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Pei Zhuang, Chithra Muraleedharan, Colleen Cowan, Sanja Turturro, Shunbin Xu; In vivo Modulation of microRNA-146 and Diabetes-induced Inflammatory Responses in Streptozotocin-induced Diabetic Rats. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):46.
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Previously we showed that microRNA-146 (miR-146) is a pivotal negative feedback regulator of multiple NF-κB activation pathways by targeting key signaling molecules of these pathways. These data suggested that miR-146 may play an important role in diabetic retinopathy (DR) and is a potential therapeutic target for treatment of DR by inhibiting diabetes-induced inflammatory response in the retina. The purpose of the current study is to test this hypothesis in vivo.
Lenti-pre-miR-146a-GFP+, which co-expresses miR-146a and GFP, lenti-miR-146-inhibitor-mCherry+, which co-expresses inhibitor of miR-146 and mCherry, as well as corresponding negative control viruses were produced and titered (Genecopoeia). Transduction efficiency was tested in primary human retinal endothelial cells (HRECs). Young male Sprague-Dawley rats were injected with a single dose of 65 mg/kg streptozotocin (STZ) to induce diabetes. One week after diabetes, animals were injected with lentivirus intravitreally (4 ul, ~106 CFU/ml) and intravenously (400 ul, ~106 CFU/ml). Three weeks (for pilot experiment) and 3 months after STZ-induced diabetes, total RNA was isolated from the retina for qRT-PCR assay. Effects on inflammatory responses and integrity of retinal microvasculature will be tested by lectin labeling of adherent retinal leukocytes, Evans blue and trypsin digestion assays.
1) HRECs were efficiently transduced by the lentiviruses in vitro; 2) lenti-pre-miR-146 increased, while lenti-miR-146-inhibitor decreased the expression of miR-146 in HRECs in vitro; downstream target genes of miR-146, CARD10 and TRAF6, were down- and up-regulated, respectively; 3) In the pilot in vivo experiment, 3 weeks after injection, lenti-pre-miR-146 and lenti-miR-146-inhibitor resulted in significant up- and down-regulation of miR-146 in the retina; 4) characterization of their effects on DR progression three months after diabetes is ongoing.
miR-146 and miR-146-inhibitor can be successfully delivered to the retina by a combined intravitreal and intravenous lentiviral injection, and modulate the expression of miR-146 and its downstream genes involved in NF-kB activation in vivo. Further investigation will provide direct evidence on whether in vivo modulation of miR-146 affects DR progression and serves as a therapeutic target for treatment of DR.
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