Altered regulation of mRNA and miRNA expression in epithelial and stromal tissue of keratoconus corneas [RNA]. 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. Overall design: MiRNA and mRNA expression profiles of epithelial and stromal cells from 8 patients with keratoconus compared to controls

研究目的：评估先天性无虹膜受试者队列中，结膜细胞（conjunctival cell）的微小RNA（microRNA）与信使RNA（mRNA）表达情况，及其与无虹膜相关角膜病（aniridia-associated keratopathy，AAK）的表型和基因型的关联。
研究方法：采用印记细胞学（impression cytology），从20名先天性无虹膜受试者与20名年龄、性别匹配的健康对照者处采集球结膜细胞（bulbar conjunctival cell）。提取RNA后，通过微阵列（microarrays）开展微小RNA与信使RNA分析。将分析结果与通过标准化临床分级量表确定的AAK患病情况及严重程度、经临床遗传学检测确定的基因型（PAX6突变？）进行关联分析。
研究结果：在所分析的2549个微小RNA中，有21个与对照组相比呈现差异表达。其中，作为角膜新生血管形成抑制剂的miR-204-5p下调幅度达26.8倍；而miR-5787与miR-224-5p分别上调2.8倍与2.4倍。在信使RNA层面，共有539个转录本呈现差异表达，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（AP-1 transcription factor complex）的组成成分。通路分析显示，无虹膜受试者体内多条富集通路存在调控异常，包括PI3K-Akt信号通路、MAPK信号通路及Ras信号通路。部分微小RNA与转录本的表达水平与AAK严重程度相关；而在存在错义突变或非PAX6编码突变的极轻度病例中，基因表达仅出现轻微改变。
研究结论：无虹膜患者的结膜细胞中，特定因子与通路存在显著调控异常，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的进展。对结膜细胞进行微小RNA与信使RNA层面的转录谱分析，是一种高效的微创手段，可用于评估眼表细胞功能障碍的调控机制。
实验整体设计：对比8名圆锥角膜（keratoconus）患者与健康对照者的上皮细胞与基质细胞的微小RNA及信使RNA表达谱。