Abstract
Purpose :
To test whether circulatory microRNAs highly expressed in surgical specimens of ocular fluids from patients with proliferative diabetic retinopathy can induce VEGF secretion in in vitro cell cultures of ARPE19 cells.
Methods :
Mimics of the 5 microRNAs and scrambled control were purchased from Ambion and transfected using RNAiMAX reagent in the ARPE19 cells. In our study, 5x104 ARPE19 cells per well were transfected with 0pmol, 10pmol, 25pmol and 50pmol miRNA mimics. The ARPE19 conditioned cell media were harvested at 12h, 24h and 48h, and VEGF concentrations were measured by ELISA, followed by analysis at MyAssays.com. Cells were harvested with addition of Qiazol, lysates were collected, total RNA isolated using Qiagen miRNeasy kit and levels of individual candidate miRNAs and VEGF mRNA were determined using Taqman qPCR assays. Pathways analysis was performed using Ingenuity Pathway Analysis (IPA) database and software.
Results :
Out of 5 top miRNA candidates (let-7b, let-7c, miR-486, miR-92a and miR-320b) 2 stimulated VEGF secretion in ARPE19 cells. Strongest stimulation of VEGF secretion was achieved at 10pmol at 12h time point for let-7b (FC>2.7) and at 10pmol at 24h for miR-486 (FC>1.75). It decreased non-significantly at higher concentrations of 25pmol and 50pmols. IPA identified 5 major pathways targeted by these miRNAs: VEGF signaling, inflammation, immune response, apoptosis and angiogenesis.
Conclusions :
It has been shown in literature that let-7 targets proinflammatory cytokines, which regulate VEGF expression. MiR-320 regulates tumor angiogenesis driven by vascular endothelial cells by silencing neuropilin 1, a co-receptor for VEGF and VEGFR. Upregulation of miR-92a is associated with endothelial inflammation and ischemia/reperfusion injury in mice, while miR-486 sustains NF-κB activity by disrupting multiple NF-κB-negative feedback loops in glioma. In this study we tested whether overexpression of these miRNAs regulate VEGF secretion in ARPE19 cells, that. Our results show that VEGF release from ARPE19 cells is highly responsive to changes in specific miRNAs concentration. Such miRNA-induced changes in retinal epithelial cells to secrete angiogenic factor may negatively impact the integrity of the microvasculature in the diabetic retina leading to angiogenesis and vascular leakage.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.