GES-1 cells, GC cell lines (MKN-45, AGS and SNU-5), and THP-1 cells were obtained from ATCC and cultured in DMEM containing 10% FBS, 1% penicillin and streptomycin (Sigma) in the condition of 37°C and 5% CO2.
Clinical sample collection
In brief, five pairs of GC and adjacent normal tissues were collected from Beijing Hospital of Traditional Chinese Medicine between June 2019 and June 2020. Each tissue sample was stored at -80°C. In addition, the serum samples were collected from the patients or healthy people. The present study was approved by the Ethics Committee of Capital Medical University. Meanwhile, the data of the patients with GC or healthy people were collected with their written informed consent.
IL-13 and IL-4 were bought from MedChemExpress.
GC cells were transfected with pcDNA3.1 or pcDNA3.1-hsa_circ_0017252 by using Lipofectamine 2000 for 48 h. After 48 h of transfection, cells were used in subsequent experiments.
For miRNA transfection, GC cells were transfected with negative control (NC) or miR-17-5p agomir by using Lipofectamine 2000 for 48 h.
Exosome extraction and identification
Exosomes were extracted from GC cell supernatants by using ultracentrifugation methods. Briefly, the complementary medium was replaced the defined medium (without serum) when cells reached 80% confluence. The supernatants were harvested and centrifuged after 48 h of culture. Subsequently, supernatants were collected to extract exosomes by ultracentrifugation (70 min, 120,000 × g, two times). TEM, NTA and western blot were performed to identify the exosomes.
Isolation and cultivation of macrophages
THP-1 cells were added with PMA (100 ng/ml). Then, cells were treated with MKN45-Exo, MKN45-Exo-hsa_circ_0017252 OE or IL4/IL-13 (20 ng/ml) for 24 h. After that, the CD206 (macrophages surface maker) and CD86 (macrophages surface maker) were tested by flow cytometry.
Fluorescence in situ hybridization
The co-localization of hsa_circ_0017252 and miR-17-5p in the cytoplasm was observed by using FISH (19).
TRIzol® reagent was applied to extract total RNA. PrimeScript RT reagent kit was used in reverse transcription. Then, SYBR premix Ex Taq II kit (Takara) was used in RT-qPCR, and Real-Time qPCRs were used three times: 2 minutes at 94°C, followed by 35 cycles (94°C for 30 s and 55°C for 45 s). The primer sequences were listed as follows: hsa_circ_0017252 forward, 5’-AAACUACUAGGUAAAGGCACUUUU-3’ and reverse, 5’-GATAAAUUUAAAGCUCUGUGCC-3’; β-actin forward, 5’-GTCCACCGCAAATGCTTCTA-3’ and reverse, 5’-TGCTGTCACCTTCACCGTTC-3’. The data were quantified by using 2−ΔΔt method. β-actin was regarded as internal control.
GC cells (5x103 cells/well) were treated for 48 h as follows: NC, hsa_circ_0017252 OE or hsa_circ_0017252 OE + miR-17-5p agomir. Subsequently, GC cells were added with CCK-8 (10 μl) for another 2 h. The absorbance of each well (450 nm) was assessed using a microplate reader.
GC cells were fixed and then incubated with anti-PKH26 (1:1,000) or anti-CD206 (1:1,000). Subsequently, the secondary antibody (1:5,000; goat anti-rabbit IgG) was used to incubate GC cells. Finally, the data was observed by a microscope.
RIPA was applied to extract protein from tissues or cell lines. BCA kit was applied to quantify the total protein. SDS-PAGE (10%) was applied to separate the proteins (40 μg per lane), and then proteins were transferred onto PVDF membranes. Subsequently, the membranes were incubated overnight at 4˚C with primary antibodies targeted against: CD63 (1:1,000), TSG101 (1:1,000), Runx3 (1:1,000), α-SMA (1:1,000), vimentin (1:1,000), N-cadherin (1:1,000), E-cadherin (1:1,000) and β-actin (1:1,000) after blocked with skimmed milk (5%) for 1 h. Following primary incubation, HRP-conjugated secondary antibodies (1:5,000) were used to incubate the membranes for 1 h. ECL kit was used to visualize the protein bands. β-actin was regarded as internal control. The densitometry analysis was performed by using IPP 6.0 (Image-Pro Plus 6.0).
Matrigel (100 μl, not included in migration assay) was used to pre-treat the upper chamber. GC cells (1.0×106 cells per chamber) were seeded into the upper chamber in medium (1% FBS). In addition, the lower chamber was supplemented with RPMI1640 (10% FBS). Subsequently, the chamber was rinsed and fixed at 4°C. Then, crystal violet (0.1%) was used to stain the chamber for 20 minutes. The data was observed under a microscope after the chamber was washed.
Dual luciferase reporter assay
Hsa_circ_0017252 containing the binding sites of miR-17-5p was cloned into the pGL6 vectors for establishment of hsa_circ_0017252 (WT/MT). Hsa_circ_0017252 (WT/MT) was transfected into GC cells with miR-17-5p/NC mimics using Lipofectamine 2000. Dual-Glo Luciferase Assay System was used to analyze the result.
Cell apoptosis detection
GC cells (5×104) were centrifuged and resuspended. Subsequently, cells were treated with Annexin V-FITC and PI at 4˚C for 15 min. Cell apoptosis was analyzed by flow cytometer, and FlowJo was used to quantify the data.
IL-10 and IL-1β levels in GC cell supernatants were assessed by ELISA kit.
In vivo study
BALB/c nude mice (aged 6-8 weeks old, n=24) were placed in a room with dedicated SPF facility. MKN45 cells co-cultured with macrophages-exo, macrophages or macrophageshsa_circ_0017252 OE were transplanted in each mouse subcutaneously. The tumor volume was measured weekly as the flowing equation: length × width2. Finally, mice were sacrificed for tissue collection. Then, tumors were weighted. All the experiments were performed in line with NIH guide. Moreover, the Ethics Committees of Capital Medical University approved the protocol of this study.
Three independent experiments were performed in each group. In addition, the mean ± standard deviation (SD) was used to express all data. The comparisons between two groups were analyzed using Student’s t-test, and the differences between multiple groups (more than 2 groups) were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test (Graphpad Prism7). P<0.05 indicates a significant change.