4.1. Clinical samples
GC and adjacent normal tissues were collected from patients with GC who had undergone surgical resection at the Department of Surgery, Gansu provincial Hospital (Gansu, China). H&E staining confirmed the histopathology features of clinical samples. This study has been performed in accordance with Declaration of Helsinki. Ethical approval for the study was granted on 9th March, 2021 by he Medical Ethics Committee of the Gansu Provincial Hospital (No. 2021-079) and informed consent was obtained from the patients.
4.2. TMT proteomics analysis
To perform the differential proteomic experiments, five GC tissue samples and matching adjacent normal tissues were obtained for the TMT-labeled quantitative proteomics analysis. After protein extraction and digestion using SDT lysis and FASP method and the quality control of the protein samples, the samples were labeled with TMT in accordance with the manufacturer's protocol(TMT10plex label reagent set, ThermoScientific, Waltham, MA, America). The proteomics analysis was performed using a Qexactiveplus mass spectrometer (Thermo, USA) by Genechem (Shanghai, China). The original map files (.raw files) generated by Q Exactivepluswere converted into mgf files by Proteome Discoverer 2.1 (Thermo Fisher Scientific, USA) and submitted to the MASCOT2.6 server for database retrieval through the software's built-in tools. In this study, we used Uniprot_HomoSapiens_20386_20180905 for our database search. The reliable protein screening criterion was peptide FDR ≤ 0.01. Student's t-test was performed to identify the significant difference in proteins in the two groups. A P-value < 0.05 and fold change > 1.2 were considered significant.
4.3. Cell lines and culture
The human gastric cancer cell lines AGS, HGC-27, KATO III, NCL-N87 and MKN-45 were purchased from Shanghai Genechem Co., LTD.ATCC previously authenticated the three cell lines viaShort Tandem Repeat (STR) typing. All cells were grown in a complete medium. Cell cultures were maintained at 37°C in a 5% CO2 incubator. In vitro assays were performed at 70–80% cell density.
4.4. Plasmids and shRNA
The GV344 lentiviral vector system, pHelper 1.0 vector, pHelper 2.0 vector, and 293T cells were used to produce lentiviruses. Human SF3B4-targeting shRNA (targeting sequence: 5’-CCCTGAGATTGATGAGAAGTT-3') and control insert sequence (5’-TTCTCCGAACGTGTCACGT-3') were designed and cloned into lentiviral vector GV344. Transfection was performed in AGSand MKN-45 cells (3–5×104/mL). shRNAs were designed and synthesized by Genechem (Shanghai, China). Moreover, the full length of VDAC1 was cloned into lentivirus vector pGC-FU-CMV-3FLAG-EF1-mCherry-T2A-puromycin (GV661) (Shanghai Genechem Co., LTD) for constructing VDAC1 over-expressed cells.
4.5. High-content screening and cell growth curve analysis.
AGS cells were transfected with ashSF3B4 or shCtrl lentivirus seeded into 96-well plates. The expression of green fluorescent protein (GFP) was observed using a fluorescence microscope. Cells were collected for further experiments when they reached 80% confluence. Cellimages were captured using a Celigo image cytometer (NexcelomBioscience, Lawrence, MA, USA) once a day for 5 days. By adjusting the input parameters, cells (2000 cells per well) could be quantified by measuring the green fluorescence signal in each well. The number of cells at each time point was compared with the cell count on day 1 to obtain a cell proliferation ratio at the corresponding time point for each experimental group, the growth curve was plotted, and the fold change in proliferation was calculated. The cell proliferation ratio was calculated as follows: fold change (shCtrl vs. experimental group) = proliferation ratio on day 5 for the shCtrl group/proliferation ratio on day 5 for the experimental group. A fold-change value equal to or more than two indicated that cell proliferation had slowed down sufficiently.
4.6. Quantitative RT-PCR
Total RNA was isolated from each sample using Trizol(Shanghai Pufei Biotech Co., Ltd, China) according to the manufacturer's protocols. The concentration and purity of the total RNA were estimated using NanoDrop 2000 (ThermoFisher, USA). cDNA was synthesized using the Promega M-MLV Reagent kit (Promega Beijing, China), following the manufacturer's instructions. Quantitative RT-PCR was performed using LightCycler® 480 II (Roche, Switzerland) according to the manufacturer's instructions, using GAPDH or β-Actin was as endogenous controls for mRNAs. Primers used for qRT-PCR are listed in Table S1.
4.7. Cell proliferation assays
Transfected AGS and MKN45 cells were incubated in 96-well plates for 24, 48, 72, 96, and 120h, and Cell Counting Kit-8 (Dojindo Laboratories, Japan) was applied to evaluate the ability of cell proliferation. Colony formation assays were performed to detect AGS and MKN45 cell cloning Capability after transfected with shSF3B4 or shCtrl. The transfected cells were seeded into a 6-well cell culture plate in 1x103/well. After 8 days of culture, the cells were stained by crystal violet solution (Sangon Biotech, China), and Colonies were graphed with a digital camera and counted. BrdU assays were performed using a BrdU cell proliferation assay kit (Roche) according to the manufacturer's protocol to assess cell proliferation after cell transfection.
