July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Investigation the role of active microRNA in the degenerating retina using Ago2 HITS-CLIP
Author Affiliations & Notes
  • Riccardo Carlo Natoli
    The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia
    The Australian National University Medical School, The Australian National University, Canberra, Australian Capital Territory, Australia
  • Nilisha Fernando
    The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia
  • Zhi-Ping Feng
    The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia
  • Hardip Patel
    The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia
  • Kate Hannan
    The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia
  • Konstantin Panov
    School of Biological Sciences, Queen’s University Belfast, Belfast, Northern Ireland, United Kingdom
  • Nadine Hein
    The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia
  • Matt Rutar
    School of Biomedical Sciences, The University of Melbourne, Melbourne, Victoria, Australia
  • Joshua Aaron Chu-Tan
    The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia
  • Footnotes
    Commercial Relationships   Riccardo Natoli, None; Nilisha Fernando, None; Zhi-Ping Feng, None; Hardip Patel, None; Kate Hannan, None; Konstantin Panov, None; Nadine Hein, None; Matt Rutar, None; Joshua Chu-Tan, None
  • Footnotes
    Support  NHMRC Project Grant 1127705
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4928. doi:
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      Riccardo Carlo Natoli, Nilisha Fernando, Zhi-Ping Feng, Hardip Patel, Kate Hannan, Konstantin Panov, Nadine Hein, Matt Rutar, Joshua Aaron Chu-Tan; Investigation the role of active microRNA in the degenerating retina using Ago2 HITS-CLIP. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4928.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : MicroRNA (miRNA) have been identified as playing an important role in the pathogenesis of Age-Related Macular Degeneration (AMD). Canonical miRNA biogenesis requires that miRNA bind to Argonaute-2 (Ago2) and then incorporate into the RNA-induced silencing complex (RISC). To identify active miRNA involved in retinal degeneration and the mRNA targets that they regulate, we have performed high-throughput sequencing of miRNA and mRNA isolated by crosslinking immunoprecipitation (HITS-CLIP) with argonaute 2 (Ago2) in the photo-oxidative damage model.

Methods : To induce central retinal degeneration, C57BL/6J mice were exposed to 5 days of 100k lux photo-oxidative damage (PD). Immunoprecipitation was performed against Ago2 protein on isolated retinas using protein A Dynabeads (Thermo Fisher Scientific) to isolate bound RNA from cross-linked tissue. The Ago:miRNA and Ago:miRNA:mRNA factions were separated with SDS-PAGE using a radiolabelled linker (32P) added to immunoprecipitated Ago2. Both miRNA and mRNA were extracted and cDNA libraries prepared using the Small RNA-seq and RNA-seq CATS (Diagenode) kits respectively. Samples were sequenced on an Illumina HiSeq 2500 with 50 bp reads, and sequence qualities checked with FASTQC. The raw reads were pre-processed and trimmed with cutadapt before being mapped using bowtie2 and bwamen to the miRBase and mm10 genomes. edgeR, voom-limma and glimma were used for differential expression analysis.

Results : Sequencing analysis of the Ago:miRNA:mRNA complexes revealed a subset of mRNA that were differentially expressed in mice which had been exposed to photo-oxidative damage (P<0.05, n=4). Among the Ago-bound miRNA, miR-124-3p was the most highly expressed in both dim-reared and PD retinas constituting the majority of the proportion of miRNA sequenced (~80%). Gene ontology and gene set enrichment analysis of Ago-bound miRNA and mRNA revealed significant roles in the modulation of miRNA in regulating biological processes such as phototransduction, inflammation, apoptosis, and complement following retinal degeneration.

Conclusions : A subset of miRNA and mRNA targets were differentially regulated as a consequence of retinal degeneration. Further characterisation of these miRNA/mRNA interactions in the context of the degenerating retina may provide an important insight into the active role these may play in diseases such as AMD.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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