Abstract
Purpose:
The PRR, which is able to bind and activate both renin and prorenin, has been identified in hRECs. To date, a large number of genes, including angiotensin II type 1 receptor (AT1R), have been shown to be regulated at the post-transcriptional level by miRNAs. miRNAs are small RNAs that regulate gene expression primarily through translational repression. The present study aimed to investigate whether PRR and its downstream molecules such as VEGF, VEGFR, and TGFβ could be regulated by miR-152 in hRECs under hyperglycemic conditions.
Methods:
hRECs were maintained at 37°C in a humidified incubator with 5% CO2 and propagated in microvascular endothelial cell growth medium (Cell Applications, Inc., San Diego, CA) containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin (LONZA, Walkersville, MD). Cells were cultured in either 5.5 mM glucose (control) or 33 mM glucose (hyperglycemia) concentration. The expression of AT1R, PRR, VEGF, VEGFR, AngII, and TGFβ was analyzed by both qRT-PCR and immunoblotting. To investigate the role of PRR, hREC cells were transfected with the siRNAs specifically targeting PRR or AT1R for 24 h.
Results:
RNAi technique using AT1R and PRR siRNAs showed that both the receptors are implicated in hyperglycemia-mediated induction of VEGF, VEGFR, and TGFβ. Luciferase assays demonstrated that transfection of miR-152 significantly suppressed the PRR 3'-untranslated region (UTR) reporter construct activity (p=002), which was prevented by miR-152 antagomirs or the mutation in the miR-152 binding site of PRR 3'-UTR. Transfection of miR-152 in hyperglycemic hRECs significantly (p<0.05) decreased the expression of PRR, VEGF, VEGFR, and TGFβ, which was significantly rescued by miR-152 antagomirs (p<0.001), as compared to miR-152-transfected cells.
Conclusions:
Our results showed that miR-152 translationally repressed the expression of PRR in hRECs. Besides the role of AT1R, hyperglycemia-induced upregulation of VEGF, VEGFR, and TGFβ is mediated through PRR, and this regulation is probably achieved through the hyperglycemia-mediated modulation of miRNAs. Our future studies will begin to study the role of miRNAs in the pathogenesis of diabetic retinopathy and age-related macular degeneration, as prorenin and PRR have emerged as a new area of research in ocular biology.
Keywords: 499 diabetic retinopathy •
533 gene/expression •
609 neovascularization