NSUN2 介导的 RNA m5C 甲基化酶调控角膜上皮伤口愈合。
The RNA m5C Methylase NSUN2 Modulates Corneal Epithelial Wound Healing.
机构信息
School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China.
State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou, Zhejiang, China.
出版信息
Invest Ophthalmol Vis Sci. 2023 Mar 1;64(3):5. doi: 10.1167/iovs.64.3.5.
PURPOSE
The emerging epitranscriptomics offers insights into the physiopathological roles of various RNA modifications. The RNA methylase NOP2/Sun domain family member 2 (NSUN2) catalyzes 5-methylcytosine (m5C) modification of mRNAs. However, the role of NSUN2 in corneal epithelial wound healing (CEWH) remains unknown. Here we describe the functional mechanisms of NSUN2 in mediating CEWH.
METHODS
RT-qPCR, Western blot, dot blot, and ELISA were used to determine the NSUN2 expression and overall RNA m5C level during CEWH. NSUN2 silencing or overexpression was performed to explore its involvement in CEWH both in vivo and in vitro. Multi-omics was integrated to reveal the downstream target of NSUN2. MeRIP-qPCR, RIP-qPCR, and luciferase assay, as well as in vivo and in vitro functional assays, clarified the molecular mechanism of NSUN2 in CEWH.
RESULTS
The NSUN2 expression and RNA m5C level increased significantly during CEWH. NSUN2 knockdown significantly delayed CEWH in vivo and inhibited human corneal epithelial cells (HCEC) proliferation and migration in vitro, whereas NSUN2 overexpression prominently enhanced HCEC proliferation and migration. Mechanistically, we found that NSUN2 increased ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) translation through the binding of RNA m5C reader Aly/REF export factor. Accordingly, UHRF1 knockdown significantly delayed CEWH in vivo and inhibited HCEC proliferation and migration in vitro. Furthermore, UHRF1 overexpression effectively rescued the inhibitory effect of NSUN2 silencing on HCEC proliferation and migration.
CONCLUSIONS
NSUN2-mediated m5C modification of UHRF1 mRNA modulates CEWH. This finding highlights the critical importance of this novel epitranscriptomic mechanism in control of CEWH.
目的
新兴的表观转录组学为各种 RNA 修饰的生理病理作用提供了新的见解。RNA 甲基转移酶 NOP2/Sun 结构域家族成员 2（NSUN2）催化 mRNA 上 5-甲基胞嘧啶（m5C）的修饰。然而，NSUN2 在角膜上皮伤口愈合（CEWH）中的作用尚不清楚。在这里，我们描述了 NSUN2 介导 CEWH 的功能机制。
方法
使用 RT-qPCR、Western blot、点印迹和 ELISA 来确定 CEWH 过程中 NSUN2 的表达和总 RNA m5C 水平。通过体内和体外实验研究 NSUN2 沉默或过表达对 CEWH 的影响。整合多组学来揭示 NSUN2 的下游靶标。MeRIP-qPCR、RIP-qPCR 和荧光素酶测定，以及体内和体外功能测定，阐明了 NSUN2 在 CEWH 中的分子机制。
结果
在 CEWH 过程中，NSUN2 的表达和 RNA m5C 水平显著增加。NSUN2 敲低显著延迟体内 CEWH，并抑制体外人角膜上皮细胞（HCEC）增殖和迁移，而 NSUN2 过表达则显著增强 HCEC 的增殖和迁移。机制上，我们发现 NSUN2 通过 RNA m5C 阅读器 Aly/REF 出口因子结合增加泛素样包含 PH 和 RING 指域 1（UHRF1）的翻译。因此，UHRF1 敲低显著延迟体内 CEWH，并抑制体外 HCEC 增殖和迁移。此外，UHRF1 过表达有效挽救了 NSUN2 沉默对 HCEC 增殖和迁移的抑制作用。
结论
NSUN2 介导的 UHRF1 mRNA 的 m5C 修饰调节 CEWH。这一发现强调了这种新型表观转录组学机制在控制 CEWH 中的重要性。
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