Long Non-Coding RNAs in Keratoconus-affected Human Cornea

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.