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• W2530316795 abstract "Osteoarthritis is a serious disease of articular cartilage. The pathogenic factors contributing to this disorder are inflammation, extracellular matrix degradation and failure to rebuild the articular cartilage. Preclinical studies suggest that microRNA-140 may play a protective role in osteoarthritis development, but little is known about the mechanism by which this occurs. Here we present the results of forced expression of microRNA-140 in an in vitro model of osteoarthritis, evaluated by global proteomics analysis. We show that inflammation was reduced through the altered levels of multiple proteins involved in the nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 pathway. microRNA-140 upregulated many of the components involved in the synthesis of hyaline extracellular matrix and reduced the levels of aggrecanases and syndecan 4, thus potentially both increasing cartilage repair and reducing cartilage breakdown. These results show how forced expression of microRNA-140 is likely to counteract all three pathogenic processes, and support the idea that intra-articular injection of microRNA-140 may benefit patients suffering from early osteoarthritis. Osteoarthritis is a serious disease of articular cartilage. The pathogenic factors contributing to this disorder are inflammation, extracellular matrix degradation and failure to rebuild the articular cartilage. Preclinical studies suggest that microRNA-140 may play a protective role in osteoarthritis development, but little is known about the mechanism by which this occurs. Here we present the results of forced expression of microRNA-140 in an in vitro model of osteoarthritis, evaluated by global proteomics analysis. We show that inflammation was reduced through the altered levels of multiple proteins involved in the nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 pathway. microRNA-140 upregulated many of the components involved in the synthesis of hyaline extracellular matrix and reduced the levels of aggrecanases and syndecan 4, thus potentially both increasing cartilage repair and reducing cartilage breakdown. These results show how forced expression of microRNA-140 is likely to counteract all three pathogenic processes, and support the idea that intra-articular injection of microRNA-140 may benefit patients suffering from early osteoarthritis. Articular cartilage covers the end of opposing bones in synovial joints. It provides smooth and almost frictionless movement of the bones against each other, and protection from damage by load and tensile forces. These properties are provided by a unique combination of extracellular matrix (ECM) molecules, of which the most important are type 2 collagen (COL2), the proteoglycan aggrecan (ACAN) and the glycosaminoglycan chondroitin sulfate (CS).1Seibel MJ Robins SP Bilezikian JP Dynamics of Bone and Cartilage Metabolism. Academic Press, San Diego2006Google Scholar Osteoarthritis (OA) is a disease that leads to gradual degradation of the articular cartilage ECM resulting in pain, stiffness and swelling of affected joints. OA is the most common form of arthritis and one of the major causes of disability in the western world, and it constitutes a huge economic burden for the society.2Centers for Disease Control and Prevention (CDC) National and state medical expenditures and lost earnings attributable to arthritis and other rheumatic conditions–United States, 2003.MMWR Morbidity and mortality weekly report. 2007; 56: 4-7PubMed Google Scholar Currently, no treatment has been shown to stop or reverse the progression of OA. In many patients, the end result will be joint replacement. There are several known risk factors associated with OA such as joint injury, inflammation, age, obesity, genetics and gender, but the molecular mechanisms behind OA are not fully understood. However, several of these pathogenic factors are thought to act through the increased secretion of interleukin 1β (IL1β) into the joint space.3Daheshia M Yao JQ The interleukin 1beta pathway in the pathogenesis of osteoarthritis.J Rheumatol. 