Identification of Novel Long Noncoding RNAs and Their Role in Abdominal Aortic Aneurysm

Abstract

Objective: Long noncoding RNAs (lncRNAs) have emerged as critical molecular regulators in various diseases. However, the potential regulatory role of lncRNAs in the pathogenesis of abdominal aortic aneurysm (AAA) remains elusive. The aim of this study was to identify crucial lncRNAs associated with human AAA by comparing the lncRNA and mRNA expression profiles of patients with AAA with those of control individuals.

Materials and methods: The expression profiles of lncRNAs and mRNAs were analyzed in five dilated aortic samples from AAA patients and three normal aortic samples from control individuals using microarray technology. Functional annotation of the screened lncRNAs based on the differentially expressed genes was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses.

Results: Microarray results revealed 2046 lncRNAs and 1363 mRNAs. Functional enrichment analysis showed that the mRNAs significantly associated with AAA were enriched in the NOD-like receptor (NLR) and nuclear factor kappa-B (NF-κB) signaling pathways and in cell adhesion molecules (CAMs), which are closely associated with pathophysiological changes in AAA. The lncRNAs identified using microarray analysis were further validated using quantitative real-time polymerase chain reaction (qRT-PCR) analysis with 12 versus 11 aortic samples. Finally, three key lncRNAs (ENST00000566954, ENST00000580897, and T181556) were confirmed using strict validation. A coding-noncoding coexpression (CNC) network and a competing endogenous RNA (ceRNA) network were constructed to determine the interaction among the lncRNAs, microRNAs, and mRNAs based on the confirmed lncRNAs.

Conclusions: Our microarray profiling analysis and validation of significantly expressed lncRNAs between patients with AAA and control group individuals may provide new diagnostic biomarkers for AAA. The underlying regulatory mechanisms of the confirmed lncRNAs in AAA pathogenesis need to be determined using in vitro and in vivo experiments.

Figure 1

Hierarchical cluster of heatmaps for the differential expression of lncRNAs and mRNAs between the AAA and control groups. (a) Hierarchical cluster of heatmaps for the lncRNAs. (b) Hierarchical cluster of heatmaps for the mRNAs. Each column represents one sample and each row represents one mRNA or lncRNA. The relative expression levels of mRNAs and lncRNAs are depicted according to the color scale. Red indicates upregulation and green indicates downregulation. The three columns on the left represent the control samples and the five columns on the right represent the AAA samples. The differentially expressed mRNAs and lncRNAs are clearly self-segregated into clusters. AAA: abdominal aortic aneurysm; lncRNAs: long noncoding RNAs.

Figure 2

Volcano plots of all the lncRNAs and mRNAs detected in the AAA and control groups. The vertical dotted line at the x-axis delimits upregulation and downregulation. The red and green plots represent the significantly upregulated and downregulated genes, respectively (fold change > 2 and P < 0.05). The black plots represent no significant changes. (a) Volcano plots of the total 35298 lncRNAs. Of them, 425 are upregulated, 1621 are downregulated, and 33252 show no differential expression. (b) Volcano plots of the total 19959 mRNAs. Of them, 599 are upregulated, 764 are downregulated, and 18596 show no differential expression. AAA: abdominal aortic aneurysm; lncRNAs: long noncoding RNAs.

Figure 3

Gene Ontology analysis of differentially expressed mRNAs between the AAA and control groups. The top 10 GO terms from the upregulated mRNAs and the top 10 GO terms from the downregulated mRNAs between two groups. In upregulated mRNAs: (a) biological process, (b) cellular component, and (c) molecular function. In downregulated mRNAs: (d) biological process, (e) cellular component, and (f) molecular function. AAA: abdominal aortic aneurysm; GO: Gene Ontology.

Figure 4

The top 18 pathways identified using KEGG pathway analysis of differently expressed mRNAs. KEGG analysis of the differentially expressed lncRNAs and mRNAs between the AAA and control groups. The top 18 significantly enriched KEGG pathway terms that correlated with the upregulated (a) and downregulated (b) genes between the two groups. AAA: abdominal aortic aneurysm; KEEG: Kyoto Encyclopedia of Genes and Genomes; lncRNAs: long noncoding RNAs.

Figure 5

The classification of lncRNAs according to their correlations with protein-coding genes. All lncRNAs were classified to six categories including intergenic, intronic antisense, natural antisense, bidirectional, intron sense-overlapping, and exon sense-overlapping. The pie chart represents the numbers and distributions of the lncRNAs detected using the microarray. lncRNAs: long noncoding RNAs.

Figure 6

Validation of confirmed lncRNAs using qRT-PCR. The results showed that the relative expression levels of ENST00000566954, ENST00000580897, and T181556 were downregulated in the AAA group (n = 11) compared with those in the control group (n = 12). All reactions were repeated three times for each lncRNA. β-Actin was used as an internal control. ^∗P < 0.05 AAAs vs. control. lncRNAs: long noncoding RNAs; AAA: abdominal aortic aneurysm.

Figure 7

lncRNA-mRNA coexpression network of the differentially expressed lncRNAs and mRNAs between the two groups. The green and pink nodes represent the lncRNAs and mRNAs, respectively. The node size represents the degree of centrality of the gene in the network, defined as the link numbers of the node. The solid lines show a positive correlation; dotted lines show a negative correlation. lncRNAs: long noncoding RNAs.

Figure 8

The lncRNA-miRNA-mRNA ceRNA regulatory network constructed based on three confirmed lncRNAs in the AAA and control groups. The nodes highlighted in red represent microRNAs, the nodes highlighted in light blue represent protein-coding RNAs, and those highlighted in light green represent lncRNAs. Edges with a T-shaped arrow represent directed relationships, while those without an arrow represent undirected relationships (ceRNA relationships). lncRNAs: long noncoding RNAs; miRNA: microRNA; ceRNA: competing endogenous RNA; AAA: abdominal aortic aneurysm.