Expression And Regulation Of MicroRNA-155in CD4~+T Cells Of Oral Lichen Planus

BackgroundOral lichen planus (OLP) is a common T cell-mediated inflammatory disease, and CD4+T cells are greatly involved in immunopathogenesis of OLP via secreting various cytokines and activating CD8+cytotoxic T cells. Some authors including us have found that in peripheral blood of OLP, CD4+T cells represented a lot of differences in the activation, differentiation and secreting function. MicroRNAs (miRNAs) are20-to25-nucleotide single-stranded molecules that control nearly1/3post-transcriptional gene expression in eukaryotes. Recently, it has become evident that abnormally expressed miRNAs play an important role in the occurrence and development of autoimmune diseases. And the abnormally expressed miRNAs in peripheral blood were regarded as the candidate biomarker in the severity assessment, diagnosis and prognosis evaluation of many autoimmune diseases in view of their stability and easily being tested.Objective1. To search the abnormally expressed miRNAs in peripheral blood of OLP patients, and to observe the relationship of the expression of these miRNAs with OLP severity as well as the serum levels of cytokines and chemokines.2. To verify the correlation of miR-155with its target SOCS1(suppressor of cytokine signaling1) in CD4+T cells of OLP.3. To determine the cytokine level alteration in the extracellular matrix of EOLP CD4+T cells, after the functional regulation of miR-155inside the cells.4. To analyse the effect of miR-155functional regulation in CD4+T cells proliferation as well as the morphology and proliferation of keratinocytes in co-cultured system.Material&Methods1. Following the inclusive&exclusive criteria, the OLP patients were recruited by Department of Oral Medicine in School and Hospital of Stomatology, Wuhan University.2. OLP severity was assessed using RAE (reticular atrophy and erosive) scoring system.3. The expression of miR-155, miR-146a, miR-125a and miR-203in peripheral blood and SOCS1mRNA in CD4+T cells of OLP was determined with SYBR Green real-time quantitative polymerase chain reaction (qRT-PCR).4. The serum levels of chemokines (CCL5and CCL26) and cytokines (IL-2, IL-4, IL-10and IFN-y) in OLP were measured with enzyme-linked immuno sorbent assay (ELISA).5. The BD IMagTM Human CD4T Lymphocyte Enrichment Set-DM was used for the negative selection of CD4T lymphocytes from PBMC.6. Using TargetScan (version5.2; http://www.targetscan.org) and PicTar (http://pictar.mdc-berlin.de/), we determined the targeting patern of miR-155with SOCS1mRNA.7. MiR-155functional regulation was conducted with miR-DownTM antagomir-155and miR-UpTM agomir-155carried by Lipofectamin2000.8. Proliferation of CD4+T cells and keratinocytes was tested with CCK8.Results1. Up-regulated miR-155and down-regulated miR-125a were found in peripheral blood of OLP patients, and their expression significantly correlated with disease severity of OLP.2. Thl type cytokine IL-2and IFN-y concentrations as well as chemokine CCL5were significantly higher, whereas Th2type cytokine IL-4and IL-10levels were significantly lower in OLP patients.3. The expression patern of miR-155in peripheral blood was the same with the serum IFN-y, but the opposite to serum IL-4among EOLP group, NEOLP group and the controls.4. SOCS1, the key target of miR-155, its mRNA was found decreased, and negatively correlated with the high expressed miR-155in CD4+T cells of EOLP.5. It was revealed that in supernatant of cultured EOLP CD4+T cells, up-regulated miR-155in EOLP CD4+T cells could result in the decrease of IL-4and IFN-y levels, while down-regulated miR-155could decrease IFN-y levels but increase IL-4levels.6. IFN-y induced EOLP CD4+T cells to up-regulate the expression of miR-155.7. Functional regulation of miR-155in EOLP CD4+T cells could change the proliferation of CD4+T cells as well as the the cellular morphology and proliferation of keratinocyte in co-culture system.Conclusion1. Up-regulated miR-155and down-regulated miR-125a in peripheral blood of OLP might be considered as the bio markers for the assessment of disease severity.2. Up-regulated miR-155might contribute to the Thl-dominated cytokine environment in peripheral blood of OLP.3. MiR-155could target at SOCS1mRNA, and formed a positive feedback with IFN-y.4. Functional regulating of miR-155in EOLP CD4+T cells could alter the proliferation of CD4+T cells as well as the morphology and proliferation of keratinocytes in the co-cultured system.