2008; 35: 2306-2312Crossref PubMed Scopus (305) Google Scholar IL1B is an inflammatory mediator that acts through the nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB) pathway to induce expression of many genes that are upregulated in OA cartilage such as IL1B, IL6, IL8, and the matrix degrading enzymes matrix metalloprotein 13 (MMP13) and a disintegrin-like and metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5), responsible for degradation of COL2 and ACAN, respectively.4Goldring MB Chondrogenesis, chondrocyte differentiation, and articular cartilage metabolism in health and osteoarthritis.Ther Adv Musculoskelet Dis. 2012; 4: 269-285Crossref PubMed Scopus (284) Google Scholar microRNAs (miRNAs) are also dysregulated in OA cartilage. miRNAs are small double stranded RNA molecules that regulate gene expression by binding to complementary sequences in mRNA molecules, leading to either degradation of the mRNA or repression of translation.5Hutvágner G Zamore PD A microRNA in a multiple-turnover RNAi enzyme complex.Science. 2002; 297: 2056-2060Crossref PubMed Scopus (1638) Google Scholar,6Lee RC Feinbaum RL Ambros V The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.Cell. 1993; 75: 843-854Abstract Full Text PDF PubMed Scopus (9658) Google Scholar This makes miRNA therapeutics a promising treatment option for several diseases.7Bader AG miR-34 - a microRNA replacement therapy is headed to the clinic.Front Genet. 2012; 3: 120Crossref PubMed Scopus (538) Google Scholar,8Lanford RE Hildebrandt-Eriksen ES Petri A Persson R Lindow M Munk ME et al.Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection.Science. 2010; 327: 198-201Crossref PubMed Scopus (1465) Google Scholar microRNA-140 (miR-140) is regarded as a cartilage specific miRNA because it is predominantly expressed in cartilaginous tissues during development.9Tuddenham L Wheeler G Ntounia-Fousara S Waters J Hajihosseini MK Clark I et al.The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells.FEBS Lett. 2006; 580: 4214-4217Crossref PubMed Scopus (374) Google Scholar,10Wienholds E Kloosterman WP Miska E Alvarez-Saavedra E Berezikov E de Bruijn E et al.MicroRNA expression in zebrafish embryonic development.Science. 2005; 309: 310-311Crossref PubMed Scopus (1337) Google Scholar Knockout studies have revealed miR-140 to be an important factor in OA development, as miR-140-/- mice showed OA-like changes such as accelerated proteoglycan loss and ECM degradation, while mice overexpressing miR-140 were protected against degradation of proteoglycans and COL2 in a model of antigen-induced arthritis.11Miyaki S Sato T Inoue A Otsuki S Ito Y Yokoyama S et al.MicroRNA-140 plays dual roles in both cartilage development and homeostasis.Genes Dev. 2010; 24: 1173-1185Crossref PubMed Scopus (458) Google Scholar In the same study the aggrecanase ADAMTS5 was shown to be a target of miR-140, and it was suggested that this could explain the protective role of miR-140 in OA. In this study we investigated the role of miR-140-5p (hereafter referred to as miR-140) using an in vitro model of OA. Human articular chondrocytes (ACs) derived from OA cartilage overexpressing miR-140 were cultured in pathophysiologically relevant concentrations of recombinant human IL1B (rhIL1B) and analyzed using real-time quantitative polymerase chain reaction (RT-qPCR), western blot and global proteomics. Mass spectrometry-based proteomics was used because miRNAs can regulate gene expression at the protein level without affecting mRNA levels. We show that that forced expression of miR-140 increased protein levels of SOX9, ACAN and chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACT1), an enzyme involved in the synthesis of CS. Decreased expression was found for a range of inflammation mediators, including IL1B, IL6 and IL8. Likewise, signaling through the NFKB pathway is likely to be inhibited by increased expression of the NFKB inhibitor IKBA. Finally, cartilage degradation may be reduced by the possible downregulation of ADAMTS5 and the increase in syndecan 4 (SDC4), an inhibitor of ADAMTS5 activation. Thus, miR-140 may act through many pathways to counteract the development of OA. To investigate the protective role of miR-140 in OA, the in vitro model of adding rhIL1B to cell cultures was first optimized and validated by dose-response and time course analysis. Figure 1a,b shows