Abstract
Purpose :
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).
Methods :
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.
Results :
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.
Conclusions :
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.