miR-96 overexpression in hRPE cells, surveying the effects on the target genes that are involved in proliferation
Mahshad Karimifard1 , Zahra-Soheila Soheili2 *, Maliheh Davari2 , Hamid Latifi Navid 2 , Shahram Samiei3
- Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
- Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
Abstract: Retinal pigment epithelium (RPE) is a monolayer of post-mitotic cells which fulfills important functions for the process of vision, including nutrients transport, phagocytosis of photoreceptors outer segments, re-isomerization of all-trans retinal into 11-cis trans retinal, secretion of growth factors and organizing the outer blood retinal barrier (oBRB). In DR, we notice degeneration of RPE cells that, make the barrier permeable and inflammation and tissue edema as the next side effects. In diabetic rats expression of the miR-183/96/182 (cluster 183) increases in retina. This may have a pivotal role in RPE cells degeneration. miR-96 targets many important genes that are involved in critical functions of the retina. We studied the outcomes of miR-96 overexpression on proliferation and survival of hRPEcell line.
Methods: To find the target pathways and related genes in DR condition, we applied miRTarBase database (http://mirtarbase.cuhk.edu.cn/php/search.php), miRWalk (http://mirwalk.umm.uni-heidelberg.de/search_genes/) and KEGG PATHWAY Database (https://www.genome.jp/kegg/pathway.html). miR-96 sequence was cloned in pAAV-GFP-intron vector by common PCR and restriction digestion methods. The resultant construct was transfected to hRPEcell line through calcium phosphate method. To perform Real-time PCR, RNA samples were extracted 48 hours and 72 hour post transfection. MTT assay and analysis of cell cycle were performed to measure proliferation rate of transfected hRPE cells after 48 hours and 72 hour.
Results: PI3K/AKT and MAPK/ERK found to be the prominent signaling pathways due to their fundamental roles in secretion of a variety of growth factors, pro-inflammatory mediators and downstream regulators in proliferation and survival of hRPE cells. Ten target genes (RELA, TIMP1, RAB5B, CHML, RAD51, PTEN, GRB2, IGF1R, PRMT5, BDNF) which showed direct and indirect relevance to prenominated pathways, were selected. miR-96 was successfully cloned in pAAV-GFP-Intron vector. Proliferation rate in miR-96 transfected cells was reduced 72 hours post treatment.
Conclusion: The results provide an insight in modulating effects of miR-96 overexpression on proliferation rate of hRPE cell line and potentially introduce a new therapeutic strategy in DR treatments.
