Clinical samples. A total of 56 matched GC and non-tumour samples (≥ 5 cm from tumor margin; 26 males and 30 females; aged 40–78 years old) were collected during surgery at the First Affiliated Hospital of Jinzhou Medical University (Jinzhou, China) from July 2010 to June 2013. The tissues were sectioned and snapfrozen using liquid nitrogen post-surgery, and stored at 80˚C. The patients were divided into circRNA_100269 high- or low-expression group was according to the mean value of circRNA_100269 expression. All the biopsies were evaluated by two independent pathologists, and the clinicopathological features of recruited patients were presented in Table I. Overall survival rates were obtained by Kaplan-Meier method. Written informed consents were signed by the patients, and all the samples were anonymized. The present study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Jinzhou Medical University.
Cell culture. Four human GC cell lines (AGS, SGC-7901, BGC-823 and MKN-45) and one normal human gastric epithelial cell line (GES-1) were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained using DMEM containing 10% fetal bovine serum (FBS), 100 µg/ml streptomycin and 100 U/ml penicillin (HyClone; GE Healthcare Life Science), and cultured at 37˚C in a incubator supplied with 5% CO2.
Cell transfection. To establish the cell model overexpressing circRNA_100269, wildtype (o/e-circRNA_100269) or mutant (o/e-NC) circRNA_100269 sequence was amplified using PCR, then subcloned into pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). In circRNA_100269 knockdown model, shRNA sequences against circRNA_100269 (sh-circRNA_100269) or negative control (sh-NC) were obtained from Genepharm Co. Ltd. (Shanghai, China). Following annealing, shRNA were inserted in lentiviral pU6-Luc-Puro vector (Genepharm Co. Ltd.). Cells without any shRNA treatment were used as the control group. Up- or downregulation of circRNA_100269 was examined by RT-qPCR. All the transfections were performed using Lipofectamine®2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Eight hours following transfection, culture media were replenished with fresh DMEM supplemented with 10% FBS. For the inhibition of PI3K signalling, cells were treated with LY294002 (10µM; Cell Signaling Technology, Beverly, USA).
RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Total RNA from clinical samples or cells was extracted by TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and then reverse transcribed into cDNA by a PrimeScript™ RT kit (Takara Biotechnology Co., Ltd., Dalian, China). The target cDNA was amplified using SYBR Green PCR Master Mix (TaKaRa Biotechnology Co., Ltd.), which was carried out using an ABI 7500 Real-Time PCR system (Thermo Fisher Scientific, Inc.) Endogenous GAPDH was used as internal control. The sequences of forward and reverse primer were as follows: circRNA_100269, 5’-CTATGTCGTTCAAGGAAGA-3’ and 5’-GTCTTGAGACTGGGTATGT-3’; PI3K, 5’-AACACAGAAGACCAATACTC-3’ and 5’-TTCGCCATCTACCACTAC-3’; E-cad, 5’-AAGAAGCTGGCTGACATGTACGGA-3’ and 5’-CCACCAGCAACGTGATTTCTGCAT-3’; vimentin, 5’-AGAACCTGCAGGAGGCAGAAGAAT-3’ and 5’-TTCCATTTCACGCATCTGGCGTT-3’; snail, 5’-TTTCTGGTTCTGTGTCCTCTGCCT-3’ and 5’-TGAGTCTGTCAGCCTTTGTCCTGT-3’; Bax, 5’-TAATCCCAGCGCTTTGGAA-3’ and 5’- TGCAGAGACCTGGATCTAGCAA-3’; cas-9, 5’-CATTTCATGGTGGAGGTGAAG-3’ and 5’-GGGAACTGCAGGTGGCTG-3’; MMP9, 5’-CAGAGATGCGTGGAGAGT-3’ and 5’-TCTTCCGAGTAGTTTTGG-3’; GAPDH, 5’-GCAAGAGCACAAGAGGAAGA-3′ and 5’-ACTGTGAGGAGGGGAGATTC-3’. PCR program was 95˚C for 5 min, followed by 45 cycles of 95˚C for 15 s, 60˚C for 20 s and 72˚C for 10 s. Relative expression was determined using 2−∆∆Cq method.
Western blotting. Total protein was extracted by radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China). The concentration of extracted protein was determined using bicinchoninic acid assay (Beyotime Institute of Biotechnology). Equal amount (30 µg) of samples were loaded on SDS-PAGE gel and subsequently transferred onto a PVDF membrane (EMD Millipore, Billerica, MA, USA). Membranes were blocked using tris-buffered saline (TBS) with 5% skimmed milk at room temperature for 2 h and incubated using correspondent primary antibodies: PI3K (1:2000; cat. no. ab140307; abcam), Akt (1:1000; cat. no. 9272; Cell Signaling Technology), p-AktS473 (1:1000; cat. no. 4058; Cell Signaling Technology), p53 (1:1000, cat. no. 9282; Cell Signaling Technology), Bcl-2 (1:1000, cat. no. 15071; Cell Signaling Technology), cyclin D1 (1:2000, cat. no. 2926; Cell Signaling Technology), E-cad (1:1000, cat. no. 3195; Cell Signaling Technology), vimentin (1:2000; cat. no. 5741; Cell Signaling Technology), snail (1:1000; cat. no. 3879; Cell Signaling Technology), Bax (1:1000; cat. no. 2772; Cell Signaling Technology), cas-9 (1:2000; cat. no. 14697; Cell Signaling Technology), MMP9 (1:1000; cat. no. 3852; Cell Signaling Technology) or GAPDH (1:1,000; cat. no. sc-47724; Santa Cruz Biotechnology Inc.) at 4˚C overnight. The membranes were subsequently incubated with corresponding horseradish peroxidase-conjugated anti-mouse (1:5,000; cat. no. sc-2371; Santa Cruz Biotechnology Inc.) or anti-rabbit IgG (1:5000; cat. no. sc-2357; Santa Cruz Biotechnology Inc.) at room temperature for 1 h. Protein bands were visualized by an enhanced ECL protein detection kit (Pierce Biotechnology; Thermo Fisher Scientific, Inc). Signals were quantified using densitometric method by Image J software (NIH, Bethesda, MD, USA).
