From June 2020 to August 2021, we collected tumors and adjacent tissues from 12 patients with gastric cancer in the Gastroenterology Department of our hospital. No patients received radiotherapy or chemotherapy before surgery, including 6 males and 6 females, 7 cases aged ≥ 60 years old, 6 cases aged < 60 years old, the specimens were stored in -196°C liquid nitrogen until western blot analysis and PCR detection. The use of specimens and research procedures have been approved by the hospital medical ethics committee (K202002146), and the patient’s informed consent was obtained.

Rabbit anti-mouse TRIAP1 monoclonal antibody (ab182858), rabbit anti-mouse GAPDH monoclonal antibody (ab8245), horseradish peroxidase labeled immunoglobulin conjugate-rabbit anti-mouse IgG conjugate (ab233006) were purchased from Abcam (United Kingdom); SuperScript IV Reverse Transcriptase (M614272), SuperScript IV One-Step RT-PCR Kit (M33253), SuperScript IV CellsDirect cDNA Synthesis Kit (M39128), RNase Inhibitor (M712192), TRIZOL (M33253), cMTT (M2003), Annexin V-FITC apoptosis detection kit (M2118), MEM (M8042), RPMI-1640 medium (R8758), FBS (F8687) were purchased from Thermo Fisher Scientific. CO₂ incubator (Shanghai Boxun, cat: BC-J160S), 4°C centrifuge (eppendorf, centrifuge 5415R), PCR machine (eppendorfrealplex), ultra-clean workbench (Shanghai Boxun model SW-CJ-2FD), inverted fluorescence electron microscope (Leica DMI3000B), electrophoresis instrument (Tanon EPS300), GloMax^® Discover microplate reader (Promega, United States), Olympus inverted microscope GX41 (Olympus, Japan), CytoFLEX_ flow cytometer (Baker Mancourt, United States).

Culture human GC cell lines NCI-N87, CRL-5822, BGC-823 and normal gastric mucosal epithelial cells GES-1 in RPMI-1640 medium supplemented with 15% fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin, and place it in a humidified incubator at 37°C and 5% CO₂.

The coding sequence of TRIAP1 was amplified by RT-PCR, and then cloned into the BstB I and Xba I sites of pCDH-EF1-MCS-T2A-Puro, and the pCD-EF1-MCS-T2A-Puro-TRIAP1 recombinant vector was transiently transfected into NCI-N87 cells to overexpress TRIAP1 and NCI-N87 cells transfected with an empty vector were used as controls. TRIAP1 small molecule interference siRNA was transfected to knock down TRIAP1 in NCI-N87 cells, and nonsense siRNA was transfected as a negative control (NC). siRNA NC, mimics NC, miR-NC mimics and inhibitors are synthesized by GenePharma (China). Inoculate NCI-N87 cells in 37°C, 5% CO₂ environment, and add 15% FBS to RPMI-1640 medium. Using lipofectamine™2000 to transfer interfere siRNA of TRIAP1,TRIAP1overexpression plasmid, miR-107 mimic and miR-107 inhibitor according to the reagent manufacturer’s instructions.

Divide the cells into nine groups: Control group (DMSO), miR-107 mimics group (miR-107 mimics), miR-107 inhibitor group (miR-107 inhibitor), NC inhibitor + mimics (NC inhibitor + NC mimics), Si-TRIAP1 group (si-TRIAP1), TRIAP1Vector group (TRIAP1Vector), NC si + Vector group (nonsense siRNA + blank plasmid), mimics + Vector group (miR-107 mimics + TRIAP1Vector), NC mimics + NC Vector group (NC mimics + NC Vector), analysis of cell proliferation, apoptosis and metastasis activity in vitro after 24 h of different transfection.

According to the instructions, use Trizol™ reagent to extract total RNA from cells, quantify RNA concentration by NanoDrop 2000 spectrometer, use RT/RI enzyme mixture and gDNA kit for cDNA reverse transcription, use TransStart^® Tip Green qPCR SuperMix (ROX) kit for qRT-PCR analysis, U6/GAPDH was used as an internal reference for TRIAP1 and miR-107. The primer sequences are shown in Table 1. TRIAP1 and miR-107 primers were synthesized by RiboBio Company. The 2^-△△CT method was used to quantify the relative intensities of TRIAP1 and miR-107 in different cells.

