Retinal miRNA Profiles of Diabetic Mice Reveal Distinct Molecular Responses of Two Candidate Drugs for Diabetic Retinopathy

Abstract

Purpose: : Alterations in the abundance of miRNAs may regulate pathways upstream as well as downstream of disease etiology. We hypothesize that restoration of altered miRNA profiles to their normal expression in mice treated with candidate therapeutic agents for diabetic retinopathy will be a powerful tool for testing drug efficacy. Our goal is to generate global miRNA profiles from retinas of normal mice and diabetic mice treated with or without inhibitors of receptor for advanced glycation end-products (RAGE) and p38 MAPK. These therapies have been found to inhibit development of early stages of the retinopathy.

Methods: : PCR amplified libraries corresponding to miRNAs were generated from retinas of 12 mice (consisting four groups of animals representing normal, streptozotocin-diabetic, diabetics treated with p38 inhibitor, and diabetics treated with RAGE inhibitor). The amplified cDNA libraries were sequenced using the Illumina platform to generate 36 base reads. The alignment of miRNA sequence reads was carried out by GERALD analysis using ELAND algorithm. The data were normalized by a scaling factor method. Alternatively, we applied SAM (significance analysis of microarrays) to RNA sequence reads to determine changes in the abundance of miRNAs.

Results: : The results from the two analytical strategies described above were not significantly different, and several miRNAs were common between the two lists. Specifically, six miRNAs were significantly upregulated in diabetic animals whereas 11 miRNAs showed decreased levels. Notable among the down-regulated miRNAs were miR-181b and miR-877, while miR-31 and miR-184 levels were considerably higher in retinas from diabetic animals compared to normal retinas. The abundance of miR-7b, miR-1224, miR-20a* and miR-421 was unaltered when compared between normal and diabetic animals. Interestingly, the levels of these miRNAs changed significantly in diabetic animals treated with RAGE inhibitor but not in p38 inhibitor treatment.

Conclusions: : Our results suggest that treatment with p38 MAPK inhibitor leads to fewer changes in miRNAs that were unaltered during diabetes. Such analyses may be potentially applied to identify new therapeutic targets to inhibit the retinopathy and decipher molecular side effects of candidate drugs.

Keywords: diabetic retinopathy • gene microarray • retina