Transcriptome profiling of microRNA by deep sequencing reveals differential microRNA expression in type 1 and type 2 diabetic corneal limbus

Abstract

Purpose : With a global prevalence of around 8.5%, diabetes mellitus is one of the most common chronic diseases. A growing evidence of tissue-specific regulatory functions of miRNAs and their role as biomarkers has prompted several studies to evaluate miRNA expression associated with diabetes mellitus. Our purpose was determining miRNA signatures of corneal limbus harboring epithelial stem cells in type 1 (T1D) vs. type 2 (T2D) diabetes.

Methods : Age-matched human autopsy normal and diabetic corneas were received from the National Disease Research Interchange in Optisol medium (Chiron Vision) within 24 hours of donor death. Ten normal and 12 diabetic (5 T1D, 5 T2D and 2 unknown classification) corneas were used for miRNA expression profiling using deep sequencing. miRNA sequencing libraries were prepared from both central and limbal corneal parts with Illumina TruSeq™ Small RNA Library Prep kit per the manufacturer’s protocol and sequenced on the Illumina GAIIx, 1x36 bp sequencing chemistry. Differential expression in two groups was calculated using one-way ANOVA or t-test. A FDR controlled p value was computed using Benjamini and Hochberg procedure for multiple hypothesis testing. To further rank differentially expressed miRNAs, the data were shifted to log2 values and fold change was calculated.

Results : Principal component analysis and hierarchical clustering showed that the miRNA expression profiles of T1D and T2D groups were differentially clustered and separated from each other. Seven miRNAs were upregulated whereas eleven miRNAs were downregulated (1.4-fold cutoff; adjusted p < 0.05) in T1D vs. T2D limbus. Both diabetic groups, T1D and T2D, also showed distinct differential expression patterns of limbus vs. central cornea. Further, comparison of normal and T1D or T2D diabetes revealed that normal samples shared less differentially expressed limbus vs. central miRNAs with T2D than with T1D samples (4 vs. 18 miRNAs).

Conclusions : The present study showed that miRNA signatures of corneal limbus are distinctly different in T1D vs. T2D. Functional correlation studies and modulation of specific differentially expressed miRNAs may pave the way to new molecular therapeutic strategies.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.