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Khaled Elmasry, Amany M Tawfik, Ahmed Ibrahim, Mohamed Al-Sayed Al-Shabrawey; Comparative miRNA Microarray Analysis of Diabetic Mice Retinas and Human Retinal Endothelial Cells Challenged With 15-HETE or High Glucose. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5207.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous studies established the role of 12/15-lipoxygenase (12/15-LO)-derived hydroxyeicosatetraenoic acids (12- and 15-HETEs) in diabetic retinopathy (DR). The goal of this study is to characterize the effect of these bioactive lipids on miRNA and related molecular pathways in human retinal endothelial cells (HRECs) compared to hyperglycemia.
RNA was extracted from retinas of age-matched normal or streptozotocin (STZ) induced diabetic mice. Also, RNA was extracted from HRECs treated with 15-HETE (0.5 µM) or its vehicle for 24 hours and high glucose (HG, 30 mM D-glucose) or osmotic control (5 mM D-glucose and 25 mM L-glucose) for 5 days. RNA was hybridized to the GeneChip miRNA 3.0 array (Affymetrix). Data were imported into Partek Genomic Suites version 6.6 (Partek, St. Louis, MO). The differential expression of miRNAs was calculated using ANOVA of Partek Package. Hierarchical clustering plots were generated using the miRNAs that showed significant changes. Data were imported into Ingenuity Pathway Analysis (IPA, Qiagen) and analyzed on Core Analysis and MicroRNA Target Filter to detect specific target genes and related pathways of the significantly changed miRNAs under various conditions.
miRNAs were filtered based on statistical significance (p-value < 0.05) and fold change (FC ≥1.5) and accordingly our data demonstrated; 1) 312 miRNAs were significantly changed in retinas of STZ diabetic mice versus control group, 129 were downregulated and 183 were upregulated by diabetes; 2) 75 miRNAs were significantly changed in HRECs subjected to 15-HETE compared to the control, 29 were downregulated and 46 were upregulated; 3) 94 miRNAs were significantly changed in HRECs subjected to HG versus control, 26 were downregulated and 68 were upregulated. The IPA linked the significantly changed miRNAs by hyperglycemia or 15-HETE to major pathways that are implicated in the development of microvascular dysfunction in DR such as ER stress, autophagy, hypoxia signaling, inflammasome and oxidative stress.
Our miRNA array demonstrated that similar to hyperglycemia, 12/15-LO-derived bioactive lipids target several miRNAs that regulate critical pathways involved in the pathogenesis of DR. Future experiment will focus on identification of specific miRNAs as potential therapeutic targets.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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