Table8_Differential methylation of microRNA encoding genes may contribute to high myopia.DOCX

Introduction: High myopia (HM), an eye disorder with a refractive error ≤−6.0 diopters, has multifactorial etiology with environmental and genetic factors involved. Recent studies confirm the impact of alterations in DNA methylation and microRNAs (miRNAs) on myopia. Here, we studied the combined aspects evaluating to the role of methylation of miRNA encoding genes in HM.
Materials and Methods: From the genome-wide DNA methylation data of 18 Polish children with HM and 18 matched controls, we retrieved differentially methylated CG dinucleotides localized in miRNA encoding genes. Putative target genes of the highest-ranked miRNAs were obtained from the miRDB and included in overrepresentation analyses in the ConsensusPathDB. Expression of target genes was assessed using the RNA sequencing data of retinal ARPE-19 cell line.
Results: We identified differential methylation of CG dinucleotides in promoter regions of MIR3621, MIR34C, MIR423 (increased methylation level), and MIR1178, MIRLET7A2, MIR885, MIR548I3, MIR6854, MIR675, MIRLET7C, MIR99A (decreased methylation level) genes. Several targets of these miRNAs, e.g. GNAS, TRAM1, CTNNB1, EIF4B, TENM3 and RUNX were previously associated with myopia/HM/refractive error in Europeans in genome-wide association studies. Overrepresentation analyses of miRNAs’ targets revealed enrichment in pathways/processes related to eye structure/function, such as axon guidance, transcription, focal adhesion, and signaling pathways of TGF-β, insulin, MAPK and EGF-EGFR.
Conclusion: Differential methylation of indicated miRNA encoding genes might influence their expression and contribute to HM pathogenesis via disrupted regulation of transcription of miRNAs’ target genes. Methylation of genes encoding miRNAs may be a new direction in research on both the mechanisms determining HM and non-invasive indicators in diagnostics.

引言：高度近视（High myopia, HM）指屈光不正≤−6.0屈光度的眼部疾病，其病因具有多因素性，涉及环境与遗传因素。近期研究证实，DNA甲基化（DNA methylation）与微小RNA（microRNAs, miRNAs）的异常对近视发生具有调控作用。本研究通过联合分析策略，探讨编码微小RNA的基因甲基化在高度近视中的作用。
材料与方法：从18例高度近视波兰儿童与18例匹配对照的全基因组DNA甲基化（genome-wide DNA methylation）数据中，筛选出位于编码微小RNA的基因内的差异甲基化CG二核苷酸。从miRDB数据库获取排名靠前的微小RNA的预测靶基因，并通过ConsensusPathDB数据库开展富集分析。利用视网膜ARPE-19细胞系的RNA测序数据，评估靶基因的表达水平。
结果：本研究发现，MIR3621、MIR34C、MIR423基因的启动子区域CG二核苷酸甲基化水平升高，而MIR1178、MIRLET7A2、MIR885、MIR548I3、MIR6854、MIR675、MIRLET7C、MIR99A基因的启动子区域CG二核苷酸甲基化水平降低。上述微小RNA的多个靶基因，例如GNAS、TRAM1、CTNNB1、EIF4B、TENM3及RUNX，此前已在欧洲人群的全基因组关联研究中被证实与近视/高度近视/屈光不正相关。对微小RNA靶基因的富集分析显示，其显著富集于与眼部结构/功能相关的通路与过程，包括轴突导向、转录调控、黏着斑，以及转化生长因子-β（TGF-β）、胰岛素、丝裂原活化蛋白激酶（MAPK）和表皮生长因子-表皮生长因子受体（EGF-EGFR）信号通路。
结论：上述编码微小RNA的基因甲基化异常，可能通过干扰微小RNA靶基因的转录调控，影响其表达并参与高度近视的发病过程。编码微小RNA的基因甲基化有望成为研究高度近视发生机制的新方向，同时也可作为无创诊断的潜在标志物。