Tumour database
The online database Gene Expression Profile Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn/index.html) was used to analyse and compare TSP2 expression levels in GC and normal gastric tissues [21]. The Kaplan–Meier Plotter prognostic analysis tool (http://kmplot.com/analysis/) was used to evaluate the effect of TSP2 expression on the prognosis of GC patients [22]. The Kaplan–Meier Plotter database was also used to analyse the correlation between the TSP2 expression level and clinical characteristics of GC patients.
Clinical data and follow-up
Using a random number table, 80 GC patients who underwent surgery in the General Surgery Department of the First Affiliated Hospital of Jinan University from January 2016 to December 2017 without prior chemotherapy or radiotherapy were selected. Eighty samples of GC tumour tissue and paired adjacent tissues (3 cm from the edge of the cancerous tissue) were collected. Eighty GC tissues and paired adjacent tissues were fixed with formalin and embedded in paraffin. The pathology department of our hospital confirmed these diagnoses. The staging was unified according to the eighth edition TNM staging standard of the International Union Against Cancer (UICC), and postoperative adjuvant treatment was performed according to the National Comprehensive Cancer Network (NCCN) GC practice guidelines. The endpoint of this study was the follow-up period of four years or the patient’s death. Overall survival (OS) was defined as the period from the day of surgery until death from any cause or the end of the follow-up. The Institutional Review Board approved this study of the First Affiliated Hospital of Jinan University, and all of the patients provided informed consent.
Immunohistochemical test
Take paraffin sections of GC tissue, make 4 μm-thick paraffin sections, and bake the slices at 65 °C for 30 min. After dewaxing, block the endogenous peroxidase with 3% H2O2, inactivate for 10 min, and rinse twice with PBS. Slices are placed 0.01 mol/L (pH 6.0) citrate buffer 90 °C–95 °C heating for 15 min to perform antigen retrieval. Wash twice with PBS. Nonspecific antigens were blocked with 5% BSA. Samples were incubated in rabbit anti-human TSP2 monoclonal antibody (BW1441, Santa Cruz) diluted 1:400 with 5% BSA. The tissue was completely covered in this solution. Samples were incubated overnight in a refrigerator at 4 °C and rinsed twice with PBS (5 min). The secondary antibody (goat anti-rabbit) was added to the glass slide to completely cover the tissue. Samples were incubated at 37 °C for 40 min and rinsed with PBS twice (5 min). DAB was used to develop colour, and the cells were observed under a microscope. The reaction time was 2–4 min, and the reaction was stopped by washing with tap water. The sample was counterstained with haematoxylin at room temperature, washed with tap water, dehydrated with gradient ethanol solution, cleared with xylene, mounted with neutral gum, and observed under a microscope. The immunohistochemical staining area was scored as follows according to the percentage of positive cells: 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%). The TSP2 staining intensity score was 0 (no staining), 1 (weak staining), 2 (medium staining), and 3 (strong staining). The final staining score is the product of two parameters divided into 2 groups: groups with a total score of 0–3 are low expression groups, and groups with a score of ≥4 are high expression groups.
Cell culture and transfection
Human HGC-27 and AGS GC cell lines were purchased from ATCC, and grown in 1640 medium supplemented with 10% foetal bovine serum and 1% penicillin-streptomycin mixture (purchased from Guangzhou Genio Biotech Co., Ltd.). All cells were cultured in a 37 °C, 5% CO2 constant temperature incubator, and the medium was changed every 2 days. Cells were observed under a microscope. After the cells reached 80–90% confluence, 0.25% trypsin was used (purchased from Guangzhou Genio Biotech Co., Ltd.) to digest and continue subculture at a ratio of 1:2. Cell transfection: Human HGC27 and AGS GC cells were cultured in 1640 medium containing 10% foetal bovine serum to a confluency of approximately 70%. Then, the target siRNA was used according to the manufacturer’s instructions (purchased from Guangzhou Genio Biotechnology Co., Ltd.). TSP2 siRNA was transfected into GC cells using Lipo3000 liposomes. The specific interference sequence and control sequence were as follows: Si-1 (5′-CCGGCCCTCCTAAGACAAGGAACATCT-3′); Si-2 (5′-CGAGATGTTCCTTGTCTTAGGAGGGTTTTTG-3′); and control group (Ctrl) (5′-CCCTCCTAAGACAAGGAACAT-3′). The obtained stably transfected cells were named Si-1 and Si-2, respectively.
Western blotting
After 48 h of cell transfection, the total protein was extracted from protein lysate. The sample and loading buffer were mixed according to the corresponding ratio, and then the sample was denatured in a boiling water bath. Each lane was loaded with an equal amount of protein (30 μg). After electrophoresis, the proteins were transferred to PVDF membranes. After blocking with 5% skimmed milk powder, blots were incubated in the corresponding primary antibody (1:1000, ab112543, Abcam) followed by the secondary antibody the next day. Then, ECL developer solution was added in a dark environment, and the blot was exposed using a gel imager. The final result is expressed as a target strip. The ratio of the optical density of the target band to that of the internal control GAPDH (1:2000, AF1186, Biyuntian) was reported as the protein expression level.
