Overexpression of miR-183/-96/-182 triggers neuronal cell fate in human retinal pigment epithelial cells in culture

Session Details

Session Title: Free Paper Session 18: New Drug & Treatment Technology

Session Date/Time: Saturday 09/09/2017 | 14:30-16:00

Paper Time: 14:54

Venue: Room 120

First Author: : S.Samiei IRAN

Co Author(s): :    M. Davari   Z. Soheili   E. Ranaei Pirmardan                       

Abstract Details

Purpose:

miR-183 cluster, composed of miR-183, miR-96 and miR-182, is highly expressed in the adult retina, particularly in photoreceptors, involving in development, maturation and normal function of neuroretina. Ectopic overexpression of miR-183/-96/-182 cluster was performed to assess reprogramming and cell fate of primary human RPE cells upon miR-183 cluster overexpression.

Setting:

Nationa Institute of Genetic Engineering and Biotechnology

Methods:

miR-183, miR-96 and miR-182 genes with their flanking sequences were amplified from genomic DNA by PCR. miR-183, -96 and -182 fragments separately and/or together, in a cluster, were cloned into pAAV.MCS vector. hRPE cells were isolated from neonate human cadaver eyes and cultured in DMEM/F12 supplemented with 10% FBS. Cultures were transfected with the constructs carrying each miR-183, -96 and -182 and/or the whole cluster. After 72 hours, total RNA was isolated, cDNA was synthesized and Real-Time PCR was performed to measure the expression levels of miR-183, -96, -182 and that of the retinal specific neuronal genes; OTX2, NRL, PDC, DCT, POU4F2, RCV, CRX, NR2E3, NGN1 and RPE65. The transfected cells were immunocytochemically examined for retina-specific neuronal markers, including rhodopsin, red opsin, Crx, Thy1, CD73, recoverin and PKCa.

Results:

Data showed that all the three miR-183, miR-96 and miR-182 were successfully overexpressed in transfected hRPE cultures (78-, 158- and 64-fold, respectively). Furthermore, qPCR results implicated that the expression of neuronal genes including OTX2, CRX, NRL, PDC and DCT was upregulated (2-, 4-, 2-, 7-, 2-fold, respectively) in hRPE cells which had been treated with miR-183 cluster. miR-183 cluster treated cells were also observed to be immunoreactive to some neuronal markers such as rhodopsin, red opsin, Crx and Thy1.

Conclusions:

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

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