Statements, human tissue specimens and cell lines
All experiments were performed in accordance with relevant guidelines and regulations. Fifty human gastric cancer samples and their adjacent normal tissues were obtained from patients who underwent surgery at the Department of Surgery of Tongji Hospital of Tongji Medical College between 2014 and 2015. We received approval for this study from the Institutional Review Board of Tongji Medical College of Huazhong University of Science and Technology, and all patients provided signed informed consent forms.
The normal gastric mucosal cell line GES-1 from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and gastric cancer cell lines from the American Type Culture Collection (ATCC; Manassas, VA, USA) were cultured in RPMI1640 (HyClone, Logan, UT, USA) supplemented with 10% foetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) at 37 °C under a humidified atmosphere with 5% CO2. The three 3 cell lines were authenticated by STR profiling and tested for mycoplasma contamination.
Oligonucleotide transfection and circular RNA plasmid construction
Both miRNA mimics and cont-miR were purchased from Sangon Biotechnology (Sangon, Shanghai, China) and transfected into cells using Lipofectamine 2000 (Invitrogen, Camarillo, CA, USA). Human cir-ITCH cDNA was PCR amplified from AGS cell gDNA, and the PCR product was purified with a miRNAVana miRNA kit (Life Technologies, New York, USA). Finally, the cir-ITCH cDNA was cloned into pCDNA3.1 (Clontech Laboratories, Inc., San Francisco, CA, USA) as previously described4. The recombinant plasmid harbouring cir-ITCH was then verified by sequencing, and the lentivirus was constructed by Geneseed (Guangzhou, China). The human miR-17 gene was also PCR amplified and then cloned into a lentiviral vector according to the manufacturer’s instructions20.
RNA extraction and quantitative real-time polymerase chain reaction (RT-qPCR)
Total miRNA was extracted with RNAiso for Small RNA (TaKaRa Bio, Otsu, Japan) following the manufacturer’s instructions. Total RNA was extracted from human tissue specimens and cultured cells using TRIzol reagent (TaKaRa Bio, Otsu, Japan) according to the protocol. Poly-A tails were added to miRNA and U6 using miRNA reaction buffer mixture (TaKaRa Bio, Otsu, Japan), after which total miRNA was reverse transcribed into cDNA using miRNA PrimeScript RT enzyme Mix (TaKaRa Bio, Otsu, Japan). cDNA was synthesized from total RNA using SuperScript III (Invitrogen). RT-qPCR was performed in a CFX96 Real-Time PCR Detection System (Bio-Rad, USA). TaqMan-based RT-qPCR was performed to assess the expression of cir-ITCH in gastric cancer tissues, while SYBR Green was used for RT-qPCR to assess the expression of cir-ITCH, linear ITCH, miRNAs and other genes in gastric cell lines. Primers were synthesized by Sango Biotech, China (Table 1). U6 and GAPDH were used as an internal reference for miRNA, mRNA and circRNA detection. All reactions were performed in triplicate.
Nucleic acid electrophoresis
The cDNA and gDNA PCR products of cir-ITCH were detected via 2% agarose gel electrophoresis at 100 V for 30 min. The DNA marker used was D0107 (Beyotime, China), and the results were visualized by UV irradiation.
RNase R digestion
Total RNA (4 µg) was incubated for 30 min at 37 °C with 10 × reaction buffer (20 mM Tris–HCl (pH = 8.0), 0.1 M KCl and 0.1 mM MgCl2) and 12 U of RNase R (Epicentre Biotechnologies, Madison, WI, USA), after which total RNA was purified by ethanol precipitation21.
RNA immunoprecipitation (RIP)
We used an EZMagna RIP kit (Millipore, Billerica, MA, USA) to perform RIP according to the manufacturer’s protocols. The gastric cancer cells lines MKN45 and AGS were lysed using RIPA lysis buffer, and then lysates were incubated with magnetic beads conjugated with different antibodies (Ago2 or IgG; Millipore) for 6 h at 4 °C. Then, the beads were rinsed and incubated with Proteinase K to remove the proteins. Subsequently, cir-ITCH expression was assessed by RT-qPCR.
Biotin-coupled miRNA capture
We transfected biotin-coupled miRNA mimics and control miRNA (Sangon, Shanghai, China) into AGS cells using Lipofectamine 2000 (Invitrogen, Camarillo, CA, USA) and harvested cells after 24 h. Subsequently, the AGS cells were washed twice with PBS and then lysed with lysis buffer. Then, 50 µl of the washed streptavidin magnetic bead samples were blocked for 2 h before being added to tubes to pull down the RNA complexes. Subsequently, the tubes were put on a rotator at low speed (10 r/min) for 4 h and then rinsed five times with lysis buffer. Finally, we used TRIzol LS (Life Technology, USA) to recover RNA, and cir-ITCH expression was assessed by RT-qPCR analysis.
Luciferase reporter assay
Wild-type cir-ITCH sequences were RT-PCR amplified, and then the sequences were inserted into the pmirGLO luciferase vector (Geneseed, Guangzhou, China). The Mutant cir-ITCH sequences were synthesized using a site-directed Gene Mutagenesis Kit (Beyotime, China) following the manufacturer’s protocol. The linear ITCH vector harbouring the wild-type or mutant 3′UTR region that can bind to miR-17, were also constructed by using a similar method. The vector and miR-17/214 mimics were cotransfected into AGS gastric cancer cells using Lipofectamine 2000. Finally, we examined the luciferase activity of cells using a dual-luciferase reporter assay system (Promega) 48 h after transfection. The relative luciferase activity was normalized to the Renilla luciferase internal control. The primers used to construct the luciferase vector are shown in Table 2.
