Materials and methods

Source of the specimen

The study included 76 wax block specimens surgically resected from confirmed patients with STAD at the Department of Pathology, Affiliated Central Hospital of Shenyang Medical College, between June 2014 and July 2016. Paracellular tissue samples from the same patients, including 53 males and 23 females,were chosen as the control group. The ages of the patients ranged from 36 to 95 years, with a mean age of 66 years. None of the patients receive radiotherapy or chemotherapy before surgery. This study was approved by the Medical Ethics Committee of the Shenyang Medical College, China.

Data gathering and analysis

Differences in CENPN expression between STAD and normal tissues were evaluated using TCGA (https://cancergenome.nih.gov) and GTEX databases.The TCGA database was used to obtain RNA sequencing data and clinical follow-up information for patients with STAD.

CENPN functional enrichment analysis and screening of co-expressed genes in STAD

TCGA-STAD data were used to perform a Pearson correlation study in CENPN and other mRNAs in gastric cancer. For enrichment analysis to assess the function of CENPN, the 300 genes with the highest positive correlations with CENPN were chosen. GO and KEGG pathway analyses were performed using Xiantao Academic.

Immune infiltration analysis

The Xiantao Academic and single-sample gene set enrichment analysis were used to examine the link between CENPN expression in STAD and 24 different types of immune cells (ssGSEA). Additionally, the relationship between CENPN expression and Th2, mast cells, Mast cells , pDC ,NK cells ,and B cells was examined using Spearman’s correlation.

Immunohistochemical staining

After roasting, dewaxing, antigen repair, and endogenous peroxidase blocker, paraffin sections were incubated with rabbit polyclonal anti-CENPN antibody (1:400 dilution,Proteintech,IL,USA) and kept in a refrigerator at 4 °C overnight. The next day, the sections were left at 25°C for 30 min, followed by incubation with the secondary antibody (Fuzhou Maixin Biotechnology Development Co, Ltd,China) at 25°C. Sections were then stained with DAB and hematoxylin, dehydrated with absolute alcohol for 10 min, and sealed with neutral gum.
Evaluating outcomes: The cytoplasm and nucleus were the main sites for CENPN expression. Two pathologists performed a blind review of the immunohistochemical results. Five visual fields at random were chosen, and the staining intensity and percentage were assessed and assigned a semi-quantitative score. The percentage scores given for stained cells were 0 (4% or less), 1 (5%–24%), 2 (25%–49%), 3 (50%–74%), or 4 (75%).The staining intensities were assigned 0 (no staining), 1 (light yellow particles), 2 (yellow), and 3. (dark-yellow particles). The staining intensity score was multiplied by the percentage score of positively stained cells in each tumor sample to yield a total score ranging from 0 to 12. A score of less than 3 was considered negative, whereas a score of 3 or higher wasconsidered positive.

Cell culture and transfection

AGS cells were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were routinely sub-cultured in F12 medium (Gibco, USA) containing 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37 °C in a cell incubator with 5% CO2.Transfection of siRNAs was carried out in accordance with the manufacturer’s instructions using the Lipo3000 reagent (Life Technology Company). After 48 h of transfection, the depletion of CENPN protein was examined by western blotting in gastric cancer cells that were in the logarithmic growth phase.

Western blot analysis

Cells were lysed using RIPA buffer according to the manufacturer’s protocol. Protein (30μg/lane) was added to the 10% SDS-PAGE gel and then transferred to a membrane, then transferred to a PVDF membrane, blocked with 5% skim milk for 1 hour in TBST, They are incubated with primary antibody (Proteintech,IL,USA) at 4°C overnight. Membranes were then washed three times andincubated with secondary antibody (Fuzhou Maixin Biotechnology Development Co, Ltd,China) for 1 h. The signal was detected using an enhanced chemiluminescence Western blot assay kit.

CCK-8 experiments

In a 96-well plate, 5000 cells were seeded into each well. To each well, 10 µl of CCK-8 (APExBio, USA) were added at 0, 24, 48, 72, and 96 h. A microplate reader (TECAN Infinite M200pro) was used to measure the optical density at 450 nm following a 1h incubation at 37 °C, and a cell growth curve was drawn.

EdU cell proliferation assay

Cells were seeded in 24-well plates at the logarithmic growth stage and cultured overnight in incubators. Transfection was performed accordantly. Each well was labelled with diluted EdU (Guangzhou Ruibo Biotechnology Co.,Ltd., China) (1:5000 dilution) and incubated for 48 h at 37 °C in a 5% CO2 incubator. The cells were fixed, penetrant added, and stained. Hoechst 33342 was then added to each well at a dilution of 1:1000. After 15 min of shaking at room temperature in the dark, the dye solution was removed, and the cells were washed with phosphate-buffered saline. Finally, an inverted Microscope was used for observation, and five different photographic fields were picked At random.

Cell cycle detection

Cell cycle progression was detected using a cell cycle detection kit (BD Pharmingen, USA) according to the manufacturer’s instructions. Cells in 6-well plates were transfected for 48 h and fixed in 75% ethanol, labeled with propidium iodide, and analyzed by flow cytometry (BD FACSCalibur). Each group consisted of three replicates.

Detection of cell apoptosis

Apoptosis was measured using an apoptosis detection kit (Vazyme). Cells were transfected for 48 h in 6-well plates, stained with annexin V and propidium iodide, and analyzed using flow cytometry (BD FACSCalibur).

Statistical analysis

GraphPad Prism 8 and SPSS 20.0 were both used for statistical analysis in this study. Every experiment included at least three independent replicates. Student’s t-test was used to calculate statistical significance and results were reported as mean ± standard deviation. Chi-square analysis was used to examine count data. P < 0.05 was considered statistically significant.