Supplementary Material for: Mechanism of myopic defocus or atropine for myopia control: different or similar ways?

Abstract Introduction: Myopia is usually caused by excessive elongation of the eye during development. This condition is common worldwide. In clinical practice, the progression of myopia is commonly controlled through optical or drug measures, but the specific mechanisms underlying these two treatments remain unclear. To verify whether the effects of these two treatments on posterior-pole tissues are similar or different, we studied a set of common transcriptional changes in chicken models. Methods: Chicks were divided into four groups, and they were given the intervention measures of plus-lens induction, minus-lens induction, minus-lens induction with atropine injection, and minus-lens induction with saline injection. Then, the genetic changes in each tissue at the posterior pole were detected, and the results of different genes were compared. A semiquantitative real-time polymerase chain reaction (RT-PCR) method was used to further study the visually induced changes in the transcription of potential candidate genes. Results: Based on RNA-sequencing (RNA-seq) analysis of the transcriptome, we identified variations between the differentially expressed transcripts (DETs) in three tissues from the two treatment groups. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, eukaryotic protein translation elongation factor 1α2 (EEF1A2) was enriched in the ‘leishmaniasis’ pathway in the choroid and showed increased expression in both the plus-lens induction and injection atropine groups. The expression levels of selected genes verified by qPCR were concordant with the RNA-seq data. Conclusions: Overlapping differentially expressed mRNAs of only one-tenth could suggest a different mechanism of myopic defocus and intravitreal injection of atropine controlling myopia. EEF1A2 might play an important role in the choroid during the treatment of myopia.摘要及引言：近视通常由发育过程中眼球过度伸长引发，该病症在全球范围内高发。临床实践中，常通过光学手段或药物手段控制近视进展，但这两类治疗方式的具体作用机制仍未明确。为验证这两类治疗方式对眼球后极部组织的作用效果是否存在异同，本研究以鸡模型为对象，分析了其中的一组共通转录变化。方法：将雏鸡分为四组，分别施加正透镜诱导、负透镜诱导、联合阿托品注射的负透镜诱导、联合生理盐水注射的负透镜诱导四种干预措施。随后检测各组眼球后极部各组织的基因表达变化，并对不同基因的检测结果进行对比分析。采用半定量实时聚合酶链反应（semiquantitative real-time polymerase chain reaction, RT-PCR）方法，进一步探究视觉诱导下潜在候选基因的转录水平变化。结果：基于转录组RNA测序（RNA-sequencing, RNA-seq）分析，本研究鉴定出两组治疗方案对应三种组织内差异表达转录本（differentially expressed transcripts, DETs）的差异情况。通过京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析，真核蛋白翻译延伸因子1α2（eukaryotic protein translation elongation factor 1α2, EEF1A2）在脉络膜的「利什曼病（leishmaniasis）」通路中富集，且在正透镜诱导组与阿托品注射组中均呈现表达上调。经定量聚合酶链反应（quantitative polymerase chain reaction, qPCR）验证的筛选基因表达水平与RNA-seq数据结果一致。结论：仅占十分之一的重叠差异表达mRNA，提示近视性离焦与玻璃体腔内注射阿托品控制近视的作用机制存在差异。EEF1A2或许在近视治疗过程中的脉络膜组织中发挥重要作用。