Author: Melinda Tea
Tea, Melinda, 2012 Heritable Influences in Experimental Retinopathy of Prematurity, Flinders University, School of Medicine
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Retinopathy of prematurity (ROP) is a potentially blinding eye condition of premature infants exposed to oxygen therapy. Different inbred rat strains exhibit differential susceptibility to oxygen-induced retinopathy (OIR), a robust animal model of ROP which mimics the pathophysiology seen in human disease. Susceptibility to OIR has previously been shown to segregate with ocular pigmentation, and is inherited in an autosomal dominant fashion in pigmented rat strains. The mode in which susceptibility to OIR is inherited in albino rat strains has not previously been determined. Using genetic cross, and backcross analysis, it was determined that susceptibility to OIR is inherited in the same autosomal dominant manner in albino rats, making it likely that genetic modifiers other than ocular pigmentation contribute to disease susceptibility. These modifiers could potentially be involved in the oxygen sensing pathway, which is central to the development of OIR and ROP. Differences in retinal gene expression are likely to underlie the differential susceptibility to OIR that is exhibited by the two inbred strains of albino rats used in this study. MicroRNAs (miRNAs) may also contribute to the OIR phenotype by regulating these changes in gene expression. To test this hypothesis, RNAs from the retinae of two different rat strains exposed to oxygen therapy for 3, 5 or 6 days were analysed for gene and miRNA expression using Affymetrix and Exiqon microarrays, respectively. A bioinformatics approach using the freely available online database, Database for Annotation, Visualisation and Integrated Discovery (DAVID), was used to assist in functional grouping of differentially regulated genes, many of which were found to be associated with response to hypoxia. Candidate genes were identified from the gene expression microarray data based on their regulation by oxygen and on a search of the literature. The gene candidates EGL nine homolog 3 (EGLN3) and EGL nine homolog 1 (EGLN1) belong to a class of oxygen-dependent prolyl hydroxylases which are responsible for regulating levels of hypoxia-inducible factor-a (HIF-a) in normoxia. HIF-a is a master transcription factor which upregulates the expression of target genes in response to hypoxia. An additional candidate, insulin-like growth factor binding protein 3 (IGFBP3) was also chosen based on evidence from the literature showing that early expression of IGFBP3 was protective against the disease in a mouse model of OIR. Expression of candidate genes was confirmed using relative quantification real-time RT-PCR analysis. miRNAs are non-coding RNAs which regulate gene expression at a post-transcriptional level and have been associated with a wide variety of physiological and pathological conditions including retinal development and OIR. Initial analysis of microarray data from all 3 time points showed a total of 15 miRNAs to be differentially expressed after correction for multiple comparisons. miRNAs were identified as candidates if they targeted oxygen-related genes, were regulated by both oxygen and strain or had significant adjusted p values (p<0.05). miRNAs which are differentially regulated by exposure to oxygen therapy in a strain-dependent manner may contribute to differences in the disease phenotype that is exhibited by these two albino rat strains. Three miRNAs of interest, miR-30e, miR-338-3p and miR-210, were chosen for confirmation by real-time RT-PCR analysis. These miRNAs are predicted to target elements of the HIF-a oxygen sensing pathway. Identifying the molecular basis of susceptibility to OIR may help to identify infants at risk of developing ROP and identify new therapeutic targets for treatment.
Keywords: experimental retinopathy of prematurity,oxygen-induced retinopathy,strain-related differences in gene and microRNA expression
Subject: Ophthalmology thesis, Medicine thesis
Thesis type: Doctor of Philosophy
School: School of Medicine
Supervisor: Professor Keryn Williams