Abstract
Purpose:
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.
Methods:
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.
Results:
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.
Conclusions:
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.