Reagents
RPMI-1640 culture solution was purchased from Gibco Company of the United States, fetal bovine serum and double antibody were both purchased from Thermophilic Technology Company. CCK-8 was purchased from Japan Tongren Chemical Company. Transwell chamber and artificial basement membrane were purchased from BD company in USA. The flow detection kit was purchased from Nanjing Keygen Biology Co., Ltd. LC-31/LC-32 (#12741,1:1000), P62 (#23214,1:1000), p-Akt (#4060, Ser 473,1:1000), Akt (#4685), p-mTOR (#5536, Ser2448,1:1000), p-ULK1 (#14202,Ser757,1:1000), ULK1 (#6439,1:1000), GAPDH (#5147,1:1000) antibodies were purchased from Cell Signaling Technology. TNFAIP8 (#ab251212) antibody was purchased from Abcam. 3-Methyladenine (3-MA, #M9281) was purchased from Sigma and 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2 (3-BDO, #R193885) was purchased from Aladdin.
Collection of gastric cancer tissues and normal tissues
147 GC tissues and corresponding adjacent non-tumorous gastric samples were obtained from Shandong Provincial Hospital Affiliated to Shandong First Medical University between 2013 and 2018. Clinical pathology information was available for all samples (supplementary. Table 1). No local or systemic treatment was conducted in these patients before the operation. The study was approved by the Research Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University. Informed consents were obtained from all patients.
Animals
24 BALB/C nu/nu male nude mice were purchased from Shanghai Sciple-Bikai Experimental Animal Co., Ltd. with the quality certificate number of SCXK (Hu) 2019-0016. The mice were 4 weeks old, weighing 15 g, and were raised under SPF conditions. All operations were carried out in accordance with the animal ethics regulations of Shandong Provincial Hospital Affiliated to Shandong First Medical University.
Cell culture
GES-1, SGC-7901, NCI-N87, MKN-28, MGC-803 were purchased from North Carolina Cell Resource Center (USA) and placed in RPMI-1640 culture solution containing 10% fetal bovine serum, and cultured in 37℃, 5% CO2 incubator. When its adherent growth reached 70%-80% confluence, it was digested and subcultured with 0.05% pancreatin.
RT-qPCR detection of TNFAIP8 gene expression in several gastric cells
GES-1, SGC-7901, NCI-N87, MKN-28, MGC-803 cells were cultured and collected to detect TNFAIP8 expression. Cell RNA was extracted according to Trizol reagent instructions and cDNA was synthesized according to cDNA reverse transcription kits instructions. RT-qPCR reaction was carried out on StepOnePlus real-time fluorescence quantitative PCR system by two-step method. The reaction conditions were: pre-denaturation at 95°C for 95°C 1 min, denaturation at 95°C for 5 s, annealing and extension at 60°C for 60°C 30 S, with a total of 40 cycles.
Cell viability
Gastric cancer cells that were silent or overexpressed TNFAIP8 were inoculated into 96-well plates with 5,000 cells per well. One 96-well plate was taken out after 24, 48 and 72 h of conventional culture. 10 uL of CCK-8 solution (5 mg/m1) was added to each well and the culture was continued for 4 h. 150 mountains of DMSO solution was added to each well, and then were shake on a shaker at low speed for 10 min. At last, it was detected at 490 nm on the microplate reader.
Establishment of human gastric cancer model in nude mice
First, nude mice were adaptively fed for 1 week, and human gastric cancer MKN-28 cells which were silent in logarithmic growth phase and overexpressed TNFAIP8 gene after pancreatin digestion were collected, centrifuged at 800 rpm for 4 minutes, and the supernatant was discarded. It was made into a single cell suspension with a concentration of 2 × 107 cells/ml. In a sterile environment, 0.2 ml/nude mouse was inoculated under the armpit, and the nude mouse was put back into the cage to continue feeding and observe the state of the nude mouse. A week later, 24 nude mice showed subcutaneous nodules of about 5 mm, the model of human gastric cancer was established.