that 0.1 ng/ml of rhIL1B induced near maximal transcription levels of IL1B, IL8, and MMP13 in ACs (i) and bone-marrow-derived mesenchymal stem cells (ii), often used in OA and cartilage research because of their chondrogenic potential. This concentration of rIL1B was used for the rest of the experiments. In a time course experiment, rhIL1B induced maximum expression level for IL1B, IL6, and IL8 at 5–8hours and for MMP13 at 20 hours in ACs (Figure 1c). IL1B, IL6, and IL8 mRNA levels were gradually decreased over 1 week in response to continuous stimulation of rhIL1B, but were still upregulated compared with unstimulated cells (Figure 1d). In contrast, MMP13 mRNA levels decreased to baseline values between days 2 and 5. IL1B activates the NFKB pathway which subsequently induces synthesis of IL1B, IL6, IL8, MMP13, and ADAMTS5.4Goldring MB Chondrogenesis, chondrocyte differentiation, and articular cartilage metabolism in health and osteoarthritis.Ther Adv Musculoskelet Dis. 2012; 4: 269-285Crossref PubMed Scopus (284) Google Scholar To validate the upregulation of these proteins, and also look for other proteins that might change following rhIL1B stimulation, mass spectrometry-based proteomics was performed on ACs from three donors. The cells were stimulated with rhIL1B for 20 hours before harvesting for analysis. IL1B, IL6, and MMP13 were confirmed to be upregulated at the protein level (Supplementary dataset 1, sheet 1). There was also a 56-, 12- and 4-fold upregulation of IL8 for the three donors, but this was not found to be statistically significant. As for all the previous experiments the mRNA for the respective proteins was upregulated in the three donors, including IL8 (see Supplementary Figure S1). In total, 91 proteins were upregulated. Several of them were involved in the NFKB pathway such as TNF receptor associated factor 1 and the NFKB transcription factors v-rel avian reticuloendotheliosis viral oncogene homolog (REL) and RELB. A total of 78 proteins were downregulated (see Supplementary dataset 1, sheet 2). The inhibitor of NFKB, NFKBIB (IKB-β), was also downregulated (see Supplementary dataset 1 for all differently expressed proteins). A gene ontology (GO) term analysis was performed, and showed that the upregulated proteins were involved in inflammatory responses, regulation of signaling, cell adhesion, carbohydrate and unsaturated fatty acid metabolism, and cartilage development while the downregulated proteins were mainly involved in metabolic processes, cell death, and cell cycle (Supplementary dataset 2, sheet 1 and 2). These results showed that addition of 0.1 ng/ml of rhIL1B had a dramatic effect on the ACs and confirmed translation of IL1B, IL6, and MMP13 mRNAs into proteins. We proceeded to test the effect of forced expression of miR-140 in this system by electroporation of a synthetic miR-140 mimic into monolayer expanded ACs on day 23 of culture. Previously we showed that liposomal transfection induced immune responses in MSCs and ACs. In contrast, no immune response was observed after electroporation.12Karlsen TA Brinchmann JE Liposome delivery of microRNA-145 to mesenchymal stem cells leads to immunological off-target effects mediated by RIG-I.Mol Ther. 2013; 21: 1169-1181Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar Electroporation was therefore used in this study. Two days after electroporation miR-140 increased ≃ 120–140 fold in the miR-140 electroporated cells compared with controls for all three donors (Figure 2a). The levels of miR-140 after electroporation corresponded perfectly to the endogenous levels of miR-140 in native cartilage (Figure 2a, donors 1 and 3). We and others have recently shown RALA, ADAMTS5, and BMP2 to be targets of miR-140. These were all reduced by miR-140 (Figure 2b), showing that the electroporation of miR-140 gave functionally appropriate results. One day after electroporation rhIL1B was added to the cells for 20 hours and RT-qPCR was used to analyze the expression of IL1B, IL6, IL8, OA-relevant chemokine (C-C motif) ligand 5 (CCL5), and ADAMTS4 mRNA.13Alaaeddine N Olee T Hashimoto S Creighton-Achermann L Lotz M Production of the chemokine RANTES by articular chondrocytes and role in cartilage degradation.Arthritis Rheum. 