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Yutao Liu, Yelena Bykhovskaya, Mariam L Khaled, Michelle Drewry, Inas F Aboobakar, Amy Estes, Xiaoyi Gao, R Rand Allingham, Michael A Hauser, Yaron S. Rabinowitz; Differential Expression of Coding and Long Noncoding RNAs in Keratoconic Corneas. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5560. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Keratoconus (KC) is the most common corneal ectasia. The purpose of this study is to investigate differential expression of coding and long noncoding RNAs (lncRNAs) in human corneas affected with KC, using stranded total RNA Sequencing.
High quality total RNA was extracted from the corneas of 10 KC patients collected during surgery and 8 normal controls from post-mortem donors. 200 ng total RNA was used to prepare sequencing libraries with the Clontech’s SMARTer Stranded RNA-Seq kit after ribosomal RNA depletion using the RiboGone - Mammalian kit. Paired-end 50bp sequences were generated using Illumina HiSeq 2500 Sequencer. Demultiplexed reads were aligned by TopHat in paired-end reading with the approximation of the median library size. Normalized counts from Cufflinks were analyzed for differential expression analysis by Cuffdiff, using a coding gene file from the Ensembl database and a lncRNA file from the NONCODE database. WebGestalt software was selected to perform pathway analysis.
Using a minimum fold change of 2 and a false discovery q-value ≤ 0.05, we identified 249 coding RNAs with differential expression, with 86 up- and 163 down-regulated, consistent with previous array-based profiles, such as TGFBR3, TIMP1, FBLN1, TIMP3, AQP5, and SFRP1. A lot of them are regulated by transcription factors such as AP1, AP4, LEF1, NFAT, and FOXO4. In addition, many appear to be targets of several cornea-expressed miRNAs, such as miR-19b, miR-200c, miR-429, miR-203, miR-30, miR-130a, and miR-301, which have been identified through cornea miRNA sequencing. Pathway analysis indicated the enrichment of genes coding for proteins involved in extracellular matrix, protein binding, glycosaminoglycan binding, and cell migration. In addition, using a minimum fold change of 2 and a false discovery q-value ≤ 0.05, we also identified 584 lncRNAs (344 up- and 240 down-regulated) differentially expressed in KC-affected cornea, which may potentially be involved in the regulation of WNT, TGF-b, collagen, and PI3 kinase pathways, such as lnc-WNT1-4, lnc-WNT4-2, TGFb2-AS1, lnc-COL9A1-3, lnc-COL9A2-1, and lnc-PIK3AP1-2.
Using total RNA sequencing, we have successfully profiled the expression of coding and long noncoding RNAs in KC-affected cornea. The differential expression data may help improve our understanding of KC etiology and eventually promote the development of novel therapeutic targets.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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