Identification ofdifferentially expressed circRNAs (DEcircRNAs)
Gastric cancer-related datasets (GSE93541, GSE89143 and GSE78092) were downloaded from the GEO database. For GSE93541 dataset, R language was utilized to analyze the DEcircRNAs in plasma samples from gastric cancer patients and healthy controls. For GSE89143 and GSE78092 datasets, R language was utilized to analyze the DEcircRNAs between gastric cancer tissues and adjacent normal tissues. The threshold value of differentially expressed genes was set at 2 times of different multiple and p<0.05. The intersection of DEcircRNAs from three datasets was performed using the Venn Diagram package.
Gastric cancer tissues and matched adjacent normal tissues were obtained from the First Affiliated Hospital of Soochow University. The written consent was obtained from each patient with gastric cancer. All samples were frozen in liquid nitrogen and stored at -80°C. This study was approved by the ethics committee of The First Affiliated Hospital of Soochow University.
Human AGS and MKN45 cells were obtained from ATCC (Manassas, VA, USA). Cells were maintained in DMEM medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS) in a 5% CO2 incubator at 37°C.
MiR-196a-3p mimics and miR-196a-3p inhibitor were designed and synthesized by Ribobio (Guangzhou, China). After that, AGS and MKN45 cells were transfected with miR-196a-3p mimics or miR-196a-3p inhibitor using the Lipofectamine 2000 kit (Thermo Fisher Scientific).
Human circPTK2 or AATK cDNA was synthesized and cloned into pcDNA3.1 vector. After that, AGS and MKN45 cells were transfected with the pcDNA3.1 control plasmid, pcDNA3.1-circPTK2 (circPTK2-OE) or pcDNA3.1 AATK (AATK-OE) using Lipofectamine 2000, followed by selected with G418.
Lentivirus-containing short hairpin RNA (shRNA) targeting circPTK2 or AATK plasmids were purchased from Hanbio (Shanghai, China). After that, 293T cells were transfected with above-mentioned lentiviral plasmids were transduced into 293 T cells to package lentivirus to infect AGS and MKN45 cells. Subsequently, the infected cells were selected by 2 µg/mL of puromycin.
RT-PCR and RT-qPCR
TRIzol Reagent (Thermo Fisher Scientific) was used to extract total RNA. Meanwhile, genomic DNA (gDNA) was isolated using the Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China). After that, cDNA was synthesized using EntiLink™ 1st Strand cDNA Synthesis Kit (ELK Biotechnology). Later on, the DreamTaq DNA Polymerase (Thermo Fisher Scientific) was used for PCR. Then, the cDNA and gDNA PCR products were analyzed 2% agarose gel electrophoresis. In addition, qPCR was performed using the EnTurbo™ SYBR Green PCR SuperMix on a Verse flow cytometry system (BD Biosciences). β-actin and U6 were used as internal controls. The primers were listed in Table 1.
Actinomycin D and RNase R treatment
AGS and MKN45 cells were incubated with Actinomycin D (2 μg/ml; Sigma) for 0, 6, 12, 18 and 24 h to assess the stability of circPTK2 and its linear isoform. In addition, total RNA (10 μg) was treated with RNase R (5 U/μg; Epicenter Technologies) for 30 min at 37°C, then, the level of circPTK2 was detected using RT-qPCR assay.
Cell viability assay
Transfected AGS and MKN45 cells were seeded onto 96 well plates and cultured for the indicated times. Later on, cell viability was determined using the Cell Counting Kit-8 (CCK-8) reagent (Dojindo Laboratories, Kumamoto, Japan). Subsequently, the absorbance was measured at a wavelength of 450 nm.
Colony formation assay
Transfected AGS and MKN45 cells were plated onto 6 well plates. After 2 weeks of incubation, cells were fixed with 4% paraformaldehyde for 20 min and then stained with 0.1% crystal violet at room temperature. After that, cell colonies were imaged and counted using a light microscope.
Transfected AGS and MKN45 cells were suspended in 200 μL serum-free medium and placed into the upper chambers (Corning, NY, USA). Later on, the lower chambers were loaded with DMEM medium (600 μL) containing 10% FBS. After 24 h of incubation, the cells on the lower surface were fixed with 4% formaldehyde, and then stained with 0.1% crystal violet solution. After that, the stained cells were imaged using a light microscope. Transwell chambers that coated with Matrigel were used for cell invasion assay.
BALB/c nude mice (5–6 weeks old) were purchased from the Jingda experimental animal Co., Ltd. (Changsha, China). This study was approved by the First Affiliated Hospital of Soochow University and conducted according to the institutional guidelines. Animals were divided into 4 groups (shRNA NC; circPTK2 shRNA2; OE NC; circPTK2 OE). AGS or MKN45 cells (5 x 106 cells/mouse) were subcutaneously injected into the right flank of each mouse. The size of the tumor was measured every 5 days. The tumor volume was calculated by the formula: (length x width2)/2. In the end of the experiment, the tumor was removed, and tumor weight was measured.
Luciferase reporter assay
The sequences including miR-196a-3p binding sites in the circPTK2 3’ UTRs and AATK 3’ UTR were cloned into the luciferase reporter vector pGL6-miR (Beyotime). After that, AGS or MKN45 cells were co-transfected with the luciferase plasmids and miR-196a-3p mimics for 48 h. Later on, the firefly and Renilla luciferase activities were measured by a dual-luciferase reporter assay system (Promega, USA).
RNA-pull down assay
The biotinylated circPTK2 or biotinylated miR-196a-3p probe were incubated with Streptavidin magnetic beads (Thermo Fisher Scientific) for 2 h at room temperature. Later on, AGS or MKN45 cells were incubated with the magnetic beads at 4°C overnight. After that, the complex was pulled down and analyzed by RT-qPCR assay.
The tumor tissues were fixed in 4% paraformaldehyde and then embedded in paraffin. Later on, tissues were sectioned (5-μm thick) and then stained with primary antibody specific for AATK (Abcam) overnight at 4°C. Images were captured by a fluorescence microscope.
Western blot assay
Protein concentration was determined by the BCA kit (Pierce, Rockford, USA). After that, equal amounts of proteins (30 μg) were separated by 10% SDS-PAGE and transferred onto a PVDF membrane. Later on, the membrane was incubated with primary antibodies against STK39 (1:1000, Abcam), AATK (1:1000, Abcam), p-STK39 (1:1000, Abcam), p-p38 (1:1000, Abcam), p38 (1:1000, Abcam), Bax (1:1000, Abcam), Bcl-2 (1:1000, Abcam), cleaved caspase 3 (1:1000, Abcam), CD81 (1:1000, Abcam), CD63 (1:1000, Abcam) and GAPDH (1:1000, Abcam) at 4°C overnight. Then, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibodies at room temperature and then visualized using the enhanced chemiluminescence reagent (Thermo Fisher Scientific).
Cells were transfected with pcDNA3.1-AATK or pcDNA3.1-STK39 plasmids After that, the transfected cells were lysed using RIPA buffer, and then cell lysates were treated with anti-AATK, anti-STK39 or anti-IgG antibodies. Later on, the samples were incubated with protein A and G Sepharose beads for 4 h at 4°C. Then, the protein binding complex was isolated and subjected to western blot assay.
Data were presented as mean ± standard deviation (S.D.). Differences between three or more groups were analyzed by One-way analysis of variance (ANOVA) and Tukey’s tests. P < 0.05 was considered statistically significant.