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A role of microRNAs, which are ~22- nucleotide non coding RNAs, has recently been recognized in human diseases. The objective of this study was to identify the expression pattern of microRNA-146 (miR-146) in cartilage from patients with osteoarthritis (OA).
The expression of miR-146 in cartilage from 15 patients with OA was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and by in situ hybridization. Induction of the expression of miR-146 by cultures of normal human articular chondrocytes following stimulation with interleukin-1β (IL-1β) was examined by quantitative RT-PCR.
All cartilage samples were divided into three groups according to a modified Mankin scale; grade I: 0 - 5, grade II: 6 - 10, grade III: 11 - 14. In OA cartilage samples of grade I, the expression of miR-146a and Col2a1 was significantly higher than that of other groups (p<0.05). In OA cartilage of grades II and III, the expression of miR-146a and Col2a1 decreased while the expression of MMP13 was elevated in grade II. These data show that miR-146a is expressed intensely in cartilage with a low Mankin grade, and that miR-146a expression decreases in accordance with level of MMP13 expression. Section in situ hybridization of pri-miR-146a revealed that pri-miR-146a is expressed in chondrocytes in all layers, especially in the superficial layer where it is intensely expressed. The expression of miR-146 was markedly elevated by IL-1β stimulation in human chondrocytes in vitro.
This study shows that miR-146 is intensely expressed in low grade OA cartilage, and that its expression is induced by stimulation of IL-1β. MiR-146 might play a role in OA cartilage pathogenesis.
Osteoarthritis (OA) is a highly prevalent disease, which is characterized by progressive degeneration of articular cartilage [1-4]. Although little is known about OA pathogenesis, an imbalance between anabolic and catabolic factors which maintains the homeostasis of cartilage is thought to lead to cartilage degradation. While there is a delicate balance between anabolism and catabolism, strictly regulating matrix turnover in normal cartilage, catabolism becomes dominant over anabolism in OA cartilage, leading to the degradation of cartilage. Several reports have demonstrated an interaction between anabolic factors such as TGF-β and catabolic factors such as matrix metalloproteinase and aggrecanase in chondrocytes, however, the molecular mechanisms involved in OA remain unclear .
MicroRNA (miRNA)s are a family of ~22-nucleotide non coding RNAs identified in organisms ranging from nematodes to humans [6-8]. Many miRNAs are evolutionarily conserved across phyla, regulating gene expression by posttranscriptional gene repression. The miRNAs regulate gene expression by binding the 3′-untranslated region of their target mRNAs leading to translational repression or mRNA degradation. Several microRNAs exhibit a tissue-specific or developmental stage–specific expression pattern and have been reported to be associated with human diseases such as cancer, leukemia, and viral infection [9-11].
Taganov et al. reported that miRNA-146a/b (miR-146a/b) is induced in response to lipopolysaccharide (LPS) and proinflammatory mediators in THP-1 cells and that its induction is regulated by nuclear factor —kappa B (NF-κB) . Nakasa et al. reported that miR-146 is expressed more intensely in synovial tissues of rheumatoid arthritis compared to that of OA and normal individuals, and its expression in rheumatoid arthritis synovial fibroblasts was induced by stimulation with inflammatory cytokines such as TNFα and IL-1β. Inflammatory cytokines also play an important role as catabolic factors in OA cartilage . Therefore, there is the possibility that miR-146a might be expressed in OA cartilage and thus participate in the anabolic and catabolic balance. The aim of this study is to identify the expression of miR-146a in OA cartilage from OA patients, and its induction by IL-1β in human chondrocytes.
Articular cartilage samples were obtained from 15 OA patients (64.3 ± 15.7 years of age, mean ± SD) undergoing operations. OA was diagnosed according to the American Rheumatism Association Criteria for OA. Nine patients with affected hips underwent total arthroplasty and six patients with affected knees underwent total knee arthroplasty with the exception of patient 1 who had secondary OA in the patellofemoral joint following trauma injury to the articular cartilage, and underwent arthroscopic debridement. Their clinical characteristics are shown in Table 1.
This clinical research was conducted in compliance with the Declaration of Helsinki. Written permission was obtained from all patients who participated in this study.
Cartilage specimens, including all cartilage layers and subchondral bone, were obtained from the load bearing sites of the femoral condyles or femoral head except for patient 1. Cartilage from patient 1 was obtained from the patella. Total cartilage loss sites were avoided. Articular cartilage was dissected into 2 parts; one was used for the isolation of RNA, the other for histology. For polymerase chain reaction (PCR) analysis, total RNA was isolated from cartilage that had been homogenized on ice with Trizol reagent (Invitrogen). For histologic analysis, cartilage samples were fixed in 4% paraformaldehyde (PFA), decalcified in 0.24 M EDTA (Sigma-Aldrich, Japan) at 4 °C until the bones were pliable, then embedded in paraffin.
