LncRNA Uc007nnj.1 Mediates Ischemia-Induced Neuron Apoptosis In Mice Retina

Purpose: Retinal ischemia/reperfusion(I/R)injury-induced retinal ganglion cells(RGCs)apoptosis is a pathological basis of various ocular diseases,which produced disastrous influences on vision.Recently,several studies reported that long noncoding RNAs(lncRNAs)play important roles in ischemia disease while the contribution of lncRNAs in I/R-induced RGCs apoptosis is still unclear.Based upon the lncRNA chip assay,we aimed to investigate the role of lncRNA uc007 nnj.1 in the pathological process of ischemia-induced RGCs apoptosis.Methods:1、 In this experiment,primary retinal ganglion cells and C57BL/6J mice were used as the research objects to establish ischemia models in vitro and in vivo.Detected the expression levels of lncRNA uc007 nnj.1 at different time points for choosing the optimal intervention time.2、 LncRNA uc007 nnj.1 siRNA and lncRNA uc007 nnj.1 plasmid was transfected into RGCs cells,and RT-qPCR was used to detect the efficiency and flow cytometry to detect the RGCs apoptosis and Western blot to detect the expression of cleaved caspase-3.3、 FISH was used to clarify the subcellular localization of lncRNA.The bioinformatics database predicted that miR-155-5p is the downstream miRNA molecule of lncRNA uc007 nnj.1.Dual-luciferase experiment verified the relationship between them.The role of miR-155-5p in ischemia-induced RGCs apoptosis was examined after transfected miR-155-5p mimics.4、 The bioinformatics database predicted that the target gene of miR-155-5p was Tle4.Dual-luciferase experiment verified their target-binding relationships of them.Western blot explored the expression of Tle4 in ischemia-induced RGCs.Using FCM and western blot detect Tle4 function in ischemia-induced RGCs apoptosis.5、 RT-qPCR and western blot were used to explore the role of miR-155-5p in lncRNA uc007 nnj.1 mediate ischemia-induced RGCs apoptosis by co-transfection of lncRNA uc007 nnj.1 siRNA and miR-155-5p inhibitor.6、 Acute intraocular hypertension animal models were performed and randomly grouped into several groups,including the scramble group,Scramble/I/R group,and lncRNA uc007 nnj.1 siRNA group;lncRNA uc007 nnj.1 siRNA/I/R group.Immunofluorescence and TUNEL were selected to observe RGCs apoptosis.Electroretinogram detects visual function in vivo.Results:1、 Our study demonstrated that lncRNA uc007 nnj.1 is highly expressed in I/R-induced RGCs.The best optimal condition in vitro was ischemia 2 hours/ reperfusion 2 hours,and the optimal intervention time in vivo was ischemia 1 hour and reperfusion 24 hours.2、 RT-qPCR results showed that compared with the Scramble group,lncRNA uc007 nnj.1 siRNA could significantly inhibit lncRNA uc007 nnj.1 expression(p<0.05),while the expression of lncRNA uc007 nnj.1 was significantly increased(p<0.05)after transfected lncRNA uc007 nnj.1 plasmid.Compared with the Scramble/I/R,knockdown lncRNA uc007 nnj.1 significantly inhibited ischemia-induced neuronal apoptosis(p<0.05)and overexpression lncRNA uc007 nnj.1 promoted ischemia-induced RGCs apoptosis(p<0.05).3、 Fluorescence in situ hybridization experiments showed that the lncRNA uc007 nnj.1 was located in the cytoplasm.The bioinformatics database predicted that miR-155-5p was the downstream target of lncRNA uc007 nnj.1,and Dual-luciferase results indicated that miR-155-5p mimic could inhibit the luciferase activity when co-transfection RGCs with lncRNA uc007 nnj.1(WT)plasmid comparing with negative control or with lncRNA uc007 nnj.1 mutant plasmid(p<0.05).The FISH assay revealed that lncRNA uc007 nnj.1 and miR-155-5p could be combined.RT-qPCR results indicated that compared with the Scramble group,silence lncRNA uc007 nnj.1significantly upregulated miR-155-5p expression(p<0.05);FCM shows that miR-155-5p mimic significantly decreased the number of RGCs apoptosis(p<0.05)and immunoblot results verified that miR-155-5p mimic significantly inhibited the activity of caspase-3(p<0.05)compared with the Scramble/I/R group.4、 The biological database indicated that Tle4 is a potential target for miR-155-5p.The dual-luciferase result showed that miR-155-5p mimic significantly reduced the luciferase activity in the Tle4 WT group compared with the Tle4 MUT group(p<0.05).Mi R-155-5p mimic could inhibit Tle4 expression compared with the Scramble group(p<0.05).Knockdown Tle4 significantly reduced the apoptosis in RGCs compared with the Scramble/I/R group(p<0.05).5、 Compared with the lncRNA uc007 nnj.1 siRNA/I/R group,knockdown miR-155-5p and lncRNA uc007 nnj.1 expression significantly increased RGCs apoptosis(p<0.05).6、 In vivo experiments showed that compared with the Scramble/I/R group,intravitreous preinjection of lncRNA uc007 nnj.1siRNA upregulated the expression of miR-155-5p(p<0.05),inhibited Tle4expression(p<0.05).Immunofluorescence staining of Tuj1 showed that compared to the Scramble/I/R group,the survival rate of RGCs was higher in the lncRNA siRNA/I/R group(p<0.05).TUNEL staining revealed that the number of TUNEL positive cells was reduced in the lncRNA siRNA/I/R group(p<0.05).Scotopic ERG demonstrated the knockdown of lncRNA uc007 nnj.1 significantly prevented I/R-induced b-wave decline.Conclusions: The research revealed that lncRNA uc007 nnj.1promote ischemia-induced RGCs apoptosis in vivo and in vitro.LncRNA uc007 nnj.1 mediates ischemia-induced neuronal apoptosis via the miR-155-5p/Tle4 axis,which provides new insights and a theoretical basis for the prevention and treatment of ischemia-induced RGCs apoptosis.