Profiles of microRNA in aqueous humor of normal tension glaucoma patients using RNA sequencing

Abstract

We aimed to identify and compare microRNAs (miRNAs) from individual aqueous humor samples between normal-tension glaucoma (NTG) patients and normal controls. Aqueous humor (80 to 120 µl) was collected before cataract surgery. Six stable NTG patients and seven age-matched controls were included in the final analysis. RNA sequencing was conducted for RNA samples extracted from the 13 aqueous humor samples, and bioinformatics analysis was employed for the miRNA targets and related pathways. Two hundred and twenty-eight discrete miRNAs were detected in the aqueous humor and consistently expressed in all samples. Eight significantly upregulated miRNAs were found in the NTG patients compared to the controls (fold-change > 2, p < 0.05). They were hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR-10b-5p, hsa-miR-375, and hsa-miR-143-3p. These miRNAs were predicted to be associated with the biological processes of apoptosis, autophagy, neurogenesis, and aging in the gene ontology categories. The related Kyoto encyclopedia of genes and genomes pathways were extracellular matrix-receptor interaction, mucin-type O-glycan biosynthesis, biotin metabolism, and signaling pathways regulating the pluripotency of stem cells. The differentially expressed miRNA in the NTG samples compared to the controls suggest the possible roles of miRNA in the pathogenesis of NTG. The underlying miRNA-associated pathways further imply novel targets for the pathogenesis of NTG.

Figure 1

Scatter plot and volcano plot of the comparison of miRNA expression in aqueous humor from subjects with NTG versus unaffected controls. (A) Up-regulated miRNAs are shown to the upper region of the scatter plot (red) and down-regulated miRNAs are shown to the lower region of the scatter plot (green). A total of 228 mature miRNAs were identified in the aqueous humor of the NTG patients. Eight miRNAs, hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR-10b-5p, hsa-miR-375, and hsa-miR-143-3p were significantly up-regulated in NTG group compared to normal control. Fold change > 2 (red dotted line), fold change <  − 2 (green dotted line); Normalized data (log2) > 4; p-value < 0.05 versus control. (B) In volcano plot, up-regulated miRNAs are shown to the right of the plot (red) and were only selected which passed the thresholds of p < 0.05 (horizontal black dotted line) and fold change > 2 (red vertical dotted line). Down-regulated miRNAs are shown to the left of the plot (green) and were only selected which passed the thresholds of p < 0.05 (horizontal black dotted line) and fold change < 2 (green vertical dotted line). After selecting only those with Normalized data (log2) > 4, the volcano plot shows eight significantly upregulated miRNAs with fold change > 2, and p-value < 0.05. Of these, eight miRNAs were significantly up-regulated compared to the controls (fold-change > 2, p < 0.05), in addition to the analysis of the fold-changes were selected which only based on the normalized array data (log2) > 4. There was no significantly down-regulated miRNAs identified in NTG patients compared to control subjects according to the same significance criteria. Plots were presented by ExDEGA v1.2.1.0 software. NTG normal tension glaucoma.

Figure 1

Scatter plot and volcano plot of the comparison of miRNA expression in aqueous humor from subjects with NTG versus unaffected controls. (A) Up-regulated miRNAs are shown to the upper region of the scatter plot (red) and down-regulated miRNAs are shown to the lower region of the scatter plot (green). A total of 228 mature miRNAs were identified in the aqueous humor of the NTG patients. Eight miRNAs, hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR-10b-5p, hsa-miR-375, and hsa-miR-143-3p were significantly up-regulated in NTG group compared to normal control. Fold change > 2 (red dotted line), fold change <  − 2 (green dotted line); Normalized data (log2) > 4; p-value < 0.05 versus control. (B) In volcano plot, up-regulated miRNAs are shown to the right of the plot (red) and were only selected which passed the thresholds of p < 0.05 (horizontal black dotted line) and fold change > 2 (red vertical dotted line). Down-regulated miRNAs are shown to the left of the plot (green) and were only selected which passed the thresholds of p < 0.05 (horizontal black dotted line) and fold change < 2 (green vertical dotted line). After selecting only those with Normalized data (log2) > 4, the volcano plot shows eight significantly upregulated miRNAs with fold change > 2, and p-value < 0.05. Of these, eight miRNAs were significantly up-regulated compared to the controls (fold-change > 2, p < 0.05), in addition to the analysis of the fold-changes were selected which only based on the normalized array data (log2) > 4. There was no significantly down-regulated miRNAs identified in NTG patients compared to control subjects according to the same significance criteria. Plots were presented by ExDEGA v1.2.1.0 software. NTG normal tension glaucoma.

Figure 2

Quantitative PCR validation of miRNA in AH of NTG patients compared with control. (A) The heatmap diagram demonstrated the results of RNA sequencing by expression of miRNAs. Red represents high relative expression and blue represents low relative expression (Z-score). Data was presented by ExDEGA v1.2.1.0 software. (B) hsa-let-7c-5p expression in the AH of NTG patients by quantitative PCR validation analysis. The expression of let-7c-5p increased significantly in the AH of the NTG patients compared to control (44.32 ± 3.63-fold, p < 0.001). Data are represented as the mean ± S.E.M. and were analyzed using Unpaired Student’s t-test (n = 7). ***p < 0.001 versus Control. AH aqueous humor, NTG normal tension glaucoma, S.E.M. standard error of the mean.

Figure 3

Percentage and number of microRNAs with significantly changed expression among gene ontology category-related microRNAs. Gene ontology (GO) category of microRNAs with relatively large expression changes are identified. Up-regulated miRNAs are shown as red graph and down-regulated miRNAs are shown as green graph. Cell death-related categories including apoptosis (3.27%) and autophagy (4.63%) occupied the major proportion. Categories related to neurogenesis (5.88%) and inflammatory response (4.47%) also mainly occupied the proportion. The percentage refers to the proportion of microRNA modified in AH of NTG patients compared to control in the total miRNAs identified by researches in each GO category. As significantly down-regulated miRNAs were not identified according to the same significance criteria with up-regulation, no green graph as down-regulated GO category is shown in this figure. Graphs were presented by ExDEGA v1.2.1.0 software.

Figure 4

KEGG pathway. Enrichment score was represented as − log10 (p-value). The higher the enrichment score value is, the more significant the pathway is. The KEGG pathways relating to ECM-receptor interaction (hsa04512; 17.03), mucin-type O-glycan biosynthesis (hsa00512; 5.40), biotin metabolism (hsa00780; 3.73) and signaling pathways regulating pluripotency of stem cells (hsa04550; 2.39) were significantly associated with miRNAs increased in the aqueous humor of NTG patients. Note that the pathway relating to ECM-receptor interaction showed the most significant association with up-regulated miRNAs in NTG patients compared to other pathways. Data was analyzed by DianaTools. KEGG Kyoto Encyclopedia Genes and Genomes, ECM extracellular matrix.