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.