Abstract
Purpose :
MHC Class I-related Chain A (MICA), a non-classical MHC molecule, can stimulate or co-stimulate CD8+ T cells or NK cells, affecting cornea allograft survival. This study investigated IFN-γ regulation of MICA expression levels in human corneal epithelium through miRNA-4448.
Methods :
MICA expression levels in human corneal epithelial cells (HCEC) stimulated with IFN-γ were detected by qRT-PCR and ELISA, and differential miRNA expression levels were measured. qRT-PCR, western blot, and immunofluorescence staining illustrated nuclear factor kappa B (NFkB)/P65 expression in IFN-γ-treated and miRNA-4448-overexpressed HCEC, and a luciferase reporter assay was used to predict their interaction. Additionally, HCECs were treated with IFN-γ or transfected with the MICA plasmid, co-cultured with NK cells and CD8+ T cells, and 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 treatedHCEC after co-culturing with NK cells and CD8+ T cells.
Results :
IFN-γ (500 ng/ml, two hours) upregulated MICA expression in HCECs in vitro. Among six differentially expressed miRNAs, miRNA-4481 levels decreased the most after IFN-γ treatment. Furthermore, the overexpression of miRNA-4481 decreased MICA expression. Additionally, miRNA-4481 regulated NFkB/P65 expression in IFN-γ-induced HCECs, and it was determined that NFkB/P65 directly targeted MICA by binding to the promotor region. A co-culture with NK cells and CD8+ T cells demonstrated that MICA overexpression enhanced HCEC apoptosis.Simultaneously, Survivin levels decreased and Caspase3 levels increased.
Conclusions :
IFN-γenhances theexpression of MICA in HCEC through modulatingmiRNA-4481 and NFkB/P65 levels, thereby contributing to HCEC apoptosis induced by NK and CD8+ T cells. The discovery may lead to new insights into the pathogenesis of corneal allograft rejection.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.