Cell transfection
MKN-45 cells were seeded into 6-well plates 1 day before transfection. Transfection was prepared when adherent cells reached a fusion degree of 40% to 60%. The original medium was removed during transfection and washed with RPMI1640 medium (serum-free) 2 times. 4 μL of lipofectamine 2000 liposomes was added to 500 μL of serum-free RPMI1640 culture solution, while 10 μL of TNFAIP8 mimics storage solution was added to 500 μL of serum-free RPMI1640 medium, and allowed to stand at room temperature for 5 minutes, and then the two were mixed and allowed to stand at room temperature for 20 minutes. The mixed solution was added to the cells, and the final concentration of mimics was 80 nmol/L. After 6 hours, the DMEM medium containing 10% fetal bovine serum was replaced, and the culture was continued to be expanded for subsequent experiments. RNA sequence of TNFAIP8 was: forward, 5′-T C C A T C G C C A C C A C C T T A-3′ and reverse, 5′-C T C T G C C T C C T T C T T G T T T T-3′; GAPDH forward, 5′-G G C A A A T T C A A C G G C A C A G T C A-3′ and reverse, 5′-G T C T C G C T C C T G G A A G A T G G T G A T-3′.
Transwell experiment
The small chamber was put into a culture plate, 300 μl of pre-heated serum-free culture medium was added into the upper chamber, and the upper chamber was left to stand for 15-30 min at room temperature to rehydrate the matrix glue; The remaining culture solution was sucked off, and the cells to be tested in each group were made into a cell suspension of 5×105 cells /ml using a serum-free medium containing bovine serum albumin (BSA). 100-200 μL of cell suspension was taken and added into Transwell upper chamber, and 500 μl of culture medium containing 20% FBS or chemokines was added into lower chamber; The cells at the bottom of the lower chamber were stained with 0. 5% crystal violet and the cells at the inner side of the upper chamber were removed with cotton swabs. Finally, cell morphology was observed under microscope and cell numbers was counted.
Flow cytometry
Cell lines stably transferring TNFAIP8, stably transferring empty carriers and non-transferring cell lines are inoculated on a 96-well plate according to a ratio of 2×104/well, each group of cells are collected and operated according to an instruction book of an apoptosis detection reagent kit, and the cells are detected by an up-flow cytometer immediately after the color was dyed. In this experimental study, in order to investigate the effect of mTOR-Akt-ULK1 signaling pathway on apoptosis, PI3K inhibitor (3-MA,10 μM, dissolved in DMSO) and MTOR kinase activator (3-BDO, 10μM, dissolved in DMSO) were added in the experiment. Annexin V staining positive cells were early apoptotic cells and PI staining positive cells were necrotic cells, and anexin V and PI staining were double positive. The cells were late apoptotic cells, and anexin V and PI staining were double negative.
Ki-67 staining
Tumor tissues were fixed with 4% formaldehyde and embedded in paraffin. All tissues were cut into 4 μm sections using a German thermoscience cryostat, and then tumor sections were incubated with primary Ki-67 antibody, sections were washed with PBS, secondary antibody were incubated according to the manufacturer's instructions.
Western blotting
The samples were minced and homogenized in ice-cold RIPA buffer containing 2 mM PMSF. Then samples were centrifugated at 12000 g for 15 min at 4°C. The total protein concentration was measured by the BCA assay kit. The proteins were subjected to SDS-PAGE electrophoresis, transferred to PVDF membranes, blocked with 5% skim dried milk for 2 h, and then probed with primary antibodies at 4 °C overnight. After that, it was incubated with HRP-conjugated secondary antibody for 2 h. The samples were visualized by an enhanced chemiluminescence (ECL) advanced kit and a gel imaging system (Tanon Science & Technology Co., Ltd., China).
Statistical analysis
All data were presented as mean ± SD. The statistical significance of differences between the means of each group was analyzed by one-way ANOVA followed by Tukey multiple comparison tests. For comparing two groups, Student’s t test was used. A p value less than 0.05 was considered statistically significant.