Tissue samples
All GC tissues used in the research were obtained from the First Affiliated Hospital of Nanjing Medical University. All GC patients received CDDP-based chemotherapy after radical gastrectomy. We followed the method published earlier to divide patients into CDDP-resistant and CDDP-sensitive groups [26]. CDDP resistance implies GC recurrence during the CDDP-based treatment. Patients who showed no tumor recurrence during CDDP-based therapy were defined as CDDP-sensitive patients. The tissues used for RNA extraction and immunochemical staining were stored in liquid nitrogen and 4% formaldehyde, respectively.
Cell lines
BGC823, SGC7901, and SGC7901CDDP cell lines were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences. BGC823CDDP cell line was established according to a published protocol [27]. The four cell lines were cultured in RPMI1640 (Gibco, USA) at 37℃ with 5% carbon dioxide in an incubator.
RNA extraction and RNase R treatment
The Cytoplasmic and Nuclear RNA Purification Kit (Norgen Biotek, Canada) was used to extract nuclear and cytoplasmic RNAs. Total RNA from GC tissues and cells was extracted using the TRIzol reagent (Invitrogen, USA). The extracted total RNA of GC cell lines was mixed with 3 U/mg RNase R for 15 min at 37°C. qRT-PCR was performed to detect the expression of circMCTP2 and MCTP2 mRNA, which indicated the stability of circMCTP2 and MCTP2 mRNA.
Quantitative real-time polymerase chain reaction
mRNA was reverse transcribed into cDNA using the Prime script RT Reagent (Takara, Japan). For reverse transcription of miRNA, we used a New Poly(A) Tailing Kit (ThermoFisher Scientific, China). qRT-PCR was carried out with an ABI StepOne Plus system using the SYBR Green Master Mix (Roche, USA). The primers are listed in Additional file 1: Table S1.
Actinomycin D assay
GC cells (5×104 cells per well) were seeded into a 24-well plate. After 24 h, GC cells were mixed with 2 mg/L actinomycin D (Sigma-Aldrich, USA) for 0, 4, 8, 12, and 24 h. The stabilities of circRNA and mRNA were examined by qRT-PCR.
Fluorescence in situ hybridization (FISH)
We followed a previously published protocol to perform the assay [28]. We used a biotin-labeled probe for circMCTP2 and a Dig-labeled probe for miR-99a-5p (Exiqon, Denmark) in this assay. The signals of the biotin-labeled probe and the Dig-labeled probe were captured using Cy5-conjugated streptavidin and a tyramide-conjugated Alexa 488 fluorochrome TSA kit (ThermoFisher Scientific, China), respectively. Nuclei were stained with DAPI.
Cell transfection
Commercially available lentivirus-circMCTP2, lentivirus-miR-99a-5p mimics, lentivirus-miR-99a-5p inhibitor, and lentivirus-shMTMR3 were purchased from GenePharma (Shanghai, China). After transfection, we used puromycin (Solarbio, China) to establish stably transfected cell lines.
Cell counting kit-8 (CCK-8) cell viability assay
CCK-8 (Dojindo, Japan) was used to determine the cell viability of GC cells. GC cells were seeded into a 96-well plate at a density of 5000 cells per well. Seeded cells were incubated for 2 h with the CCK-8 reagent before measurement.
EDU assay
We performed the EDU assay to assess DNA synthesis, which indicated the cell proliferation of GC cells, using an EDU assay kit (RiboBio, China). Stained GC cells were photographed using a microscope (Nikon, Japan).
Flow cytometric analysis
GC cells were seeded at a density of 2×105 cells per well in a 6-well plate. After treating with CDDP for 48 h, a PI/ Annexin V Apoptosis Detection Kit (BD, USA) was used to stain the collected GC cells. The proportion of apoptotic GC cells was detected using a flow cytometer (Gallios, Beckman, USA).
Colony formation assay
GC cells were seeded into a 6-well plate at a density of 1000 cells per well. After being cultured for 14 days, crystal violet (Kaigen, China) was used to stain the fixed GC cells. The cells were washed with PBS and then the number of colonies were counted.
Luciferase reporter assay
Wild-type (wt) and mutant (mut) sequences of circMCTP2 were designed and inserted into the pGL-3 luciferase reporter vector (Realgene, China). BGC823CDDP and SGC7901CDDP were co-transfected with luciferase reporter plasmids and miR-99a-5p mimics. Firefly and renilla luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega, USA).
