Scleral micro-RNA signatures in adult and fetal eyes

Abstract

Introduction: In human eyes, ocular enlargement/growth reflects active extracellular matrix remodeling of the outer scleral shell. Micro-RNAs are small non-coding RNAs that regulate gene expression by base pairing with target sequences. They serve as nodes of signaling networks. We hypothesized that the sclera, like most tissues, expresses micro-RNAs, some of which modulate genes regulating ocular growth. In this study, the scleral micro-RNA expression profile of rapidly growing human fetal eyes was compared with that of stable adult donor eyes using high-throughput microarray and quantitative PCR analyses.

Methods: Scleral samples from normal human fetal (24 wk) and normal adult donor eyes were obtained (n=4 to 6, each group), and RNA extracted. Genome-wide micro-RNA profiling was performed using the Agilent micro-RNA microarray platform. Micro-RNA target predictions were obtained using Microcosm, TargetScan and PicTar algorithms. TaqMan® micro-RNA assays targeting micro-RNAs showing either highest significance, detection, or fold differences, and collagen specificity, were applied to scleral samples from posterior and peripheral ocular regions (n=7, each group). Microarray data were analyzed using R, and quantitative PCR data with 2^-deltaCt methods.

Results: Human sclera was found to express micro-RNAs, and comparison of microarray results for adult and fetal samples revealed many to be differentially expressed (p<0.01, min p= 6.5x10(11)). Specifically, fetal sclera showed increased expression of mir-214, let-7c, let-7e, mir-103, mir-107, and mir-98 (1.5 to 4 fold changes, p<0.01). However, no significant regionally specific differences .i.e., posterior vs. peripheral sclera, were observed for either adult or fetal samples.

Conclusion: For the first time, micro-RNA expression has been catalogued in human sclera. Some micro-RNAs show age-related differential regulation, higher in the sclera of rapidly growing fetal eyes, consistent with a role in ocular growth regulation. Thus micro-RNAs represent potential targets for ocular growth manipulation, related to myopia and/or other disorders such as scleral ectasia.

Figure 1. Volcano plots of microarray data showing the relationship between fold change in micro-RNA expression and significance for all pair-wise comparisons (n=4 to 6 per group).

The x-axis shows fold change (log₂ ratio scale) and the y-axis, the negative log₁₀ of p-values (higher values indicate greater significances). The two vertical red lines demarcate the area outside of which there is at least a two-fold difference in expression levels between fetal and adult scleras. The single horizontal red line marks the threshold for an unadjusted p-value of 0.05; data points above this line can be considered statistically significant at p-value of 0.05, without correction for multiple testing. Shown in red are data points achieving statistical significance after correction for a 5% false discovery rate (FDR); those not reaching statistical significance are in blue.

Figure 2. The PCA plot illustrating the correlation of expression between samples.

Clustering on the PCA plot indicates a strong correlation. The x-axis shows the first principal component (PC1) revealing variations of expression between ‘adult’ and ‘fetal’ groups. The y-axis shows the second principal component (PC2) revealing variations of expression between ‘posterior’ and ‘peripheral’ groups.

Figure 3. Patterns of micro-RNA expression differences examined by cluster analysis.

The false discovery rate controlled p-values were k-means clustered to identify major patterns driving the differences in micro-RNA expression between different samples. The k-means cluster centers were themselves hierarchically clustered to identify the relationships between the different clusters.

Figure 4. Results from Taqman micro-RNA PCR assays.

(Top) Relative fold changes in micro-RNA expression in adult compared to fetal posterior sclera (n=7); all micro-RNAs except miR-let7-b were significantly up-regulated in fetal compared to adult samples. (Bottom) Relative fold changes in micro-RNA expression in adult compared to fetal peripheral sclera (n=7); all micro-RNAs except hsa-let7b and hsa-let7c were significantly up-regulated in fetal compared to adult samples. (Y-axis in log scale to the base 2).