2001; 44: 1633-1643Crossref PubMed Scopus (107) Google Scholar,14Verma P Dalal K ADAMTS-4 and ADAMTS-5: key enzymes in osteoarthritis.J Cell Biochem. 2011; 112: 3507-3514Crossref PubMed Scopus (272) Google Scholar All genes except MMP13 were downregulated by miR-140 (Figure 3a). The synovial membrane is also known to be inflamed in OA patients, and synovial fibroblasts (SFs) are thought to contribute to OA development by secreting inflammatory cytokines. It is therefore desirable that miR-140 reduce inflammation also in the SFs. Figure 3b shows that miR-140 also inhibited IL1B, IL6, and IL8 in rhIL1B-stimulated SFs. Again MMP13 was not affected. This is consistent with previous findings where treatment with anti-miR-140 did not affect MMP13 protein15Tardif G Hum D Pelletier JP Duval N Martel-Pelletier J Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes.BMC Musculoskelet Disord. 2009; 10: 148Crossref PubMed Scopus (204) Google ScholarFigure 3Results of miR-140 overexpression in rhIL1B stimulated ACs. The cells were electroporated with a negative control sequence or miR-140. The following day the cells were stimulated with rhIL1B for 20 hours before analysis. RT-qPCR analysis of IL1B, IL6, IL8, CCL5, ADAMTS4, and MMP13 in ACs (a) and synovial fibroblasts (SFs) (b). Numbers in parentheses indicate fold change values relative to results for control transfection plus IL1B stimulation. Differentially shaded bars indicate the different genes. IL1B, interleukin 1β; RT-qPCR, real time-quantitative polymerase chain reaction; rhIL1B, recombinant human IL1B; MMP13, matrix metalloprotein 13; ACs, articular chondrocytes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We proceeded with proteomics analysis to look for differentially expressed proteins in the miR-140 transfected ACs. Mass spectrometry analysis showed that miR-140 decreased 43 and increased 114 proteins (Supplementary dataset 1, sheet 3, and 4). Selected proteins are listed in Table 1. IL1B and IL6 were downregulated, consistent with the RT-qPCR results. IL8 was downregulated 1.9-, 3.2-, and 17.5-fold in the three donors, but this was not statistically significant. IL8 is therefore not included in the lists. Several other proteins involved in inflammation, NFKB signaling and OA were downregulated (Table 1 and Figure 4). In particular, annexin 6 (ANXA6), E3 ubiquitin-protein ligase ZFP91 (ZFP91), MAGE family member D1 (MAGED1), tripartite motif containing 4 (TRIM4), and DEAD-box helicase 41 (DDX41) are known to be involved in the activation of the NFKB pathway. This suggested that miR-140 inhibited inflammation by inhibiting the NFKB pathway. IKB is known to be the most potent inhibitor of the NFKB pathway. IKBA was found to be upregulated in donor 2 and 3 only, and thus is not on the list of significantly upregulated proteins. As activation of the NFKB pathway happens within minutes after stimulation, we performed western blotting to look at the IKBA levels at early time points. Two AC donors were used, including donor 1 that was not significantly upregulated according to the proteomics (Figure 5a). In both donors IKBA was increased in miR-140-electroporated unstimulated cells and after 30 minutes of rIL1B stimulation. IKBA was also increased after 20 hours, but with minor differences in donor 2. This shows that miR-140 inhibited activation of NFKB also by increasing IKBA. Finally, integrin alpha 2 (ITGA2), known to facilitate inflammatory degradation of articular cartilage, was greatly reduced (Table 1).16Peters MA Wendholt D Strietholt S Frank S Pundt N Korb-Pap A et al.The loss of α2β1 integrin suppresses joint inflammation and cartilage destruction in mouse models of rheumatoid arthritis.Arthritis Rheum. 