RNAseq of the peripheral cornea and limboscleral tissues of rats treated with systemic spironolactone in a model of corneal neovascularization

The aim of this study was to analyse the transcriptional regulation in the corneal and limbal tissues of rats treated with spironolactone in a model of corneal neovascularization (CNV) induced by limbal difficiency. CNV was induced by total corneal de-epithelialization and limbal resection. Rats were injected subcutaneously with spironolactone (25mg/kg/day) diluted in 90% olive oil and 10% DMSO or vehicle (90% olive oil and 10% DMSO) for 14 days. On day 3 (early inflammatory stage) and day 7 (angiogenic stage), rats were euthanized and the peripheral cornea and the limboscleral tissues were exised for RNA-sequencing. Total RNA samples were sequenced at the iGenSeq transcriptomic platform of the Brain and Spine Institute (ICM, Paris, France). RNA quality was checked by capillary electrophoresis (Agilent 2100 Bioanalyzer system) and RNA with integrity numbers (RIN) ranging from 7.8 to 8.2 was accepted for library generation. Quality of raw data has been evaluated with FastQC. Illumina DRAGEN bio-IT Platform (v3,8,4) was used to align reads on reference genome rd6 and quantification with genecode vM25 annotation gtf. Library orientation, library composition and coverage along transcripts were checked with Picard tools. differential analysis has been also conducted with edgeR. Multiple hypothesis adjusted p-values were calculated with the Benjamini-Hochberg procedure to control FDR. We identified genes differentially regulated by spironolactone vs. vehicle on day 3 and day 7.本研究旨在分析角膜缘缺陷诱导的角膜新生血管（corneal neovascularization, CNV）模型中，经螺内酯处理的大鼠角膜及角膜缘组织的转录调控情况。该模型通过全角膜上皮刮除联合角膜缘切除术诱导CNV生成。大鼠以皮下注射方式给予螺内酯（25mg/kg/天，溶于90%橄榄油与10%二甲基亚砜混合溶液）或溶剂对照（仅含90%橄榄油与10%二甲基亚砜的混合溶液），持续给药14天。分别于给药第3天（炎症早期）及第7天（血管生成期）安乐处死大鼠，切除外周角膜及角巩缘组织用于RNA测序（RNA-sequencing）。总RNA样本于法国巴黎脑与脊髓研究所（ICM, Paris, France）的iGenSeq转录组学平台完成测序。通过毛细管电泳（Agilent 2100 Bioanalyzer系统）检测RNA质量，选取RNA完整性指数（RNA Integrity Number, RIN）介于7.8至8.2之间的样本用于文库构建。使用FastQC对原始数据质量进行评估。采用Illumina DRAGEN生物信息IT平台（v3.8.4）将测序读段比对至参考基因组rd6，并基于GENCODE vM25注释GTF文件进行定量分析。使用Picard工具检测文库方向、文库组成及转录本覆盖度。采用edgeR进行差异表达分析。通过Benjamini-Hochberg法计算多重假设检验校正后的P值，以控制错误发现率（False Discovery Rate, FDR）。本研究鉴定出给药第3天及第7天，螺内酯组与溶剂对照组间存在差异调控的基因。