The prognostic value of COL3A1/FBN1/COL5A2/SPARC-mir-29a-3p-H19 associated ceRNA network in Gastric Cancer through bioinformatic exploration

Abstract

Increasing studies on malignant tumors have proposed a new competing endogenous RNA (ceRNA) regulatory mechanism that mRNA, miRNA and lncRNA interact with each other. However, the mRNA-miRNA-lncRNA associated ceRNA network in gastric cancer remains unknown. We used online bioinformatic softwares to predict the hub genes and their upstream miRNAs and lncRNAs in gastric cancer, and then performed survival analyses. After collecting gastric cancer tissue samples and performing PCR experiments, the correlations among predicted mRNA, miRNA and lncRNA were further verified. A total of 101 up-regulated significant differentially expressed genes (DEGs) and 219 down-regulated significant DEGs in gastric cancer were confirmed. Functional enrichment analyses of these significant DEGs indicated that they were potentially enriched in some pathways involved in tumor malignant biological processes or metabolism. Then, we identified 20 hub genes in the PPI networks. Combined with expression and survival analyses, 8 up-regulated genes and 1 down-regulated gene were identified as central genes and acted as important prognostic roles in gastric cancer. 17 miRNAs were confirmed that might potentially regulate the expressions of these central genes. But only 8 out of them indicated better outcome in gastric cancer. Further, 79 lncRNAs were predicted that might have the potence to combine with the 8 central miRNAs. The lncRNA H19 was eventually defined as a central lncRNA by survival analyses. Stimultaneously, we found that there were certain interactions among lncRNA, miRNA and mRNAs in 50 gastric cancer tissues by qRT-PCR. Moreover, the high expression of H19 is associated with advanced TNM stage, primary tumor and lymph nodes, indicating a poor prognosis. In summary, we uncovered the prognostic value of COL3A1/FBN1/COL5A2/SPARC-mir-29a-3p-H19 ceRNA network in gastric cancer.

Keywords: bioinformatic analysis; competing endogenous RNA (ceRNA); gastric cancer; long noncoding RNA (lncRNA); microRNA (miRNA); prognosis.

Figure 1

Screening for significant DEGs in gastric cancer. (A-D) Volcano plots from four datasets (GSE13911, GSE79973, GSE19826 and GSE54129). X axis represents the p-value of the logarithmic transformation, and Y axis represents the average gene expression differences between gastric cancer samples and normal samples. The four volcano plots showed all DEGs; red dots and green dots represent the up-regulated and down-regulated genes in gastric cancer samples, respectively; black dots represent genes that are not differentially expressed between gastric cancer samples and controls. |log₂FC| >1 and p-value < 0.05 were set as the cut-off criteria. (E-F) Venn diagrams of up-regulated and down-regulated DEGs from four GSE datasets. The genes taking the intersection are considered to be significant DEGs.

Figure 2

Function analysis of the significant DEGs. (A) The enriched GO biological processes (BP) of the up-regulated significant DEGs. (B) The enriched GOBP of the down-regulated significant DEGs. (C) The enriched KEGG pathways of the up-regulated significant DEGs. (D) The enriched KEGG pathways of the down-regulated significant DEGs.

Figure 3

The top 30 hub genes determined in PPI networks. (A) The PPI networks of the up-regulated significant DEGs. (B) The top 30 hub genes of the up-regulated significant DEGs. (C) The PPI networks of down-regulated significant DEGs. (D) The top 30 hub genes of the down-regulated significant DEGs.

Figure 4

Ascertainment of central genes in gastric cancer. (A-B) Ascertainment of central genes among the top 10 hub genes of the significant up-regulated and down-regulated DEGs by integrating expression and prognosis analyses base on GEPIA and Kaplan Meier-plotter databases, respectively. (C-K) Expressions and prognostic values of the central genes (FN1, COL3A1, FBN1, BGN, COL5A2, THBS2, COL5A1, SPARC and ATP4A) in gastric cancer.

Figure 5

Identification of upstream central miRNAs of central genes. (A) Establishment of miRNA-gene network based on Cytoscape software. (B-I) Prognostic values of central miRNAs (miR-200c-3p, miR-200b-3p, let-7g-5p, miR-140-3p, miR-29a-3p, let-7b-5p, miR-27b-3p and miR-135b-5p) in gastric cancer.

Figure 6

Identification of upstream central lncRNAs of central miRNAs. (A) Identification of central lncRNAs among the predicted lncRNAs by integrating expression and prognosis analyses using GEPIA and UALCAN databases, respectively. (B) Expression and prognostic value of H19 in gastric cancer. (C) The map of the novel mRNA-miRNA-lncRNA ceRNA network associated with prognosis of gastric cancer.

Figure 7

Preliminary validation of the mRNA-miRNA-lncRNA network in gastric cancer tissue samples. (A) H19 expression in GC tissues (n = 50) compared with normal tissues (n = 50) was examined by qRT-PCR and normalized to GAPDH expression. (B) Kaplan-Meier overall survival curves according to H19 expression levels. (C) The relationships between H19 expression and clinicopathological characteristics (TNM stage, primary tumor and lymph nodes). (D) Spearman correlation analysis among the expression of H19, miR-29a-3p and mRNAs (COL3A1/FBN1/COL5A2/SPARC).