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
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.
Nationa Institute of Genetic Engineering and Biotechnology
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.
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.
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.