The TCF-LEF reporter was purchased from SABiosciences (Qiagen, Inc., Valencia, CA, USA), and we examined reporter activity using a dual-luciferase reporter assay following the manufacturer’s protocol22.
Antibodies and immunoblotting
Antibodies against ITCH (#ab220637), β-catenin (#ab16051), Wnt3a (#ab219412), β-actin (#ab179467), phospho-Dvl (#ab124933) and Dvl (#ab233003) were purchased from Abcam (Cambridge, UK). HRP-conjugated goat anti-rabbit IgG (#sc-2004) was purchased from Santa Cruz Biotechnology. Gastric cancer cells and gastric cancer tissues were lysed using RIPA lysis buffer (Beyotime, China) following the manufacturer’s protocols. Subsequently, the proteins in samples were separated via 10% SDS polyacrylamide gel electrophoresis and then transferred onto PVDF membranes. The membranes containing total proteins were blocked in 5% non-fat milk in TBST for 1 h and then incubated with primary antibodies overnight. Then, we washed the membranes with TBST 3 times and incubated them with HRP-conjugated secondary antibodies for 1 h. Finally, the protein bands were detected using enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA).
Cell viability and clonability assays
The transfected cells were seeded into 96-well plates at a density of 1 × 104 cells/well, and cell viability was measured using the cell counting kit-8 (CCK-8) system (Dojindo, Japan) following the manufacturer’s protocols. CCK-8 solution (10 μl) was added to each well, and the plate was incubated at 37 °C for 1 h. Then, the absorbance of each well at 450 nm was measured using a microplate reader (Tecan, Switzerland). For the colony formation assay, cells were cultured at a low density (1000 cells/plate) for approximately 2 weeks, after which they were stained with Giemsa, and the colonies were counted.
Migration and invasion assays
For migration assays, 1 × 104 cells were transferred to a transwell insert with 8-µm pores (BD Biosciences). For invasion assays, 2 × 105 cells were transferred to the top chamber of a transwell with a Matrigel-coated membrane (BD Biosciences). Then, medium supplemented with 10% serum was added to the lower chamber, and cells were cultured in serum-free medium in the top chamber. After incubating the cells in a tissue culture incubator for 16 h, we used cotton-tipped swabs to remove the cells from the upper sides of the membrane. Finally, we stained the migrated/invaded cells with crystal violet and counted the cells.
In vivo tumour growth assays
Male athymic nude mice were purchased from the Animal Experimental Center of Tongji Hospital of Tongji Medical College and were acclimated for 2 weeks before being injected with AGS gastric cancer cells. This experiment was undertaken according to the guidelines for the Care and Use of Laboratory Animals of Tongji Medical College. All procedures were approved by the Committee on the Ethics of Animal Experiments of Tongji Hospital of Tongji Medical College. We used sodium pentobarbital anaesthesia to minimize the suffering of nude mice throughout the assay. During the experiment, the investigator was blinded to the group allocation. Male nude mice were randomly divided into 3 groups (n = 3). Equal numbers of AGS cells (106) overexpressing cir-ITCH and with or without miR-17 restoration were suspended in 100 μl of PBS and subcutaneously injected into the right rear flank of each mouse. We used the formula V = 1/2 a × b2 (a: longest tumour axis; b: shortest tumour axis) to calculate the tumour volume. During the animal experiments, if nude mice died or the xenograft tumour did not grow, we excluded those mice from subsequent experiments. After 5 weeks, all nude mice were sacrificed, and the tumours were excised and preserved in formalin or liquid nitrogen.
Tumour engraftment and PDTX maintenance
We chose one primary gastric cancer sample and cut it into fragments (1 mm3) in Matrigel Basement Membrane Matrix (0.1 ml, 50%, BD Biosciences). Then, the fragments were implanted into the dorsal flank of nude mice (n = 5). Nude mice with palpable tumours were divided into 2 groups, the 1.5 mg/kg empty vector group or the cir-ITCH vector group, with mice in each group receiving an intratumoural injection twice a week for 2 weeks. After 2 weeks, all nude mice were sacrificed, and the tumours were weighed and preserved in formalin or liquid nitrogen.
Paraffin-embedded blocks were sectioned onto positively charged microscope slides and then were deparaffinized with xylene. After deparaffinization, the slides were hydrated with absolute ethanol and pretreated with citrate buffer for 20 min in a 98 °C steamer to retrieve the antigens. Then, the slides were incubated with antibodies against ITCH (1:200, Abcam, Cambridge, UK) or β-catenin (1:250, Abcam, Cambridge, UK) at 4 °C overnight. Subsequently, an UltraSensitive S-P Detection kit (KIT-9720, Maixin, Fuzhou, China) was used to perform immunostaining, and a DAB kit (PW017, Sangon Biotech, Shanghai, China) was used to develop colour. Finally, the samples were counterstained with haematoxylin, after which the integrated optical density (IOD/Area) in different groups were examined using Image-Pro Plus 6.0.
The data are presented as the means ± standard error of the mean (s.d.). Difference between two groups were analysed by Student’s t-test. Spearman’s rank test was used to evaluate the relationships between the relative expressions of cir-ITCH and linear ITCH in gastric cancer tissues. Kaplan–Meier (log-rank test) analysis was performed to calculate overall survival time. Differences were considered significant at p < 0.05.