Total RNA yields were calculated and quality was determined using absorption spectrochemical analysis. One microgram of total RNA was reverse-transcribed using the QuantiTect® Reverse Transcription Kit (Qiagen, Chatsworth, CA) according to the manufacturer’s protocol. The genomic DNA elimination reaction was carried out using 2μl of gDNA wipeout buffer, 1μg (1μl) template RNA and 11μl RNase-free water at 42°C for 2 min. Reverse transcription was performed in 1μl quantiscript reverse transcriptase, 4μl quantiscript RT buffer, 1μl RT primer mix and 14μl template RNA (the entire genomic DNA elimination reaction) at 42°C for 15 min and 95°C for 3 min and then the cDNA product was maintained at 4°C.
Quantitative reverse transcription—PCR (RT-PCR) assays were performed using a TaqMan microRNA assay kit (Applied Biosystems, Foster City, CA) for the mature miR-146a and a Taqman Gene Expression Assay for hCol2a1, hMMP13, and hGAPDH (Applied Biosystems). RT reactions for mature miR-146a contained a sample of total RNA, 50 nM stem-loop RT primer, 10× RT buffer, 100 mM each dNTPs, 50 units/μl of MultiScribe reverse transcriptase, and 20 units/μl of RNase inhibitor. Reaction mixtures (15μl) were incubated in a thermal cycler (BioRad, Hercules, CA) for 30 minutes at 16°C, 30 minutes at 42°C, and 5 minutes at 85°C and then maintained at 4°C.
Real-time PCR was performed using a Mini Opticon Real-time PCR System (BioRad, Hercules, CA) in a 10 μl PCR mixture containing 2μl of RT product, 5μl of 2× TaqMan Universal PCR Master Mix, 0.2 μM TaqMan probe, 15 μM forward primer, and 0.7 μM reverse primer.
All reactions were incubated in a 48-well plate at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute; all were performed in triplicate. The U18 and GAPDH genes were used as controls to normalize differences in total RNA levels between samples. A threshold cycle (Ct) was observed in the exponential phase of amplification, and quantification of relative expression levels was performed using standard curves for target genes and the endogenous control. Geometric means were used to calculate the Δ Δ Ct values and were expressed as 2–ΔΔCt. The value of each control sample was set at 1 and was used to calculate the fold change in target genes for the induction of miR-146a by IL-1β.
Each paraffin embedded cartilage sample was sectioned at 5 μm and every tenth section was stained with Safranin O-fast green staining. Two independent assessors (TN and KY) graded each sample using a modified Mankin scale [15, 16]. For in situ hybridization, primary miR-146a fragments were derived from PCR products, cloned using the Qiagen PCR cloning kit into the pDrive vector (Qiagen, Chatsworth, CA), and sequenced. Primer sequences for primary miR-146a were 5′-TAT-TGG-GCA-AAC-AATCAG-CA-3′ (forward) and 5′-GCC-TGA-GAC-TCT-GCCTTC-TG-3′ (reverse). Digoxigenin (DIG)—labeled riboprobes were transcribed with a DIG RNA labeling kit and T7 polymerase (Roche, Mannheim, Germany). After deparaffinization, each section was fixed in 4 % paraformaldehyde for 10 minutes at room temperature, washed 3 times in phosphate buffered saline (PBS) for 3 minutes, and subsequently treated with 600 μg of proteinase K for 10 minutes at room temperature. After treatment in 0.2 % glycine-PBS for 10 minutes, sections were refixed in 4 % paraformaldehyde for 10 minutes, washed 3 times in PBS for 3 minutes each, and acetylated with 0.25 % acetic anhydride in 0.1 M triethanolamine hydrochloride for 10 minutes. After washing in PBS for 30 minutes, sections were prehybridized for 1 hour at 65°C with prehybridization buffer (50 % formamide and 5× saline—sodium citrate [SSC]). Hybridization with DIG-labeled riboprobes was performed overnight at 65°C in hybridization buffer (50 % formamide, 5× SSC, 5× Denhardt’s solution, and 250 μg/ml of baker’s yeast transfer RNA). After hybridization, sections were washed in 5× SSC for 30 minutes at 65°C, 0.2× SSC for 2 hours at 65°C, and 0.2× SSC for 5 minutes at room temperature. Blocking was performed overnight at 4°C with 4 % horse serum and alkaline phosphatase—conjugated Fab anti-DIG antibody (Roche) in 1 % sheep serum. Staining was performed using BCIP and nitroblue tetrazolium (NBT; Roche).
