The Role Of MicroRNA-122 And Its Target Gene CPEB1 In Corneal Allograft Rejection

Purposes:To investigate the role of micro RNA-122 and its target gene CPEB1 during penetrating coneral transplantation rejection.Methods: 1.Establishing mice penetrating keratoplasty model; 2. Real-time PCR was used to analyze the expression of mi R-122 and CPEB1 in three layers of cornea tissue and immune cells. 3.Over expression agomir/antagomir-122 analyzing CPEB1 expression and apoptosis. 4.Corneal stromal cell line was cultured and infected with lentivirus containg mi R-122-binding-site wild type or mutated CPEB1. Cells were then treated with inflammatory cytokines(TNFα/IL-1β/TNFγ) for 48 hours. Apoptosis of corneal stromal cells and NO generation was determined using annexinV staining and NO kit.Results:1.Detection of the percentage of immune cells by flow cytometry after penetrating corneal transplantation: the percentage of immune cells is significantly higher in the non-rejection group(P<0.05), no siginificant difference was found between the rejection group and control group(P>0.05). 2. Real-time PCR analysis: the expression of both mi R-122 and CPEB1 is significantly higher in the corneal stromal layer compared with stromal layer and endodermis layer(P<0.0001); mi R-122 was not expressed in the immune cells.3. The stromal cells were infected with lentivirus containg mi R-122-binding-site wild type or mutated CPEB1. The infection efficiency was more than 90%. 4. Over expression agomir/antgomir-122 analysis: Transfection agomir-122, the expression of CPEB1 is lower than control and reduced apoptosis; transfection antagomir-122, the expression of CPEB1 is higher than control and increased apoptosis(P<0.05). 5. Apoptosis and NO detection: over expression of CPEB1 promote corneal stromal cells apoptosis and NO generation(P<0.05); All samples treated with inflammatory cytokines showed increased apoptosis(P<0.05);After treated with cytokine, the stromal cells expressing mi R-122-binding-sitemutated CPEB1 showed significantly higher rate of apoptosis comparing with stromal cells expressing mi R-122-binding-site wild type CPEB1(P<0.05); The apoptosis is similar between unused cytokine treated stromal cells expressing mi R-122-binding-site wild type or mutated CPEB1; Regardless of cytokine stimulation, the stromal cells expressing mi R-122-binding-site-mutated CPEB1 showed significantly higher rate of NO generation comparing with stromal cells expressing mi R-122-binding-site wild type CPEB1(P<0.05).Conclusions: mi R-122 and CPEB1 were highest expressed in the corneal stromal layer and expression of mi R-122 was not detected in the immune cells; When the expression of mi R-122 was down-regulated, the expression of its target, CPEB1, was increased. This leads to increased apoptosis and NO generation of corneal stromal cells and promotes corneal transplantation rejection.