Integrated analysis of long non-coding RNA-microRNA-mRNA competing endogenous RNAregulatory networks in thromboangiitis obliterans

Abstract

Thromboangiitis obliterans (TAO) is a non-atherosclerotic, segmental, chronic vascular inflammatory disease. Our aim was to explore the underlying mechanisms of long non-coding RNA (lncRNA)-related competing endogenous RNAs (ceRNAs) in TAO. Six blood samples were collected from patients with TAO and healthy individuals (three for each category). Total RNA was extracted from the blood of each participant and sequenced. Differentially expressed lncRNAs (DE-lncRNAs) and miRNAs (DE-miRNAs) were screened, and ceRNA networks associated with TAO were constructed. Thereafter, the genes in the ceRNA network were subjected to functional analyses. Finally, a ceRNA relationship (lncRNA NEAT1-hsa-miR-1-3p-mRNA GNA12) was selected for further validation. Analysis revealed that 347 DE-lncRNAs (150 downregulated and 197 upregulated) and 16 DE-miRNAs (3 downregulated and 13 upregulated) were identified in TAO. Further, TAO-associated ceRNA networks, which included 219 lncRNAs, 6 miRNAs, and 53 mRNAs, were proposed and subjected to gene annotation and pathway analysis. Additionally, NEAT1 and GNA12 levels were significantly upregulated, while miR-1-3p levels were evidently downregulated in TAO patients, as compared with those in healthy controls. Dual luciferase reporter assays showed that NEAT1, miR-1-3p, and GNA12 interacted with each other. We report potential TAO-associated ceRNA regulatory networks and suggest activation of NEAT1/miR-1-3p/GNA12 signaling as a novel mechanism for TAO progression.

Keywords: Thromboangiitis obliterans; ceRNA network; lncRNA; miRNA.

Figure 1.

Analysis of thromboangiitis obliterans (TAO)-associated differentially expressed lncRNAs (DE-lncRNAs). (a) Volcano plot of DE-lncRNAs in TAO patients vs. healthy subjects. Blue and red represented the downregulated and upregulated genes, respectively. (b) The heatmap of DE-lncRNAs based on the expression level of DE-lncRNAs in TAO patients vs. healthy subjects. (c) Gene Ontology (GO) terms analysis of DE-lncRNAs. (d) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of DE-lncRNAs

Figure 2.

Analysis of TAO-associated differentially expressed miRNAs (DE-miRNAs). (a) The heatmap of DE-miRNAs, including 3 downregulated and 13 upregulated genes in TAO patients vs. healthy subjects. (b) GO terms analysis of DE-miRNAs. (c) KEGG pathway enrichment of DE-miRNAs

Figure 3.

Construction of TAO-associated competitive endogenous RNA (ceRNA) regulatory networks. Rhombuses, triangles, and circles represent lncRNAs, miRNAs, and mRNAs, respectively. Red and green represent significant upregulation and downregulation in TAO patients vs. healthy subjects, respectively

Figure 4.

Functional analyses of the genes in the TAO-associated ceRNA networks. (a) GO terms analysis of the genes in the ceRNA networks involved in TAO progression. (b) KEGG pathway enrichment of the genes in the TAO-linked ceRNA networks

Figure 5.

Validation of sequencing results and the ceRNA relationship of lncRNA NEAT1-hsa-miR-1-3p-mRNA GNA12 in TAO progression. The relative expressions of lncRNAs NEAT1 (a), MALAT1 (b), RAD51-AS1 (c), and SNHG22 (d) in healthy individuals and TAO patients. The relative levels of miR-1-3p (e), miR-6786-3p (f), miR-3124-5p (g), and miR-33a-3p (h) in healthy individuals and TAO patients. (i) The relative expression of GNA12 in healthy individuals and TAO patients. *p< 0.05 as compared with healthy individuals. (j) LncRNA NEAT1 interacts with miR-1-3p. *p< 0.05 as compared with negative control (NC) mimics. (k) GNA12 directly binds with miR-1-3p. *p< 0.05 as compared with NC mimics