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. 2015:2015:846501.
doi: 10.1155/2015/846501. Epub 2015 Mar 1.

MicroRNA-146b-3p regulates retinal inflammation by suppressing adenosine deaminase-2 in diabetes

Sadanand Fulzele  1 , Ahmed El-Sherbini  2 , Saif Ahmad  3 , Rajnikumar Sangani  1 , Suraporn Matragoon  4 , Azza El-Remessy  4 , Reshmitha Radhakrishnan  2 , Gregory I Liou  2
Affiliations

MicroRNA-146b-3p regulates retinal inflammation by suppressing adenosine deaminase-2 in diabetes

Sadanand Fulzele et al. Biomed Res Int. 2015.

Abstract

Hyperglycemia- (HG-) Amadori-glycated albumin- (AGA-) induced activation of microglia and monocytes and their adherence to retinal vascular endothelial cells contribute to retinal inflammation leading to diabetic retinopathy (DR). There is a great need for early detection of DR before demonstrable tissue damages become irreversible. Extracellular adenosine, required for endogenous anti-inflammation, is regulated by the interplay of equilibrative nucleoside transporter with adenosine deaminase (ADA) and adenosine kinase. ADA, including ADA1 and ADA2, exists in all organisms. However, because ADA2 gene has not been identified in mouse genome, how diabetes alters adenosine-dependent anti-inflammation remains unclear. Studies of pig retinal microglia and human macrophages revealed a causal role of ADA2 in inflammation. Database search suggested miR-146b-3p recognition sites in the 3'-UTR of ADA2 mRNA. Coexpression of miR-146b-3p, but not miR-146-5p or nontargeting miRNA, with 3'-UTR of the ADA2 gene was necessary to suppress a linked reporter gene. In the vitreous of diabetic patients, decreased miR-146b-3p is associated with increased ADA2 activity. Ectopic expression of miR-146b-3p suppressed ADA2 expression, activity, and TNF-α release in the AGA-treated human macrophages. These results suggest a regulatory role of miR-146b-3p in diabetes related retinal inflammation by suppressing ADA2.

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Figures

Figure 1
Figure 1
ADA2 is localized in the activated monocytes or macrophages in human retina with diabetes. Immunolabeling of ADA2 (green) and Iba-1 (red), a marker of activated microglia or macrophages, was made in human retinas under normal and diabetic conditions. Colocalization of ADA2 and Iba1, as marked by the orange-stained cells in the inner-retina produced by the merged red and green on the cell surface, is only identified in retinas with diabetes. The figures represent one of three donor eyes in each diabetic and normal group.
Figure 2
Figure 2
ADA2 is a target of miR-146b-3p. (a) Database searches of the TargetScan sites showed that human ADA2 mRNAs have conserved miR-146b-3p recognition sites. Alignment of the predicted miRNA binding sites in the 3′-UTR of the human ADA2 mRNA was made. Pairing of target region (top) and miRNA (bottom) is shown. (b) Secrete-Pair Dual Luminescence assay system was used to determine if ADA2 expression might be suppressed directly by miR-146b-3p. GLuc luciferase reporter gene linked with or without the 3′-UTR of the ADA2 gene was cotransfected with miR-146b-3p mimic, miR-146b-5p mimic, or negative control (NC, nontargeting miRNA) in differentiated U937 cells. Luciferase activities normalized with SEAP activities were determined with a luminometer. The results are mean ± SD from three independent experiments. * P < 0.05.
Figure 3
Figure 3
miR-146b-3p is dysregulated in diabetes: ectopic expression of miR-146b-3p inhibits ADA2 and TNF-α expression. PMA-differentiated U937 macrophages were transfected with miR-146b-3p mimic or negative control (NC, nontargeting miRNA). Expression was evaluated by qRT-PCR for (a) ADA2 mRNA and (b) TNF-α quantification (ELISA). (c) ADA2 activity using standard quantification method and (d) miR-146b-3p expression (qRT-PCR). The results are presented as the mean ± SD from three independent experiments. * P < 0.05.
Figure 4
Figure 4
ADA2 is inversely associated with miR-146b-3p: AGA treatment upregulates TNF-α and ADA2 and downregulates miR-146b-3p. PMA-differentiated U937 macrophages were exposed to AGA and media and cell lysates were processed for the following analyses. (a) ADA2 activity was determined by a standard assay (Diazyme Laboratories). (b) ADA2 mRNA expression by qRT-PCR, (c) miR-146b-3p expression by qRT-PCR, and (d) TNF-α release were determined by ELISA. The results are mean ± SD from three independent experiments. * P < 0.05.
Figure 5
Figure 5
miR-146b-3p is dysregulated in diabetes: 8 donor eyes with type 2 diabetes and 4 nondiabetic donor eyes were evaluated for ADA2 activity and miR-146b-3p expression in the vitreous.* P < 0.05.
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