July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Long Non-Coding RNAs in Keratoconus-affected Human Cornea
Author Affiliations & Notes
  • Yutao Liu
    Department of Cellular Biology and Anatomy, Augusta University, Augusta, Georgia, United States
    James & Jean Culver Vision Discovery Institute, Augusta, Georgia, United States
  • Mariam Lotfy Khaled
    Department of Cellular Biology and Anatomy, Augusta University, Augusta, Georgia, United States
  • Yelena Bykhovskaya
    Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Hanzhou Li
    Department of Cellular Biology and Anatomy, Augusta University, Augusta, Georgia, United States
  • W Daniel Stamer
    Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina, United States
  • Hongyan Xu
    Department of Population Health Sciences, Augusta University, Augusta, Georgia, United States
  • R. Rand Allingham
    Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina, United States
  • Michael A Hauser
    Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina, United States
    Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States
  • Yaron Rabinowitz
    Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Yutao Liu, None; Mariam Khaled, None; Yelena Bykhovskaya, None; Hanzhou Li, None; W Daniel Stamer, None; Hongyan Xu, None; R. Rand Allingham, None; Michael Hauser, None; Yaron Rabinowitz, None
  • Footnotes
    Support  This work was supported in part by National Institutes of Health (NIH; Bethesda, MD, USA) grants R01EY023242, R01EY023646, R01EY009052, and P30 EY005722.
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 4391. doi:
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      Yutao Liu, Mariam Lotfy Khaled, Yelena Bykhovskaya, Hanzhou Li, W Daniel Stamer, Hongyan Xu, R. Rand Allingham, Michael A Hauser, Yaron Rabinowitz; Long Non-Coding RNAs in Keratoconus-affected Human Cornea. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4391.

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

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Abstract

Purpose : Keratoconus (KC) is the most common corneal ectasia. We aimed to investigate differential expression of long noncoding RNAs (lncRNAs) in human corneas affected with KC.

Methods : 200 ng total RNA from the corneas of 10 KC patients and 8 non-KC normal controls was used to prepare sequencing libraries with the Clontech’s SMARTer Stranded RNA-Seq kit. Ribosomal RNA was depleted using the RiboGone - Mammalian kit. Paired-end 50bp reads were generated using Illumina HiSeq 2500 Sequencer. We aligned the reads using TopHat and normalized counts with Cufflinks. We identified differentially expressed lncRNAs with Cuffdiff with a lncRNA file from the NONCODE database. To annotate these lncRNAs using R, we performed correlation analyses between these lncRNAs and differentially expressed mRNAs in the controls and KC patients separately, examined their significances, and calculated the false discovery rate (FDR) with Benjamini-Hochberg approach. Correlations with FDR<0.05 were imported into Cytoscape to build gene networks to identify functional hubs. Based on the known function and the network analysis, we selected 7 differentially expressed lncRNAs for validation using droplet digital PCR (ddPCR).

Results : Using |fold change| ≥ 2 and a FDR < 0.05, we have identified 433 coding RNAs and 586 lncRNAs with differential expression. We successfully validated the differential expression of five selected lncRNAs, including lnc-WNT4-2:1, MEG3, lnc-BLID-5:1, lnc-SCP2-2:1, and lnc-ALDH3A2-2:1. By binding to distal regulatory elements, MEG3 has been reported to regulate TGF-β genes, which are involved in the pathogenesis of KC. Our correlation analysis identified 352 differentially correlated expression pairs between normal control and KC groups. The top significant correlation pairs (FDR value < 1x10-4) were NONHSAT215306 vs. ADCY6, NONHSAT216711 vs. lnc-SLCO5A1-5:1, NONHSAT209018_1 vs CEACAM6, and NONHSAT206355 vs. CACNA2D1. Expression network analyses identified approximately 20 network hubs, including the ddPCR validated lncRNAs and many coding RNAs, such as MUC16, RGS2, ELOVL5, CCND2, A2M, APOD, lnc-BLID-5:1, lnc-SCP2-2:1, and lnc-ALDH3A2-2:1.

Conclusions : We identified a set of differentially expressed lncRNAs in KC-affected human cornea. Our correlation-based analysis reveals several KC-related expression networks containing these lncRNAs. Future studies with larger number of samples are necessary to validate our pilot findings.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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