MicroRNA (miRNA) are a well-established class of small (∼22 nucleotides) endogenous noncoding RNAs that influence the stability and translation of messenger RNA (mRNA) (1). Using various computational and experimental approaches, hundreds of miRNA have been identified in numerous animal species. The miRNA genes are transcribed by RNA polymerase II as primary miRNA (pri-miRNA) (2, 3). The RNase III enzyme Drosha then processes the nuclear pri-miRNA, yielding a ∼70-nucleotide molecule known as precursor miRNA (pre-miRNA) (4), which is exported from the nucleus. Maturation of the pre-miRNA into miRNA is then mediated by the cytoplasmic enzyme Dicer (5), after which the mature miRNA is loaded into the RNA-induced silencing complex (RISC) (6). Once loaded, the miRNA guides the RISC complex to the 3′-untranslated region (3′-UTR) of target mRNA. The so-called “seed region” (nucleotides 2–8) of miRNA is most important for target recognition and silencing (7, 8). MicroRNA usually bind with imperfect complementarity to their target, which is called the “seed sequence” (7). Association of miRNA with their target mRNA silence expression via at least 3 mechanisms: inhibition of translation, inhibition of the initiation of translation, and destabilization of target mRNA (1).
Recent advances have shown that miRNA expression during development is highly tissue-specific (9–12), which suggests that miRNA may be involved in specifying and maintaining tissue identity. For example, the expression of miR-124a is restricted to the brain and spinal cord in the fish and the mouse, and to the ventral nerve cord in the fly (13). In those tissues, it contributes to the differentiation of neural progenitors into mature neurons through degradation of non-neuronal transcripts (14). In non-neuronal cells, miR-124a is targeted by the repressor element 1–silencing transcription factor (REST). Its conserved sequence and expression across species suggest that miR-124a is an ancient molecule that acts in both muscle and brain development. In addition to tissue development, miRNA also appear to be involved in metabolism, in cell differentiation, growth, and death, and in carcinogenesis (1).
Rheumatoid arthritis (RA) is a chronic disease of unknown cause that presents a characteristic constellation of features, including synoviocyte hyperplasia, which results in pannus formation and joint destruction (15–17). The rheumatoid synovium consists of epithelial cells, which include 2 types of synovial lining cells, fibroblast-like synoviocytes (FLS) and macrophage-like synoviocytes, as well as infiltrating leukocytes, which include T cells, B cells, and dendritic cells, among others (18). The local production of cytokines and chemokines by these cells accounts for many of the pathologic and clinical manifestations of RA (18). In culture, RA FLS proliferate and secrete a variety of cytokines/chemokines/angiogenic factors, including fibroblast growth factor, granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1α (MIP-1α), and they present adhesion molecules, such as selectins, vascular cell adhesion molecules, and intercellular adhesion molecules, on their surfaces (19).
Our aim in the present study was to investigate the extent to which specific miRNA are involved in the pathogenesis of RA by comparing miRNA expression profiles in FLS from RA patients with those in FLS from osteoarthritis (OA) patients. Our findings suggest that miR-124a plays a key role in regulating the proliferation and chemokine production of RA FLS.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Notable features of miRNA include their redundancy with respect to their target binding sequences in the 3′-UTR of mRNA and their relatively small total number, which is speculated to range from 500 to 1,000. For example, analysis using miRanda 3.0 predicted the 3′-UTR of 1,300 human mRNA as potential targets of miR-124a. These unique properties prompted us to speculate that an miRNA could regulate a number of molecules involved in the pathogenesis of RA. However, miRNA are regarded as negative regulators of the translation of mRNA. Therefore, there are bidirectional possibilities for the involvement of miRNA in the pathogenesis of RA: not only the down-regulation of specific miRNA binding to the 3′-UTR of mRNA, which generates proinflammatory proteins, but also the up-regulation of specific miRNA binding to the 3′-UTR of mRNA, which generates antiinflammatory proteins, might make a contribution. In this regard, both elevated and suppressed levels of miRNA are important in RA FLS.
In trying to detect miRNA whose expression differed in RA and OA FLS, we found that the level of miR-124a in RA FLS was less than one-sixth of that seen in OA FLS. Although the mechanism by which the expression of miR-124a is regulated is not yet clear, our findings suggest that low levels of miR-124a expression during RA pathogenesis could have significant effects on synovial cell proliferation, leukocyte chemotaxis (MCP-1), and angiogenesis (angiogenin and VEGF).
In addition, our reporter assays showed that cotransfection with pre-miR-124a decreased luciferase levels from the reporters that were fused to the wild-type 3′-UTRs, but not those that were fused to the mutant 3′-UTRs, of CDK-2 and MCP-1 mRNA, whereas Western analyses showed that overexpression of miR-124a in RA FLS suppressed the expression of CDK-2 and the secretion of MCP-1 by synoviocytes. These results strongly suggest that CDK-2 and MCP-1 are the direct targets of miR-124a in RA synoviocytes. Using ELISAs, we also found that overexpression of miR-124a in synoviocytes leads to the down-regulation of angiogenin and the up-regulation of VEGF. It is not clear, however, how miR-124a influences the expression of these angiogenic chemokines, since miRanda 3.0 analyses detected no seed sequences for miR-124a in either angiogenin or VEGF mRNA, nor did any other available miRNA database program, and miR-124a did not suppress luciferase activity driven by the 3′-UTR of angiogenin. It may be that miR-124a acts indirectly on various molecules, but that idea remains to be tested.
