Ethics approval
The clinical research protocol was approved by the Institutional Ethics Committee of the Henan Tumor Hospital on March 2, 2021, and informed consent was obtained from all patients (n = 114).
Tissue samples collection
Tumor tissues (n = 114) and their adjacent normal tissues (n = 112) of GC patients were collected from the Henan Tumor Hospital. The inclusion criteria of the samples were the survival period of GC patients and none of them received any local or systemic treatment before surgery.
Immunohistochemical staining
The tissue samples were fixed in 4% paraformaldehyde, hydrated through ethanol solution, embedded in paraffin and incubated with 3% hydrogen peroxide. Afterwards, the tissues samples were incubated with the primary anti-PHF5A (1:100 dilution, Abcam, ab193115) anti-FOS (1: 1:500 dilution, Abcam, ab184938), and anti-Ki67 (1:200 dilution, Abcam, ab16667) overnight at 4 °C. After incubation with secondary antibody HRP goat anti-rabbit IgG (1:200 dilution, Beyotime, A0208) at room temperature for 1 h, DAB and hematoxylin was stained. Scoring the intensity of tissue staining based on the criteria provided in the reference [22]. A score greater than or equal to the median of immunohistochemistry indicated high expression of PHF5A, otherwise low expression.
Cell culture
The human normal gastric mucosa cells GES1 and GC cell lines MGC-803, AGS, SGC-7901, BGC-823 were purchased from Chinese Academy of Sciences (Shanghai, China). All of the cell lines were maintained the Dulbecco’s modified eagle medium supplemented with 10% fetal bovine serum (Invitrogen Gibco) and incubated at 37 °C in a humidified environment with 5% CO2.
RNA interference and cell transfection
According to the gene sequence, small, interfering, specifically targeting human PHF5A (shPHF5A), FOS (shFOS), amplified sequence of PHF5A (PHF5A), and non-specific negative control (shCtrl)were synthesized (Additional file 1: Table S1). AGS and MGC-803 cells were incubated for 24 h and transfected with lentivirus shPHF5A, shFOS and PHF5A (1 × 108 TU/mL) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, USA) at a MOI (multiplicity of infection) of 10. Notably, GC cells transfected with lentivirus PHF5A and NC-shFOS for overexpressing PHF5A [PHF5A + NC(KD)]; the cells transfected with lentivirus shFOS and NC-PHF5A for downregulating FOS [shFOS + NC(OE)]; the cells transfected with shFOS and PHF5A for simultaneously downregulating FOS and overexpressing PHF5A [shFOS + PHF5A]; NC(OE + KD) was the cells transfected with empty plasmid, as negative control.
RNA extraction and qRT-PCR
Total RNA was purified from cultured GC cell lines using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The concentration and purity of RNA samples was assessed with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). RNA was reverse transcribed into cDNA by Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). We conducted quantitative real-time polymerase chain reaction (qRT-PCR) by using the SYBR Green master mix (Thermo Fisher Scientific). Expression of PHF5A and FOS was assessed by threshold cycle (CT) values and analyzed using the 2ΔΔCt method. Primer sequences were designed (Additional file 2: Table S2) and synthesized by Sheng gong (Guangzhou, China).
Western blot analysis
Total proteins were purified using RIPA lysis buffer (Beyotime, Jiangsu, China) and the concentrations were detected by protein assay kit (Beyotime, Jiangsu, China). Protein lysates were subjected to 10% SDS-PAGE, transferred to PVDF membrane (Millipore), hybridized with corresponding primary antibody (Additional file 3: Table S3) overnight at 4 °C. The next day, the membranes were incubated with secondary antibody HRP goat anti-rabbit IgG (Beyotime, A0208) at room temperature for 2 h. The coloration of the membrane was performed with chemiluminescence ECL kit (Thermo Fisher Scientific) and protein signal was visualized by Odyssey Infrared scanning system (Li-Cor, Lincoln, NE, USA).
Co-immunoprecipitation (Co-IP) assay
A target protein-specific anti-PHF5A or anti-FOS antibody (Additional file 3: Table S3) in conjunction with Protein A/G affinity beads (Santa Cruz Biotechnology) for 30–60 min at 4 °C. The bead-antibody complexes were suspended with protein lysate. The beads were washed 3 times with extraction buffer, and collected by centrifugation at 3000g. Subsequently, the immunoprecipitants were subjected to western blot.
Celigo cell counting assay
AGS and MGC-803 cells were transfected with lentivirus and cultured in 96-well plates at a density of 2000 cells per well. The cells were counted at 24, 48, 72 and 96 h by Celigo (Nexcelom) and the cell growth curve was plotted for the 5 days.
