In the current study we showed that the basal expression of miR-203 is increased in RASFs compared to OASFs and that overexpression of miR-203 in these cells leads to increased production of MMP-1 and IL-6. By demonstrating this, we identified miR-203 as another miRNA with altered expression in RASFs.
Pathologic expression of miR-203 has mainly been identified in various malignant tumors, where its expression levels are mostly found to be higher than in normal tissue (25
). In some malignancies, however, miR-203 levels are down-regulated (14
), indicating that it can also function as a tumor suppressor. In particular in hematopoietic malignancies, restoration of miR-203 leads to decreased expression of the oncoprotein BCR-ABL1 (13
). In accordance with our data, studies of cancer cells have also shown that the expression of miR-203 is regulated by CpG methylation and can be modulated by demethylating agents (13
Apart from tumor pathology, miR-203 has been shown to play an important role in keratinocyte differentiation in the skin and was found to be highly expressed in psoriatic skin plaques (30
). Most interestingly, cultured dermal cell suspensions from lesional psoriatic skin spontaneously produce more IL-6 protein than do nonlesional psoriatic or healthy cells (32
). Our findings, together with reports of increased miR-203 expression in psoriasis (32
), suggest that the link between IL-6 and miR-203 might also apply in other chronic inflammatory diseases.
In earlier studies we demonstrated for the first time that expression of miR-155 and miR-146a is altered in RA synovium and synovial fibroblasts and provided evidence of a functional role of miR-155 as a modulator of MMP-3 and MMP-1 expression in RASFs (10
). Recently, published data by another group revealed that miR-124a is down-regulated in RASFs and contributes to the regulation of cell proliferation and the production of monocyte chemoattractant protein 1 (11
). Based on these reports a complex picture of altered expression of miRNA in RASFs emerges, and its functional relevance to the development of the disease phenotype becomes evident.
Previously, our group suggested that the longstanding, inflammatory milieu in RA leads to epigenetic changes in synovial fibroblasts and showed that DNA in RASFs is globally hypomethylated (21
). In the present study, we demonstrated that demethylating treatment of synovial fibroblasts leads to increased expression of miR-203. Therefore, we propose that the constitutively high production of miR-203 in RASFs may be due to changes in epigenetic modifications in the promoter of the miR-203 gene. The variable levels of miR-203 that we measured in RASFs from patients with symptoms of very recent onset (<3 months) support the hypothesis that this epigenetic switch is turned on during the development of the disease, which may eventually lead to the imprinted activated phenotype of synovial fibroblasts seen in RA (33
). Interestingly, the fact that 2 of the patients with early RA already exhibited levels of miR-203 as high as those in patients with established RA suggests that miR-203 is involved in the pathogenesis of the disease in its very early stage. It must be stressed, however, that further in vivo experiments are needed to confirm this hypothesis.
The finding that expression of miR-203 was not increased in the synovial tissue of RA patients can be explained by the pronounced heterogeneity and vast interindividual differences in the cellular composition of inflamed synovial tissue. If increased miR-203 levels are a specific feature of hypomethylated synovial fibroblasts, the high numbers of inflammatory and vascular cells in synovial tissue lysates might conceal differences between the fibroblast populations.
IL-6 is one of the major proinflammatory cytokines present in abundance in rheumatoid joints, and recently, therapeutic regimens targeting IL-6 have been successfully introduced into clinical practice (34
). Of interest, resident cells of the joint, i.e., synovial fibroblasts, are recognized as a major source of this cytokine, and even more interestingly, RASFs in vitro are characterized by higher spontaneous production of IL-6 as compared to OASFs (22
). To better understand the contribution of miR-203 to the modulation of IL-6 levels in RASFs, knowing that NF-κ
B is the major transcription factor driving basal expression of IL-6 in synovial fibroblasts, we analyzed the influence of miR-203 on the NF-κ
B signaling pathway and were able to clearly show that miR-203 is dependent on NF-κ
B activity to modulate IL-6 levels. Therefore, our study identifies miR-203 as a novel endogenously altered modulator of IL-6 expression in RASFs, which is upstream of NF-κ
B. However, using miR target prediction algorithms, we could not identify the direct miR-203 target involved in this mechanism.
In recent years it has become clear that epigenetic modifications play an important role in the regulation of microRNA and that dysregulated expression of microRNA occurs in a variety of pathologic conditions (36
). Herein we have shown that the high expression of miR-203 in RASFs leads to increased secretion of IL-6 and MMP-1, two molecules that are highly associated with RA pathogenesis and contribute importantly to chronic inflammation and joint destruction in the disease. Given recent reports of successful approaches to targeting miRNA in vivo (39
), our data suggest that miR-203 may be a new therapeutic target in RA.