Alter Expression And Function Of MicoRNA In CD4~+ T Cell Of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a common chronic inflammatory disease which is characterized by radiographic joint destruction, severe functional deterioration, and work disability. RA targets the synovial lining of joints, bursae and tendon sheaths. At least in the early phase, RA has been known as distinction from other organ-specific autoimmune disease in that. Rather than causing cell death and tissue destruction at the outset, this disease is characterized by the activation and proliferation of stromal tissues in the target organ.When the shared epitope in human leukocyte antigen (HLA)-DR genes is found in about 80% of RA patients, CD4~+ T cells has been noticeable based on the evidence of genetic predisposition. Migration of activated T cells into the joint, proliferation in situ, or inhibition of T cell death lead to persistence of these infiltrating cells. Activated by surrounding cytokines or autoantigen, synovium CD4~+ T cells interact with other cells such as macrophage-like or fibroblast-like synoviocytes by cytokines or direct contact. The complex cell-cell interactions form functional networks, release proinflammatory cytokines such as IL-1,TNF-αand IL-17, and eventually cause tissue destruction. Above all, CD4~+ T cells may be the key participants in RA. CD4~+ T cells isolated from synovial tissue and fluid appear to be dysfunctional, which respond poorly to stimulation with mitogen or antigens in vitro. On the other hand, although CD4~+ T cells in rheumatoid synovial fluid are found to express phenotypes which suggest susceptibility to apoptosis, however, these CD4~+ T cells are strongly resistant to apoptosis in synovium fliud which contain amounts of apoptosis factors such as bioactive FasL, TRAIL and TNF-α.. So we hypothesised that synovial microenvironment factors probably induced the variance of intracellar regulation of CD4~+ T dysfunction. It is significant to explore the new mechanism of intracellar regulation.MicroRNAs (miRNAs) are～22 nt noncoding RNAs that can suppress the expression of protein-coding genes by targeting cognate mRNAs for degradation or translational repression. MiRNAs account for 3% of all genes, which make them to be the most abundant class of regulators in the human genome. To date, miRNAs have been known to be novel players in the regulation of normal immune function and inflammation. T cells also have been found to express a different miRNAs subset, which link to the cell development (miR-142s), mature (miR-150s), activation, differentiation (miR223) and function.Furthermore, deregulation of miRNA expression may contribute to human autoimmunity diseases. A set of alter expression miRNAs were identified from chronic skin inflammation of psoriasis and synovium tissue of RA. While miRNAs are known to be an important regulator in several autoimmunity diseases, neither their expression nor roles have been characterized in CD4~+ T cells of rheumatoid arthritis patients.In our research, we demonstrated that CD4~+ T cells from either synovium fluid or peripheral blood of RA patients was characterized by a specific miRNAs expression profile, which differed from that of healthy human. There were the up-regulation of hsa-miR-146a and the down-regulation of hsa-miR-363 in CD4~+ T cells from both peripheral blood and synovium, and the down-regulation of hsa-miR-498 in CD4~+ T cells from peripheral blood, compared with normal CD4~+ T cells. And another miRNA expression, hsa-miR-150, increased in CD4~+ T cells from peripheral blood, compared with that from synovium fluid. In order to analyse whether these miRNAs contribute to RA pathogenesis, we detected the level of TNF-α, IFN-γ, IL-2, IL-4, IL-6 and IL-10 in serum and synovium fluid at the same time. The significant positive correlation between hsa-miR-146a and TNF-αin both peripheral blood and synovium fluid has been found by dependability statistical analysis. As we know, in inflammatory joint, TNF-αare abundant, which may be the factor of stimulating the increase expression of has-miR-146a. Next we verificated this hypothesis by the experiment that TNF-αinduced the up-regulation of has-miR-146a in Jurkat T cells and normal CD4~+ T cells as dose-dependent in vitro, and SF also stimulated the up-regulation of has-miR-146a in these cells.Furthermore, has-miR-146a was transfected by lentivirus vector into Jurkat T cells in order to evaluation its function and targets. First of all, we predicted the targets by bioinformatics methods, and ultimately TRAF6 was the top of score and then identificated as the real target of has-miR-146a in Jurkat T cells by Western blot. However, we could not found any obvious effects on cell proliferation, activation and apoptosis when we transfected hsa-miR-146a into Jurkat T cells.We also constructed hsa-miR-146a transgenic mice in order to research the function of hsa-miR-146a in vivo. The transgenic mice were verificated by PCR, fluorescence microscope, Real-Time PCR and FACs. These mice appeared healthy and viable after birth. But hepatauxe and big blood clot in the chest cavity were found in the 10 weeks old transgenic mice. Subsequent histopathological examination showed signs of systemic imflammation, including lymphocytes infiltrates in lung cell compartment and glomcrulus. The most surprising phenomenon was mass multinucleated giant cell in spleen of transgenic mice. All of these pathognomonic feature hinted that there was the multisystem inflammatory disease .In addition, T cell phenotypic analysis showed that percentages of CD4~+ T cell and CD8~+ T cell were normal. Frequency of Treg, CD4~+ CD69~+ T cells and CD8~+ CD69~+ T cells were also not changed. But transgenic mice showed signs of hyperactive T cell due to percentage of central memory T cells increased in spleen, and percentage of effector memory T cells increased in peripheral blood. Subsequently, no considerable differences in percentages of BMDC were observed. BMDC from transgenic mice showed normal expression of CD86 and MHC II molecule after treatment with LPS. Maybe the maturation and function of BMDC were not impacted by has-miR-146a. These appearances in has-miR-146a transgenic mice were similar to TRAF6 deletion specific to T cells mice.Thus, these findings defined a previously unrecognized role for T-cell up-regulated has-miR-146a by suppress translation of TRAF6. A new layer of regulatory mechanisms in the pathogenesis of RA was hinted by our results. Further studies are required to elucidate the function of miR-146a in CD4~+ T cells and autoimmunity disease.