Abstract
Purpose :
There is growing awareness of the regulatory functions of long noncoding RNAs (lncRNAs). We hypothesize that by applying differential expression (DE) analysis to transcriptome data collected from normal and diseased eyes, we will attain a clearer understanding of the role of lncRNAs in the development of AMD and its phenotypes.
Methods :
Human eyes (N=15) were collected within 6 hours of donor death. One eye was preserved with RNA Later while the other eye of each pair was used for histologic evaluation. cDNA libraries were made from the retina (R) and RPE/choroid/sclera (RPCS) of each eye (8 normal, 7 AMD, with phenotypes of early (E), geographic atrophy (GA) and neovascular (NVAMD) assigned) and sequenced on an Illumina HiSeq. We calculated the expression levels of all RefSeq non-coding genes using the Fragments Per Kilobase of gene per Million mapped fragment (FPKM) metric and considered a gene differentially expressed (DE) if the false discovery rate (FDR) p-value is <0.05. Differential expression (AMD vs normal) was assessed for both tissue layers (R, RPCS).
Results :
An initial analysis was carried out for DE lncRNAs with high expression (FPKM > 1.0 for R and/or RPCS). A total of 46 DE genes were identified. For early AMD, 7 DE genes were specific to R, 3 were specific to RPCS and 2 were common to both tissue layers; for geographic atrophy the DE genes were R=13, RPCS=1 and R/RPCS=1, and for NVAMD the DE genes were R=12. The DE genes were then analyzed for overlap between phenotypes (Fig. 1). LINC00599 (RNCR3) is down-regulated in R for all phenotypes (p < 5X10-5); RNCR3 is the main precursor for microRNA Mir-124, which is essential to prevent retinal degradation. MEG3 is down-regulated in R and RPCS for the E and GA phenotypes (p < 5X10-5) ; MEG3 is in the VEGF pathway and modulates angiogenesis. PROX1-AS1 is up-regulated in E/RPCS (p < 5X10-5); this gene is antisense and could have regulatory function on PROX1 transcription factor, a protein implicated in retinal regeneration. Also, while our focus is on lncRNAs, we note that microRNA Mir-145 is up-regulated in GA/RPCS (p < 5X10-5); this miRNA has been shown to inhibit angiogenesis.
Conclusions :
Our results support our hypothesis that DE analysis is a useful tool for identifying transcriptome variations involved in the development of AMD, and moreover can distinguish patterns of expression unique to specific phenotypes of the disease.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.