Setd7靶向p21调控角膜上皮损伤修复的分子机制研究

Corneal epithelial wound healing (CEWH) plays a critical role in maintaining corneal integrity, but the mechanisms involved in this process remain to be investigated in depth. Histone methylation modifications are important components of epigenetic regulation. We performed preliminary studies showing that during wound healing there are significant changes occurring in H3K4 methyltransferase Setd7, H3K4me and p21 expression during this process. Such changes suggest that Setd7-mediated H3K4me targeted p21 can be important effectors of the CEWH process. On the basis, we first established a mice CEWH model to extract epithelial histones. Two dimensional-nano-LC-MS/MS analysis was performed along with western blot and real time qRT-PCR to identify the differences in histone methylation expression patterns between normal control epithelial cells and those involved in the wound healing process. Human corneal epithelial cell specific for Setd7 losses or gains of function were generated to determine their effects on these epithelial cellular responses to injury. Additionally, molecular mechanisms of Setd7-targeted p21 will be identified by ChIP. The functional mechanisms of specific Setd7 on CEWH will also be verified in an in vivo model. This study will clarify the molecular mechanisms of Setd7-mediating control of H3K4 methylation status during CEWH as well as identify novel potential drug targets for more selective intervention and in treatment of patients with corneal wounds compromising the CEWH process.

角膜上皮的损伤修复对于维持角膜的完整性至关重要，其分子调控机制尚未明了。组蛋白甲基化修饰是一种极为重要的表观遗传调控机制，我们的前期工作发现：角膜上皮损伤修复中组蛋白甲基化转移酶Setd7以及H3K4me下调，同时p21也明显降低，提示Setd7介导的H3K4me靶向p21调控角膜上皮的损伤修复。在此基础上，本项目首先联合采用质谱和生物信息学等技术，系统分析小鼠角膜损伤修复模型中组蛋白甲基化修饰图谱。进而以人角膜上皮细胞为模型，通过基因干扰和过表达技术，明确Setd7的生物学功能。通过ChIP明确Setd7 靶向p21的表观调控机制以及H3K4me修饰的靶位点。最后通过小鼠模型在体验证Setd7对角膜上皮的损伤修复的效应机制。本研究将阐明Setd7介导的组蛋白甲基化在角膜上皮损伤修复中的分子调控机制，为角膜损伤相关疾病的临床治疗提供新的思路。

角膜上皮损伤修复（CEWH）对于维持角膜的完整性至关重要，其分子调控机制尚未明了。组蛋白甲基化修饰是一种极为重要的表观遗传调控机制。本项目的研究发现H3K4me水平以及相应的甲基化酶Setd7的表达在CEWH中均明显下调。体内实验显示下调的Setd7可显著加速CEWH进程；体外实验显示敲减Setd7明显促进人角膜上皮细胞（HCEC）的迁移和增殖，而过表达Setd7明显抑制了HCEC的迁移和增殖。Setd7抑制HCEC增殖的机制是细胞周期阻滞在G1期，而非诱导细胞凋亡。敲减Setd7可通过降低p21启动子H3K4me水平降低其表达进而抑制HCEC细胞的迁移和增殖。角膜上皮损伤引起p21的下调，并降低p21启动子甲基化水平。我们还通过STING数据库鉴定到了与Setd7高度互作的表观调控分子DNA甲基化转移酶1 （DNMT1）和H3K9me3甲基转移酶1（SUV39H1）。然后在体内/外实验中阐明了DNMT1介导DNA甲基化靶向miR-200a和p15 (CDKN2B)促进CEWH的详细机制，继而也揭示了Suv39h1通过H3K9me3介导p27沉默而调控CEWH的功能机制。此外，考虑到CEWH涉及的细胞功能表型类似于眼内葡萄膜黑色素瘤（UM）的生长特性，本项目研究在成功解析RNA m6A修饰靶向c-Met调控UM增殖和迁移的基础上，还发现了RNA m6A修饰和RNA m5C修饰在调节CEWH中起了重要作用。本项目的研究不仅阐明了传统的表观遗传学—组蛋白修饰和DNA甲基化—在CEWH中的生物学功能和分子机制，也初步探讨了前沿表观遗传学—RNA m6A和RNA m5C—在CEWH中的重要作用，为角膜损伤相关疾病的临床治疗提供新的思路。