Integrated Analysis of lncRNA-miRNA-mRNA ceRNA Network in Mixed Dry Eye Disease

Abstract

In order to investigate the relationship between inflammation and lncRNA in mixed dry eye disease (DED), this study establishes competitive endogenous RNA (ceRNA) network in mixed DED. Microarray analysis of cornea from mixed DED mice is performed to screen for differences in lncRNA and target genes, and miRNA bioinformatics were predicted based on the ceRNA hypothesis. The ceRNA network, which consists of 96 relationship pairs, is constructed using the top 10 upregulated lncRNAs and all upregulated mRNAs and two pairs of lncRNA-miRNA-mRNA pairs (NONMMUT047964.2-miR-671-5p-Egr-1andNONMMUT054540.2-miR-1934-5p-Grm2) are selected for RT-qRCR verification in mouse corneal epithelial cells under high osmotic pressure and the samples for microarray. Meanwhile, mouse corneal epithelial cell lines (MCECs), transfected siRNA of NONMMUT047964.2 under high osmotic pressure, shows a decrease in apoptosis rate and a decrease in expression of IL-1β and IL-6. The experimental results show that the NONMMUT047964.2-miR-671-5p-Egr-1 axis may regulate the inflammation and promote the apoptosis of corneal epithelial cells under hypertonic condition.

Figure 1

Identification of DED in mouse model: (a) administration of subcutaneous injection of scopolamine for 4 weeks in DED group. Examinations were taken at week 0, week 2, and week 4; (b) representative images of fluorescein staining on week 0, week 2, and week 4 in mixed DED group compared with images of the controls; (c) the fluorescein staining score of DED mice's corneas were significant increasing during molding process at week 0, week 2 and week 4 (P < 0.01); (d) representative images of wetted cotton thread in mixed DED group compared with the controls (P < 0.05); (e) tear production of mice in DED group was significantly decreased after injection of scopolamine and was much smaller than that in control group during the whole modeling process; and (f) HE staining images of mouse corneal epithelium after model establishment. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. DED, dry eye disease.

Figure 2

. Bioinformatic analysis and qPCR verification of differentially expressed lncRNAs and mRNAs: (a) heatmap of expression levels of tissue-specific lncRNAs; (b) heatmap of expression levels of tissue-specific mRNAs; (c) volcano plot filtering identified 856 lncRNAs differentially expressed in DED group; (d) volcano plot filtering identified 700 mRNAs differentially expressed in DED group; (e) lncRNA NONMMUT047964.2, NONMMUT054540.2, ENSMUST00000229379.1 expression in DED and control mouse corneal tissues determined by qPCR analysis; and (f) the expression of Grm2 and Egr-1 in the tissue sample via RT-qPCR analysis. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. DED, dry eye disease.

Figure 3

GO and KEGG analysis of the DE mRNAs: (a) the barplot of top 30 enrichment pathway by upregulating DE mRNA by go analysis; (b) the bubble plot of top 30 enrichment pathway by upregulating DE mRNA by go analysis; and (c) the bubble plot of top 30 enrichment pathway by upregulating DE mRNA by KEGG analysis. GO, Gene Ontology. KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 4

The ternary regulatory network of lncRNA-miRNA-mRNA.

Figure 5

Verification of ceRNA relation pairs: (a) the expression of miR-671-5p and miR-1934-5p in mouse cornea; (b) the expression of NONMMUT054540.2, miR-1934-5p, and Grm-2 in hypertonic MCECs compared with control group; and (c) the expression of NONMMUT047964.2, miR-671-5p, and Egr-1 in hypertonic MCECs compared with control group.

Figure 6

Validation of lncRNA NONMMUT047964.2, regulating the inflammation and apoptosis in vivo: (a) flow cytometric analysis of MCECs apoptosis after treatment; (b) barplot of apoptosis after treatment; (c) the expression of IL-1β; (d) the expression of IL-6; (e) the expression of NONMMUT047964.2; (f) the expression of miR-671-5p; and (g) the expression of Egr-1 in MCECs following NaCl treatment.