Long noncoding RNAs (lncRNAs) previously thought to be “dark matter” of the genome, play key roles in various biological processes. The lncRNA ANRIL is located at a genetic susceptibility locus for coronary artery diseases and type 2 diabetes. We examined the role of ANRIL in diabetic retinopathy, through study of its regulation of VEGF both in vitro and in vivo.
Researchers at Western University subjected human retinal endothelial cells (HRECs) to incubation in high glucose to mimic diabetes. ANRIL expression was measured with or without small interfering RNA (siRNA) knockdown in HRECs. ANRIL knockout mice, with or without streptozotocin-induced diabetes, were also investigated. Cell and tissues were measured for VEGF mRNA and protein expression. Functional alterations in VEGF were determined through tube formation, cell proliferation, and retinal vascular permeability assays. Vascular endothelial growth factor regulation through ANRIL’s interactions with polycomb repressive complex 2 (PRC2) components and p300 were studied thorough PRC2 blocker, siRNA, and RNA immunoprecipitation (RNA-IP) assays.
High glucose and diabetes caused ANRIL upregulation in HRECs and in the retina. Glucose-mediated elevation of ANRIL, on silencing, prevented VEGF expression. Such regulation involved ANRIL-mediated control of the PRC2 components p300 and miR200b. Direct binding of ANRIL to p300 and enhancer of zeste homolog 2 (EZH2; a PRC2 component) were elevated following exposure to high glucose levels.
(A) Volcano plot of differentially expressed lncRNAs in NG versus HG in HRECs. Vertical lines correspond to 1.2-fold changes up and down, respectively, and the horizontal line represents a P value of 0.05. Red points indicate differentially expressed genes with statistical significance. Arrow represents location of ANRIL. (B) Array data were validated by real-time RT-PCR analysis of ANRIL, which confirmed its increased expression on exposure to HG (48 hours). No alterations were seen following incubation with 25 mM l-glucose (osmotic control, Osm). (RNA expressions are presented as a ratio of β-actin, normalized to NG.) (C) Fluorescence in situ hybridization using probes against human ANRIL showed increased expression (nuclear and perinuclear cytoplasmic) in HRECs exposed to HG versus NG (48 hours). (D) Quantitative data following analysis using ImageJ confirmed glucose-induced ANRIL upregulation (MFI, mean fluorescence intensity). Images quantified by ImageJ (*P < 0.05 versus NG. n = 4 or more/group).