4.8. Apoptosis assay
In transfected AGS and MKN45 cells grown to 80% confluence, the suspension cells were collected, and adherent cells on the dish were digested by trypsin into single cells. We collected all cells for apoptosis detection using Annexin V-APC apoptosis detection kit (eBioscience, USA) and flow cytometry analysis (BD, USA).
4.9. Western blot analysis
A RIPA lysis buffer (Beyotime Institute of Biotechnology) containing protease inhibitors was used for lysing cells on ice as directed by the manufacturer. Detecting protein concentrations was performed with a BCA Protein Assay kit (Beyotime Institute of Biotechnology). The total cellular proteins were isolated using SDS-PAGE (10%) and then transferred to PVDF membranes. The membranes were blocked with TBST solution containing 5% non-fat milk for 60 min at room temperature and then incubated with primaryantibodies, including Anti-SF3B4 (1:500), anti-FOXO3(1:1000), anti-VDAC1(1:1000), anti-DNAJA3(1:500), anti-DRAM2 (1:500),anti- ATG7 (1:10000), anti-ATG5 (1:3000), anti- ATG9A (1:1000) and anti- ATG12 (1:1000)at4°C overnight. After washing, the membranes were incubated with secondary antibodies. All blots were developed with 20X LumiGLO® Reagent and 20x peroxide (#7003, Cell Signaling Technology, Inc). Visualized images were obtained using a Tanon4600 detector (Shanghai, China).
4.10. Immunohistochemistry (IHC)
We obtained 38 pairs of gastric cancer and adjacent normal tissue specimens from surgical resections performed at the Gansu provincial hospital between May 2018 and May 2020. The Medical Ethics Committee of the Gansu provincial Hospital approved this study. The sections were deparaffinized, heat-mediated antigen repair was performed using citrate buffer, blocked by peroxidase blocking solution (maxim, Biotechnology, Fuzhou, China) for 10 min at room temperature, and treated by normal non-immunized animal serum according to the manufacturer's protocol. Next, the staining of the tissue sections by diluted primary anti-SF3B4 or anti-VDAC1 was performed at 4°C overnight. Slides were washed with PBS and incubated with biotinylated secondary antibody (maxim, Biotechnology, Fuzhou, China) per the manufacturer's instructions for 1h.Tissue sections were washed, stained with 3, 3-diaminobenzidine (DAB), counterstained with hematoxylin, dehydrated with ethanol, and mounted with resin. Images were captured using NanoZoomer 2.0-HT (Hamamatsu, Japan). The SF3B4 expression levels in each spot were assessed by the immunoreactive scores (IRS), which were calculated by multiplying the score of intensity and the extent score. Each staining intensity was scored with a score of 0–3 (0 = negative;1 = weak; 2 = moderate; 3 = strong). The extent of positively stained cells was also scored into 5 categories: 0(0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). Using the IRS, staining patterns were classified as low (IRS: 0–6) and high (IRS: 8–12).
4.11. In vivo tumorigenicity assays
4–6-weeks female nude BALB/c mice were purchased from Shanghai Lingchang Biological Technology Co., Ltd. MKN-45 cells were transfected with shSF3B4 and shCtrl, respectively. Transfected MKN45 cells (4×106) were subcutaneously injected into the right underarm of each mouse (n = 12). The mice were cultured for 19 days post-injection, and data collection began 7 days post-injection. A measurement of thevolume (V) was made with calipers by measuring the length (L) and width (W) and calculating the volume using the formula V (mm3) =π/6×L×W×W. The volume of tumors was estimated at 7, 10, 14, 17, and 19 days post-injection. Moreover, nineteen days after cell injection, a dose of 10µL/g of D-luciferin (15 mg/mL, Qcbio, China) was injected and live images were acquired using an in vivo imaging system(Perkin Elmer, America). In the end, mice were sacrificed with pentobarbital sodium injections and their tumors were removed for taking photos and weighting. The animal experiments were complied with the ARRIVE guidelines and the Medical Ethics Committee of the Gansu provincial Hospital approved this study.
4.12. RNA-Seq Analysis
The RNA-sequencing analysis was performed by Genechem (Shanghai, China). Briefly, total RNA was isolated from shSF3B4 and shCtrl-transfected AGS cells using Trizol (Shanghai Pufei Biotech Co., Ltd, China)following the manufacturer's instructions. Purity of RNA was determined by the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA), and integrity was assessed by the RNA Nano 6000 Assay Kit (Agilent Technologies, CA, USA). Sequencing libraries were constructed using NEBNext®UltraTMRNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer's instructions, and index codes were added to attribute sequences to each sequencing sample. The clustering of the index-coded samples was performed on a cBotCluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. A 150 bp paired-end read was generated from the library preparations after cluster generation using an IlluminaNovaseq platform. Differential expression analysis of two groups was performed using the DESeq2 R package (1.16.1), Genes with an adjusted p-value < 0.05 found by DESeq2 were assigned as differentially expressed genes (DEGs). IPA (Qiagen, Hilden, Germany) was performed using all the DEGs to analyze the enriched functional annotations.
4.13. Statistical analysis
Results for continuous variables are presented as means ± sd unless otherwise stated. Student's t-Test and Chi-square test were used during the data analysis. The 2−ΔΔCt method was used during the qPCR assays. SPSS 19.0 was used for statistics, and p < 0.05 was considered significant. GraphPad Prism 6.04 was used to generate graphs.