Abstract
Major histocompatibility complex class I-related chain A (MICA), a non-classical major histocompatibility complex molecule, can stimulate or co-stimulate CD8+ T cells or natural killer (nk) cells, thus affecting cornea allograft survival. This study investigated IFN-γ regulation of MICA expression levels in human corneal epithelium by miRNA4448. MICA expression levels in human corneal epithelial cells (HCECs) stimulated with IFN-γ were detected by qRT-PCR and an enzyme-linked immunosorbent assay, and differential miRNA expression levels were measured. qRT-PCR, Western blotting, and immunofluorescence staining revealed nuclear factor kappa B (NFκB)/P65 expression in IFN-γ-treated and miRNA4448-overexpressed HCECs. A luciferase reporter assay was used to predict the interaction between NFκB and MICA. Additionally, HCECs were transfected with MICA plasmid or treated with IFN-γ and NKG2D-mAb and cocultured with NK cells and CD8+ T cells. Cell apoptosis was measured using Annexin V/PI staining. qRT-PCR detected the expression of anti-apoptosis factor Survivin and apoptosis factor Caspase 3 in MICA-transfected and IFN-γ-treated HCECs after co-culturing with NK cells and CD8+ T cells. IFN-γ (500 ng/ml, 24 h) upregulated MICA expression in HCECs in vitro. Among six differentially expressed microRNAs, miRNA4448 levels decreased the most after IFN-γ treatment. The overexpression of miRNA4448 decreased MICA expression. miRNA4448 downregulated NFκB/P65 expression in IFN-γ-induced HCEC, and it was determined that NFκB/P65 directly targeted MICA by binding to the promotor region. A coculture with NK cells and CD8+ T cells demonstrated that MICA overexpression enhanced HCEC apoptosis, which could be inhibited by NKG2D-mAb. Simultaneously, Survivin mRNA expression decreased and Caspase3 mRNA expression increased upon the interaction between MICA and NK (CD8+ T) cells in HCECs. IFN-γ enhances the expression of MICA in HCECs by modulating miRNA4448 and NFκB/P65 levels, thereby contributing to HCEC apoptosis induced by NK and CD8+ T cells. This discovery may lead to new insights into the pathogenesis of corneal allograft rejection.
Highlights
Corneal transplantation is one of the most common human organ transplantations worldwide
With the IFN-γ treatment for 24 h, the expression levels of MICA protein and MICA messenger RNA (mRNA) in HCECs were upregulated in a concentration-dependent manner (Figure 1)
Little is known about the expression of MICA and its regulation mechanisms in relation mRNA levels (D) and protein levels after pyrrolidine dithiocarbamate (PDTC) treatment for 2 h. (E,F) PDTC decreased both MICA mRNA and protein expression levels in human corneal epithelial cells, as demonstrated by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) and Enzyme-Linked Immunosorbent Assay (ELISA), respectively
Summary
Corneal transplantation is one of the most common human organ transplantations worldwide. The 1-year allograft survival rate is currently as high as 90%, more than half of transplantation patients suffer various types of corneal rejection, such as epithelial rejection, chronic stromal rejection, and endothelial rejection. Major histocompatibility complex (MHC) is thought to play a significant role in corneal immune status. MHC molecules are not expressed in normal corneal endothelial cells, and the restricted distribution of MHC molecules in corneal epithelial cells has called into question the role of MHC in corneal rejection [2]. A large, multi-center follow-up study showed no significant correlation between the human leukocyte antigen, the gene complex encoding MHC, and clinical outcomes [3]. The role of other immune pathways (beyond the classical MHC antigen) in corneal allograft rejection requires further investigation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