Articular cartilage was harvested from femoral condyles and tibial plateaus of human tissue donors. All articular cartilage samples were graded according to a modified Mankin scale, and we used only chondrocytes from normal articular cartilage. Human chondrocytes were isolated and cultured as described previously . The cartilage tissue was incubated with trypsin at 37°C for 10 minutes. After the trypsin solution was removed, the tissue slices were treated for 12 to 16 hours with type IV clostridial collagenase in Dulbecco’s modified Eagle’s medium (DMEM) with 5% fetal calf serum (FCS). After initial isolation, the cells were kept in high-density cultures in DMEM (high glucose) supplemented with 10% CS, L-glutamine, and antibiotics. After the cells had grown to confluence, they were split once (passage 1) and grown to confluence again for use in the experiments.
Cells were seeded at 1.0×105/well into a 6-well plate containing 2 ml of DMEM plus 5% FCS and 1% penicillin/streptomycin. After cells became adherent, they were treated with recombinant human IL-1β (5 ng/ml) (Pepro Tech, Rocky Hill, NJ) and then incubated for 24 hours under an atmosphere of 5 % CO2. Cells were washed twice with cold PBS, and then total RNA was isolated with Trizol reagent. Real-time PCR was performed in triplicate with the TaqMan microRNA assay kit for mature miR-146a or the Taqman Gene Expression Assay for MMP13.
One-way analysis of variance (ANOVA) followed by Tukey’s post hoc analysis was used to compare gene expression between the three groups. The Mann-Whitney U test was used to compare the gene expression between two groups. P values less than 0.05 were considered to be statistically significant. All statistical analyses were performed on a personal computer using the Stat View version 5.0 statistical package (Abacus Concepts, Berkeley, CA).
The expression of mature miR-146a and of mRNA for Col2a1 and MMP13 were screened by real time PCR in all samples to investigate miR-146a expression and its relationship to the extent of degradation of cartilage in articular cartilage (Figure 1A). All cartilage samples were divided into three groups according to a modified Mankin scale; grade I: 0 - 5, grade II: 6 - 10, grade III: 11 - 14. In OA cartilage samples of grade I, the expression of miR-146a and col2a1 was significantly higher than that of other groups (p<0.05) (Figure 1B). In addition, the expression of MMP13 was significantly lower than that of other groups (p<0.05). In OA cartilage samples of grades II and III, the expression of miR-146a and col2a1 decreased while the expression of MMP13 increased in grade II. In OA cartilage of grade III, MMP13 expression decreased, and there was a significant difference between grades I and II (p<0.05). These data indicate that miR-146a is expressed intensely in cartilage with a low grade of the Mankin scale, and miR-146a expression decreases with increasing MMP13 expression.
To examine the distribution of cells expressing miR-146a in OA cartilage, we performed in situ hybridization (ISH) of primary-miR-146a. ISH revealed that miR-146a is expressed in superficial, middle, and deep layers (Figure 2A-E). The chondrocytes expressing miR-146a were frequently located in the superficial and middle zones, where proteoglycans were depleted from the matrix (Figure 2F-H). In cartilage with the low expression level of miR-146a together with a high level of expression of MMP13, the cells expressing miR-146a were sparsely distributed in comparison with those in cartilage with a high expression level of miR-146a. In clustered chondrocytes, miR-146a also expressed (Figure 2C). In cartilage with a high grade of the Mankin scale, there were few miR-146a expressing cells (Figure2I, J).
To confirm the induction of expression of miR-146a in normal human articular chondrocytes following stimulation with IL-1β, we conducted real time PCR to investigate the expression of mature miR-146a and MMP13 (Figure 3). The expression of miR-146 was markedly increased in human chondrocytes after stimulation with IL-1β (Figure 3A). There was a significant difference between cells treated with and without IL-1β. MMP-13 expression was also induced after IL-1β stimulation, in the same way as miR-146 (Figure 3B).
Recently, it has been well established that miRNAs play a crucial role in the pathogenesis of human diseases [9-11,13]. 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 . 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 
miR-146a/b has been described as one of the key molecules in the inflammatory response and oncogenesis [12,13,20-24]. 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 . 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 , while Stanczyk et al. reported that miR-155 and 146a were intensely expressed in rheumatoid arthritis synovial fibroblasts and synovial tissues . 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 .
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 . 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 . 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 . 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, 21, 22]. There are several studies showing that chondrocytes in OA cartilage secrete MMP13 in response to IL-1 [26, 29, 30]. 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 [31,32]. Further functional analysis of miR-146 in OA pathogenesis could provide a novel and reasonable system for OA treatment.
This work was supported in part by Grant-in-Aid for Young Scientists (B) from the Japan Ministry of Education, Culture, Sports, Science and Technology (No.20791044) (T.N.), except for in vitro study supported by NIH (AR50631), Research on Publicly Essential Drugs and Medical Device (The Japan Health Sciences Fundation) (H. A.)