Dynamics Of Rna Editing In Laser-Induced Choroidal Neovascularization In Mice

Objective: Choroidal neovascularization(CNV)is a major pathological feature of various vision-threatening eye diseases,such as age-related macular degeneration(AMD).Epigenetic regulation plays an important role in the development of CNV.Nevertheless,such regulation mechanism has not been fully elucidated.RNA editing is an important epigenetic regulation mechanism.This study can not only improve our understanding in the feature and mechanism of RNA editing events during the development of CNV,but also propose a novel strategy for exploring the pathogenesis of CNV.Methods: CNV was induced in the right eyes(OD)in C57BL/6 mice using laser photocoagulation.Retinal flat mount was prepared for immunostaining using Isolectin B4.The total RNA of the retina was extracted,from which libraries were constructed using the poly A method,and sequenced on the Illumina Nova-Seq 2000.RNA sequencing reads mapped to the UCSC mouse genome(mm10)with splice junctions identified using RNA STAR,and gene expression was analyzed using Subreads and edge R.RNA editing sites were identified using Varscan2,and validated using PCR followed by Sanger sequencing.Gene ontology and pathway enrichment analysis were conducted using the Enrichr website.Human retinal microvascular endothelial cells(HRMECs)were transfected with the wildtype plasmid or edited plasmid of selected edited genes using Lipofectamine 3000.Cell proliferation,migration,and tube formation were assessed by CCK-8 assays,scratch assays and endothelial cell tube formation assays.RNA and protein expression level of genes and the molecular mechanisms of signal pathways were analyzed by RT-q PCR and Western Blot.Results: 1.we identified a total of 185,415,251,216 differential RNA editing(DRE)sites at the time of Day 0,Day 3,Day 7 and Day 14.The majority of DRE sites were up-regulated in CNV retina compared to controls at these four time points.The majority of DRE sites occurred in 3′ UTRs.The nucleotide neighbor 3’ of A-to-I DRE site showed preferred G,and G neighbor 5’ of A-to-I DRE site was generally not favored.the nucleotides 5′ and 3′ to C-to-U DRE site had a strong preference for AT enrichment.Apobec1,Apobec3 and Adar were differentially expressed between CNV and control cases,with increased expression in the CNV cases.DRE genes from the four time points exhibited significant gene ontology(GO)enrichment in synaptic and neural functions.Furthermore,we observed significant enrichment of pathway involved in the occurrence and development of CNV.2.Highly conserved RNA editing in the antizyme inhibitor 1(AZIN1)gene was found in the retinas of both CNV mice and AMD patients.Overexpression in HRMECs showed that the edited AZIN1 resulted in upregulation of vascular endothelial growth factor A(VEGFA)and ornithine decarboxylase(ODC)protein,and in turn accelerate proliferation,migration and tube formation,and thus could promote angiogenesis and CNV.Conclusion: In current study,we carried on a systematic research to the feature of RNA editing events in CNV mice from different time points and then demonstrated the role and mechanism of AZIN1 RNA editing in CNV by in vivo and in vitro experiments.This study can not only propose a novel strategy for exploring the pathogenesis of CNV,but also provide important references for the follow-up research of RNA editing event in CNV mice.