Cell migration and invasion experiments
After transfection for 48 h, HGC-27 and AGS cell lines were digested with 0.25% trypsin. Then, the digestion was terminated, and the culture medium was discarded by centrifugation. The cells were resuspended in serum-free 1640 medium, and the cell density was adjusted to 1 × 106/ml. After repeated pipetting and mixing of the cell suspension, 0.2 ml of the cell suspension was added to the upper chamber of the Transwell chamber. Then, 0.6 ml of 1640 medium containing 10% serum was added to the lower chamber of the 24-well plate. Then, the plate was gently shaken and placed in an incubator for 24 h. The Transwell chamber was removed, and the culture medium in the well was discarded. The cells in the upper chamber were gently removed with a cotton swab. Cells were rinsed 3 times with PBS and fixed with 4% paraformaldehyde for 25 min. Then, the chamber is properly dried, and the cells are stain with 0.1% crystal violet for 20 min. Cells are washed thrice in PBS, and the chamber is air dried. The chamber is placed under a microscope. Five fields of view are randomly selected to observe the cells, take pictures, and count them. In the invasion experiment, Matrigel was added to the Transwell chamber, and the treatment method was the same as described above. Then, 0.2 ml of cell suspension was added to the upper chamber, and the remaining methods were the same as described above.
EdU assay
A total of 5000 HGC-27 cells were plated in each well of 6-well plates and treated with ethanol (50 μM, 48 h), transfected with Ctrl (100 nM, 48 h), or transfected with si-1 or si-2 (100 nM, 48 h). An EdU staining proliferation kit was purchased from Abcam (ab222421). EdU solution was added to the plates. Plates were incubated for 3 h and then treated with 4% formaldehyde. After the process, the cells were stained with DAPI and viewed under an inverted microscope (Olympus, Japan). Each experiment was repeated three times.
Flow cytometry
After transfection for 48 h, the cells were collected for single-cell suspension and centrifuged at 1500 r min − 1 for 5 min after rinsing three times with cold PBS. The supernatant was discarded, and the cells were resuspended in PBS at a density of 1 × 106/mL. The cells were fixed by adding − 20 °C precooled 75% ethanol at 4 °C for 1 h followed by centrifugation. After rinsing twice with PBS, the supernatant was discarded, and the cells were incubated with 100 μL RNase in darkness followed by a 30-min water bath. Subsequently, 400 μL propidium iodide (PI) (Sigma, 5 mg/100 mL) was added, and the mixture was incubated in darkness at 4 °C for 30 min for detection. The cells (1 × 104) were evaluated using flow cytometry (6HT, Wuhan Cellwar Biotechnology Co., Ltd., Wuhan, China) with a 350 mesh sieve. Fluorescent signal intensity at an excitation wavelength of 488 nm was recorded to evaluate the cell cycle.
After transfection for 48 h, cells were digested with trypsin without ethylenediaminetetraacetic acid (EDTA), collected in a flow tube, and centrifuged at 2000 rnmin− 1 for 8 min at room temperature. After being washed, the cells were resuspended in precooled PBS (4 °C) and centrifuged at 2000 r min − 1 for 5 min, and the supernatant was discarded. The cells were collected and stained according to the Annexin-V-FITC cell apoptosis detection kit (Sigma) with Annexin-V-FITC/PI staining solution containing Annexin-V-FITC, PI, and HEPES at a ratio of 1:2:50. Briefly, 100 μL staining solution was used to resuspend 1 × 106 cells. Once the solution was completely mixed and incubated at room temperature for 15 min, 1 mL HEPES buffer solution was added to the cells and mixed. Flow cytometry was used to evaluate cell apoptosis with an excitation wavelength of 488 nm. The procedure was repeated thrice.
Real-time quantitative polymerase chain reaction (PCR)
After that, the cells were collected, and RNA was extracted using the TRIzol method. After the purity and integrity of the RNA had been determined, cDNA was prepared through reverse transcription. The PCR conditions were the following: predenaturation at 95 °C for 1 min; denaturation at 95 °Cfor 15 s, annealing at 58 °C for 20 s, extension at 72 °C for 20 s, for 40 cycles; then, extension at 72 °C for 5 min to terminate the reaction. After the completion of real-time quantitative PCR (RT-qPCR), the reliability of the melting curve and amplification curve results obtained by PCR was quantitatively analyzed, and the cycle threshold (Ct) was set. There were three duplicate holes in each group, and the test was repeated 3 times. The sequence details:TSP2(Forward: 5′-GGG GAC ACT TTG GAC CTC AAC-3′;Reverse: 5′-GCA GCC CAC ATA CAG GCT A-3′);GAPDH (Forward: 5′-ACA ACT TTG GTA TCG TGG AAGG-3′;Reverse: 5′-GCC ATC ACG CCA CAG TTT C-3′).
Statistical analysis
SPSS 22.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 7 (GraphPad Software, Inc., San Diego, CA, USA) were used for data analysis and graphing. The differences between groups were assessed using a t-test or one-way analysis of variance. The expression level of related genes and the characteristic clinicopathological parameters were compared using Fisher’s exact test or χ2 test. Kaplan–Meier survival curves were used to analyse the relationship between TSP2 expression level and OS. P < 0.05 indicates a statistically significant difference.