The gastric cancer and adjacent tissue samples were washed with PBS several times and broken into fragments less than 1mm3. The transfected NCI-N87 cell pellet was suspended in 50 μl RIPA buffer and lysed on ice for 30 min at 4°C. The supernatant protein was collected by centrifugation at 12,000 rpm for 15 min, and the protein concentration was measured by Bradford assay. The same amount of protein in each group was boiled for 5 min and separated on a 10% SDS-PAGE gel. The protein bands were transferred to the nitrocellulose membrane Above, block with TBST (50 mM Tris, 100 mM NaCl and 0.1% Tween-20, pH) containing 5% (w/v) skimmed milk, and with the primary antibodies TRIAP1 and GAPDH at 4°C overnight, and then with HRP-conjugated Affinipure goat anti-rabbit IgG was incubated for 2 h at room temperature. The protein bands were visualized by ECL reagent, and imaged and quantified using the Bio-Rad ChemiDoc™ MP system.

Construct a wild-type or mutant plasmid at TRIAP1-3′-UTR site that binds to miR-107 for dual-luciferase reporter gene analysis, amplify and insert the psiCHECK™2.0 dual-luciferase vector containing XhoI and NotI, the recombinant expression vector of wild-type TRIAP1-3′-UTR is named psiCHECK-2-TRIAP1-3′-UTR-WT, and for mutant TRIAP1-3′-UTR, it is psiCHECK-2-TRIAP1-3′-UTR -MUT. Suspend 5×10⁴ NCI-N87 cells in 1 ml of RPMI-1640 containing 15% FBS, inoculate them into 6-well plate wells, and incubate them at 37°C and 5% CO2 for 24 h. Then, each group of NCI-N87 The cells were co-transfected with 2 μg psiCHECK-2-TRIAP1-3′-UTR-WT and MUT, which contained 25 μM miR-107 mimics or negative control, in the presence of 4 μl lipofectamine™2000, the transfection was carried out at 37°C and 5% CO₂ for 8 h. After the culture medium was discarded, the culture was incubated for 16 h, centrifuged at 1,000 rpm for 5 min to collect the sediment of each group, and Lysis in 200 μl lysis buffer at 4°C for 15 min. Finally, put 20 μl of the lysate supernatant of each group into the luminometer tube, first mix it with 100 μl Luciferase Assay Reagent II, and then mix with 100 μlStop&Glo Reagent and use Dual-Luciferase reporter gene assay system to detect luciferase activity.

Inoculate each group of cells (5×10³ cells) into a 96-well plate, and incubate at 37°C and 5% CO2 for 24, 48, 72 and 96 h. Add 0.5 mg/ml MTT (3-(4,5-Dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) solution to each well of cells, incubate in the dark at 37°C and 5% CO₂ for 4 h. After removing the supernatant, add 150 μl of DMSO to each well to dissolve the formazan, and microplate reader was used to measure the absorbance at 490 nm for cell activity quantification.

The cells were plated in BioCoat Matrigel Invasion Chambers at a density of 1×10⁵ cells/well, the chambers were inserted into the wells of the 24-well plate, and incubated in RPMI 1640 medium containing 10% fetal calf serum. After 4 h, the invaded cells were fixed and stained with 0.1% crystal violet and the number of cells in five random fields was counted.

Twenty 6-week-old BALB/c athymic nude mice (females, weighing 16–20 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China) and were cultured in animal centers under pathogen-free conditions. All animal experiments are in compliance with the “Guidelines for the Care and Utilization of Laboratory Animals.” Suspend NCI-N87 cells in 100 ml phosphate buffer solution, and then subcutaneously inoculate them into the right abdomen of nude mice (5×10⁶ cells/mouse). The transplanted nude mice were randomly divided into two groups (8 small mice in each group). Rats), the Contorl (normal saline) and miR-107 mimics (miR-107 mimics) groups were injected once a day at a dose of 3 nmol per mouse and injected into the implanted tumor. Monitor the tumor volume (V) by measuring the length (L) and width (W) with a vernier caliper, and use formula V (cm³) = W²×L/² to calculate. After 4 weeks, all experimental mice were euthanized, and all tumors were removed and Weigh and perform immunohistochemistry.

