Results
Expression of CENPN in STAD
tissue
Using the GEPIA database, we first examined the expression of CENPN in
STAD in TCGA and GTEx databases. STAD tissues had considerably higher
levels of CENPN mRNA expression than their neighboring tissues (Figure
1A, P < 0.05). We used the TCGA database to assess the
expression of CENPN in STAD paired samples, and the results revealed
that in 27 samples, the expression of CENPN in STAD was higher than that
in matched normal tissues (Figure 1B, P < 0.001). In
addition, the ROC curve showed that CENPN expression had good predictive
ability, with an area of 0.950 under the curve (95% confidence interval
[CI]=0.926–0.973), and STAD tissues could be distinguished from
normal tissues (Figure 1C).
To further validate CENPN expression in STAD, immunohistochemical
stainingwas performed on 76 tumor specimens and adjacent paracellular
tissues to assess the differential expression of CENPN. CENPN was highly
expressed in STAD tissues, with low or no expression in paracellular
tissues, and its positive expression sites were primarily in the
cytoplasm and nucleus (Figure 1D). The positivity rate in STAD tissues
was 76.3%, whereas it was 18.4% in adjacent tissues (Table 1).
Relationship between CENPN expression in STAD and
clinicopathological parameters
We investigated the relationship between CENPN expression and various
clinicopathological parameters in patients with STAD to better
understand the significance and possible molecular mechanisms of CENPN
expression in the development of STAD. The expression of CENPN was
significantly different in STAD patients with varying degrees of
invasion, TNM stage, and lymph node metastasis ( P <
0.05). However, there was no statistically significant difference in
CENPN expression according to sex, age, tumor size, or degree of
differentiation ( Table 2 ) .
CENPN functional enrichment analysis and co-expression gene
screening in
STAD
To further investigate the functions and pathways affected by CENPN, we
used TCGA data to examine the correlation between CENPN and other genes
in STAD. The top 300 genes most strongly associated with CENPN were
chosen for enrichment analysis, and the top 20 genes are displayed in
the heat map (Figure 2A). Using the Xiantao database, we examined
potential functional pathways based on the top 300 genes. GO and KEGG
enrichment analysis revealed that CENPN plays important roles in the
cell cycle, DNA replication, chromosome separation, and nuclear
division, as well as several other key signaling pathways (Figure 2B).
Correlation between immune cell infiltration and CENPN
expression
Immune cells that infiltrate tumors play a significant role in the tumor
microenvironment and is linked to the development, spread, and
metastasis of cancer20, 21. Therefore, we examined the
connection between CENPN expression in STAD and the degree of immune
cell infiltration (Figure 3A). A favorable correlation was determined
between CENPN expression and Th2 cells and NK CD56dim cells (Figure
3B,C). Mast cells , pDC ,NK cells ,and B cells were negatively
correlated with the expression of CENPN (Figure 3D-G).
Effect of CENPN on STAD cell
proliferation
To better understand the regulatory function of CENPN on the
proliferation of STAD cells, we transiently transfected siRNAs to
downregulate CENPN expression in AGS cell lines. The transfection effect
was confirmed using western blotting. The CENPN expression in AGS cells
in the transfection group was significantly lower than that in the
control group (Figure 4A, P < 0.01). We then determined
the proliferation and growth of AGS cells after CENPN downregulation
using the CCK-8 assay. The results showed that CENPN downregulation
significantly reduced the proliferation of AGS cells compared to the
control group (Figure 4B, P < 0.05). To determine the
effect of CENPN on STAD cell proliferation, an EdU test was also
performed. The findings demonstrated that CENPN siRNA-transfected AGS
cells had considerably lower proliferation ratios than the control group
(Figure 4C, P < 0.05). In conclusion, the
downregulation of CENP may inhibit the proliferation of STAD cells.
Effect of CENPN on the cell cycle of STAD
cells
To further investigate the mechanism by which CENPN regulates the
proliferation of human AGS cells, flow cytometry was used to detect cell
cycle changes. CENPN knockdown in AGS cells resulted in an increased
percentage of G0-G1 cells compared to the controls, while the percentage
of cells in the S and G2-M phases decreased significantly (Figure 5,P < 0.05). These findings imply that CENPN may promote
cell proliferation by regulating the cell cycle.
Effect of CENPN on STAD cell
apoptosis
We used flow cytometry to investigate the role of CENPN in cell
apoptosis in STAD. The percentages of early, late, and complete
apoptosis in AGS cells were 23.67 % (12.7% in the control group), 7.39
% (2.98% in the control group), and 31.06 % (15.68% in the control
group), respectively. After CENPN knockdown,the apoptosis rate of AGS
cells was considerably higher than that of the control group (Figure 6,P < 0.05). Therefore, we concluded that CENPN knockdown
promotes apoptosis in STAD cells.