Cao Qian, Li Yunchuan, Li Yong, Li Lan
Department of Ophthalmology, The Affiliated Calmette Hospital of Kunming Medical University, Kunming, China.
Ann Transl Med. 2021 Sep;9(18):1410. doi: 10.21037/atm-21-2054.
Worldwide, corneal transplantation (CT) is the most common type of tissue replacement and the increased rate of corneal graft rejection (CGR) after CT is a critical problem. Corneal endothelium cells (CECs) are often targets of the immune response mediated by graft-attacking effector T cells. However, the molecular mechanism underlying CGR remains poorly understood.
The differentially expressed microRNAs (miRNAs) and mRNA of graft-fail corneas were measured by transcriptome sequencing (RNA-Seq). real-time quantitative polymerase chain reaction was used to measure gene expression levels. Western blot and immunofluorescence staining were used to measure protein expression levels. Kaplan-Meier survival curves were constructed to assess corneal graft survival. Hematoxylin and eosin staining was used for histopathological examination. CCK-8 and ELISA staining were used to detect cell viability and inflammatory cytokines levels, respectively. Flow cytometry was used to detect cell apoptosis and the population of Treg and Th17. Transwell migration and wound-healing assays were used to measure cell migration.
We identified 453 miRNAs and 4,279 mRNAs aberrant expression in the corneas showing CGR. The differentially expressed miR-151-5p and its potential target gene [interleukin 2 receptor subunit alpha ()] were selected from the RNA-Seq microarrays. The levels of miR-151-5p and IL-2Rɑ were respectively downregulated and upregulated in the CGR. The luciferase activity assay suggested that IL-2Rɑ is a target of miR-151-5p in 293 T cells. In addition, the miR-151-5p inhibitor, si-IL-2Rɑ, and oe-IL-2Rɑ transfection tests in CECs further confirmed that miR-151-5p downregulation and IL-2Rɑ overexpression promoted apoptosis of CECs and inhibited CEC migration, tight junction-related protein ZO-1 and Claudin-5 expression, and PI3K/AKT signaling pathway activity; however, downregulation of IL-2Rɑ abolished the inhibitor effect of miR-151-5p. Similarly, upregulation of miR-151-5p alleviated CGR via activation of the PI3K/AKT signaling pathway and balancing of Th17/Treg, and upregulation of IL-2Rɑ abolished the alleviating effect of miR-151-5p.
Upregulation of miR-151-5p alleviated CGR by activating the PI3K/AKT signaling pathway and balancing Th17/Treg via targeting of IL-2Rɑ, which contributes to improving the results of CT.
在全球范围内，角膜移植（CT）是最常见的组织置换类型，而CT后角膜移植排斥反应（CGR）发生率的增加是一个关键问题。角膜内皮细胞（CECs）常常是由攻击移植物的效应T细胞介导的免疫反应的靶标。然而，CGR潜在的分子机制仍知之甚少。
通过转录组测序（RNA-Seq）检测移植失败角膜中差异表达的微小RNA（miRNAs）和信使核糖核酸（mRNA）。采用实时定量聚合酶链反应检测基因表达水平。采用蛋白质免疫印迹法和免疫荧光染色法检测蛋白质表达水平。构建Kaplan-Meier生存曲线评估角膜移植存活情况。采用苏木精-伊红染色进行组织病理学检查。分别采用CCK-8法和酶联免疫吸附测定法检测细胞活力和炎性细胞因子水平。采用流式细胞术检测细胞凋亡以及调节性T细胞（Treg）和辅助性T细胞17（Th17）的比例。采用Transwell迁移实验和伤口愈合实验检测细胞迁移情况。
我们在出现CGR的角膜中鉴定出453个miRNAs和4279个mRNA异常表达。从RNA-Seq微阵列中筛选出差异表达的miR-151-5p及其潜在靶基因[白细胞介素2受体α亚基（IL-2Rɑ）]。在CGR中，miR-151-5p水平下调，IL-2Rɑ水平上调。荧光素酶活性测定表明，在293T细胞中IL-2Rɑ是miR-151-5p的靶标。此外，在CECs中进行的miR-151-5p抑制剂、小干扰RNA（si）-IL-2Rɑ和过表达载体（oe）-IL-2Rɑ转染实验进一步证实，miR-151-5p下调和IL-2Rɑ过表达促进CECs凋亡，抑制CECs迁移、紧密连接相关蛋白闭锁小带蛋白1（ZO-1）和闭合蛋白5（Claudin-5）表达以及磷脂酰肌醇-3-激酶/蛋白激酶B（PI3K/AKT）信号通路活性；然而，IL-2Rɑ下调消除了miR-151-5p的抑制作用。同样，miR-151-5p上调通过激活PI3K/AKT信号通路和平衡Th17/Treg减轻CGR，而IL-2Rɑ上调消除了miR-151-5p的减轻作用。
miR-151-5p上调通过靶向IL-2Rɑ激活PI3K/AKT信号通路和平衡Th17/Treg减轻CGR，这有助于改善角膜移植效果。
