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Manuela Bartoli, Chaunte Stampley, Sean Shaw, Pamela Martin, Folami Lamoke; MicroRNA-21 (miR-21) Induces Down-regulation Of PEDF In Retinal Pigmented Epithelial Cells By Suppression Of PPAR Alpha. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1594.
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Micro-RNA 21 (miR-21) is up-regulated in the ischemic retina and hypoxia-induced miR-21 expression contributes to increased matrix metalloproteinases activity (MMP2 and MMP9) in retinal endothelial cells. Increased MMP2 and MMP9 activity in the ischemic retina has been linked to the proteolytic degradation of pigmented epithelial derived factor (PEDF), a key retinal angiostatic factor. Here we have investigated the effects of miR-21 on PEDF expression and protein stability in retinal pigmented epithelial cells (ARPE19) and in the ischemic retina.
Immunoblotting and quantitative RT-PCR were conducted to determine PEDF and peroxisome proliferator activated receptor alpha (PPARalpha) expression at protein and mRNA levels, respectively, in ARPE19 after transfection with miR-21 or a scramble micro RNA (miR-s). The same assays were also used to determine the expression of PEDF and PPAR alpha in a model of ischemic retinopathy. Mice were exposed from postnatal day 7 to 12 (P7 and OIR12 respectively) to 73% oxygen tension. At OIR12 the mice were returned to normoxic conditions and analyzed at postnatal day 14 and 17 (OIR14 and OIR17, respectively). In situ hybridization was conducted using locked nucleic acid-based probes to determine miR-21 sites of production in the ischemic murine retina.
Transfection of ARPE19 with miR-21 significantly decreased protein and mRNA expression of PEDF. We have identified a PPAR alpha responsive element on the PEDF promoter region. PPAR alpha has shown to be a potential target for miR-21. Transfection of ARPE19 with miR-21 down-regulated PPARalpha expression at protein and mRNA levels. In the ischemic retina, maximal expression of miR-21 (OIR14 and 17) correlated with maximal downregulation of PEDF and PPAR-alpha protein levels. In situ hybridization further confirmed production of miR-21 by the retinal pigmented epithelial cells in the ischemic retina.
Expression and activity of small non-coding RNAs is emerging as critical regulation of retinal gene expression in normal and pathological conditions. Our results demonstrate a novel role for miR-21 in down-regulating PEDF expression by its ability to block PPAR alpha expression, thus halting PPAR alpha mediated-PEDF expression.
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