A total of 107 pairs of GC and normal tissues were collected from May 2013 to December 2014 in Lanzhou University Second Hospital. The sample collection was approved by the Ethics Committee of Lanzhou University Second hospital. All patients included in the study did not receive any treatments before surgery.
Paraffin embedded tissue sections were dewaxed and hydrated. The sections were heated in a 10mM sodium citrate solution at 98°C for antigen retrieval. They were soaked in 0.3% H2O2 for 25 min to block endogenous peroxidase. Anti-YWHAG (1:200, Abcam, USA) and anti-Ki67 (1:300, Abcam, USA) were incubated with sections overnight at 4°C. Then sections were stained with diaminobenzidine solution and counterstained with hematoxylin. IHC results were independently evaluated and recorded by two senior pathologists using a blind method. The score was determined based on the percentage of cells stained (0 = 0%-5%, 1 = 6%-25%, 2 = 26%-50%, 3 = 51–75%, 4 = 76% -100%) and staining intensity (0 = no staining,1 = weak staining,2 = moderate staining, and 3 = strong staining). The product of the two scores obtained the final YWHAG and Ki67 scores.
The transcription data of GC were obtained from The Cancer Genome Atlas (TCGA) database (https://ancergenome.nih.gov/), which included 375 tumor samples and 32 normal samples. YWHAG gene expression analysis was performed with R software. Gene enrichment analysis (GSEA) software was used to reveal the potential function of YWHAG in GC.
Human GC cell lines MKN45, HGC27, NCI-N87, MKN28, KAO-III, AGS and human normal gastric mucosal epithelial cells (GES-1) were purchased from Cell Culture Center of Chinese Academy of Medical Sciences (Beijing, China). All cell lines were identified by short tandem repeats (STR). They were maintained in RPMI 1640 medium (Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, USA) at 37℃ in a humidified incubator with 5% CO2.
YWHAG-overexpression and knockdown lentivirus (GeneChem, Shanghai, China) were transfected into NCI-N87 and MKN45 cell lines. An empty lentivirus vector was used as a negative control. After 72 hours of transfection, the cells were selected with puromycin (Biofroxx, German) for 3 days. The transfection efficiency was detected by Western blot.
Cell counting kit-8 (CCK8) assay
The YWHAG knockdown, overexpression cells and the corresponding negative transfected control cells were seeded in a 96-well plate and cultured in an incubator at 37°C. At the specific time points, 10ul CCK8 solution was added and then incubated for 1 hour. Finally, the absorbance value was measured at the wavelength of 490nm.
Cells were seeded in 96-well plates at a density of 2×103 cells per well and incubated with EdU (RiboBio, Guangzhou, China) for 2 hours. After that, 4% paraformaldehyde was used for fixation, and staining was performed according to the reagent manufacturer's instructions. Finally, the Operetta CLS High Content Screening System (PerkinElmer, Waltham, MA).
Cell cycle and apoptosis analysis
Cells were resuspended in 100ul binding buffer, and the cell concentration was adjusted to 2x105 cell/tube. Cells were fixed in 75% ethanol at 4 ° C overnight to evaluate the cell cycle. Then cells were stained with propidium iodide (PI, BD Bioscience, USA). For cell apoptosis, each sample was added with Annexin V-FITC /PI (BD Bioscience, USA) and incubated in the dark at room temperature. Flow cytometry was applied.
The invasion ability of YWHAG on GC was evaluated by an invasion assay of trans-well coated with matrix (BD Bioscience, USA). 5×104 cells were resuspended into a serum-free medium and added to the upper chamber. The medium containing 10% FBS was added to the lower chamber as a chemical attractant. The medium was removed after 24 hours, and cells on the membrane surface were wiped clean with a cotton swab. Cells migrated to the lower compartment were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The number of invaded cells was measured under a microscope.
After the cells seeded in a 6-well plate were fused to 90%, a sterile 200ul pipette tip was used to form a scratch wound on the cell monolayer. The cells were washed twice with PBS and cultured in serum-free medium. The width of the wound was measured at 0, 24, 48 and 72 hours.
The cells were lysed by radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitor on ice. The same amount of protein was separated by 10%SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane, which was then sealed with blocking solution for 2 hours. Next, the membranes were incubated with the corresponding primary antibody overnight at 4℃. The primary antibodies used were: anti-GAPDH (1:10000, Proteintech, China), anti-YWHAG (1:1000, Abcam, USA), anti-cyclinD1 (1:1000, Abcam, USA), anti-CDK2 (1:1000, Abcam, USA), anti-E-cadherin (1;1000, Proteintech, China), anti-vimentin (1:2000, Proteintech, China), anti-AKT (1:1000, Abcam, USA), anti-p-AKT (1:1000, Abcam, USA) and anti-p-PI3K (1:1000, Abcam, USA). After incubation with the secondary antibodies at room temperature for 1 hour, the blots were exposed with a chemiluminescent detection system.
Tumor xenograft model
BALB/c nude mice aged 5–6 weeks were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. (Hangzhou, China). All mice were raised in a specific pathogen-free environment. The experimental protocol was approved by the ethics committee of Lanzhou University Second hospital. MKN45 cells with YWHAG knockdown and MKN45 cells were injected subcutaneously into the left side of BALB/c nude mice (five mice per group). NCI-N87 and NCI-N87 cells with YWHAG overexpression performed the same models. After one week, the tumor volumes (length×width2/2) were measured every 3 days. On 35th day, all mice were sacrificed under deep anesthesia, and tumors were removed.
The results are analyzed using GraphPad Prism8 software. One-way analysis of variance (ANOVA) was used to analyze the differences between multiple groups, and student’s t-test was for differences between two groups. Chi-square test was applied to analyze the correlation between YWHAG and clinicopathological characteristics. P < 0.05 was considered statistically significant.