Abstract Form

Title: Overexpression of miR-183 cluster induces neuronal cell fate in Human Retinal Pigment Epithelial (hRPE) Cells
Author(s): Maliheh Davari, Zahra-Soheila Soheili, Shahram Samiei, Zohreh Sharifi, Ehsan Ranaei Pirmardan
Presentation Type: Oral
Subject: Retina and Retinal Cell Biology
Presenting Author:
Name: Maliheh Davari
Affiliation :(optional) National Institute of Genetic Engineering and Biotechnology
E mail: Maliheh_Davari@yahoo.com
Mobile: 09128152826
Abstract (Max 200 words)
Purpose: 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.
Methods: 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.
Results: 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 25┬Ám.
Conclusion: 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.