Comprehensive analysis of the transcriptome-wide m6A methylome in pterygium by MeRIP sequencing. Comprehensive analysis of the transcriptome-wide m6A methylome in pterygium by MeRIP sequencing

Aim: Pterygium is a common ocular surface disease, which is affected by a variety of factors. Invasion of the cornea can cause severe vision loss. N6-methyladenosine (m6A) is a common post-transcriptional modification of eukaryotic mRNA, which can regulate mRNA splicing, stability, nuclear transport, and translation. To our best knowledge, there is no current research on the mechanism of m6A in pterygium. Methods: We recruited 24 pterygium patients from Shanghai Yangpu Hospital, and the level of m6A modification was detected using an m6A RNA Methylation Quantification Kit. Expression and location of METTL3, a key m6A methyltransferase, were identified by immunostaining. Then we used m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq), RNA sequencing (RNA-seq) and bioinformatics analyses to compare the differential expression of m6A methylation in pterygium and normal conjunctival tissue. Results: We identified 2949 dysregulated m6A peaks in pterygium tissue, of which 2145 were significantly upregulated and 804 were significantly downregulated. The altered m6A peak of genes were found to play a key role in the Hippo signaling pathway and endocytosis. Joint analyses of MeRIP-seq and RNA-seq data identified 72 hypermethylated m6A peaks and 15 hypomethylated m6A peaks in mRNA. After analyzing the differentially methylated m6A peaks and synchronously differentially expressed genes, we searched the Gene Expression Omnibus database and identified five genes related to the development of pterygium (DSP, MXRA5, ARHGAP35, TMEM43, and OLFML2A). Conclusion: Our research shows that m6A modification plays an important role in the development of pterygium and can be used as a potential new target for the treatment of pterygium in the future. Overall design: mRNA profiles and m6A profiles in pterygium

研究目的：翼状胬肉是一种常见眼表疾病，受多种因素影响，当其侵入角膜时可引发严重视力丧失。N6-甲基腺嘌呤（N6-methyladenosine，m6A）是真核mRNA常见的转录后修饰方式，可调控mRNA剪接、稳定性、核运输及翻译过程。据目前所知，尚无针对m6A在翼状胬肉中作用机制的相关研究。研究方法：本研究从上海杨浦医院招募24名翼状胬肉患者，采用m6A RNA甲基化定量试剂盒检测样本的m6A修饰水平；通过免疫染色鉴定关键m6A甲基转移酶METTL3的表达与定位。随后，我们利用m6A修饰RNA免疫沉淀测序（m6A-modified RNA immunoprecipitation sequencing，MeRIP-seq）、RNA测序（RNA sequencing，RNA-seq）及生物信息学分析，对比翼状胬肉组织与正常结膜组织中m6A甲基化的差异表达情况。研究结果：本研究在翼状胬肉组织中鉴定出2949个差异表达的m6A峰，其中2145个显著上调，804个显著下调。差异m6A峰相关基因在Hippo信号通路与胞吞过程中发挥关键调控作用。对MeRIP-seq与RNA-seq数据进行联合分析后，共鉴定出mRNA上72个高甲基化m6A峰及15个低甲基化m6A峰。在分析差异甲基化m6A峰与同步差异表达基因后，我们检索了基因表达综合（Gene Expression Omnibus，GEO）数据库，筛选出5个与翼状胬肉发生发展相关的基因：DSP、MXRA5、ARHGAP35、TMEM43及OLFML2A。研究结论：本研究证实m6A修饰在翼状胬肉的发生发展中扮演重要角色，未来可作为翼状胬肉治疗的潜在新型靶点。整体实验设计：翼状胬肉组织中的mRNA表达谱与m6A修饰谱