The contribution of cell cycle–related proteins, such as cyclins, CDKs, and CDK inhibitors, to carcinogenesis has been intensively investigated. Recent reports have shown that in human cancers, the expression of specific miRNA closely related to the regulation of cell growth and apoptosis differs from that in normal tissues (25, 26). RA is characterized by pronounced synovial hyperplasia and by synovial fibroblasts that appear to be transformed (27, 28). In animal models of RA, this transformed appearance of RA synoviocytes could be mitigated by transferring the CDK inhibitor genes p16INK4a and p21Cip1 into inflamed joints (29, 30). CDK-2, which is inhibited by p21Cip1 and p27Kip1, is another key CDK: CDK-2/cyclin E complexes are required for the G1-to-S phase transition and initiation of DNA synthesis, whereas CDK-2/cyclin A complexes function during the progression of cells through S phase (31). Our in vitro finding that overexpression of miR-124a caused G1 phase arrest in RA FLS is indicative of a direct suppression of CDK-2 mRNA by miR-124a.
The microRNA miR-124a was initially identified as a crucial regulator involved in neurogenesis (13). Our findings suggest that miR-124a takes on other functions at different stages of human development. Recently, Pierson et al (32) reported that the level of miR-124a expression, which is enriched in brain tissue, is low in medulloblastomas. They also showed by luciferase assay that the 3′-UTR of CDK-6 mRNA is a direct target of miR-124 and that CDK-6 expression is suppressed by miR-124 overexpression in medulloblastoma cell lines. We confirmed that the expression of CDK-6 protein was higher in RA FLS than in OA FLS and that CDK-6 expression was suppressed when pre-miR-124a was transfected into E11 and RA FLS–like CDK-2 (data not shown). Since CDK-6 is also known as a G1/S phase regulator, as is CDK-2, we think that miR-124a is an important regulator of the G1/S transition in synovial tissue as well as in tumors.
We also showed that MCP-1 is down-regulated by miR-124a. The pivotal role played by MCP-1 in RA in humans is highlighted by the findings of enhanced production of MCP-1 in serum and/or synovial fluid from patients with RA (33–35). Moreover, data from studies of animal models suggest that MCP-1 is involved in the pathogenesis of RA (36–38), and MCP-1 was recently reported to be a sensitive marker of disease activity in patients with juvenile RA (39). MCP-1 attracts memory T lymphocytes and natural killer cells, which are major contributors to the pathogenesis of RA (40). In addition, MCP-1 mediates angiogenesis via VEGF (41). We identified a putative binding site for miR-124a in the 3′-UTR of MCP-1 mRNA by database analysis and demonstrated miR-124a–specific suppression of MCP-1 secretion from RA FLS, which suggests that down-regulating miR-124a in RA FLS would facilitate MCP-1 secretion, thereby enhancing its chemotactic effects.
There have been studies showing that the expression of specific miRNA is altered by extracellular signals, such as cytokines (TGFβ, IFNγ, TNFα, and IL-1β) and Toll-like receptor ligands (LPS and poly[I-C]) (42–44). Those studies demonstrated that the miRNA induced by extracellular signals regulate the mRNA of proteins that are closely linked to cell type–specific functions. Considering our findings as well as the inflammatory environment of RA synovium, it is therefore possible that some extracellular signals may regulate the expression of miR-124a in synoviocytes. However, in our screening tests, we identified no cytokine/chemokine that affected miRNA-124a expression.
Investigating the constituents of the culture supernatants may be helpful, since RA fibroblasts continuously produce epidermal growth factor and platelet-derived growth factor in an autocrine manner over several months of culture (45). This suggests that the reduction in miR-124a expression may be caused by an epigenetic event that may not be simulated by short-term exposure to cytokines. Consistent with this idea, Lujambio et al (46) recently reported that miR-124a is a proliferation-associated miRNA and that it is silenced by the hypermethylation of the miR-124a gene in a variety of cancer cells (46). Since our data showed that the proliferation of RA FLS was suppressed by the introduction of pre-miR-124a, it would be of interest to investigate the methylation status of the miR-124a gene in RA FLS.
Transcription factors that regulate the activity of miRNA promoters have recently been described (47, 48). Moreover, several miRNA have been shown to regulate the 3′-UTR of mRNA that encode transcription factors (49), and a circuit that sequentially involves miRNA and transcription factors in a mutual negative feedback loop has been described (48, 49). As for the transcription factors, it has been reported that REST inhibits miR-124a expression in non-neuronal cells. However, the role of REST has not been investigated aside from neuronal development, let alone its role in synovial cell biology or RA pathogenesis. Many genes that encode miRNA have configurations similar to those of standard gene loci that generate mRNA and proteins, and interestingly, one such group of gene loci is the miR-124a (50). By taking a different approach, such as searching for transcription factors that bind to the promoter region of miR-124a, it may be possible to identify the transcription factor(s) that regulates miR-124a expression in RA FLS.
Taken together, our findings suggest that the pathogenesis of RA will be better understood when miRNA are added to the big picture that illustrates the molecular kinetics of RA. We anticipate that miRNA will be considered in future strategies aimed at diagnosing and treating RA.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Kawano had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Nakamachi, Kawano, Kumagai.
Acquisition of data. Nakamachi, Kawano, Sakai, Chin, Saura, Kurosaka.
Analysis and interpretation of data. Nakamachi, Kawano, Takenokuchi, Kumagai.
Manuscript preparation. Nakamachi, Kawano.
Statistical analysis. Nakamachi, Nishimura.