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Clinical Trial
. 2018 Apr 1;27(7):1263-1275.
doi: 10.1093/hmg/ddy040.

Differentially expressed microRNAs in the aqueous humor of patients with exfoliation glaucoma or primary open-angle glaucoma

Michelle D Drewry  1 , Pratap Challa  2 , John G Kuchtey  3 , Iris Navarro  2 , Inas Helwa  1 , Yanzhong Hu  4 , Hongmei Mu  5 , W Daniel Stamer  2 , Rachel W Kuchtey  3   6 , Yutao Liu  1   7   8
Affiliations
Clinical Trial

Differentially expressed microRNAs in the aqueous humor of patients with exfoliation glaucoma or primary open-angle glaucoma

Michelle D Drewry et al. Hum Mol Genet. .

Abstract

Both exfoliation glaucoma (XFG) and primary open-angle glaucoma (POAG) have been linked to decreased conventional outflow of aqueous humor (AH). To better understand the molecular changes in the AH content under such conditions, we analyzed the miRNA profiles of AH samples from patients with POAG and XFG compared to non-glaucoma controls. Individual AH samples (n = 76) were collected from POAG and XFG patients and age-matched controls during surgical procedure. After RNA extraction, the miRNA profiles were individually determined in 12 POAG, 12 XFG and 11 control samples. We identified 205, 295 and 195 miRNAs in the POAG, XFG and control samples, respectively. Our differential expression analysis identified three miRNAs (miR-125b-5p, miR-302d-3p and miR-451a) significantly different between POAG and controls, five miRNAs (miR-122-5p, miR-3144-3p, miR-320a, miR-320e and miR-630) between XFG and controls and one miRNA (miR-302d-3p) between POAG and XFG. While none of these miRNAs have been previously linked to glaucoma, miR-122-5p may target three glaucoma-associated genes: OPTN, TMCO1 and TGF-ß1. Pathway analysis revealed that these miRNAs are involved in potential glaucoma pathways, including focal adhesion, tight junctions, and TGF-ß signaling. Comparison of the miRNA profile in AH to unrelated human serum (n = 12) exposed potential relationships between these two fluids, although they were not significantly correlated. In summary, we have successfully profiled the miRNA expression without amplification in individual human AH samples and identified several POAG or XFG-associated miRNAs. These miRNAs may play a role in pathways previously implicated in glaucoma and act as biomarkers for disease pathogenesis.

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Figures

Figure 1.
Figure 1.
Venn diagram for the distribution of miRNAs between the control, POAG and XFG samples. The miRNA content of the samples groups was organized into a Venn diagram, with the values in the figure representing the number of unique miRNAs identified within and among each sample group.
Figure 2.
Figure 2.
Expression of the most abundantly expressed miRNAs in the control, POAG and XFG samples. The most abundant miRNAs were defined as those within the top 95th percentile. Represented here are the mean number of normalized counts per sample group for each of abundantly expressed miRNA, with * indicating a P-value ≤ 0.05.
Figure 3.
Figure 3.
Expression of the DE miRNAs in POAG, XFG, and between XFG and POAG. Differential analyses indicated that three miRNAs were DE in POAG, five in XFG and one between XFG and POAG. The mean number of normalized counts per sample group for the eight DE miRNAs are represented in this figure, with * denoting P ≤ 0.05 and ** for P < 0.001.
Figure 4.
Figure 4.
Heatmap of the KEGG pathways enriched for the target genes of the POAG DE miRNAs. This heatmap illustrates the number of target genes of the POAG DE miRNAs involved in each enriched pathway, with shades of blue for the up-regulated miRNAs and shades of gray for the down-regulated miRNAs. The pathways are clustered based on their representations in the target genes.
Figure 5.
Figure 5.
Heatmap of the KEGG pathways enriched for the target genes of the XFG DE miRNAs. This heatmap illustrates the number of target genes of the XFG DE miRNAs involved in each enriched pathway, with shades of blue for the up-regulated miRNAs and shades of gray for the down-regulated miRNAs. The pathways are clustered based on their representations in the target genes.
Figure 6.
Figure 6.
Validation of AH miRNA expression profiles using ddPCR. Measuring miRNA concentration of POAG (n = 17), XFG (n = 14), and control (n = 10) AH with ddPCR (A) gave a similar relative expression profile to that produced with the NanoString technology (B) for five of the DE miRNAs: miR-122-5p, miR-125b-5p, miR-320a, miR-320e and miR-451a, with * denoting P ≤ 0.05.
Figure 7.
Figure 7.
Validation of serum miR-122-5p expression using ddPCR. Expression of miR-122-5p in non-diseased serum (n = 4) and control AH (n = 10) were measured using ddPCR (A) and the NanoString technology (B). Similar trends in expression levels were seen between the two techniques.
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