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chao jiang, Xue Chen, Chen Zhao; MicroRNA-302d promotes dedifferentiation of the retinal pigment epithelium by targeting the CDKN1A. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5358.
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MicroRNA plays a critical role in dedifferentiation of retinal pigment epithelium (RPE) cells. In our previous studies, we found the hsa-miR-302d-3p was down-regulated in RPE differentiation process. However, the microRNA mediated dedifferentiation in human RPE cells has not been thoroughly reported. In our study, we aim to analyze the role of miR-302d in RPE dedifferentiation
We respectively transfected hsa-miR-302d mimic and inhibitor into hiPSC-RPE at 30 day to see its role on cell differentiation. The related rpe genes and proteins expression in the rpe differentiation process were observed by Q-PCR and western blotting. We also observe the expression of ZO-1 in ips-rpe cells according to immunofluorescence. We next determined whether miR-302d would promote proliferation and migration in ARPE-19 cells. Furthermore, cell cycle were observed by flow cytometry after transfected hsa-miR-302d mimic and inhibitor. We confirm that CDKN1A as a direct target of miR-302d in ARPE-19 cells. Also, we established an AMD model for the treatment of hydrogen peroxide with H2O2. We observed the phagocytosis and apoptosis of RPE cells after transfected hsa-miR-302d mimic and inhibitor
We found relative mRNA expressions were significantly decreased of RPE65, RLBP1, MERTK, BEST1, CTNNB1 and TJP1 in hiPS-RPE at 30 days after transfected with miR-302d mimic compared to NC mimic. Also the relative mRNA expressions were significantly increased of RPE65, RLBP1, MERTK, BEST1, and TJP1 in hiPSC-RPE at 30 days transfected with miR-302d inhibitor compared to NC inhibitor. Both proliferation and migration were promoted by miR-302d overexpression after transfection with ARPE19 cells. And the luciferase reporter gene assay showed that CDKN1A is the target gene of microRNA302d. After transfected with miR-302d minics and inhibitor, we then treated the cells with 200uM H2O2. We found that with the oxidative stress model, the cell apoptosis rates significantly increased after transfected with miR-302d inhibitor compared to NC inhibitor
Mir-302d is associated with RPE cells proliferation and migration as well as apoptosis under oxidative stress, indicating that Mir-302d negatively regulates RPE cells differentiation.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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