MicroRNA-761 Regulates Programmed Necrosis Of Retinal Ganglion Cells Through Targeting FADD

Purpose: Glaucoma is one of the leading blind-causing disease in the world.Glaucoma is a chronic optic neuropathy characterized by retinal ganglion cells(RGCs)death,resulting in damage to optic nerve head and retinal nerve fiber layer.Apoptosis was once considered to be the only mode of programmed death of RGCs.However,multiple data shows that in addition to apoptosis,programmed death also includes necroptosis recently.The purpose of this study is to investigate the regulation of FADD gene on necroptosis of RGCs in mice.Methods and results: We show here that RGCs’ programmed death includes necroptosis and FADD plays a crucial role in this part.We establish a mouse optic nerve crush(ONC)model and observe the death pattern of RGCs in 0-17 days after ONC by immunofluorescence staining.Mice are intraperitoneally injected with vehicle(PBS)or programmed necrosis inhibitor Necrostatin-1(Nec-1)2 weeks before ONC.This experiment confirms that programmed necrosis occurs in RGCs 14 days after ONC and Nec-1 can antagonize programmed necrosis effectively.Mice are intravitreal injected with AAV2-FADD virus to enhance FADD expression or AAV2-NC as a comparison.We find that FADD overexpression can reduce programmed necrosis in RGCs and improve RGCs survival in ONC model.We predict the upstream micro RNA(mi RNA)of FADD and the long non-coding RNA(lnc RNA)of the micro RNA by using bioinformatics software.We verify their binding by dual-luciferase reporter system.Mi RNA-761 can effectively regulate the expression of FADD and lnc RNA-NR＿045396 can control mi RNA-761’s expression.We demonstrate this process with mice models and the detail protocols will be described in methods of each part.We find that the decreased activity of mi RNA-761 can effectively increase the expression of FADD in RGCs,thus reducing the necroptosis after RGCs injury and improving the survival of RGCs.Lnc RNA-NR＿045396can regulate mi RNA-761 in a similar way to achieve the similar effect.Conclusion: 1.Programmed death of mouse RGCs includes programmed necrosis.2.FADD overexpression can reduce programmed necrosis in RGCs and improve RGCs survival in ONC model.3.Signal pathway lnc RNA-NR＿045396-mi RNA＿761-m RNA＿FADD can regulate FADD gene expression and improve RGCs survival by inhibiting programmed necrosis in ONC model.