利用CRISPR/Cas9技术研究Ret受体泛素化对HSV-1感染性角膜炎角膜上皮细胞凋亡的调控机制

Herpes simplex keratitis (HSK) is the leading cause of blindness in corneal infective disease. The antiapoptotic characterisics of herpes simplex virus-1 (HSV-1) in infected host cells is the vital mechanism of virus replication and propagation, and the deterioration of corneal injury. We found previously that Ret expression was reduced in human corneal epithelial cells (HCE) infected with HSV-1. Furthermore, cell apoptosis was down-regulated in Ret knock-down HCEs, while cell apoptosis was up-regulated in Ret overexpressed HCEs, which indicating that Ret promotes the apoptosis of HCE infected with HSV-1. Besides, ubiquitinated Ret was found increased upon infection. Therefore, we hypothesize that the enhancement of Ret ubiquitination is a vital mechanism of the antiapoptotic characterisics of HSV-1 in HCE. However, the specific ubiquitin-protein ligase (E3) of Ret and the regulatory mechanism on HCE apoptosis still remain unknown. Therefore, we aim to conduct the below studies: (1) screening the specific ubiquitin ligase (E3) of Ret using the customized CRISPR Knock -Out pooled sgRNA library of E3. (2) investigating the regulatory role of specific E3 of Ret on apoptosis of HCE in HSK. (3) assessing the effects of specific E3 inhibitors on the apoptosis of HCE and the replication of virus in vivo and in vitro experiments. This project would provide new insights into the mechanisms of the antiapoptotic characterisics of specific E3 of Ret in HSK. In addition, the outcome of this study may lead to a new therapeutic strategy using E3 inhibitors to treat HSK.

单纯疱疹病毒性角膜炎（HSK）是致盲率最高的角膜病变，抗宿主细胞凋亡是单纯疱疹病毒-1（HSV-1）在角膜组织复制传播，病情进展的重要机制。我们的前期研究发现，在HSV-1感染的角膜上皮细胞中Ret受体表达下调，进一步敲除/过表达Ret，细胞凋亡相应减少/增加，提示Ret具有促凋亡作用；此外，我们发现HSK中泛素化Ret水平增高。据此，我们推测Ret泛素化降解增加是HSV-1抑制角膜上皮细胞凋亡的重要机制。但Ret泛素化降解的特异性泛素连接酶（E3）及作用机制尚不清除，本课题拟开展如下研究：（1）文库筛选：使用CRISPR文库筛选技术明确Ret降解的特异性E3酶；（2）功能验证：过表达或者敲除筛选出的E3基因，验证其对HSK中角膜上皮细胞凋亡的作用；（3）靶向干预：在HSV-1体内外模型中观察E3抑制剂对细胞凋亡和病毒复制的干预作用。本课题的研究将为靶向抑制病毒复制和治疗HSK提供新策略。

单纯疱疹病毒性角膜炎（HSK）为致盲率最高的角膜病变，也是世界范围内感染性单眼盲的首位病因。抗宿主细胞凋亡是单纯疱疹病毒-1（HSV-1）在角膜组织复制传播，病情进展的重要机制。因此研究HSV-1的抗宿主细胞凋亡机制，寻找有效的干预手段，对于限制病毒复制和控制病情进展具有重要意义。.. 我们的研究发现，感染HSV-1的人角膜上皮细胞（HCE）Ret表达下调；进一步用CRISPR/Cas9技术敲除Ret基因，细胞凋亡减少，病毒复制增加，而过表达Ret则细胞凋亡增加，病毒复制减少，提示HSV-1感染状态下Ret对角膜上皮凋亡有促进作用；另外我们发现Ret在角膜上皮细胞中通过泛素-蛋白酶体通路降解，且HSV-1感染后泛素化Ret水平增高。这些研究结果提示，Ret泛素化降解增加可能是HSV-1抗角膜上皮细胞凋亡的重要机制。.. Cbl(Casitas B-lineage lymphoma)蛋白是一类在生物体内广泛分布的细胞内蛋白，属于泛素连接酶E3系。c-Cbl在细胞内广泛表达，但主要存在于细胞质中，在细胞信号通路和蛋白泛素化中具有重要的生物学作用。我们的实验结果证实c-Cbl在角膜上皮中表达；Ret和c-Cbl蛋白相结合，这种结合被Mg132抑制；c-Cbl基因敲除后，角膜上皮细胞中泛素化Ret减少，Ret表达增加，角膜上皮细胞中HSV-1病毒复制减少。以上结果证实c-Cbl是角膜上皮细胞Ret降解的泛素-蛋白连接酶E3，其表达下降可使Ret泛素化水平下降，降解减少，表达上调，从而促进HSV-1感染的角膜上皮细胞凋亡，抑制病毒复制。我们将进一步寻找对细胞凋亡和病毒复制具有潜在的靶向干预作用的c-Cbl抑制剂，从而为HSK的治疗提供新思路和新手段。