Colony formation assay
MGC-803 cells were transfected with lentivirus and cultured in six-well plates at a density of 1000 cells per well. Cell colonies were washed twice by using cold phosphate buffered saline (PBS), fixed with 75% ethanol and stained with 0.1% crystalline purple. They were cultured until the colonies were visible, counted and photographed.
Cell apoptosis detection
AGS and MGC-803 cells were transfected with lentivirus and inoculated into six-well plates (2 mL/well) for 5 days. The cells were centrifuged for 5 min, the cell precipitates were successively eluted with precooled D-hanks (pH = 7.2–7.4) and 1×binding buffer (eBioscience), resuspended by 200 µL 1× binding buffer, stained with 10 µL Annexin V-APC (eBioscience) at room temperature in the dark for 15 min and detected by flow cytometry (Guava easyCyte HT, Millipore).
Transwell assay
Transwell chambers (24-well, 8-mm pore, MA, USA) were used to measure the migration ability of the cells in 24-well plates. AGS and MGC-803 cells were transferred into the upper chamber with 200 µL serum-free medium and medium with 30% FBS was added to the lower chamber. The cells on the upper chamber were removed, while the cells adhering to the Polycarbonate membrane were fixed with 4% precooled paraformaldehyde for 30 min and stained with 0.1% crystal violet for 20 min at room temperature. Finally, the migrated cells were photographed from five randomly selected fields under a 200 × microscope.
Wound-healing assay
AGS and MGC-803 cells were cultured into 6-well plates (100 µL/well) at a density of 4000 cells per well. The cells were eluted with PBS, fixed with 3.7% paraformaldehyde (Corning) for 15 min, stained with 1% crystal violet for 10 min. Wound healing was observed at 0 h, 8 and 72 h under a microscope for image acquisition and Image J software (National Institutes of Health) was used to quantify the distance (µm) between the scratches.
Sphere-forming assay
Sphere-forming ability is an important method for the identification of tumor stem cells in vitro, and is indicated by the Sphere Formation Efficiency (SFE). AGS and MGC-803 cells balls were collected and cultured by 70 μm cell sieve for 10–14 days and digested into single cells using trypsin. An appropriate number of cells were spread into 96-well plates for further culture for 10–14 days, the number of cell balls was counted, and SFE was calculated by the formula: SFE = Number of cell balls with diameter greater than 75 μm per well/total number of original inoculated cells per well.
Xenograft tumor assay
BALB/c nude mice (age, 4–6 weeks; weight, 16–20 g) were ordered from the animal laboratory center (Vitong Lihua, Beijing). The experimental procedures performed on mice were approved by the Ethics Committee of Henan Tumor Hospital and the Animal Protection Association. MGC-803 cells transfected with lentivirus Control or PHF5A were subcutaneously injected into the right armpit of mice, respectively (4 × 106 cells/mouse). Subsequently, mice in PHF5A group were given intravenous Pladienolide B at a daily dose of 5 mg/kg and kept fed normally. A week after injection, tumor volume was monitored 1 to 2 times a week and calculated using the following formula: Volume (mm3) =π/6 × L × W × W (L: longest dimension, W: dimension perpendicular to length). In addition, the mice were anesthetized by intraperitoneal injection of 0.7% pentobarbital sodium at a dose of 10 µL/g, and tumor burden was observed (emission wavelength, 510 nm) under in vivo imager system (Perkin Elmer, IVIS Spectrum). After 24 days, mice were sacrificed by cervical dislocation and the tumors were subjected to detection of PHF5A, Ki67 expression using IHC staining.
Microarray analysis
RNA was purified from MGC-803 cells transfected with lentivirus shCtrl or shPHF5A, and analyzed by A Affymetrix Human Gene Microarray Prime View (Affymetrix Scanner 3000 scan) to identify differentially expressed genes (DEGs). The screening criteria of DEGs was |Fold Change| ≥ 1.3 and false discovery rate (FDR) ≤ 0.05. Significant enrichment of DEGs in classical pathways, disease and function, and interaction networks was explored based on Ingenuity Pathway Analysis (IPA).
Statistical analysis
Data were obtained from three independent experiments which are presented as the means ± standard deviation (SD) and a P-value < 0.05 was considered significant. Comparisons between different groups were analyzed with the t test. The significance of differences between groups was assessed by GraphPad Prism V8.0 (GraphPad, CA, USA) and SPSS 20.0 (IBM, SPSS, IL, USA).