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Mehrnoosh Saghizadeh, Jordan Brown, Alexander Ljubimov, Vincent Funari; miRNA EXPRESSION PROFILING IN CENTRAL AND LIMBAL DIABETIC AND NORMAL HUMAN CORNEAS USING DEEP SEQUENCING. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3229.
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To identify and characterize miRNA expression by deep sequencing analysis in central and limbal parts of normal and diabetic human corneas.
Total RNA was extracted from age-matched human autopsy normal (n=10) and diabetic (n=12) 8.5 mm-trephined central corneas and their limbi using mirVana miRNA Ambion kit. From 44 total RNA samples that passed quality control standards small RNAs (22-28 bp) were gel isolated and miRNA libraries were prepared using Illumina small RNA Sample Preparation Kit according to the manufacturer’s protocol. Samples were multiplexed and pooled into two lanes for a 35 bp sequencing run. Upon completion of sequencing, FASTQ files were converted to FASTA files, and adapters were trimmed from each sequencing. FASTA files for sequencing were parsed by sample and aligned using BLAST with 90% similarity to miRBase v19. Student’s t-test was performed for each of four comparisons: normal central vs. normal limbus, normal central vs. diabetic central, normal limbus vs. diabetic limbus, and diabetic central vs. diabetic limbus. For each comparison, significant miRNAs were identified (fold change > 2, p-value < 0.05). A knowledge base (Ingenuity Pathway Architect) was searched for biochemical pathways that could be regulated by differential miRNAs.
A number of miRNAs with significant differential expressed in diabetic vs. normal corneas and in central vs. limbal parts were quantitatively identified; some were not present in the mirbase 19. Comparison of normal central corneas vs. normal limbi and diabetic central corneas vs. diabetic limbi yielded a few differential miRNA species. Many more miRNAs had differential expression between normal vs. diabetic central corneas (90) and especially, normal vs. diabetic limbi (141). In the central corneas, about equal numbers of miRNAs were either up- or downregulated in normal vs. diabetic tissue. However, in the diabetic limbus, 133 miRNAs were downregulated compared to normal, with only 8 upregulated.
The results are consistent with previously observed significant changes in the expression of stem cell markers in diabetic limbal epithelial cells. Abnormal miRNA expression may be an important mechanism of diabetic changes affecting corneal epithelial stem cell compartment and wound healing. Manipulating miRNA levels in diabetic corneas could potentially help alleviate symptoms of diabetic keratopathy.
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