Construction and Comprehensive Analyses of a Competing Endogenous RNA Network in Tumor-Node-Metastasis Stage I Hepatocellular Carcinoma

Abstract

Background: Long noncoding RNAs (lncRNAs) can function as competing endogenous RNAs (ceRNAs) and interact with microRNAs (miRNAs) to regulate target gene expression, which can greatly influence tumor development and progression. Different tumor-node-metastasis (TNM) stages of hepatocellular carcinoma (HCC) defined by the American Joint Committee on Cancer (AJCC) have different clinical results. Our purpose was to comprehensively analyze differentially expressed (DE) lncRNAs, miRNAs, and mRNAs in stage I HCC and identify prognosis-associated RNAs.

Methods: RNA-seq data were obtained from The Cancer Genome Atlas (TCGA) database. A stage I HCC-associated miRNA-lncRNA-mRNA network was constructed. Next, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analyses of ceRNA-associated DEmRNAs were performed using Database for Annotation, Visualization, and Integrated Discovery (DAVID) 6.8 and Clusterprofile in the R package. The protein-protein interaction (PPI) network of the above mRNAs was then constructed using STRING. Finally, the association between lncRNAs and mRNAs in the ceRNA network and prognosis of patients was further analyzed. Linear regression analysis of the above lncRNAs and mRNAs associated with overall survival was performed.

Results: After a comparison between HCC and adjacent nontumor tissues, 778 lncRNAs, 1608 mRNAs, and 102 miRNAs that were abnormally expressed were identified. The ceRNA network was composed of 56 DElncRNAs, 14 DEmiRNAs, and 30 DEmRNAs. Functional analysis results showed that 30 DEmRNAs were enriched in 14 GO biological process categories and 6 KEGG categories (false discovery rate (FDR) < 0.05). A PPI network was composed of 22 nodes and 58 edges. We detected 4 DElncRNAs (BPESC1, AC061975.6, AC079341.1, and CLLU1) and 6 DEmRNAs (CEP55, E2F1, E2F7, EZH2, G6PD, and SLC7A11) that had significant influences on the overall survival (OS) of stage I HCC patients (P < 0.05). lncRNA BPESC1 was positively correlated with mRNA CEP55 via miR-424, and lncRNA AC061975.6 was positively correlated with mRNA E2F1 via miR-519d.

Conclusion: Our study identified novel lncRNAs and mRNAs that were associated with the progression and prognosis of stage I HCC and further investigated the regulatory mechanism of lncRNA-mediated ceRNAs in the development of stage I HCC.

Figure 1

Specific lncRNA-related ceRNA network and characteristics of the constituent lncRNAs in stage I HCC patients. The volcano plot shows the expression profiles of mRNAs (a), lncRNAs (b), and miRNAs (c). Red dots indicate upregulated RNAs, and green dots indicate downregulated RNAs. Heatmaps of differentially expressed RNAs in different samples: (d) mRNAs, (e) lncRNAs, and (f) miRNAs. The y-axis represents RNAs and the x-axis represents patient samples; red denotes upregulation and green denotes downregulation. ceRNA: competing endogenous RNA; HCC: hepatocellular carcinoma; lncRNA: long noncoding RNA; miRNA: microRNA.

Figure 2

The ceRNAs network in stage I HCC patients. (a) Venn diagram of DEmRNAs involved in the ceRNA network. (b) The ceRNA network of lncRNAs-miRNAs-mRNAs involved in HCC. Diamonds represent IncRNAs, round rectangles represent miRNAs, and triangles represent mRNAs. The nodes highlighted in red and blue indicate up- and downregulation, respectively. DEmRNA: differentially expressed mRNA.

Figure 3

Enrichment analysis of DEmRNAs involved in the ceRNA network. (a) The top 10 significantly enriched pathways identified in the DEmRNA GO enrichment analysis. (b) Significantly enriched KEGG pathways of DEmRNAs (FDR < 0.05). GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; FDR: false discovery rate.

Figure 4

Identification of hub genes from the PPI network. (a) The PPI network of ceRNA network-associated DEmRNAs. (b) Information about nodes in the PPI network. PPI: protein-protein interaction.

Figure 5

Kaplan–Meier survival curves of 4 DElncRNAs involved in the ceRNA network associated with overall survival in patients with stage I HCC (P < 0.05). DElncRNA: differentially expressed lncRNA.

Figure 6

Kaplan–Meier survival curves of 6 DEmRNAs involved in the ceRNA network associated with overall survival in patients with stage I HCC (P < 0.05).

Figure 7

Correlation analysis of DEGs linear regression analysis between lncRNAs and mRNAs associated with overall survival. Linear regression of BPESC1 (a–d), AC079341.1 (e–h), and AC061975.6 (i–k) versus related DEmRNA expression level (P < 0.05). The red line represents the linear model fitted by the dots in each figure. DEGs: differentially expressed genes.

Figure 8

Flow chart of the bioinformatics analysis.