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Survival Improvement in Human Retinal Pigment Epithelial Cells via Fas Receptor Targeting by miR-374a
Nooshin Tasharrofi1,2,3, Fatemeh Kouhkan3, Masoud Soleimani4
1Faculty of Pharmacy, Department of Pharmaceutical Nanotechnology, Tehran University of Medical Sciences, Tehran, Iran.
Abstract:
Oxidative conditions of the eye could contribute to retinal cells loss through activating the Fas-L/Fas pathway. This phenomenon is one of the leading causes of some ocular diseases like age-related macular degeneration (AMD). By targeting proteins at their mRNA level, microRNAs (miRNAs) can regulate gene expression and cell function. The aim of the present study is to investigate Fas targeting by miR-374a and find whether it can inhibit Fas-mediated apoptosis in primary human retinal pigment epithelial (RPE) cells under oxidative stress. So, the primary human RPE cells were transfected with pre-miR-374a pLEX construct using polymeric carrier and were exposed to H2 O2 (200 μM) as an oxidant agent for induction of Fas expression. Fas expression at mRNA and protein level was evaluated by quantitative real-time PCR and Western blot analysis, respectively. These results revealed that miR-374a could prevent Fas upregulation under oxidative conditions. Moreover, Luciferase activity assay confirmed that Fas could be a direct target of miR-374a. The cell viability studies demonstrated that caspase-3 activity was negligible in miR-374a treated cells compared to the controls. Our data suggest miR-374a is a negative regulator of Fas death receptor which is able to enhance the cell survival and protect RPE cells against oxidative conditions. J. Cell. Biochem. 118: 4854-4861, 2017. © 2017 Wiley Periodicals, Inc.
Insights
MicroRNA-374a (miR-374a) protects retinal pigment epithelial (RPE) cells from oxidative stress by inhibiting the Fas death receptor pathway. This finding offers potential therapeutic strategies for preventing vision loss in ocular diseases like AMD.
Area of Science:
- Ophthalmology
- Molecular Biology
- Cell Biology
Background:
- Oxidative stress in the eye can lead to retinal cell loss via the Fas-L/Fas pathway, a key factor in age-related macular degeneration (AMD).
- MicroRNAs (miRNAs) regulate gene expression post-transcriptionally, offering a mechanism to control cellular processes.
Purpose of the Study:
- To investigate if miR-374a targets Fas.
- To determine if miR-374a inhibits Fas-mediated apoptosis in human retinal pigment epithelial (RPE) cells under oxidative stress.
Main Methods:
- Primary human RPE cells were transfected with pre-miR-374a and exposed to hydrogen peroxide (H₂O₂).
- Fas expression was measured using quantitative real-time PCR and Western blot.
- Luciferase activity assay confirmed direct targeting of Fas by miR-374a.
- Cell viability was assessed by measuring caspase-3 activity.
Main Results:
- miR-374a prevented the upregulation of Fas under oxidative conditions.
- Fas was confirmed as a direct target of miR-374a.
- Caspase-3 activity was significantly reduced in cells treated with miR-374a, indicating enhanced cell survival.
Conclusions:
- miR-374a acts as a negative regulator of the Fas death receptor.
- miR-374a enhances RPE cell survival and provides protection against oxidative damage.
- These findings suggest miR-374a as a potential therapeutic agent for oxidative stress-related ocular diseases.