Cell lines and cell culture
All human gastric cancer cell lines (MKN45, HGC27), and human embryonic kidney (HEK) 293T cells were obtained from American Type Culture Collection. The above cells were cultured in1640 and DMEM medium with 10% foetal bovine serum (FBS). All cell lines were authenticated by short-tandem repeat analysis. These cell lines were mycoplasma-free (checked by using a Mycoplasma Stain Assay Kit named C0296, Beyotime Biotechnology, Shanghai, China), and cultured in a humidified environment supplemented with 5% CO2 at 37°C.
Reagents, antibodies, and clinical tissue samples
MG132 and CHX were obtained from Sigma (Shanghai, China). A rabbit-enhanced polymer detection system (#PV-9001) for immunohistochemistry (IHC) was purchased from ZSGB-Bio (Beijing, China). The antibodies used include the following: 19602-1-AP (anti-TRIP13), 10528-1-AP (anti-DDX21), 10122-1-AP (anti-CDK2), 11224-1-AP (anti-α-Tubulin), 11554-1-AP (anti-CCNE1), 51064-2-AP (anti-HA-Tag), 20543-1-AP (anti-Flag-Tag), 27309-1-AP (anti-Ki67), 20874-1-AP (anti-E-cadherin), 22018-1-AP (anti-N-cadherin), 10197-1-AP (anti-HDAC1) and 13099-1-AP (anti-Snail) were purchased from Proteintech (Wuhan, China). Clinical gastric cancer tissue samples were purchased from Zhongke Guanghua (Xi'an) Intelligent Biotechnology Co., Ltd.
Immunohistochemistry staining
Paraffin-embedded tumors were cut into slices with a thickness of 6 mm, and then the paraffin sections were dewaxed and hydrated. Then, paraffin slices were put into citrate buffer (pH 6.0) and heated in a microwave oven to 95°C for 20 min to facilitate antigen retrieval. Then, endogenous peroxidase activity was quenched, which was followed by blocking with normal goat serum. Then, the TRIP13, Ki67 and DDX21 antibodies were diluted with PBS (1:150), and the antibodies were added to the paraffin sections and incubated overnight at 4°C. Then, a horseradish peroxidase-linked secondary antibody was added and incubated with the sections, which was followed by the addition of a DBA reagent. The results were observed under a microscope before counterstaining with haematoxylin.
Plasmids, lentivirus packaging, and infection experiments
Small-hairpin shRNAs for TRIP13, HDAC1 and DDX21 and a negative control shRNA (shGFP) were obtained from Gene Pharma Co. Ltd. (Shanghai, China) and were inserted into the pLKO.1 vector. Flag-TRIP13, MYC-DDX21 and HA-UB were cloned into the pCDH-CMV-MCS-EF1-GFP-Puro vectors and pRK5 vectors respectively, which were purchased from Youbao Company (Changsha, China). For lentivirus packaging and infection assays, the target plasmid (500 ng) and packaging plasmid (PLP1, PLP2, and VSVG)/500 ng were co-transfected into 293T cells in one six-well plate by using the transfection reagent Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Lentiviruses were collected 48 h later and used to infect gastric cancer cells twice, 12 h per infection. The infected cells were screened by treatment for 48 h with puromycin, and the surviving cells were frozen and stored in liquid nitrogen for subsequent assays.
Animal models and tumor xenograft
Four-week-old female nude mice were purchased from Huafukang Biotechnology Co., Ltd. Mice were randomly assigned to receive subcutaneous injection and divided into 4 groups (6 mice/group) and 2 groups (5 mice/group). In short, stably transfected gastric cancer cells MKN45 (shGFP, shTRIP13, shTRIP13/TRIP13, shTRIP13/DDX21, and shDDX21) (1×106 cells) and resuspend them on 100µl Inject PBS into subcutaneous tissue of mice. Before subcutaneous injection, anesthetize the mice with an isoflurane nasal anesthesia system to alleviate pain. Before collecting tumors, anesthetize mice with an isoflurane nasal anesthesia system and euthanize them through cervical dislocation. Then, collect the mouse corpses and store them at -20°C, and send them to the limited company Leibit Biotechnology Co., Ltd. for incineration. The tumor volume is calculated using the following formula: V= (length ×width 2)/2. The tumors were collected and photographed for subsequent immunohistochemical experiments. All animal treatments are approved by the Animal Protection and Ethics Committee of Henan University of Traditional Chinese Medicine. All experiments were conducted in accordance with the Animal Health and Use Guidelines (2006) of the Ministry of Science and Technology of China.
Western blot assay
Cells were lysed with RIPA lysis buffer, and the released proteins were separated on 10% and 12% polyacrylamide electrophoresis gel respectively. Transfer the gel onto the polyvinylidene fluoride membrane, which is sealed with 5% skimmed milk powder at room temperature for 1 h. After that, incubate the polyvinylidene fluoride membrane overnight (4°C), and then incubate it with the second antibody (peroxidase labeled anti rabbit antibody) at room temperature for a second time for 1 h. Finally, analyze the results with ECL Prime Protein Blot (WB) detection system (GE Healthcare).
