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Venkata R M Chavali, Anita Bowman, Harini V Gudiseva; Profiling of noncoding RNAs in age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4793.
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© ARVO (1962-2015); The Authors (2016-present)
To explore the total non-coding RNA (ncRNA) landscape and identify the differentially expressed ncRNA in the peripheral retina (PR) and peripheral retinal pigment epithelium-choroid (PRPC) tissues obtained from normal and age-related macular degeneration (AMD) donor eyes.
Donor eye globes from 8 normal and 8 AMD Caucasian donors were enucleated and preserved in RNAlater. For each globe, 8mm punches were made from peripheral regions. PR and PRPC were separated into individual tubes. Total RNA was isolated from these tissues using AllPrep RNA/DNA mini kit (Qiagen). Sequencing libraries were prepared using the TruSeq Stranded Total RNA Sample Prep Kit and sequenced on the HiSeq 2000 (Illumina) generating 100bp-paired end reads. Contamination in samples was determined by looking for PRPC specific transcripts in PR and vice versa. Analysis of the total ncRNA transcriptome on combined sequencing data was done using both open-source and custom bioinformatics tools (including GSNAP, Cufflinks Ver 2.2). Identification of protein-coding genes cis to DE ncRNA (co-localization analysis) was done using GREAT and these colocalized genes were submitted for GO,pathway, and protein-protein interaction (PPI) analyses using Consensus-PathDB. Conservation analysis was done using pre-computed PhastCons scores.
Sequencing libraries were generated from high quality RNA obtained from PR and PRPC tissues (RIN>8.5). 332 and 251 DE ncRNA were found between normal PR vs AMD PR and normal PRCS vs AMD PRCS, respectively. Among the DE ncRNA species, pseudogene expression was predominant followed by antisense transcripts and lncRNA. We also observed significant differences in the level of conservation of the ncRNAs upregulated in AMD samples when compared to the normal state. Protein coding genes colocalized to DE ncRNA from normal vs AMD PRPC samples revealed involvement in the connexin-36/connexin 45 gap junction complex as well as canonical Wnt and VEGF receptor complexes. Protein coding genes colocalized to DE ncRNA from normal vs AMD PR samples revealed involvement in the NF-kappaB p50 dimer/IKBA complex.
Several DE ncRNA in PR and PRCS tissues in both normal and AMD diseased states were identified. GO, pathway and PPI analyses suggest the putative regulatory roles of ncRNAs in both retinal homesostasis and in AMD. Further functional characterization of various DE ncRNA will elucidate their role in etiology of AMD.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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