miR-183 cluster, composed of miR-183, miR-96 and miR-182, is highly expressed in the neuroretina. As a key miRNA cluster in differentiation of photoreceptors, miR-183 cluster was overexpressed in multipotent hRPE cells to evaluate its potential to induce hRPE reprogramming toward neuron fate in vitro.

hRPE cells were transfected with the construct carrying miR-18/-96/-182 genes and subjected to measure the expression levels of miR-183 cluster members and that of retina-specific neuronal genes such as OTX2, NRL, PDC, DCT, POU4F2, RCV, CRX, NR2E3 and NGN1. The hRPE cells expressing miR-183 cluster were also immunocytochemically examined for retina-specific neuronal markers including Rhodopsin, red opsin, CRX, Thy1, CD73, recoverin and PKC to identify the developing neuron-like cells.

Data showed that hRPE cells overexpressing miR-183, miR-96 and miR-182 (78-, 158- and 64-fold, respectively) also demonstrated the upregulation of neuronal genes such as OTX2, NRL, PDC and DCT (2-, 2-, 7-, 2-fold, respectively) (Figure 1). Furthermore, some cells immunoreactive for neuronal markers including Rhodopsin, red opsin, CRX and Thy1 were found in miR-183 cluster-treated hRPE cultures (Figure 2). Fig. 1. Relative expression of miR-183 cluster and retina-specific neuronal genes in transfected hRPE cells. (A) miR-96/-183/-182 genes were overexpressed 158-, 78- and 64-fold, respectively, in hRPE cells transfected with miR-183 cluster (B) hRPE cells overexpressing miR-183 cluster showed upregulation of several retina-specific neuronal genes including OTX2, NRL, PDC and DCT. Fig. 2. hRPE cells overexpressed miR-183 cluster were led toward retinal neuron fate. A population of cells in hRPE cultures transfected with pAAV.miR-183 cluster.EGFP were immunoreactive to retina-specific neuronal markers including Rhodopsin, CRX and red opsin (A, B, C), and Thy1 (D). They also displayed morphological features resembled neurons (Arrows in A, B and D). Cells transfected with control vector not developed signals for neuronal markers (E, F, G, and H). Scale bars are 25µm.

Both transcriptional and translational upregulation of neuronal genes in miR-183 cluster-treated hRPE cells suggests that in vitro overexpression of miR-183 cluster members could pave the reprogramming way of cultured hRPE cells to retinal neuron-like fate.