Targeted ablation of miR-183/96 led to significant retinal degeneration in mice

Abstract

Purpose: MicroRNA 183 cluster (miR-183/96/182) is highly expressed in mammalian retina and other sensory organs. To understand its biological roles in vivo, we generate knockout (KO) mice and determine associated phenotypes. Previously we created miR-182 deficient mice, in the present study we generate miR-183/96 KO mouse.

Methods: miR-183/96 KO mice were generated using classical gene targeting strategy. PCR assay was used to determine the genotype. Electroretinogram (ERG), spatial vision testing, fundus photographs and fluorescein angiography were carried out. Optical coherence tomography (OCT) and retinal histology were performed to investigate the retinal structure. Other multisensory behavior examinations were also conducted, including buried food pellet, tail suspension and open field testing. Gene expression profiling was performed through RNA sequencing of isolated retina. Potential target genes predicted by computational programs were further validated by luciferase reporter assay in vitro and western blotting assay.

Results: Successful generation of the miR-183/96 KO mice was validated by genotyping. The KO mice showed an aberrant photopic response, with a decrease of b-wave amplitude, implying the dysfunction of cone cell. Spatial vision testing demonstrated significant visual impairment. Fundus photographs and fluorescein angiography of the KO mice exhibited thinner blood vessel and increased autofluorescent spots. Both OCT and HE staining demonstrated a decreased retina thickness. Multisensory behavior testings indicated that the KO mice had impaired olfactory ability, abnormal emotion and were more sensitive to pain response. A series of assays identified that slc-x is a target gene of miR-183/96.

Conclusions: Our data suggested that targeted ablation of miR-183/96 led to significant retinal degeneration and multi-sensorial abnormalities.