Integrative Analysis of Dysregulated lncRNA-Associated ceRNA Network Reveals Functional lncRNAs in Gastric Cancer

Abstract

Mounting evidence suggests that long noncoding RNAs (lncRNAs) play important roles in the regulation of gene expression by acting as competing endogenous RNA (ceRNA). However, the regulatory mechanisms of lncRNA as ceRNA in gastric cancer (GC) are not fully understood. Here, we first constructed a dysregulated lncRNA-associated ceRNA network by integrating analysis of gene expression profiles of lncRNAs, microRNAs (miRNAs), and messenger RNAs (mRNAs). Then, we determined three lncRNAs (RP5-1120P11, DLEU2, and DDX11-AS1) as hub lncRNAs, in which associated ceRNA subnetworks were involved in cell cycle-related processes and cancer-related pathways. Furthermore, we confirmed that the two lncRNAs (DLEU2 and DDX11-AS1) were significantly upregulated in GC tissues, promote GC cell proliferation, and negatively regulate miRNA expression, respectively. The hub lncRNAs (DLEU2 and DDX11-AS1) could have oncogenic functions, and act as potential ceRNAs to sponge miRNA. Our findings not only provide novel insights on ceRNA regulation in GC, but can also provide opportunities for the functional characterization of lncRNAs in future studies.

Keywords: DDX11-AS1; DLEU2; competing endogenous RNA; gastric cancer; long non-coding RNA; network analysis.

Figure 1

The heatmap of lncRNAs (DELs), mRNAs (DEMs), and miRNAs (DEMis) in gastric cancer (GC). The samples were represented in columns, and the genes were represented in rows, with different colors. The expression value for each row was normalized by the z-score. Red indicates high relative expression and blue indicates low expression of genes as shown in the scale bar.

Figure 2

Global view and function analysis of the ceRNA network in GC. (A) Global view of the ceRNA network in GC. The lncRNA, mRNA, and miRNA were colored red, yellow, and green. There were 28 lncRNAs, 130 mRNAs, 16 miRNAs, and 173 edges in this network; (B) The top 10 significant gene ontology (GO) terms in GC-related ceRNA network; (C) The top 10 significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in GC-related ceRNA network. The count indicates the number of genes associated with a GO term in biological processes (BP) or a KEGG pathway.

Figure 3

The top highest 17 BC nodes in ceRNA network. The left Y-axis denotes the betweenness centrality (BC) value of node, and the right Y-axis denotes the cumulative percentage of the BC. These nodes made up 80.5% of all the BCs in the ceRNA network.

Figure 4

The ceRNA subnetworks and functional enrichment analysis of the hub lncRNAs. (A) The RP5-1120P11-associated ceRNA subnetwork; (B) DLEU2-associated ceRNA subnetwork; (C) DDX11-AS1-associated ceRNA subnetwork. The lncRNA, mRNA, and miRNA were colored red, yellow, and green; (D) The enrichment analysis result of RP5-1120P11-associated ceRNA subnetwork; (E) The enrichment analysis result of DLEU2-associated ceRNA subnetwork; (F) The enrichment analysis result of DDX11-AS1-associated ceRNA subnetwork. The count indicates the number of genes associated with a GO term in BP or a KEGG pathway.

Figure 5

The expression levels and correlation of DLEU2 related DEMi and DEMs in GC. (A) The boxplot showed the expression levels of DLEU2 and DLEU2 related DEMs. * denotes p < 0.001; (B) The scatter plots showed the correlation of expression between DLEU2 and hsa-miR-30-5p; (C) The scatter plots showed the correlation of expression between DLEU2 and DLEU2 related DEMs; (D) The scatter plots showed the correlation of expression between DLEU2 related DEMs and hsa-miR-30-5p. All the data were obtained from GDC.

Figure 6

Functional validation of the predicted hub lncRNAs in GC. (A) The qRT-PCR results showed that the expression levels of DLEU2 and DDX11-AS1 in 12 pairs of GC tissues (GTs) were significantly higher than the adjacent normal tissues (NTs); (B) The qRT-PCR results showed DLEU2 and DDX11-AS1 knockdown in BGC823/MKN45 cells after transfection with NC (negative control), siRNA 1 (si-1), and siRNA 2 (si-2). The CCK-8 assays showed that knockdown of DLEU2 and DDX11-AS1 suppressed cell proliferation in BGC823/MKN45 cells; (C) The colony-forming assays showed that knockdown of DLEU2 and DDX11-AS1 suppressed cell proliferation in BGC823/MKN45 cells; (D) In BGC823 and MKN45 cells, knockdown of DLEU2 and DDX11-AS1 increased hsa-miR-30-5p and hsa-miR-145-5p expression, respectively. Biological experimental results were analyzed using paired or unpaired t-test, * denotes p < 0.05.