TCGA database
Gastric cancer tissue samples with mRNA expression data and corresponding clinicopathological data were downloaded from the cancer genome atlas (TCGA) database. Samples were divided into tumor (n=375) and healthy tissues (n=32) to evaluate the diffential expression of MARCH1. Some patients had incomplete clinicopathological characteristics data. These patients were included in the analysis of clinicopathological characteristics they possessed, but excluded in the analysis of information they lacked.
Cell culture
AGS cell line were originally owned by the laboratory, which was cultured in RPMI-1640 medium (Biological Industries, China) supplemented with 10% fetal bovine serum (Biological Industries, China) and 1% penicillin-streptomycin solution (Hyclone, US) at 37℃ in 5% CO2 atmosphere. 0.25% trypsin (Biological Industries, China) was used to digest and passage the cell untill they reached 90% confluence.
Cell transfection
Cells grown in the logarithmic phase were plated with 1×105 per well in a six-well plate, or 5×103 in a 96-well plate the day before transfection. After 24h the cell density is about 60%~80% and then the cells were transfected with siRNAs or plasmids using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocol. Two siRNAs against MARCH1, negative control (NC) siRNAs and the MARCH1 overexpression plasmid were synthesized by Genepharma (Shanghai, China). The sequences for the MARCH1 siRNA were : siMARCH1-1, 5′‐CAGGAGGUCUUGUCUUCAUTT‐3′ and 5′‐AUGAAGACAAGACCUCCUGTT‐3′; siMARCH1-2, 5′‐GGUAGUGCCUGUACCACAATT‐3′ and 5′‐UUGUGGUACAGGCACUACCTT‐3′. The NC siRNA sequences were: 5′‐UUCUCCGAACGUGUCACGUTT‐3′ and 5′‐ACGUGACACGUUCGGAGAATT‐3′.
Western blotting
Whole-cell protein lysates were extracted using cell lysis buffer (Beyotime, China), and the protein concentrations were determined by the BCA assay (Solarbio, China). The lysates were boiled in SDS polyacrylamide gel electrophoresis (SDS‐PAGE) sample loading buffer (EpiZyme, China) for 5‐10 minutes at 99°C and separated on SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) by electroblotting, and after blocking in 5% nonfat milk (Sangon Biotech, China) for 2-3h. Then the membrane was incubated with primary antibodies against anti-MARCH1 (immunoway, China), anti-β-catenin (immunoway, China), anti-tubulin α (immunoway, China) at 4℃ overnight and then left with the secondary antibodies peroxidase-conjugated goat anti-rabbit (BOSTER, China) for 60 minutes at room temperature. Finally the membranes were quantified using an enhanced chemiluminescence signal (EpiZyme, China). Photometric analyses of immunoblots were carried out using the Image Lab software package. The quantitative analysis through ImageJ software.
Cell proliferation assay
Cell proliferation assays were performed using a Cell Counting Kit-8 (CCK-8, MCE, China). According to the manufacturer’s instructions, 5000 cells were seeded in 96-well plates overnight before treatment and per group set 5 auxiliary holes. Transfect the plasmid or siRNA according to the instructions, stop the culture at 0h, 24h, 48h, 72h after transfection. CCK-8 reagent was added to each well, and after incubation with the reagent for 2h at 37℃. The absorption and reference wavelength was measured at 450nm. Then we draw proliferation graphs of different groups over time.
Transwell assay
Cell migration and invasion assay were performed using 6.5mm transwell insert chambers with a 8.0µm pore polycarbonate membrane. The cells (3000), which were transfected after 24h were cultured in serum-free medium, placed in the upper chambers and coated with Matrigel basement membrane matrix (Corning, USA) for 2h at 37℃ before the cells were added. The medium with 20% FBS was added to the down chamber. The cells were incubated for 48h, and cells which did not migrate through the pores were removed with a cotton swab. Then the upper chambers were fixed in 4% paraformaldehyde, stained with 0.5% crystal violet (Sangon Biotech, China) and counted under a photo microscope. However, there was no need for the Matrigel coating for the cellular migration assay.
Cell apoptosis assay
The apoptosis assays were performed using an Annexin V, FITC Apoptosis Detection Kit (Dojindo, Japan) according to the manufacturer’s instructions. We collected at least 10,000 cells after transfection, washed them twice with cold PBS, and used 100µL Annexin V Binding Solution to make cell suspension. The cell suspension was incubated with 5μL of Annexin V-FITC and 5μL of PI for 15 minutes, followed by apoptosis analysis by flow cytometry (BD Biosciences, USA).
Statistic analysis
Statistical analysis was performed by SPSS 26.0. GraphPad Prism7.0 software was used for statistical drawing. The Mann‑Whitney U test was used to compare MARCH1 expression in normal and tumor tissues. The Wilcoxon signed‑rank test was used to compare MARCH1 expression in paired tissues. To examine the association between clinicopathological characeristics and MARCH1 expression (after excluding normal tissue sample data), the Wilcoxon signed‑rank test was performed for comparison between two groups [distant metastasis (M stage) and alive status], and the Kruskal-Wallis test was performed for multi-group comparison [TNM Stage, Grade, tumor stage (T stage) and lymph node metastasis (N stage)]. The median value of MARCH1 expression was selected as the cut-off value, and
patients with gastric cancer were subsequently divided into high and low‑expression groups. Kaplan‑Meier survival curves were constructed and log rank tests performed to evaluate the association between MARCH1 expression and overall survival (OS). The independent-sample t-test is used for comparison between the two groups, the one-way analysis of variance (ANOVA) is used for the comparison between multiple groups. *p < 0.05, **p < 0.01 and ***p < 0.001 were considered statistically significant.