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Bing Jiang, Lili Xie, Mao Mao; MicroRNA-761 regulates programmed necrosis of retinal ganglion cells through targeting FADD. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2540.
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© ARVO (1962-2015); The Authors (2016-present)
Programmed necrosis is involved in progression of various diseases including degenerative eye diseases such as glaucoma. Recent studies have demonstrated that programmed necrosis is dependent on activation of receptor-interacting serine/threonine-protein kinases, RIPK1 and RIPK3 which may be negatively regulated by Fas-associated protein with death domain (FADD). MicroRNAs plays important roles in degenerative eye diseases, and the purpose of this study was to determine if microRNA-761 can regulate RIPK1- and RIPK3-dependent necrosis through targeting FADD in retinal ganglion cells (RGCs).
To induce programmed necrosis of RGCs in vitro and in vivo, primary mouse RGCs were treated with H2O2 and optic nerve crush (ONC) injury was performed in adult C57BL/6 mice . Apoptosis/necrosis was tested using the Annexin V/PI staining and TUNNEL/PI staining. Moreover, the dual luciferase assay was performed to test if microRNA-761 could regulate the FADD promoter activity and microRNA-761 mimics or inhibitors were transfected and the mRNA and protein levels of FADD were quantified to test if microRNA-761 can regulate FADD expression. Finally, whether the interaction of RIPK1 and RIPK3 can be influenced by the expression of FADD was tested through co-immunoprecipitation in the cellular model.
Our results showed that the apoptosis rate of RGCs was 51.2% when they were treated with 700μmol/L H2O2 for 24h. The necrosis rate was 89.1% when RGCs were treated with 900μmol/L H2O2 for 48h and the protein expression of FADD was significantly down-regulated, whereas the mRNA expression of FADD was up-regulated (p<0.01). Also, RGCs necrosis occurred in the mouse ONC model and the protein expression of FADD was significantly decreased (p<0.05) after 17 day of injury. Furthermore, up-regulation the expression of FADD could significantly decrease the necrosis rate of RGCs in both the cellular model and mouse ONC model (p<0.01). Finally, we also demonstrated that microRNA-761 could directly regulate FADD expression and FADD overexpression could inhibit the formation of RIPK1 and RIPK3 complexes in RGCs.
Our results reveal a novel model of RGC necrosis regulation, which is composed of microRNA-761 and FADD. Modulation of their expression levels may provide a new approach to prevent RGCs necrosis.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
RGCs necrosis occurred in cell culture model and mouse ONC model
microRNA-761 regulates FADD expression.
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