The data is expressed as the mean ± SD of three independent experiments. GraphPad Prism 6.0 (La Jolla, California, United States) was used for statistical analysis. The difference between the two groups was evaluated by Student’s t test analysis, When p < 0.05, the difference is statistically significant.

Compared with paracancer tissues, the levels of TRIAP1 protein and mRNA in the tumor tissues of patients were significantly increased (Figure 1A), while the levels of miR-107 were significantly reduced (Figure 1B), and the expression of miR-107 and TRIAP1 in tumor tissues of GC patients was negative correlated (R = −0.922, Figure 1C). Further in vitro experiments showed that compared with normal gastric epithelial mucosal cells GES-1, the expression level of TRIAP1 protein and mRNA significantly increased in human GC cell lines NCI-N87, CRL-5822 and BGC-823 cells (Figures 1D,E), while the expression level of miR-107 was significantly down-regulated (Figure 1E), due to the relatively high level of TRIAP1 protein in NCI-N87 cells, this cell line was used for later experiments.

After miR-107 mimics transfection, the level of miR-107 mRNA was significantly increased, and the level of TRIAP1 protein was significantly reduced. After transfection of miR-107 inhibitor, the level of miR-107 mRNA in NCI-N87 cells was significantly decreased, and the level of TRIAP1 protein was significantly reduced. (Figures 2A,B), further dual-luciferase reporter gene detection showed that miR-107 mimics transfection can significantly inhibit the fluorescence of NCI-N87 cells transfected with psiCHECK-2-TRIAP1-3′-UTR-WT, and it has no significant effect on fluorescein activity in NCI-N87 cells transfected with psiCHECK-2-TRIAP1-3′-UTR-MUT (Figure 2C).

After transfection with si-TRIAP1 or TRIAP1 vector, the transfection efficiency was detected by Western blot. The results showed that TRIAP1 expression in NCI-N87 cells transfected with siTRIAP1 was significantly knocked down, while TRIAP1 expression was significantly up-regulated after TRIAP1 vector transfection (Figure 3A); Transwell and MTT analysis showed that compared with the control group after transfection with si-TRIAP1, cell migration and invasion capabilities were significantly reduced, proliferation activity was significantly inhibited, and apoptosis was significantly increased. Compared with the control plasmid, overexpression of TRIAP1 can significantly enhance the migration and invasion capabilities of NCI-N87 cells. Promote the proliferation activity of NCI-N87 cells and inhibit the apoptosis of NCI-N87 cells (Figures 3B–D).

MTT and apoptosis analysis showed that compared with NCI-N87 cells in the control group or NC inhibitor + mimics group, after transfection with miR-107 mimics, cell proliferation was significantly reduced, and cell apoptosis was significantly increased. Further Transwell analysis showed that, compared with the NCI-N87 cells transfected with NC mimics, the miR-107 mimic transfection has significantly reduced cell migration and invasion capabilities (Figures 4A–D), while miR-107 inhibitor transfection significantly enhanced the migration and invasion ability of NCI- N87 cells, promotes cell proliferation activity and inhibits the apoptosis of NCI-N87 cells (Figures 4A–D).

Compared with the miR-107 mimics group, overexpression of TRIAP1 in NCI-N87 cells can significantly promote the expression of TRIAP1, reverse the cell migration and invasion ability inhibited by miR-107 mimics, promote cell proliferation activity, and inhibit cell apoptosis induced by miR-107 mimics (Figures 5A–D).

Compared with the mice in the control group, the tumor growth was significantly slower after miR-107 mimics treatment, the weight difference between the two groups of mice did not change significantly. Further immunohistochemical and western blot analysis showed that after miR-107 mock treatment, the expression of miR-107 in tumors was significantly increased, while the expression of TRIAP was significantly decreased, the expression of tumor growth marker Ki67 was significantly decreased, and the expression of apoptosis-related proteins was significantly (Bad, CCS-3) increased, suggesting that miR-107 could inhibit tumor progression in mice (Figures 6C–F).