2012; 64: 1359-1368Crossref PubMed Scopus (52) Google Scholar The GO term analysis showed that the downregulated proteins were associated with regulation of response to stimulus, biological adhesion, cell activation, regulation of vascular endothelial growth factor production, and hyaluronan metabolic process (Supplementary dataset 2, sheet 3).Table 1Differentially expressed proteins after miR-140 transfection and rhIL1B stimulationDownregulated by miR-140ProteinProtein nameFold changeBiological processesITGA2Integrin, α2−15.4ECM, OA, inflammationDDX41Probable ATP-dependent RNA helicase DDX41−15.2TRIM4Tripartite motif-containing protein 4−10.4SDC4Syndecan-4−9.2IL1BInterleukin-1β−7.5ZFP91E3 ubiquitin-protein ligase ZFP91−5.9ANXA6Annexin 6−4.7MAGED1Melanoma-associated antigen D1−4.5IL6Interleukin-6−4.4FBN1Fibrillin-1−3.2LAMB3Laminin subunit β-3−2.2ANKRD17Ankyrin repeat domain-containing protein 17−2.0PVRL2Poliovirus receptor-related protein 2−2.0EEF1DEukaryotic translation elongation factor 1Δ−4.7Cell cycleMCCColorectal mutant cancer protein−2.8ERHEnhancer of rudimentary homolog−2.5NUMBProtein numb homolog−2.0PDS5ASister chromatid cohesion protein PDS5 homolog A−2.0TJP1Tight junction protein ZO-1−5.3Intracellular membrane trafficking and cytoskeletonCERCAMProbable inactive glycosyltransferase 25 family member 3−4.2ARFIP2Arfaptin-2−3.8GOLGA1Golgin subfamily A member 1−3.7RND3Rho-related GTP-binding protein RhoE−2.8SNF8Vacuolar-sorting protein SNF8−2.3FBLIM1Filamin-binding LIM protein 1−2.1PI4KBPhosphatidylinositol 4-kinase-β−2.1Upregulated by miR-140CSGALNACT1Chondroitin sulfate N-acetylgalactosaminyltransferase 111.6ECM, OA, inflammationcarbohydrate metabolismDOLKDolichol kinase3.9GALNT5Polypeptide N-acetylgalactosaminyltransferase 53.6NFATC2Nuclear factor of activated T-cells, cytoplasmic 23.6XYLBXylulose kinase3.6PTX3Pentraxin-related protein PTX33.0ALG12Dol-P-Man:Man(7)GlcNAc(2)-PP-Dol alpha-1,6-mannosyltransferase2.8FUCA1Tissue alpha-L-fucosidase2.8ACANAggrecan core protein2.4THY1 (CD90)Thy-1 membrane glycoprotein2.4NFATC1Nuclear factor of activated T-cells, cytoplasmic 12.3C1GALT1Glycoprotein-N-acetylgalactosamine 3-β-galactosyltransferase 12.1HGSNATHeparan-alpha-glucosaminide N-acetyltransferase2.2HAT1Histone acetyltransferase type B catalytic subunit2.0MMP13Collagenase 32.0MPDU1Mannose-P-dolichol utilization defect 1 protein2.0PAPD5PAP-associated domain-containing protein 52.8Cell cycleFGFR1Fibroblast growth factor receptor 12.7FAM208AProtein FAM208A2.6GSPT2Eukaryotic peptide chain release factor GTP-binding subunit ERF3B2.5CHAMP1Chromosome alignment-maintaining phosphoprotein 12.3GTF2H2CGeneral transcription factor IIH subunit 2-like protein2.3MCM4DNA replication licensing factor MCM32.3LARP4BLa-related protein 4B2.2NEK3Serine/threonine-protein kinase Nek32.2EIF2DEukaryotic translation initiation factor 2D2.1CDK2Cyclin-dependent kinase 22.0CRNKL1Crooked neck-like protein 12.0FBXL20F-box/LRR-repeat protein 202.8Protein degradationPOMPProteasome maturation protein2.4PSMG1Proteasome assembly chaperone 12.4ARIH2Rho GTPase-activating protein 222.2UBE2Q2Ubiquitin-conjugating enzyme E2 Q22.2LRSAM1E3 ubiquitin-protein ligase LRSAM12.1 Open table in a new tab Figure 5Western blot analysis of inflammation and chondrogenesis proteins. (a) IKB protein levels were analyzed in unstimulated cells, after 30 minutes (min) and 20 hours (h) following rhIL1B stimulation. (b) IL1B, IL6, IL8, ACAN, SOX9, and CSGALNACT1 in cells electroporated with control or miR-140 for 1 day and subsequently stimulated with rhIL1B for 20 hours. ACTB was used as loading control in a and b. (c) RT-qPCR analysis of ACAN, SOX9, and CSGALNACT mRNA levels after electroporation with miR-140 and stimulation with rhIL1B. D1, Donor 1; D2, Donor 2; D3, Donor 3. IL1B, interleukin 1β; RT-qPCR, real time-quantitative polymerase chain reaction; rhIL1B, recombinant human IL1B.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Of the upregulated proteins, many are known to play important roles in ECM and carbohydrate metabolism, OA, inflammation, cell cycle, and protein degradation (Table 1). Among the most interesting upregulated proteins were ACAN, CSGALNACT1, nuclear factor of activated T-cells (NFAT)C1, NFATC2, MMP13, and histone acetyltransferase type B catalytic subunit (HAT1). ACAN is the most important proteoglycan in articular cartilage while CSGALNACT1 is very important for synthesis of CS and normal development of articular cartilage in mice.17Sakai K Kimata K Sato T Gotoh M Narimatsu H Shinomiya K et al.Chondroitin sulfate N-acetylgalactosaminyltransferase-1 plays a critical role in chondroitin sulfate synthesis in cartilage.J Biol Chem. 2007; 282: 4152-4161Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar,18Sato T Kudo T Ikehara Y Ogawa H Hirano T Kiyohara K et al.Chondroitin sulfate N-acetylgalactosaminyltransferase 1 is necessary for normal endochondral ossification and aggrecan metabolism.J Biol Chem. 2011; 286: 5803-5812Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar,19Zheng J Wu C Ma W Zhang Y Hou T Xu H et al.Abnormal expression of chondroitin sulphate N-acetylgalactosaminyltransferase 1 and Hapln-1 in cartilage with Kashin-Beck disease and primary osteoarthritis.Int Orthop. 