Background: 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.
Methods: 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.
Results: 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 (IL-2Rɑ)] 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.
Conclusions: 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.
Keywords: Corneal transplantation (CT); PI3K/AKT signaling pathway; Th17/Treg; corneal allograft rejection (CGR); miR-151-5p/IL-2Rɑ.
2021 Annals of Translational Medicine. All rights reserved.