Purchase this article with an account.
Qi Wang, Svetlana Bozack, Maria Grant, Michael Boulton, Walter Esselman, Julia Busik; MicroRNA-15a Is Involved In Ceramide Inflammatory Pathways by Targeting the Acid Sphingomyelinase (ASM) In Diabetic retina. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3692.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level through translational repression and/or target messenger RNAs degradation in a sequence-dependent manner and modulate several cellular processes. Acid sphingomyelinase (ASM) is important early responder in inflammatory cytokine signaling that catalyze the hydrolysis of sphingomyelin to the proinflammatory and proapoptotic second messenger ceramide. Here, we investigated the role of miR-15a, an ASM-targeting miRNA in diabetes.
Type 1 STZ-induced diabetic mice and age matched controls were used. Mice were sacrificed and their retinas were harvested for RNA analysis. The human retinal pigment epithelial cell line ARPE-19 cultured under normal and elevated glucose conditions and the human retinal vascular endothelial cells (HREC) from diabetic (n=6) and control donors (n=6) were collected for analysis. The mRNA expressions of miR-15a, ASM, VEGF-A, VEGF-C were measured. ASM, VEGF-A, VEGF-C, NF-κB protein levels with or without miR-15a mimic or antagomir transfection were examined. The ceramide immunofluorescence intensity with or without miR-15a mimic or antagomir transfection was detected. A luciferase assay was performed to detect miR-15a’s binding to ASM 3'-untranslated region (UTR). In situ hybridization was used to localize retinal miR-15a.
ASM is a predicted target of miR-15a. We demonstrated that miR-15a directly binds to ASM 3'-UTR in luciferase binding assay. miR-15a messenger RNA expression was decreased in the retinas from type 1 STZ-induced diabetic mice compared with control mice. HREC isolated from diabetic donors and ARPE-19 cells treated with 25 mmol/L glucose had decreased miR-15a expression and increased ASM expression compared with non-diabetic 5 mmol/L glucose treated cells. Delivery of miR-15a mimic in ARPE-19 cells suppressed the expression levels of ASM, VEGF-A, VEGF-C, NF-κB and ceramide production, while miR-15a antagomir transfection caused ASM, VEGF-A, VEGF-C, NF-κB and ceramide upregulation.
The results of this study demonstrate that dowregulation of miR-15a in diabetic retina contributes to diabetes-induced upregulation of ASM and thus pathogenesis of diabetic retinopathy.
This PDF is available to Subscribers Only