Large-Scale microRNA Expression Profiling Identifies Putative Retinal miRNA-mRNA Signaling Pathways Underlying Form-Deprivation Myopia in Mice

Abstract

Development of myopia is associated with large-scale changes in ocular tissue gene expression. Although differential expression of coding genes underlying development of myopia has been a subject of intense investigation, the role of non-coding genes such as microRNAs in the development of myopia is largely unknown. In this study, we explored myopia-associated miRNA expression profiles in the retina and sclera of C57Bl/6J mice with experimentally induced myopia using microarray technology. We found a total of 53 differentially expressed miRNAs in the retina and no differences in miRNA expression in the sclera of C57BL/6J mice after 10 days of visual form deprivation, which induced -6.93 ± 2.44 D (p < 0.000001, n = 12) of myopia. We also identified their putative mRNA targets among mRNAs found to be differentially expressed in myopic retina and potential signaling pathways involved in the development of form-deprivation myopia using miRNA-mRNA interaction network analysis. Analysis of myopia-associated signaling pathways revealed that myopic response to visual form deprivation in the retina is regulated by a small number of highly integrated signaling pathways. Our findings highlighted that changes in microRNA expression are involved in the regulation of refractive eye development and predicted how they may be involved in the development of myopia by regulating retinal gene expression.

Fig 1. Form-deprivation myopia in C57BL/6J mice.

Form-deprivation myopia was induced in C57BL/6J mice by applying a diffuser to the right eye of P24 animals. Ten days of visual form deprivation induced -6.93 ± 2.44 D (p < 0.000001, n = 12) of myopia in the right eyes compared to the contralateral control eyes.

Fig 2. Hierarchical cluster analysis of 53 miRNAs differentially expressed in the myopic retina versus control retina.

Logarithmic values (base 2) of Agilent total gene signal for differentially expressed miRNAs (cutoff: FC > 2, FDR-adjusted p-value < 0.05) were quantile normalized, shifted to mean of zero, scaled to standard deviation of 1.0 and subjected to hierarchical clustering using Euclidean dissimilarity and average linkage. The color scale indicates transcript abundance relative to the mean of zero: red identifies an increase in relative miRNA abundance; blue identifies a decrease in relative miRNA abundance. Columns show individual samples, whereas rows show individual miRNAs. Control samples c1, c2, and c3 correspond to myopic samples m1, m2, and m3 respectively. The “co-clustering” of the control sample c1 and myopic sample m3 resulted from the clustering algorithm that was used to generate the cluster and reflects individual differences in gene expression as well as differences in the myopic response to visual form deprivation between animals. It appears that the myopic response in the animals comprising experimental group 1 (samples c1 and m1) was the weakest among the 3 groups. However, the relationship between control sample c1 and the corresponding myopic sample m1 follows the same pattern as in the other two experimental groups even though samples c1 and m1 fall within the same color scheme. Consistent up- or down-regulation of the specific miRNAs in the myopic eyes versus corresponding control eyes across all samples is reflected by the low p-values shown in Table 1.

Fig 3. Overlap between miRNAs differentially expressed in the myopic retina and miRNAs differentially expressed in the retina versus sclera.

Venn diagram shows overlap between 53 miRNAs, which were differentially expressed in the myopic retina, 136 miRNAs, which were up-regulated in the retina versus sclera, and 109 miRNAs, which were up-regulated in the sclera versus retina. Eighteen differential miRNAs were equally expressed in both retina and sclera, 20 differential miRNAs were up-regulated in the retina versus sclera and 15 differential miRNAs were down-regulated in the retina versus sclera.

Fig 4. Gene ontology categories affected in myopic retina.

Graph shows top 18 biological processes which were modified in the myopic retina.

Fig 5. Overlap between miRNA-regulated signaling pathways affected in myopic retina.

Diagram depicts miRNA contributions to the 9 miRNA-mRNA signaling cascades associated with form-deprivation myopia in mice.