Profiling of Micro RNAs Involved in Retinal Degeneration Caused by Selective Müller Cell Ablation

Abstract

Dysfunction ofMüller cells has been implicated in the pathogenesis of several retinal diseases. In order to understand the potential contribution ofMüller cells to retinal disease better, we have developed a transgenicmodel in which foci ofMüller cell ablation can be selectively induced. MicroRNAs (miRNAs), small non-coding RNAs that are involved in post-transcriptional modulation, have critical functions in various biological processes. The aim of this study was to profile differential expression of miRNAs and to examine changes in their target genes 2 weeks after Müller cell ablation.We identified 20 miRNAs using the miScript HC PCR array. Data analysis using two target gene prediction databases (TargetScan and mirTarBase) revealed 78 overlapping target genes. DAVID and KEGG pathway analysis suggested that the target genes were generally involved in cell apoptosis, p53, neurotrophin, calcium, chemokine and Jak-STAT signalling pathways. Changes in seven target genes including Cyclin D2, Caspase 9, insulin-like growth factor 1, IL-1 receptor-associated kinase (IRAK), calmodulin (CALM) and Janus kinase 2 (Jak2), were validated with qRT-PCR and western blots. The cellular localisation of cleaved-caspase 9, Cyclin D2, Jak2 and CALM was examined by immunofluorescence studies. We found that the transcription of somemiRNAs was positively, rather than negatively, correlated with their target genes. After confirming that overexpressed miR-133a-3p was localised to the outer nuclear layer in the damaged retina, we validated the correlation between miR-133a-3p and one of its predicted target genes, cyclin D2, with a luciferase assay in 661 photoreceptor cells. Results revealed bymiRNA profiling, target gene analysis and validation were generally consistent with our previous findings that selective Müller cell ablation causes photoreceptor degeneration and neuroinflammation. Our data on alterations ofmiRNAs and their target gene expression after Müller cell ablation provide further insights into the potential role of Müller cell dysfunction in retinal disease.