MicroRNA (miRNA) play a significant role in the pathogenesis of complex neurodegenerative diseases including age-related macular degeneration (AMD), acting as post-transcriptional gene suppressors through their association with argonaute 2 (AGO2) - a key member of the RNA Induced Silencing Complex (RISC). Identifying the retinal miRNA/mRNA interactions in health and disease will provide important insight into the key pathways miRNA regulate in disease pathogenesis and may lead to potential therapeutic targets to mediate retinal degeneration. To identify the active miRnome targetome interactions in the healthy and degenerating retina, AGO2 HITS-CLIP was performed using a rodent model of photoreceptor degeneration. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data was performed to identify the cellular location of AGO2 and key members of the microRNA targetome in the retina. AGO2 findings were verified by in situ hybridization (RNA) and immunohistochemistry (protein). Analysis revealed a similar miRnome between healthy and damaged retinas, however, a shift in the active targetome was observed with an enrichment of miRNA involvement in inflammatory pathways. This shift was further demonstrated by a change in the seed binding regions of miR-124-3p, the most abundant retinal AGO2-bound miRNA, and has known roles in regulating retinal inflammation. Additionally, photoreceptor cluster miR-183/96/182 were all among the most highly abundant miRNA bound to AGO2. Following damage, AGO2 expression was localized to the inner retinal layers and more in the OLM than in healthy retinas, indicating a locational miRNA response to retinal damage. This study provides important insight into the alteration of miRNA regulatory activity that occurs as a response to retinal degeneration and explores the miRNA-mRNA targetome as a consequence of retinal degenerations. Further characterisation of these miRNA/mRNA interactions in the context of the degenerating retina may provide an important insight into the active role these miRNA may play in diseases such as AMD.

中文翻译：

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功能性 microRNA 靶标在视网膜中经历变性诱导的转变


MicroRNA (miRNA) 在包括年龄相关性黄斑变性 (AMD) 在内的复杂神经退行性疾病的发病机制中发挥着重要作用，通过与 argonaute 2 (AGO2)（RNA 诱导沉默的关键成员）相关，充当转录后基因抑制因子复杂（RISC）。识别健康和疾病中视网膜 miRNA/mRNA 相互作用将为了解 miRNA 在疾病发病机制中调节的关键途径提供重要见解，并可能导致介导视网膜变性的潜在治疗靶点。为了确定健康和退化视网膜中活跃的 miRnome 靶组相互作用，使用光感受器退化的啮齿动物模型进行了 AGO2 HITS-CLIP。对公开的单细胞 RNA 测序 (scRNAseq) 数据进行分析，以确定 AGO2 的细胞位置和视网膜中 microRNA 靶组的关键成员。 AGO2 结果通过原位杂交 (RNA) 和免疫组织化学 (蛋白质) 进行了验证。分析显示，健康和受损视网膜之间存在相似的 miRnome，然而，随着参与炎症途径的 miRNA 的富集，观察到活性靶标组的变化。 miR-124-3p（最丰富的视网膜 AGO2 结合 miRNA）种子结合区域的变化进一步证明了这种转变，并且在调节视网膜炎症中具有已知的作用。此外，光感受器簇 miR-183/96/182 都是与 AGO2 结合的最丰富的 miRNA 之一。损伤后，AGO2 表达定位于视网膜内层，并且 OLM 中的表达量高于健康视网膜，表明 miRNA 对视网膜损伤有定位反应。 这项研究为视网膜变性反应中 miRNA 调节活性的改变提供了重要的见解，并探索了视网膜变性导致的 miRNA-mRNA 靶标组。在退化视网膜的背景下进一步表征这些 miRNA/mRNA 相互作用可能会为了解这些 miRNA 在 AMD 等疾病中可能发挥的积极作用提供重要的见解。