Abstract
Purpose: :
Studies in various cell systems demonstrated the importance of microRNAs in cell differentiation. In the RPE, miR-204 and -211 are the two most highly expressed miRNAs and are important for epithelial function (Wang et al, FASEB J, 2010). The goal of this study was to investigate the role of miR-204/211 in RPE differentiation.
Methods: :
Human fetal RPE (hfRPE) grown on transwells were used as a model system. MicroRNA microarray was performed using human miRNA Microarray (V2) from Agilent Technologies. hfRPE cells were transfected (Dharmafect 4) with miR-204/211 mimics or inhibitors (AppliedBiosystems) once during seeding and another three days later. Total RNA of samples were extracted using mirVana kit and reverse transcribed using superscript III (Invitrogen). Changes in cell morphology were evaluated using immunofluorescence confocal microscopy and protein expression was analyzed by western blotting.
Results: :
In primary hfRPE cultures, seeding at low density induced RPE dedifferentiation with a concomitant downregulation of miR-204, -211, and RPE-specific genes. Transfection of hfRPE cells seeded at low density with miR-204/211 mimics prevented dedifferentiation and cells exhibited characteristic RPE morphology as well as upregulation of genes and proteins that are critical for RPE function (RPE65, CRALBP, MCT3). On the other hand, transfecting hfRPE cells seeded at high density with miR-204/211 inhibitors prevented differentiation as characterized by downregulation of RPE genes and loss of RPE morphology. Microphthalmia-associated Transcription Factor (MITF) is a master regulator of RPE differentiation and its expression was downregulated in dedifferentiated hfRPE cells. Silencing MITF with siRNA resulted in decreased expression of miR-204/211 and loss of epithelial morphology. Importantly, co-transfection of miR-204/211 mimics with MITF siRNA rescued the RPE phenotype.
Conclusions: :
Our data demonstrate that miR-204/211 play a key regulatory role in RPE differentiation and suggest that miRNA-based therapy could be a viable approach for the treatment of ocular diseases that involve RPE dedifferentiationsuch as PVR and AMD.
Keywords: differentiation • proliferative vitreoretinopathy • gene microarray