Altered regulation of mRNA and miRNA expression in epithelial and stromal tissue of keratoconus corneas [RNA]

Purpose: To evaluate conjunctival cell microRNA and mRNA expression in relation to observed phenotype and genotype of aniridia-associated keratopathy (AAK) in a cohort of subjects with congenital aniridia. Methods: Using impression cytology, bulbar conjunctival cells were sampled from 20 subjects with congenital aniridia and 20 age and sex-matched healthy control subjects. RNA was extracted and microRNA and mRNA analysis was performed using microarrays. Results were related to the presence and severity of AAK determined by a standardized clinical grading scale and to the genotype (PAX6 mutation?) determined by clinical genetics. Results: Of the 2549 microRNAs analyzed, 21 were differentially expressed relative to controls. Among these miR-204-5p, an inhibitor of corneal neovascularization, was downregulated 26.8-fold, while miR-5787 and miR-224-5p were upregulated 2.8 and 2.4-fold relative to controls, respectively. At the mRNA level, 539 transcripts were differentially expressed, among these FOSB and FOS were upregulated 17.5 and 9.7-fold respectively, and JUN by 2.9-fold, all components of the AP-1 transcription factor complex. Pathway analysis revealed dysregulation of several enriched pathways including PI3K-Akt, MAPK, and Ras signaling pathways in aniridia. For several microRNAs and transcripts, expression levels aligned with AAK severity, while in very mild cases with missense or non-PAX6 coding mutations, gene expression was only minimally altered. Conclusion: In aniridia, specific factors and pathways are strongly dysregulated in conjunctival cells, suggesting that the conjunctiva in aniridia is abnormally maintained in a pro-angiogenic and proliferative state, promoting the aggressivity of AAK in a mutation-dependent manner. Transcriptional profiling of conjunctival cells at the microRNA and mRNA levels presents a powerful, minimally-invasive means to assess the regulation of cell dysfunction at the ocular surface. MiRNA and mRNA expression profiles of epithelial and stromal cells from 8 patients with keratoconus compared to controls

目的：本研究旨在评估先天性无虹膜患者队列中结膜细胞的微小RNA（microRNA）与信使RNA（mRNA）表达特征，并分析其与无虹膜相关性角膜病（aniridia-associated keratopathy, AAK）的表型及基因型的关联。
方法：采用印迹细胞学（impression cytology）技术，分别采集20例先天性无虹膜患者与20例年龄、性别匹配的健康对照者的球结膜细胞。提取总RNA后，通过微阵列（microarray）技术开展微小RNA与信使RNA表达分析。将检测结果与采用标准化临床分级量表评估的AAK患病状态及严重程度，以及经临床遗传学检测确定的基因型（PAX6突变？）进行关联分析。
结果：在纳入分析的2549个微小RNA中，共有21个在患者与对照组间存在差异表达。其中，作为角膜新生血管形成抑制剂的miR-204-5p表达下调26.8倍；miR-5787与miR-224-5p则分别较对照组上调2.8倍与2.4倍。在信使RNA层面，共筛选出539个差异表达转录本，其中FOSB与FOS分别上调17.5倍与9.7倍，JUN上调2.9倍，三者均为AP-1转录因子复合物的核心组成成分。通路富集分析显示，无虹膜患者体内存在多条调控异常的富集通路，包括PI3K-Akt、MAPK及Ras信号通路。部分微小RNA与转录本的表达水平与AAK严重程度呈显著相关性；而在仅携带错义突变或非PAX6编码区突变的极轻度AAK患者中，基因表达仅发生轻微改变。
结论：先天性无虹膜患者的结膜细胞中存在特定因子与通路的显著调控异常，提示无虹膜患者的结膜组织异常维持于促血管生成与增殖状态，以突变依赖的方式加剧AAK的侵袭性进展。对结膜细胞开展微小RNA与信使RNA层面的转录谱分析，是一种高效且微创的手段，可用于评估眼表细胞功能异常的调控机制。与健康对照组相比，8例圆锥角膜（keratoconus）患者上皮细胞与基质细胞的微小RNA及信使RNA表达谱