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Zeljka Smit-McBride, Kingsley Okafor, Anthony T Nguyen, Matthew Bordbari, Amar P Patel, Allan A Hunter, Lawrence S Morse; Comparative expression analysisin plasma ofcirculating microRNAs identified in ocular fluids as putative biomarkers for diabetic retinopathy . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5170.
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© ARVO (1962-2015); The Authors (2016-present)
Our goal was to establish whether miRNA biomarkers, which we identified in the ocular fluids as putative biomarkers for diabetic retinopathy, are also differentially expressed in plasma.
Plasma, aqueous and vitreous samples were collected from 10 controls and 20 patients with diabetes undergoing vitreoretinal surgery [diabetes Type I (DR-I), Type II (DR-II) and Type II, no retinopathy (NoDR-II)]. Samples were stored at -80oC. MicroRNAs from plasma were isolated using Exiqon RNA purification kit, quantified on BioAnalyzer, labeled with FlashTag kit and profiled on Affymetrix GeneChip miRNA 3.0 microarrays. Data analysis was performed using Expression Console (EC) and Transcription Analysis Console (TAC) (Affymetrix) and for pathway analysis Ingenuity Pathway Analysis software (IPA) was used. qPCR using Taqman assays is used to confirm the candidate microRNAs.
Our comparison of circulatory microRNAs from aqueous and vitreous humor with corresponding plasma samples from the same patients showed that we can detect statistically significant dysregulation of some of the members from the let-7 and miR-320 families (p<0.05; -1.5>Fold Change (FC)>1.5). In DR-I group let-7 microRNAs are upregulated in the ocular fluids (FC=1.5-16) and downregulated in plasma (FC=-1.41 to -4.38), while miR-320 microRNAs are upregulated both in vitreous (FC=5.1) and plasma (FC=1.24). In DR-II group let-7 microRNAs are upregulated in ocular fluids (FC=2.12-6.1), and not detectable in the significant cut off in the plasma. In NoDR II group we observed upregulation of let-7 family members both in aqueous (FC=3.69) and in plasma (FC=3.81). Pathway analysis of the let-7 family of miRNAs shows that some of the biological pathways that they regulate are TGF-beta, Insulin Receptor Signaling, Apoptosis and VEGF Receptor Signaling. MiR-320 family has been assigned biological process by GO as cellular response to glucose stimulus and has a role in apoptosis, migration, proliferation, and signaling in diabetes mellitus.
The plasma profiling of circulatory microRNAs shows that DR biomarkers identified in aqueous and vitreous fluids are also differentially regulated in plasma. A number of circulatory microRNAs have shown differential presence in normal vs. diabetic retinopathy, offering promise for further study on use of plasma for diagnostic purposes.
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