Identification of Long Noncoding RNA Associated ceRNA Networks in Rosacea

Abstract

Rosacea is a chronic and relapsing inflammatory cutaneous disorder with highly variable prevalence worldwide that adversely affects the health of patients and their quality of life. However, the molecular characterization of each rosacea subtype is still unclear. Furthermore, little is known about the role of long noncoding RNAs (lncRNAs) in the pathogenesis or regulatory processes of this disorder. In the current study, we established lncRNA-mRNA coexpression networks for three rosacea subtypes (erythematotelangiectatic, papulopustular, and phymatous) and performed their functional enrichment analyses using Gene Onotology, KEGG, GSEA, and WGCNA. Compared to the control group, 13 differentially expressed lncRNAs and 525 differentially expressed mRNAs were identified in the three rosacea subtypes. The differentially expressed genes identified were enriched in four signaling pathways and the GO terms found were associated with leukocyte migration. In addition, we found nine differentially expressed lncRNAs in all three rosacea subtype-related networks, including NEAT1 and HOTAIR, which may play important roles in the pathology of rosacea. Our study provided novel insights into lncRNA-mRNA coexpression networks to discover the molecular mechanisms involved in rosacea development that can be used as future targets of rosacea diagnosis, prevention, and treatment.

Figure 1

LncRNA and mRNA expression profile of normal group, ETR, PhR, and PPR groups. (a) Heat map of lncRNAs expression in normal group, ETR, PhR, and PPR groups. Red represents upregulated lncRNAs and green represents downregulated lncRNAs. (b) Heat map of mRNAs expression in normal group, ETR, PhR, and PPR groups. Red represents upregulated mRNAs and green represents downregulated mRNAs. (c) Volcano plots of lncRNAs for normal group, ETR, PhR, and PPR groups. The horizontal axis represents fold change (log 2) and the vertical axis is p value (–log 10). Red points (fold change > 1) indicate upregulated lncRNAs and blue points (fold change < –1) indicate downregulated lncRNAs. (d) Hierarchical clustered heat maps showing the log 10 transformed expression values for differently expressed lncRNAs among normal group, ETR, PhR, and PPR groups. The horizontal axis represents samples, while the vertical axis represents the biological elements studied. Red represents higher expression and blue represents lower expression. (e) Volcano plots of mRNAs for normal group, ETR, PhR, and PPR groups. The horizontal axis is fold change (log 2) and the vertical axis is p value (–log 10). Red points (fold change > 1) indicate upregulated mRNAs and blue points (fold change < –1) indicate downregulated mRNAs. (f) Hierarchical clustered heat maps showing the log10 transformed expression values for differently expressed mRNAs among normal group, ETR, PhR, and PPR groups. Red represents higher expression and blue represents lower expression.

Figure 2

(a) The overlapping Venn diagram of lncRNAs among ETR, PhR, and PPR groups. There are 6 upregulated lncRNAs and 3 kinds of downregulated lncRNAs. (b) The overlapping Venn diagram of mRNAs among ETR, PhR, and PPR groups. (c) Gene ontology (GO) analysis of differentially expressed lncRNAs in rosacea patients and normal group. The horizontal axis represents the proportion of those genes accounted for in all the GO annotated genes, the left side of the vertical axis represents the annotation terms, and the right side of the vertical axis represents biological process (BP) terms, cellular component (CC) terms, and molecular function (MF) terms. Bubble scale represents the number of genes in each GO term; depth of bubble color represents p value. (d) The annotation terms are displayed as an interaction network using the BinGO plug-in for Cytoscape. Bubble scale represents number of genes; depth of bubble color represents p value. (e) The enriched GO biological process terms of differentially expressed mRNAs involved in the lncRNAs network.

Figure 3

(a) Clustering dendrogram of genes. The dissimilarity of genes is based on the topological overlap. The genes are assigned to different modules and are identified using different colors. (b) Interactions between genes in the coexpression modules. The different colors on the horizontal and vertical axis represent different groups and modules. The colors in the middle represent the relativity among each module. (c) Gene set enrichment analysis (GSEA) in differentially expressed mRNAs linked to lncRNAs in the ETR group. Ferroptosis regulated by GPX4, MAP1LC3B, VDAC3 and PPAR signaling pathway regulated by PPARG, CD36, MMP1 were involved. The horizontal axis represents the rank in all the ordered dataset, and the vertical axes represent enrichment score and ranked list metric. (d) Ferroptosis regulated by GPX4, MAP1LC3B, VDAC3, NOD-like receptor signaling pathway regulated by STAT1, IL1B, NLRX1, IL6, VDAC3, and PPAR signaling pathway regulated by PPARG, CD36, MMP1 were involved in PhR group. (e) NF-kappa B signaling pathway regulated by IL1B, TNFSF13B, VCAM1, NOD-like receptor signaling pathway regulated by STAT1, IL1B, IL6, NLRX1, and PPAR signaling pathway regulated by MMP1, PPARG, CD36 were involved in PPR group.

Figure 4

Associations of consensus module eigengenes and the clinical phenotype of rosacea. The module name is displayed in the left-hand panel. Numbers in the table indicate the correlations of the corresponding module eigengenes and clinical phenotype, with p values displayed below the correlations in brackets. The intensity and direction of associations are indicated on the right side of the heatmap (red, positively correlated; blue, negatively correlated). ME, module eigengene.

Figure 5

(a) Global view of the ceRNA network in the turquoise module. The circle depicts mRNA, triangle depicts miRNA, and diamond depicts lncRNA. Red and blue depict up- and downregulated genes, respectively. (b) The GO analyses. (c) The KEGG pathway enrichment. (d) GO terms that were associated in the ceRNA network are displayed as an interaction network. Lines indicate GO terms. Bubble scale represents the number of genes. The depth of bubble color represents log 2 fold change.

Figure 6

(a) The relative expression of NEAT1, HOTAIR, and ZNF667-AS1 in rosacea lesions by qPCR and microarray. (b) FISH staining (Merge) confirmed the expression of NEAT1 and decreased expression of HOTAIR and ZNF667-AS1 in rosacea compared to normal adjacent specimens.