Recently, it has been well established that miRNAs play a crucial role in the pathogenesis of human diseases [9
]. Marcucci et al.
identified that the microRNA-181 family plays an important role in acute myeloid leukemia (AML) and encodes proteins involved in pathways of innate immunity mediated by toll-like receptors and interleukin-1β [10
]. Hébert et al.
reported that increased expression of proteins like APP or BACE1 beta-secretase may also be associated with genetic Alzheimer’s disease, and that miR-29a, -29b-1, and -9 can regulate BACE1 expression in vitro
]. Bruneau et al.
recognized that dysregulation of miR-1 or other developmentally important miRNAs might result in congenital heart disease in humans [19
miR-146a/b has been described as one of the key molecules in the inflammatory response and oncogenesis [12
]. Taganov et al.
reported that miR-146a/b is an NF-κB dependent gene which inhibits the expression of IRAK 1 and TRAF 6 by binding to the 3′ UTR of their mRNAs, and its expression is induced by inflammatory cytokines [12
]. They proposed that miR-146a/b might regulate cytokine signaling in the immune response through a negative feedback regulation loop involving down-regulation of IRAK 1 and TRAF 6. Monticelli et al.
demonstrated that miR-146 expression is higher in Th1 cell than in Th2 or naïve T cells [25
], while Stanczyk et al.
reported that miR-155 and 146a were intensely expressed in rheumatoid arthritis synovial fibroblasts and synovial tissues [24
]. Nakasa et al.
demonstrated that miR-146a is expressed in RA synovial tissues and showed that the miR-146a expressing cells were primarily CD68+ macrophages, but also included several CD3+ T cell subsets and CD79a+ B cells [13
Inflammatory cytokines also play a key role in OA cartilage degeneration. One of the most prominent catabolic cytokines playing a crucial role in OA is IL-1β. IL-1β not only promotes the release of degenerative enzymes such as MMPs and aggrecanases, but also inhibits the synthesis of extracellular matrix proteins by chondrocytes [26
]. In the current study, we confirm that miR-146 is expressed following stimulation by IL-1β in chondrocytes isolated from normal cartilage. This strongly supports the hypothesis that miR-146a expression is induced in OA pathogenesis.
In our preliminary studies, miR-146a in OA cartilage was expressed at a significantly greater level than in normal cartilage when analyzed using real time PCR (data not shown). In the present study, we divided OA cartilage into three grades according to a modified Mankin score. MiR-146 was expressed intensely in low grade OA cartilage, and decreased with increasing cartilage degeneration. In each case, miR-146a was likely to be expressed at a higher level in the cartilage with lower expression of MMP13, and the expression of miR-146a decreased with increasing expression of MMP13. MiR-146a induced by inflammatory cytokines might play a role in repression of catabolic factors such as MMP13 through miR-146 negative feedback including down regulation of IRAK1 and TRAF6 in early OA cartilage. In late stage OA cartilage with low expression levels of miR-146a, cartilage degradation might progress due to loss of miR-146a acting as a repressor of catabolic signals. In situ
hybridization of miR-146a in our study revealed that a greater number of miR-146a expressing chondrocytes were observed in the superficial zone with matrix degenerative changes. Proinflammatory cytokines such as IL-1β and TNFα were reported to be expressed by chondrocytes in the superficial zone, and these cells are sparsely distributed in the deep zone in OA specimens [27
]. Far fewer miR-146a expressing chondrocytes were observed in the deep zone where the matrix appeared normal. In contrast, clustered chondrocytes surrounded by normal matrix expressed miR-146a. These results suggest that miR-146a is not expressed in normal chondrocytes, but starts to be expressed in chondrocytes which begin to undergo degenerative changes. We were unable to clarify the reason why miR-146a is expressed abundantly in early OA cartilage, and its expression is decreased as the cartilage degrades. The target genes for miRNAs are estimated to range from one to hundreds based on target predictions using bioinformatics approaches [28
]. Mir-146a might therefore have other target genes apart from IRAK1 and TRAF6 in cartilage, and play a role in the progression of OA.
To our knowledge, the present study is the first report to focus on miRNA expression in OA cartilage. Our results revealed that miR-146a is expressed intensely in low grade OA cartilage, and is induced by IL-1β. Our study shows that miRNA could be a novel player in the anabolic and catabolic signals of cartilage homeostasis. However, the function of miR-146 in OA pathogenesis still remains unclear. MiR-146 is reported to be a negative regulator in the inflammatory response, its expression induced by inflammatory cytokines [12
]. There are several studies showing that chondrocytes in OA cartilage secrete MMP13 in response to IL-1 [26
]. These reports support our speculation that miR-146 is a negative feedback regulator of MMP13 in OA cartilage. However, our results also raise the possibility that miR-146 may be an activator in early OA cartilage because of the high degree of chondrocyte activation at local sites in the early stages of OA. Direct proof is necessary to substantiate our speculation. This might be difficult to prove in human cartilage samples from individual patients that are sampled at one point in time, which is the limitation of our study. Quite recently, several therapeutic trials to regulate the endogenous miRNAs related to various diseases were conducted [31
]. Further functional analysis of miR-146 in OA pathogenesis could provide a novel and reasonable system for OA treatment.