Increasing evidence indicates that aberrant expression of miRNAs is implicated in the pathogenesis of autoimmune diseases. Accumulating data have suggested that the proper regulation of miRNA expression is important in the maintenance of normal immune functions and prevention of autoimmunity. A number of miRNAs have been found to show a tissue-specific pattern during cancer development and viral infection. Previous studies have shown that increased miR-17-92 expression in lymphocytes leads to the development of lymphoproliferative disorder and autoimmune disease in mice [30
]. Recently, upregulated miR-146a has been found in activated primary T cells and memory T cells [31
]. Moreover, increased miR-146a expression has been detected primarily in PBMC and synoviocytes from RA patients [22
]. Although deregulation of miRNA expression has been observed in human autoimmune diseases, it is still largely unclear how miRNAs affect autoimmune pathogenesis in patients. Although a set of altered expression miRNAs are recently identified in both PBMC and synovial tissue from RA patients, neither miRNAs expression profile nor their roles have been fully characterized in CD4+
T cells of RA patients.
In this study, we have selected the RA patients who did not have any DMARDs therapy because it is known that drug treatment can affect expression of miRNAs [32
]. We have found CD4+
T cells from both SF and peripheral blood of RA patients exhibiting a specific miRNAs expression profile. Although miR-146a expression was not found to be associated with the disease activity index in the current study, our results have shown a significant positive correlation between miR-146a and TNF-α in both peripheral blood and SF by dependability statistical analysis. Furthermore, we have found that miR-146a expression is upregulated in CD4+
T cells in response to TNF-α or SF stimulation in vitro
. Thus the inflammatory milieu might alter miR-146a expression in infiltrated T cells. TNF-α is a critical mediator of the inflammatory pathway in the rheumatoid joints. As TNF-α inhibition therapy appears to dramatically reduce markers of inflammation and slow joint structural damage. Our findings of overexpression of miR-146a in T cells may have functional implications in eliciting joint inflammation of RA patients.
In addition to its increasingly recognized function in modulating innate immunity, miR-146a has been shown to be involved in Th1/Th2 polarization and regulatory T cell development [33
], indicating a potential role for miR-146a in autoimmune response. Notably, miR-146a was found to be up-regulated in skin lesions of psoriasis, PBMC and synovial tissue of RA, whereas it was down-regulated in PBMC of systemic lupus erythematosus [20
]. Although the increased expression of miR-146a in T cells does not show any affect on cytokine production in this study, available data on overexpression of miR-146a in several types of cells of RA suggests that miR-146a is possibly involved in modulating functions of T cells and other cells in RA pathogenesis. Although our current data confirm the increased miR-146a levels in synovial and peripheral blood CD4+
T cells of RA patients, we were not able to differentiate the expression pattern of miR-146a levels in various CD4+
T cell subpopulations due to the limited numbers of synovial and peripheral blood CD4+
T cells. Further studies are warranted to clarify whether miR-146a expression is increased in activated T cells or preferentially upregulated in memory T cells of RA patients.
The current understanding of the critical roles of miRNAs in regulating cellular functions mainly depends on the identification of their target genes. TRAF6, a known target of miR-146a in macrophages and PBMCs, plays a key role in mediating signals from TNF receptor and IL-1 receptor in innate immunity. The suppressed TRAF6 expression by miR-146a indicated that miR-146a may be a negative regulator in the TNF-α signal pathway [22
]. TRAF6 was also identified as a T cell-intrinsic negative regulator in mice with T cell-specific TRAF6 deletion, in which signs of hyperactive humoral immunity, including increased serum levels of immunoglobulin and DNA autoantibodies [37
], were observed with similar clinical features of RA.
It has now become clear that certain miRNAs operate through targeting single genes while others act broadly through regulating the expression of multiple targets [40
]. Up to now, the identification of new targets usually depends on bioinformatic analysis and experimental screening. Previously, gene expression analysis has facilitated the identification of targets upon overexpressed plant miRNAs [41
]. In contrast to plants, vertebrate miRNAs are believed to exert their functions mainly through translational repression rather than mRNA cleavage [42
]. However, it has been recently found that miRNAs can induce the degradation of mRNAs bearing fully complementary target sites by similar mechanisms of small-interfering RNA [43
]. Transcriptome analysis can generate huge datasets with expression levels for all currently known genes.
Studies on the changes of mRNA expression induced by miRNAs have been considered as useful methods for uncovering their new targets and new downstream signal molecules [44
], but only the degraded targets can be identified by transcriptome analysis. As many genes such as IRAK1, IRAK2, TRAF6, FADD, IRF-5, STAT-1 and PTC1 have been found to be targets of miR-146a in human disease [20
], the targets and functions of miR-146a may be different in specific cells. In our experimental system, transcriptome analysis of miR-146a with up-or down-regulation in Jurkat T cells has not identified any known targets for miR-146a.
It is plausible to reason that miR-146a may mainly act through transcriptional repression rather than degradation of targets by imperfect basepair with the 3'-untranslated region of the targets. FAF1 is known to bind to the intracellular portion of the apoptosis signal transducing receptor Fas/Apo-1 and caspase-8 and shows similar characteristics of Fas-associated death domain protein, which can enhance Fas-mediated apoptosis [47
]. We show that overexpression of FAF1 induces significant apoptosis in Jurkat T cells, consistent with previously reported findings [49
]. Interestingly, the ectopic expression of miR-146a has been found to protect Jurkat T cells from activation-induced cell death, whereas FADD is identified as one of its targets [31
]. Here, we further demonstrate that overexpression of FAF1 in miR-146a-overexpressing Jurkat T cells abolished the suppressive effect of miR-146a on T cell apoptosis. It becomes clear that activation-induced cell death is mainly mediated via the Fas/FasL pathway, which plays an important role in the immunity and induction of peripheral tolerance to self-antigens. A study by Ryu and colleagues has identified FAF1 as a member of Fas death-inducing signaling complexes such as FADD [50
]. However, our transcriptome analysis of miR-146a failed to validate FADD as its target. Currently, it is unclear whether this is due to the detection limitation of the technique used or the possibility that miR-146a regulates FAF1 in an FADD-independent fashion. Interestingly, the expression of two targets of miR-146, TRAF6 and IL-1 receptor-associated kinase 1, remains unchanged although miR-146 is upregulated in PBMC from RA patients [22
]. Thus, it remains to be elucidated whether miR-146 exerts its effect on various targets in a cell-type-dependent manner. Nevertheless, it is noteworthy that FAF1 does not seem to be a direct target of miR-146a according to our bioinformatics analysis [51
]. Further studies are needed to identify genes involved in mediating the effect of miR-146a on FAF1 expression. Dysregulated T cell apoptosis is closely associated with autoimmunity diseases, especially RA. Thus, our findings that miR-146a upregulation in CD4+
T cells is correlated with increased TNF-α levels may suggest that miR-146a acts as a critical factor in eliciting and maintaining the inflammation via suppressing T cell apoptosis during RA pathogenesis.