Genome-Wide Scleral Micro- and Messenger-RNA Regulation During Myopia Development in the Mouse

Abstract

Purpose: MicroRNA (miRNAs) have been previously implicated in scleral remodeling in normal eye growth. They have the potential to be therapeutic targets for prevention/retardation of exaggerated eye growth in myopia by modulating scleral matrix remodeling. To explore this potential, genome-wide miRNA and messenger RNA (mRNA) scleral profiles in myopic and control eyes from mice were studied.

Methods: C57BL/6J mice (n = 7; P28) reared under a 12L:12D cycle were form-deprived (FD) unilaterally for 2 weeks. Refractive error and axial length changes were measured using photorefraction and 1310-nm spectral-domain optical coherence tomography, respectively. Scleral RNA samples from FD and fellow control eyes were processed for microarray assay. Statistical analyses were performed using National Institute of Aging array analysis tool; group comparisons were made using ANOVA, and gene ontologies were identified using software available on the Web. Findings were confirmed using quantitative PCR in a separate group of mice (n = 7).

Results: Form-deprived eyes showed myopic shifts in refractive error (-2.02 ± 0.47 D; P < 0.01). Comparison of the scleral RNA profiles of test eyes with those of control eyes revealed 54 differentially expressed miRNAs and 261 mRNAs fold-change >1.25 (maximum fold change = 1.63 and 2.7 for miRNAs and mRNAs, respectively) (P < 0.05; minimum, P = 0.0001). Significant ontologies showing gene over-representation (P < 0.05) included intermediate filament organization, scaffold protein binding, detection of stimuli, calcium ion, G protein, and phototransduction. Significant differential expression of Let-7a and miR-16-2, and Smok4a, Prph2, and Gnat1 were confirmed.

Conclusions: Scleral mi- and mRNAs showed differential expression linked to myopia, supporting the involvement of miRNAs in eye growth regulation. The observed general trend of relatively small fold-changes suggests a tightly controlled, regulatory mechanism for scleral gene expression.

Figure 1

Differences among patterns of miRNA expression examined by cluster analysis. Cluster analyses were performed on 67 log2-transformed miRNA probes with P < 0.05, using Gene Cluster 3.0. Data were adjusted by centering median, and centered correlation was used as distance measurement (n = 7).

Figure 2

Patterns of mRNA expression differences examined by cluster analysis. Cluster analyses were performed on 691 log2-transformed mRNA probes with P < 0.05, using Gene Cluster 3.0. Data were adjusted by centering median, and centered correlation was used as distance measurement (n = 7).

Figure 3

Quantitative PCR validation of microarray analysis results are shown for 3 miRNAs (top) and 3 mRNAs (bottom) were revealed to be differentially expressed in mouse sclera. Scleras from 2-week form deprived eyes and fellow control eyes were compared.