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

Abstract

PURPOSEKeratoconus (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.\n\nMETHODS200ng total RNA from the corneas of 10 KC patients and 8 non-KC normal controls was used to prepare sequencing libraries with the SMARTer Stranded RNA-Seq kit after ribosomal RNA depletion. Paired-end 50bp sequences were generated using Illumina HiSeq 2500 Sequencer. Differential analysis was done using TopHat and 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.\n\nRESULTSUsing |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 with cell migration/motility, extracellular space, cytokine response, and cell adhesion, suggesting the potential functions of these correlated lncRNAs, especially those with multiple pairs of correlations.\n\nCONCLUSIONSOur RNA-Seq based differential expression and correlation analyses have identified many potential KC contributing coding and noncoding RNAs.