Cell proliferation assay. Cells were harvested 24 h post-transfection, and 1 × 104 cells were placed in 96-well plates. The proliferation of cells was examined using MTT assay (Sigma-Aldrich; Merck KGaA) at day 1, 2, 3 and 4. Briefly, 20 µl of MTT solution was added into each well and incubated at 37˚C for 4 h, the absorbance at 450 nm was detected using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Wound healing assay. Cells were seeded onto 6-well plates at a density of 4 × 105 cells/well and transfected with corresponding vectors. After the cells reached the confluency of 80–100%, the cell monolayer was scratched in a straight line with a sterile micropipette tip and washed three times with PBS, which was replaced with fresh DMEM. Subsequently, the scratch width changes were observed immediately following the scratch and at 6, 12 and 24 h. The images were captured using a fluorescence microscope (magnificationx100, Olympus Corporation, Tokyo, Japan). The migration of cells was determined by ImageJ 6.0 using the following formula: Migration area ratio = proportion of closed wound area/entire field of view area.
Transwell assay. A total of 1 × 105 cells were suspended using FBS-free culture medium and seeded onto the Matrigel®-pre-coated (Sigma-Aldrich, St. Louis, MO, USA) upper chamber (BD Biosciences, Franklin Lakes, New Jersey, USA). Subsequently, 500 µl of culture medium containing 10% FBS was added into the lower chamber. Following overnight incubation, non-invasive cells were detached using a cotton swab, while invaded cells in the lower chamber were fixed using 4% paraformaldehyde and stained by 0.5% crystal violet. The numbers of invasive cells were counted in five randomly selected fields using an inverted light microscope (magnificationx200, Olympus Corporation, Tokyo, Japan).
Cell cycle and apoptosis analysis. Cells were inoculated onto 6-well plates with a density of 4 × 105 cells/well following the treatments with o/e-circRNA_100269 or o/e-NC, respectively. Then, cells were collected using low-speed centrifugation (1000 rpm) at 4˚C for 5 min. Cell pellets were rinsed and re-suspended in PBS, subsequently fixed with 70% pre-chilled ethanol and stored at 4˚C for two days. Cells were lysed prior to flow cytometry, centrifuged and then re-suspended using propidium iodide (PI, Sigma-Aldrich, USA) staining buffer containing 50 µl/ml of PI with 250 µl/ml RNase A. Cell cycle distributions were determined by a flow cytometer (BD Biosciences, USA) and then analysed using Flowjo version 7.6 software (Flowjo LLC, USA). To evaluate cell apoptosis, the suspended cells was incubated in dark at 4˚C for 30 min and stained with 5 µl annexin V-FITC (JingMei Biotech, Beijing, China), and apoptosis was examined using a a flow cytometer (BD Biosciences, USA) and then analysed using Flowjo version 7.6 software (Flowjo LLC, USA).
In vivo nude mouse xenograft. Female BALB/C nude mice (4-5-week-old) with the weight of ~ 19 g were purchased from The Laboratory Animal Research Centre of Nantong University (Nantong, China). The mice were routinely housed in a temperature-controlled environment (22 ± 2˚C) with 60% relative humidity, under a 12-h dark/light cycle with libitum access to food and water for at least three days before the experiments. Mice were randomly grouped (n = 5 in each group) and injected with AGS cells transfected with sh-NC or sh-circRNA_100269. Briefly, a total of 1 × 107 cells were suspended in 200 µl PBS and injected into the back subcutaneously. Mice with developing tumors were monitored four times a week. Six weeks post-injection, the mice were sacrificed, and the tumor tissues were removed and examined. Tumor volume was calculated as follows: V (mm3) =(length x width2)/2. To initiate metastasis, 1 × 105 cells were suspended in 20 µl PBS and then injected in the lateral tail vein of mice. Experiments contained 10 mice inoculated with tumor cells on day 0. Followed injection, mice were randomly sorted into experimental groups to be evaluated after 42 days. The protocol of animal experiment was approved by the Ethics Committee of our Hospital.
Statistical analysis. Data were presented as means ± standard error of mean and analysed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). The significance of differences was analysed using one-way analysis of variance (ANOVA) or the Student’s t-test. A student-Newman-Keuls test was carried out after ANOVA. The association between RNA expression was determined using Spearman’s correlation analysis. P < 0.05 was considered to indicate a statistically significant difference.