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.