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.

引言：近视通常是由发育过程中眼球过度伸长所引发的疾病，在全球范围内发病率较高。临床实践中，常通过光学手段或药物疗法控制近视进展，但这两类治疗方式的具体作用机制仍不明确。为验证这两种干预手段对眼球后极部组织的作用效果是否存在异同，本研究以鸡为模型，对其转录组的共性变化展开了研究。
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，以及联合生理盐水注射的负透镜诱导四种干预方式。随后检测各组眼球后极部各组织的基因表达变化，并比较不同组间的基因表达差异。此外，本研究采用半定量实时聚合酶链反应（RT-PCR）进一步探究视觉诱导下潜在候选基因的转录变化情况。
结果：基于转录组的RNA测序（RNA-seq）分析，本研究明确了两个治疗组三类组织中的差异表达转录本（DETs）之间的表达差异。通过京都基因与基因组百科全书（KEGG）富集分析发现，真核生物蛋白质翻译延伸因子1α2（EEF1A2）在脉络膜组织中富集于"利什曼病"通路，且在正透镜诱导组与阿托品注射组中均呈现表达上调。经qPCR验证的部分候选基因的表达水平与RNA-seq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注射阿托品控制近视的作用机制可能存在差异。EEF1A2或许在近视治疗过程中的脉络膜组织中发挥重要作用。