Immunoprecipitation (IP) assay
The IP experiment was conducted as previously described [33]. Briefly, collect cells and lyse them through IP lysis buffer. Then, incubate protein A/G magnetic beads with different antibodies. Incubate the cell lysate overnight (4°C) with antibody conjugated beads. Wash the beads, then denature them at 100°C and detect the proteins by Western blotting.
Ubiquitination and turnover assay
The constructed plasmids shGFP, shTRIP13, Flag-HECTD3 and HA-UB plasmid transfection into 293T cells. 48 hours after transfection, 50 µg/ml proteasome inhibitor MG132 was added to the cells and incubated for 8 hours. The cells were collected and lysed using IP lysis buffer. The cell lysate was incubated at 4°C with antibody conjugated beads. The beads were washed, denatured at 100°C, and the proteins were detected by Western blotting. For turnover assay, infected cells were subjected to 48 hours and treated with CHX at a concentration of 50 µg/ml cells. Finally, cells were collected, lysed, and analyzed using Western blotting.
Edu staining
According to the manufacturer's recommendation, Edu staining assay was performed to detect cell proliferation [34]. 2 × 104 cells were placed in a 24 well plate and cultured overnight. The next day, the cells were first incubated with 10mM Edu for 2 hours, followed by incubation with 4% paraformaldehyde (PFA) for 15 minutes, incubation with 0.3% Triton X-100 for 10 minutes, and incubation with 5% bovine serum albumin (BSA) for 1 hour. Before taking photos under a microscope for processing, the nuclei were stained with DAPI at room temperature for 30 minutes.
Cell proliferation detection and plate colony formation experiment
To detect cell proliferation ability, 1 × 103 stable transfected MKN45 and HGC27 cells were cultured in 96 well plates and 6 well plates for 6 and 15 days, respectively. As mentioned previously [35], cell viability was detected by cell counting and MTT assay. After staining with crystal violet, the scanning plate clone formation assay was used, and the absorption rate of 560 was finally measured using absolute ethanol to measure nm for statistical analysis. All experiments were conducted independently three times.
Quantitative reverse transcriptional PCR (qRT-PCR)
As mentioned previously [36], using TRIzol reagent (Invitrogen)™), Reverse transcribe 2µg of RNA into cDNA. SYBR qPCR SuperMix Plus is used for qRT PCR (Novoprotein, China). The primer pairs are shown in supplementary Table 1. Results were calculated using the ΔΔCt method with glyceraldehyde-3-phosphate dehydrogenase serving as the internal control [37].
Luciferase reporter assay
The promoter region of TRIP13 is connected to the pGL3 vector and obtained from Changsha Youbao Biotechnology Co., Ltd. Then, by transfecting with Lipofectamine 2000, the pGL3 plasmid, pRL-TK internal control vector (Promega), and shHDAC1 vector were co-transfected into MKN45 and HGC27 cells. After 48 hours, perform luciferase reporter gene testing according to the manufacturer's instructions (Promega). Each group has three repeated experiments.
Proximity ligation assay (PLA) and immunofluorescence (IF) assay
Growth of on a cover glass slide in a 24 well plate 3 × 104 MKN45 and HGC27 cells were transiently transfected, followed by fixation in 4% paraformaldehyde for 20 minutes and permeabilization in 0.3% Triton X-100 at room temperature for 15 minutes. Then, after sealing in 5% goat serum at room temperature for 1 hour, incubate the cover glass with primary antibody overnight at 4 ° C. Next, the subsequent experiments were conducted using the PLA assay kit [38]. Finally, the cells were visualized and photographed using a confocal fluorescence microscope.
Migration and wound healing assays
Cell migration and invasion experiments were conducted using transwell chambers (pore size 8 µm, Corning, Beijing, China). For intrusion experiments, cover the membrane with Matrix (BD Biosciences). Add 1640 medium with 10% fetal bovine serum to the lower part of the chamber, and add cells from serum-free 1640 medium to the upper part of the chamber. After 25 and 29 hours, stable transfected MKN45 and HGC27 cells were fixed with 4% paraformaldehyde for 15 minutes, and then stained with crystal violet. Calculate the average number of cells from at least three randomly selected microscope images. For cell wound healing determination, culture cells in a six well plate and make the wound using the tip of a 10 µl pipette. Finally, observe the healing process of cells through a microscope.
Patient database analysis
All data sets were obtained from GEPIA (http://gepia.cancer-pku.cn/index.html) databases, TIMER2 (http://timer.cistrome.org/), R2 databases (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi), gastric cancer (Kaplan–Meier Plotter) databases and Protein Atlas database (http://www.proteinatlas.org/).
Statistical analysis
Triplicates at least were designed and performed in each of the above experiments. Statistical parameters (sample size and significance analysis) are shown in the legend. Data were analysed and shown as mean ± SD. Statistics analyses were performed using GraphPad Prism 8.0. Two-tailed Student’s t tests were performed for paired samples. *p < 0.05, **p < 0.01, and ***p < 0.001 were considered to indicate statistical significance.