Expression Analysis of CST1
CST1 are expressed in a wide range of cancers according to The Cancer Genome Atlas (TCGA) UALCAN database. CST1 expression is lowest in the Brain lower grade glioma (LGG) dataset (median value 0) and highest in the Pancreatic adenocarcinoma (PAAD) dataset (median value 7.181), CST1 expression in gastric cancer(GC) is second only to PAAD(Fig1A).compared with normal tissues, most tumor types show significantly higher expression of CST1,and is most evident in GC(Figure1B). Next, we used oncomine database data to analyze the expression level of CST1 in cancer. The results showed that there were 388 studies involving in cancer, 38 of them were statistically different, and 37 of them had increased expression of CST1 in tumors(Figure2A). The results of 4 studies on gastric cancer suggested that the expression of CST1 in cancer tissues was significantly higher than that in normal tissues. There was significant difference in Cho gastric (Fig2B,p<0.0001),Cui gastric(Figure2C,p<0.0001),Derrico gastric (Fig2D,p<0.005),and Wang gastric (Fig2E,p < 0.0001).
The expression of CST1 in different types of gastric cancer
We used a ualcan online database to detect the expression level of CST1 in the classification of different influencing factors of gastric cancer. The results suggested that the expression of CST1 in primary tumor vs normal (Figure3A), age vs normal (Figure3B) ,gender vs normal (Figure3C),grade vs normal (Fig3D), stage vs normal (Figure3E), histological subtypes vs normal(Fig3F) was higher than that in normal tissues (p < 0.05).
Promoter methylation levels of CST1 in different types of gastric cancer
Next, We used a ualcan online database to detect the promoter methylation levels of CST1 in the classification of different influencing factors of gastric cancer. The results suggested that the promoter methylation levels of CST1 in primary tumor vs normal (Figure4A), gender vs normal (Figure4B), age vs normal (Figure4C), grade vs normal (Figure4D), TNM vs normal (Figure4E), stage vs normal (Figure4F) was lower than that in normal tissues (p < 0.05).
Correlation Between the Expression of CST1 and Prognosis
We employed the KM plotter database to explore the effect of CST1 expression on the survival of patients of GC, we detected a significant correlation between prognosis and CST1 expression (Figure5). We revealed that a higher CST1 expression level was significantly related to a poorer prognosis in GC (OS, HR = 1.7, 95% CI = 1.42–2.02, p =2.9e-09; First progression survival (FPS), HR = 1.63, 95% CI = 1.32–2.01, p = 3.9e-06; post progression survival (PPS), HR = 1.92, 95% CI = 1.51–2.43, p = 4.4e-08;) (Figures5A, Figure5B, Figure5C).
CST1 expression and prognosis in TCGA GC dataset
The data of 354 patients with gastric cancer expressing CST1 were downloaded from the TCGA database, and the expression of CST1 in patients with gastric cancer was analyzed. CST1 expression level was most significantly upregulated (Fold-change =20.94,p<0.001)( Figure6A, Figure6B).The patients were divided into two groups according to the median expression of CST1 (35.107): high expression of CST1 (n = 177) and low expression of FBXO22 (n = 177). The clinicopathological features of patients with gastric cancer were shown in Table1. The results showed that the expression of CST1 was positively correlated with the OS(Figure6C).
Functional Annotations and Predicted Signaling Pathways
To identify the CST1-related pathways activated in GC, we conducted GSEA between low and high CST1 expression datasets. Significant differences (false-discovery rate <0.25, p < 0.05) in the enrichment of the Molecular Signature database Collection are shown in Figure 7. The results showed that CST1 was mainly involved in Coagulation(Figure7A) and Epithelial-Mesenchymal-Transition(Figure7B), Complement(Figure7C), Apoptosis(Figure7D), Glycolysis(Figure7E). These results showed that high expression of CST1 was closely associated with malignancy in GC.
Enrichment Analysis of CST1-Related Genes and CST1,CST2 and CST4 Genetic Alterations in Patients with GC
We investigated the interaction network for CST1 by the GeneMANIA database. Figure 8A shows the top 20 genes and their relationship with CST1. Among them, CST2 and CST4 of the same family of CST1 are extremely correlated with it. Next, we investigated the frequency of alterations CST1,CST2 and CST4 in five groups of GC cases via the cBioPortal web resource(Figure8A). Overall, 1460 samples from TCGA GC database were studied (Figure8B,Figure8C), and the mutation rate was found to be low (1.36–3.16%) in Gastric Cancer (OncoSG, 2018),Stomach Adenocarcinoma (TCGA, Nature 2014),Stomach Adenocarcinoma (TCGA, Firehose Legacy),Stomach Adenocarcinoma (TCGA, PanCancer Atlas),Stomach Adenocarcinoma (Pfizer and UHK, Nat Genet 2014). The type as well as the location of distinct mutations are indicated in Figure8D.
KM plotter database analysis of the relationship between CST2 and CST4 expression and prognosis in gastric cancer
We employed the KM plotter database to explore the effect of CST2 and CST4 expression on the survival of patients of GC, we detected a significant correlation between prognosis and CST1 expression (Figure9). We revealed that a higher CST2 expression level was significantly related to a poorer prognosis in GC (OS, HR = 1.82, 95% CI = 1.53–2.18, p =1.8e-11; First progression survival (FPS), HR = 1.84, 95% CI = 1.5–2.25, p = 2.7e-09; post progression survival (PPS), HR = 2.07, 95% CI = 1.65–2.61, p = 2.2e-10;) (Figures9A, Figure9B, Figure9C). Higher CST4 expression level was significantly related to a poorer prognosis in GC (OS, HR = 1.52, 95% CI = 1.28–1.8, p =1.5e-06; First progression survival (FPS), HR = 1.54, 95% CI = 1.24–1.9, p =5.9e-05; post progression survival (PPS), HR = 1.56, 95% CI = 1.23–1.99, p = 0.00025;) (Figure9D, Figure9E, Figure9F).
Knockdown of CST1 expression inhibited the proliferation, colony formation, migration and invasion ability of gastric cancer cells.
We first examined the protein expression level of CST1 in four gastric cancer cell lines and found that CST1 was expressed at a higher level in AGS and NCI-N87 than in MGC803 and SGC7901 (Figure10A). Secondly, we knocked down the expression of CST1 in AGS and NCI-N87 cells (Figure10B). Anchorage-independent colony-formation assay showed that knockdown of CST1 significantly inhibited the clonogenic ability of AGS and NCI-N87 cells (Figure10C, Figure10D). We then used Transwell assay to detect the effect of CST1 on the migration and invasion ability of gastric cancer cells, and found that inhibition of CST1 expression significantly inhibited the migration and invasion ability of AGS and NCI-N87 cells (Figure11A, Figure11B).