Effects of miR-96 overexpression on Pigment epithelium-derived factor (PEDF) expression in hRPE cells
Zeynab Hosseinpoor1 *, Zahra-Soheila Soheili2 , Maliheh Davari2 , Shahram Samie3 , Hamid Ahmadieh4
- National Institute of Genetic Engineering and Biotechnology
- National Institute of Genetic Engineering and Biotechnology
- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
- Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Abstract: Pigment epithelium-derived factor (PEDF) is an anti-angiogenic protein produced by RPE cells. Decreased level of PEDF in retina is associated with retinal neovascularization in proliferative diabetic retinopathy (PDR). There is also an inverse correlation between PEDF and VEGF expression under high glucose condition. Furthermore, miR-96 is one of the most highly expressed miRNAs in diabetic retina. In this study, we investigated miR-96 overexpression effects on PEDF expression level in hRPE cell line.
Methods: miR-96 was cloned into pAAV-MCS-eGFP-intron vector. hRPE cells were cultured in DMEM/F12 media supplemented with 10% FBS and transfected with pAAV-miR96-eGFP-intron construct. Total RNA was isolated 48 hours and 72 hours post transfection. The expression level of PEDF gene (SerpinF1) in hRPE cell line was examined by RT-qPCR.
Results: miR-96 was upregulated by more than 82-fold in hRPE cells after 48 and 72 hours. The expression level of PEDF was increased (more than 2-fold) initially. Interestingly, the mRNA level of the gene was significantly decreased (0.3-fold) after 72 hours of transfection.
Conclusion: Although, PEDF was first upregulated as an antioxidant factor, our results revealed that, high expression of miR-96 for 72 hours significantly decreased PEDF expression in hRPE cells. According to the important role of PEDF in regulation of angiogenesis, our results suggest that miR-96 overexpression may aggravate DR signs by downregulating PEDF in an indirect manner.
