Differential Expression of Coding and Long Noncoding RNAs in Keratoconus-Affected Corneas

Abstract

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

Methods: From the corneas of 10 KC patients and 8 non-KC healthy controls, 200 ng total RNA was used to prepare sequencing libraries with the SMARTer Stranded RNA-Seq kit after ribosomal RNA depletion, followed by paired-end 50-bp sequencing with Illumina Sequencer. Differential analysis was done using TopHat/Cufflinks with a gene file from Ensembl and a lncRNA file from NONCODE. Pathway analysis was performed using WebGestalt. Using the expression level of differentially expressed coding and noncoding RNAs in each sample, we correlated their expression levels in KC and controls separately and identified significantly different correlations in KC against controls followed by visualization using Cytoscape.

Results: Using |fold change| ≥ 2 and a false discovery rate ≤ 0.05, we identified 436 coding RNAs and 584 lncRNAs with differential expression in the KC-affected corneas. Pathway analysis indicated the enrichment of genes involved in extracellular matrix, protein binding, glycosaminoglycan binding, and cell migration. Our correlation analysis identified 296 pairs of significant KC-specific correlations containing 117 coding genes enriched in functions related to cell migration/motility, extracellular space, cytokine response, and cell adhesion. Our study highlighted the potential roles of several genes (CTGF, SFRP1, AQP5, lnc-WNT4-2:1, and lnc-ALDH3A2-2:1) and pathways (TGF-β, WNT signaling, and PI3K/AKT pathways) in KC pathogenesis.

Conclusions: Our RNA-Seq-based differential expression and correlation analyses have identified many potential KC contributing coding and noncoding RNAs.

Figure 1

Differentially expressed coding genes in KC-affected human corneas identified by RNA sequencing (A) and ddPCR (B). For panel A, **FDR < 0.01, *0.01< FDR < 0.05. For panel B, ***P value < 0.001, **P value < 0.01, * 0.01< P value < 0.05. Error bars: panels A and B represent standard error.

Figure 2

Differentially expressed lncRNAs in KC-affected human corneas identified using by RNA-Seq (A) and ddPCR (B). For panel A, **FDR < 0.01, *0.01 < FDR < 0.05. For panel B, ***P value < 0.001, **P value < 0.01, *0.01< P value < 0.05. Error bars: panels A and B represent standard error.

Figure 3

Correlation networks of the differentially expressed coding RNAs and lncRNAs displayed by Cytoscape. The nodes represent mapped RNAs colored and sized by the degree of correlations with other RNAs. The edges are sized and colored based on the significant differences (i.e., FDR) between controls and cases. Correlation networks were shown for the Lnc-ALDH3A2-2:1 (A), APOD (B), CLIC4 (C), SLC2A1 (D), and A2M (E) genes.