The RNA-seq QC data.

BackgroundThe axonal growth capacity of retinal ganglion cells decreases dramatically within the first day of birth, and the axonal regeneration after injury in mature mammals is very limited. Here, this study aimed to delineate the transcriptomic changes associated with altered axonal growth capacity and to identify the key genes associated with axonal regeneration by the RNA sequencing (RNA-Seq) analysis.MethodsThe whole retinas from the mice of embryonic day (E) 20, postnatal day (P) 1 and P3 were collected at 6 hours after optic nerve crush (ONC). Differentially expressed genes (DEGs) for ONC or ages were identified by the RNA-Seq analysis. K-means analysis was conducted for the clustering of DEGs based on expression patterns. Enrichment of functions and signaling pathways analysis were performed based on Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and Gene Set Enrichment analysis (GSEA). Quantitative real time polymerase chain reaction (qRT-PCR) was used to validate the DEGs selected from the RNA-Seq analysis.ResultsIn total, 5,408 DEGs were identified for ages, and 2,639 DEGs in neonatal mouse retina after ONC. K-means analysis revealed 7 clusters in age-DEGs and 11 clusters in ONC-DEGs. The GO, KEGG and GSEA pathway analyses identified significantly enrichment of DEGs in the visual perception and phototransduction for the age effect, and the break repair, neuron projection guidance, and immune system pathway for the ONC. PPI analysis identified hub genes in the axon-related gene cluster. The expressions of Mlc1, Zfp296, Atoh7, Ecel1, Creb5, Fosb, and Lcn2, thought to be involved in RGC death and axonal growth were validated by qRT-PCR.ConclusionsThis study, for the first time, delineated the gene expression changes following ON injury in embryonic and neonatal mice, providing a new resource of age- and injury-driven data on axonal growth capacity.背景：视网膜神经节细胞（retinal ganglion cells）的轴突生长能力在出生后第一日便急剧下降，成熟哺乳动物视神经损伤后的轴突再生能力亦极为有限。本研究借助RNA测序（RNA sequencing, RNA-Seq）技术，旨在阐明与轴突生长能力改变相关的转录组变化，并筛选出参与轴突再生的关键基因。 方法：分别采集胚胎第20天（E20）、出生后第1天（P1）及出生后第3天（P3）的小鼠在视神经钳夹损伤（optic nerve crush, ONC）后6小时的全视网膜组织。通过RNA-Seq分析，分别鉴定出不同年龄组及ONC处理组的差异表达基因（differentially expressed genes, DEGs）。基于基因表达模式，采用K-means聚类分析对DEGs进行聚类分组。依托基因本体（Gene Ontology, GO）、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）数据库及基因集富集分析（Gene Set Enrichment Analysis, GSEA），开展功能富集与信号通路分析。采用实时定量聚合酶链式反应（quantitative real time polymerase chain reaction, qRT-PCR）对RNA-Seq筛选得到的DEGs进行实验验证。 结果：本研究共鉴定得到5408个年龄相关DEGs，以及2639个ONC处理后新生小鼠视网膜中的DEGs。K-means聚类分析显示，年龄相关DEGs可划分为7个聚类簇，ONC相关DEGs则可划分为11个聚类簇。GO、KEGG及GSEA通路分析结果表明，年龄相关DEGs显著富集于视觉感知与光转导通路，而ONC相关DEGs显著富集于断裂修复、神经元投射导向及免疫系统通路。蛋白质相互作用（protein-protein interaction, PPI）分析筛选得到轴突相关基因簇中的核心基因（hub genes）。经qRT-PCR验证，Mlc1、Zfp296、Atoh7、Ecel1、Creb5、Fosb及Lcn2的表达水平与RNA-Seq结果一致，上述基因被认为参与视网膜神经节细胞死亡及轴突生长过程。 结论：本研究首次阐明了胚胎期与新生期小鼠视神经损伤后的基因表达变化，为轴突生长能力相关的年龄及损伤依赖性转录组数据提供了全新的研究资源。