Patients and serum samples
A total of 94 serum samples were obtained from GC patients who were diagnosed with primary GC confirmed by pathological diagnosis in Nanjing Medical University Affiliated Suzhou Hospital from April 2019 to March 2022. We also collected serum samples from 17 patients with benign gastric diseases including gastritis, gastric ulcer, benign tumor, etc. In addition, 21 serum samples were obtained from healthy volunteers as control subjects. We have gotten informed consent from all of the mentioned patients and volunteers above. Also, this study was carried out after approval by the Institutional Ethical Committee of Nanjing Medical University on 2022 − 02–23. The number of the Ethics Committee decision is No. (2022)169.
Cell lines and cell culture
Human GC cell line HGC-27 (Guangzhou Cellcook Biotech Co., Ltd., Guangzhou, China) was cultured in complete medium, which contains 90% RPMI 1640 medium (Invitrogen, Carlsbad, CA) and 10% fetal bovine serum (FBS) in a CO2 incubator at 37°C. Primary NFs and CAFs were collected from tumor tissues and paired normal samples. Briefly, the tissue was washed 3 times with PBS, soaked in 10000 U of penicillin and streptomycin for 15 min, and cut into small pieces of 4 mm 2. And then 20% FBS-containing fibroblast medium (ScienCell Research Laboratories, San Diego, CA) was added for culture until the fibroblasts around the tissue block are confluent, and then they were digested by trypsin and passaged. In this study, we used NFs and CAFs of passages 5–8.
Exosome extraction
To exclude the effect of exosomes in serum, we centrifuged at 100,000g for 18h before extracting exosomes in the cell culture supernatant. The cell culture supernatant was collected and filtered using an exosome enrichment column (Merck Millipore Ltd. Darmstadt, Germany). Exosomes were then extracted using exosome extraction reagents (System Bioscience, LLC Palo Alto, CA) according to the protocol provided by the manufacturer.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted via TRIzol reagent (Invitrogen) and then a HiFiScript cDNA Synthesis Kit (Cwbio, Beijing, China) was used to produce cDNA. Subsequently, RealSYBR Mixture (Cwbio) was applied in real-time qPCR performed in a real-time PCR Detection System (LightCycler 480, Roche, Switzerland). U6 was used as an internal reference.
Transmission electron microscopy (TEM)
Transmission electron microscopy was applied for exosome morphology. Briefly, 1% glutaraldehyde was used to fix exosomes in PBS and then a drop (20µL) of exosomes was placed on 200-mesh Formvar/carbon-coated nickel grids, which were stained with 3% (W/V) aqueous phospphotungstic aid. The morphology of exosomes was recorded by transmission electron microscopy (Tecnai 12; Philips).
Nanoparticle size analysis
The exosome solution was diluted 10000 times before being loaded on the nanoparticle size analyzer (#NanoSight NS300, Malven, England). It was carried out in accordance with the operating procedures provided by the manufacturer.
Immunofluorescence
Cells were fixed with 4% paraformaldehyde for 15 min and washed three times with PBS for 5 min each. Subsequently, cells were blocked with 0.3% Triton™ X-100 buffer for 60 min at room temperature. After blocking, the primary antibody was added and incubated overnight at 4°C. Then, rinse three times with PBS for 5 minutes each before adding secondary antibody and incubating specimens for 1 hour in the dark. Before photographing with a fluorescence microscope, three washes with PBS were performed for 5 min each.
Treatment of supernatant of cell culture medium
After fresh cell culture supernatant was collected, cell debris was removed by centrifugation at 1500 g for 10 min. When the cells were treated, an equal volume of the complete medium of the treated cells was added, and the cells were cultured at 37°C with a 5% CO2 atmosphere for 48h.
Cell transfection
For relevant materials and procedures of cell transfection, please refer to our previous article[11].
Cell counting assay and cell colony formation assay
After a 36 h transfection, 5× 10 4 cells were seeded in 5 wells equally in 24-well plates. The total cell number was then counted every 24h. GraphPad 5.0 was applied to draw growth curves depending on cell number. Also, cells were seeded in 6-well plates for colony formation assay (1×104 per well) and cultured t 37°C with a 5% CO2 atmosphere for 7 days. Subsequently, cells were fixed using 4% paraformaldehyde and then stained with crystal violet. The colony number was finally calculated using a microscope.
Transwell migration and invasion assay
After a 36 h transfection, 2× 105 cells were seeded into the top chamber of Transwell chambers (Chemicon, Temecula, CA, USA) resuspended in a 200 µL serum-free medium. Complete medium (600 µL) was added into the bottom chamber. For invasion assay, the top chamber was added with 20% Matrigel matrix 8 h before seeding cells. Finally, cells were fixed using 4% paraformaldehyde, stained with crystal violet, and counted under the microscope after the upper layer cells were carefully removed.
Wound healing assay
A total of 2 x 104 cells were seeded in a 24 well plate overnight, and then a straight line was drawn in the middle of every well with a 10 µL pipette tip. After being washed three times with PBS, cells were changed to a serum-free medium. Cells were then photographed and recorded after 0, 4, and 8 h in a CO2 incubator at 37°C.
Western blot analysis
RIPA buffer (Beyotime, Shanghai, China) was mixed with 0.1% protease inhibitor PMSF (Beyotime) and applied to lyse cells. The protein samples were denatured by heating and mixed with loading buffer before electrophoresis. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to separate proteins. Then target proteins were electrophoretically transferred at 360 mA current for 120 min and collected on 0.22-µm polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). 5% skim milk was used to block non-specific binding sites for 60 min at room temperature. Primary antibody was applied to bind the target protein on PVDF membranes overnight at 4°C. The membrane was washed three times for 5 min each with TBS/T and incubated with the secondary antibody for 60 min at 37°C. Scientific SuperSignal chemiluminescent HRP substrates (Thermo Fisher, Waltham, USA) was then applied matched with an enhanced chemiluminescence kit according to the operating procedures provided by the manufacturer. GAPDH was applied as an internal reference.
Statistical analysis
Mainly, we used SPSS 22.0 (Chicago, IL, USA) for statistical analyses and GraphPad Prime 5 (GraphPad, San Diego, CA) for statistical charts. All experiments mentioned in this study were repeated thrice at least. Values recorded in this study are performed as mean ± SD. Student’s t-test was applied to analyze the statistical difference between paired groups. Pearson χ2 test was used to analyze if there was any correlation between serum exosomal LINC00691 level and clinicopathological features of GC patients. For survival time analysis, we applied Kaplan–Meier test and log-rank test. P < 0.05 was considered as a statistically significant difference.