RNA pull-down assay
The RNA pull-down assay was performed as per previously described methods [29]. RiboBio (Guangzhou, China) designed and synthesized the biotin-labeled probe specific to circMCTP2. GC cells were collected and sonicated to produce cell lysates. The lysate was incubated with circMCTP2 probe that was pre-bound on streptavidin-coupled Dynabeads (Invitrogen, USA) and oligo probe. The RNA mixture bound to the magnetic beads was rinsed with wash buffer and then extracted using the RNeasy Mini Kit (QIAGEN, Germany). We performed qRT-PCR on the RNA product. Biotinylated-miR-99a-5p and biotinylated-miR-NC were produced by GenePharma (Shanghai, China). After 48 h, the constructs were transfected into GC cells, and the cells were lysed. The lysate was incubated with streptavidin-coated magnetic beads and then rinsed with PBS. The biotin-coupled RNA complex was pulled down and subjected to qRT-PCR.
RNA immunoprecipitation assay
A Magna RNA immunoprecipitation (RIP) kit (Millipore, USA) was used to perform RIP assay. We used RIP buffer to lyse GC cells and the cell lysate was then incubated with magnetic beads conjugated with anti-Ago2 antibody (Millipore, USA) or IgG antibody. Finally, the immunoprecipitated RNA was extracted and subjected to qRT-PCR.
Transmission electron microscopy (TEM)
Prepared GC cells were harvested and a 2.5% solution of glutaraldehyde was used to fix GC cells overnight. Then, the GC cells were fixed with 1% OSO4 for 1 h. Samples were dehydrated with increasing concentrations of ethanol, followed by embedding in Epon. Sections were cut with a ultramicrotome and then stained with 0.3% lead citrate. A JEM-1010 electron microscope (JEOL, Japan) was used to observe autophagy in GC cells.
Confocal microscopy
GC cells transfected with GFP-mRFP-LC3 lentivirus (GeneChem, China) were seeded into a 35-mm culture dish for confocal microscopy. Hoechst was used to stain nucleus. Red and yellow puncta representing autolysosomes and autophagosomes, respectively, were detected by confocal microscopy (Carl Zeiss, Germany). Three random fields were selected for the quantification of puncta.
Western blotting
Total proteins were extracted from GC cells and tissues. The proteins were transferred to PVDF membrane (Millipore USA) after SDS-PAGE electrophoresis. After blocking in TBST buffer with 5% skimmed milk, the membranes were incubated with primary antibodies overnight at 4°C. The membranes were rinsed thrice with TBST and then incubated with secondary antibodies at room temperature for 2 h. After washing with TBST thrice, the bands were detected using an enhanced chemiluminescence detection system with Chemiluminescence HRP Substrate (Millipore, WBKL0100). Anti-Bcl2, anti-Bax, anti-caspase3, anti-c-caspase3, anti-β-actin, anti-P62, anti-Beclin1, anti-LC3, and anti-MTMR3 were purchased from Abcam (Cambridge, UK). Anti-rabbit IgG-HRP and anti-mouse IgG-HPR antibodies were obtained from Santa Cruz (Dallas, TX, USA).
Nude mice xenograft model
Female BALB/c nude mice (5 weeks old) were purchased from the Department of Laboratory Animal Center of Nanjing Medical University. Stably transfected GC cells (1×106) were injected subcutaneously into each armpit of a nude mouse. A week later, CDDP (5 mg/kg) was injected intraperitoneally into nude mice three times per week. All the nude mice were sacrificed at day 35.
TUNEL assay
We performed TUNEL assay to detect the rate of apoptosis in GC cells in nude mice subcutaneous tumors. The assay was carried out using a Cell Death Detection Kit (Roche, USA) and the TUNEL-positive cells were counted using a microscope (Nikon, Japan).
Immunochemical staining
Tissue samples from GC patients and subcutaneous tumors from nude mice were fixed with 4% formalin and embedded in paraffin. The sections were incubated with anti-ki67, anti-P62, and anti-MTMR3 overnight at 4°C. Then the sections were incubated with secondary antibody for 1 h at room temperature and developed with DAB solution for 3 min. The sections were counterstained with hematoxylin.
Statistical analysis
Statistical analysis was performed using SPSS software (version 19.0). P < 0.05 (*) or P < 0.01 (**) was considered statistically significant. We repeated all experiments thrice. The results are shown as mean ± standard deviation (SD). The Student’s t-test and the Pearson’s χ2 test were also used in the statistical analysis.