Integrated profiling identifies SLC5A6 and MFAP2 as novel diagnostic and prognostic biomarkers in gastric cancer patients

Abstract

Gastric cancer (GC) is one of the leading causes of malignancy‑associated mortality worldwide. However, the underlying molecular mechanisms of GC are unclear and the prognosis of GC is poor. Therefore, it is important and urgent to explore the underlying mechanisms and screen for novel diagnostic and prognostic biomarkers, as well as therapeutic targets. In the current study, scale‑free gene co‑expression networks were constructed using weighted gene co‑expression network analysis, the potential associations between gene sets and clinical features were investigated, and the hub genes were identified. The gene expression profiles of GSE38749 were downloaded from the Gene Expression Omnibus database. RNA‑seq and clinical data for GC from The Cancer Genome Atlas were utilized for verification. Furthermore, the expression of candidate biomarkers in gastric tissues was investigated. Survival analysis was performed using Kaplan‑Meier and log‑rank test. The predictive role of candidate biomarkers in GC was evaluated using a receiver operator characteristic (ROC) curve. Gene Ontology, gene set enrichment analysis and gene set variation analysis methods were used to interpret the function of candidate biomarkers in GC. A total of 29 modules were identified via the average linkage hierarchical clustering. A significant module consisting of 48 genes associated with clinical traits was found; three genes with high connectivity in the clinical significant module were identified as hub genes. Among them, SLC5A6 and microfibril‑associated protein 2 (MFAP2) were negatively associated with the overall survival, and their expression was elevated in GC compared with non‑tumor tissues. Additionally, ROC curves indicated that SLC5A6 and MFAP2 showed a good diagnostic power in discriminating cancerous from normal tissues. SLC5A6 and MFAP2 were identified as novel diagnostic and prognostic biomarkers in GC patients; both of these genes were first reported here in connection with GC and deserved further research.

Figure 1

Cluster dendrogram for 15 gastric cancer samples from the GSE38749 dataset. Classification is according to American Joint Committee on Cancer; with stage reported as stage III, green; stage IIIa, yellow; stage IIIb, red; and status reported as survival, red; and death, green.

Figure 2

Determination of the soft-threshold in weighted genes co-expression network analysis. (A) Analysis of the scale-free fit index for various soft-thresholds determining scale independence. (B) Analysis of the mean connectivity for various soft-thresholds.

Figure 3

Identification of modules associated with the clinical traits of gastric cancer. (A) Cluster dendrogram of all differentially expressed genes clustered on a dissimilarity measure. (B) Heatmap of the correlation between module eigengenes and clinical traits of gastric cancer. (C) Distribution of average gene significance errors in the modules associated with tumor prognosis of gastric cancer. (D) Connectivity of gene significance with module membership in the 'black’ module.

Figure 4

Correlation of modules based on their eigengenes. Visualization of hierarchical clustering dendrogram of the eigengenes (top); the eigengene network represents the relationships among modules. Heat map of the eigengene adjacency (bottom); the colored bars on the left and below indicate the module for each row or column.

Figure 5

Pathway analysis of hub genes. Significantly enriched genes using Gene Ontology annotations for the 'black’ module, including (A) biological processes, molecular function and cellular components and (B) Kyoto Encyclopedia of Genes and Genome pathways. (C) Weighted co-expression network for the 'black’ module genes, with nodes displayed according to the connectivity of genes.

Figure 6

Correlation of three genes with module membership in the 'black’ module. Associations between module membership for the 'black’ module and the expression of (A) EVA1A, (B) SLC5A6 and (C) MFAP2 are plotted. MFAP2, microfibril-associated protein 2.

Figure 7

Overall survival of patients with gastric cancer associated with hub gene expression. Curves were created using the Kaplan Meier-plotter and patients were stratified into high- and low-level expression groups according to median expression. Survival is plotted for (A) EVA1A, (B) SLC5A6 and (C) MFAP2 expression. MFAP2, microfibril-associated protein 2.

Figure 8

Validation of SLC5A6 and MFAP2 expression in gastric cancer. mRNA expression at different stages for (A) SLC5A6 and (B) MFAP2, with P>0.01 for all comparisons (ANOVA). mRNA expression in tumor and normal tissues for (C) SLC5A6 and (D) MFAP2. ^*P<0.0001 (paired Student's t-test). MFAP2, microfibril-associated protein 2.

Figure 9

ROC curves for hub genes. Area under the ROC curve were determined for (A) SLC5A6 and (B) MFAP2. ROC, receiver operating characteristic; AUC, area under the curve; MFAP2, microfibril-associated protein 2.

Figure 10

Immunohistochemistry analysis of SLC5A6 expression in gastric cancer samples. Data were obtained from the Human Protein Atlas. (A-E) Protein levels of SLC5A6 in normal tissue (staining: not detected; intensity: negative; quantity: negative). (F) Protein levels of SLC5A6 in tumor tissue (staining: medium; intensity: moderate; quantity: 25%~75%). (G) Protein levels of SLC5A6 in tumor tissue (staining: medium; intensity: moderate; quantity: 25~75%). (H) Protein levels of SLC5A6 in tumor tissue (staining: not detected; intensity: weak; quantity: <25%). (I) Protein levels of SLC5A6 in tumor tissue (staining: not detected; intensity: weak; quantity: <25%). (J) Protein levels of SLC5A6 in tumor tissue (staining: not detected; intensity: weak; quantity: <25%). Scale bar, 200 µm. Normal tissue (n=6), tumor tissue (n=12).

Figure 11

Immunohistochemistry analysis of MFAP2 expression in gastric cancer samples. Data were obtained from the Human Protein Atlas. (A-E) Protein levels of MFAP2 in normal tissue (staining: not detected; intensity: negative; quantity: negative). (F-J) Levels of MFAP2 in tumor tissue (staining: not detected; intensity: negative; quantity: negative). Scale bar, 200 µm. MFAP2, microfibril-associated protein 2. Normal tissue (n=5), tumor tissue (n=12).