All GC tissues used in this study were obtained from the First Affiliated Hospital of Nanjing Medical University. All GC patients received CDDP-based chemotherapy after radical gastrectomy. Tumor marker evaluation was performed after each cycle of chemotherapy. Abdominal contrast-enhanced CT was performed after every two cycles of chemotherapy. Based on the results of tumor marker examination and imaging examination, we could judge whether the tumor recurred. CDDP resistance was defined as tumor recurrence during CDDP-based chemotherapy after radical gastrectomy, and CDDP sensitivity was defined as no tumor relapse during CDDP-based therapy . The tissues used for RNA extraction and immunochemical staining were stored in liquid nitrogen and 4% formaldehyde, respectively.
The BGC823, SGC7901, and SGC7901CDDP cell lines were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences. The BGC823CDDP cell line was established according to a published protocol . The four cell lines were cultured in RPMI 1640 (Gibco, USA) at 37°C 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 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 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 . 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 (Thermo Fisher Scientific, China), respectively. Nuclei were stained with DAPI.
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.
We performed the EdU assay to assess DNA synthesis, which indicated GC cell proliferation, 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 treatment 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 was 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 cotransfected 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 per previously described methods . RiboBio (Guangzhou, China) designed and synthesized a 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 prebound to 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 the 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 an ultramicrotome and then stained with 0.3% lead citrate. A JEM-1010 electron microscope (JEOL, Japan) was used to observe autophagy in GC cells.
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 nuclei. 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.
Total protein was extracted from GC cells and tissues. The proteins were transferred to PVDF membranes (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 three times with TBST and then incubated with secondary antibodies at room temperature for 2 h. After the membranes were washed with TBST three times, 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 mouse 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 nude mice were sacrificed at day 35.
We performed TUNEL assays to measure the rate of GC cell apoptosis rate in nude mouse subcutaneous tumors. The assay was carried out using a Cell Death Detection Kit (Roche, USA), and TUNEL-positive cells were counted using a microscope (Nikon, Japan).
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 antibodies 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 Product and Service Solutions (SPSS) software version 19.0 was used for statistical analysis. All experiments were performed in triplicate, and the results are expressed as the mean value ± standard deviation (SD). Student’s t-test was used to determine significant differences between two independent groups. Pearson’s c2 test was used to analyze the relationship between the circMCTP2 expression level and clinicopathological features of the GC patients. The survival analysis was performed with the Kaplan-Meier method. The differences in survival between the two groups were assessed using the log-rank test. Linear correlation analysis was performed to determine the correlations between gene expression levels. P < 0.05 (*) or P < 0.01 (**) was considered statistically signiﬁcant.