Detection of microRNAs in the mammalian retina using a modified in situ hybridization protocol.

Abstract

Purpose: Diabetic retinopathy (DR) is a leading cause of blindness involving neoangiogenesis and blood retinal barrier breakdown. Our goal is to identify novel genetic targets involved in DR. microRNAs (miRNAs) are non-coding RNAs that regulate gene expression by translational repression, target degradation or gene silencing. miRNA are known to modulate several pathologic diseases and processes, we and others have found perturbations of retinal miRNAs in a hyperglycemic and hypoinsulinemic mouse model. Yet the spatial localization of these miRNAs is currently unknown. Here, we characterize the location of several miRNAs using a modified approach.

Methods: Heterozygous male Ins2^Akita , genetically obese mice Ob/Ob were age matched with C57BL6/J mice. Then, we measured miRNA abundance in the retina and choroid/retinal pigmented epithelium (RPE) at several time points using multiplexed libraries of Ins2^Akita, Ob/Ob and wild-type mice. Next, we choose unregulated and de-regulated miRNAs including -124, -126, -143, and -184. We designed, synthesized and hybridized antisense locked nucleic acid (LNA) probes that targeted the specific miRNAs. Prior to hybridization, we cross-linked the small RNAs in fresh frozen tissues with formaldehyde and EDC fixation, the hybridized the LNA probes, and used colorimetric or fluorescent detection systems.

Results: miRNA profiles were generated for Ins2^Akita, Ob/Ob and control mice using multiplex next generation sequencing which showed down regulation of several miRNAs including miR-126 and miR-143. We found that conducting ISH without EDC fixation, the signal was undetectable; however, with additional EDC fixation, we located several miRNAs including miR-124, miR-126, and miR-143 in the retina and choroid. ISH studies showed cell-type specific staining in several retinal layers that contain the retinal or choroidal vasculature.

Conclusions: In this study, we further characterized the miRNA spatial localization in the wild-type C57Bl6 mouse, Ins2Akita and Ob/Ob mouse model. We found that ISH studies showed that several miRNAs are cell type specific and potentially involve the retinal vasculature. The miRNAs identified in our screen offer potential targets for genetic modulation of hyperglycemic induced pathology in the eye. These miRNAs and downstream pathways may open the way for novel small RNA therapeutics for diseases such as DR.