-27 cell line was infected with lentivirus si-NC or si-circ_0035277. A 10 mm incision was performed on the mice abdomen after intraperitoneal anaesthesia with 50 mg/kg of sodium pentobarbital. HGC-27 cells (1×106) mixed with Matrigel matrix were injected into the subserosal region of the stomach similar to a previous study . Lastly, the stomach was reinserted into the peritoneal cavity to close the incision. Luciferase activity of fireflies was observed via bioluminescence imaging. BALB/c nude mice were euthanised with 200 mg/kg of pentobarbital. Animal experiments were approved by the Animal Care and Use Committee of The Affiliated Suqian First People’s Hospital of Nanjing Medical University.
Haematoxylin and eosin staining
Lung tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin and sectioned. Lung slices were immersed in 0.5% haematoxylin for 5 minutes and eosin solution for 3 minutes. Images of stained sections were observed under a light microscope (Nikon, Japan).
Data were analysed via GraphPad 8.0 software, presented with mean±SD and repeated at least three times. Student’s unpaired t-test was used to compare differences between the two groups. One-way ANOVA, followed by post hoc Bonferroni test, was utilised to assess differences amongst multiple groups. P < 0.05 was considered significantly different.