2013; 37: 2051-2059Crossref PubMed Scopus (22) Google Scholar In mice, loss of NFATC1 and NFATC2 leads to OA.20Greenblatt MB Ritter SY Wright J Tsang K Hu D Glimcher LH et al.NFATc1 and NFATc2 repress spontaneous osteoarthritis.Proc Natl Acad Sci USA. 2013; 110: 19914-19919Crossref PubMed Scopus (53) Google Scholar,21Wang J Gardner BM Lu Q Rodova M Woodbury BG Yost JG et al.Transcription factor Nfat1 deficiency causes osteoarthritis through dysfunction of adult articular chondrocytes.J Pathol. 2009; 219: 163-172Crossref PubMed Scopus (67) Google Scholar HAT1 is an inhibitor of NFKB signaling.22Sadler AJ Suliman BA Yu L Yuan X Wang D Irving AT et al.The acetyltransferase HAT1 moderates the NF-κB response by regulating the transcription factor PLZF.Nat Commun. 2015; 6: 6795Crossref PubMed Scopus (52) Google Scholar Previously, we showed that inhibition of miR-140 reduced both ACAN and SOX9 protein levels.23Karlsen TA Jakobsen RB Mikkelsen TS Brinchmann JE microRNA-140 targets RALA and regulates chondrogenic differentiation of human mesenchymal stem cells by translational enhancement of SOX9 and ACAN.Stem Cells Dev. 2014; 23: 290-304Crossref PubMed Scopus (101) Google Scholar However, SOX9 was not differently expressed in the proteomic analysis. In addition, IL8 was not significantly downregulated. SOX9 and IL8 were therefore included as a part of the western blot validation experiments for the proteomic analysis (Figure 5b). The results confirmed downregulation of IL1B, IL6, and IL8; and upregulation of ACAN, SOX9, and CSGALNACT1 (two known isoforms) in all the three donors. Interestingly, the upregulation of ACAN, SOX9, and CSGALNACT1 protein occurred in the absence of similar changes in mRNA levels (Figure 5c). ADAMTS5 mRNA was reduced following miR-140 transfection (Figure 2b), but ADAMTS5 was not detected in the proteomics analysis, presumably for technical reasons. Western blotting of ADAMTS5 showed a number of bands, of which only the smallest band (25 kDa) was downregulated in two of the three donors (Figure 5b). However, SDC4 binds to ADAMTS5 and has been shown to be essential for the activation of ADAMTS5.24Echtermeyer F Bertrand J Dreier R Meinecke I Neugebauer K Fuerst M et al.Syndecan-4 regulates ADAMTS-5 activation and cartilage breakdown in osteoarthritis.Nat Med. 2009; 15: 1072-1076Crossref PubMed Scopus (262) Google Scholar SDC4 was greatly reduced following miR-140 transfection (Table 1), which could lead to reduced ADAMTS5 activity in the absence of greatly reduced ADAMTS5 protein. According to GO term analysis the upregulated proteins were mainly associated with different metabolic processes, protein modification, cellular component organization, and development process (Supplementary dataset 2, sheet 4). Several of the downregulated proteins are predicted targets of miR-140 (Table 2), which suggests that downregulation of these proteins is mediated through the canonical pathways for miRNA bioactivity. Three such predicted targets are the mRNAs transcribed from the IL1B, IL6, SDC4 genes. To validate these mRNAs as targets for miR-140, luciferase reporter constructs containing the 3′-untranslated region (UTR) of IL1B, IL6, SDC4 or three control constructs containing the 3′UTR of ACTB, a random 3′UTR and a vector without 3′UTR (EMPTY), were cotransfected into HEK293 cells together with a negative control miR or miR-140 mimic (Figure 6). A lower luciferase signal was detected for IL1B, IL6, and SDC4 in the miR-140 transfected cells, but not in the three control vectors. This shows that IL1B, IL6, and SDC4 are targets of miR-140. We also performed a search in the PITA database to assess the binding strength (represented as an energetic score-ddG) of the 3′UTR of IL1B, IL6, and SDC4 to the miR-140 seed sequence. The binding strength scores were moderate and comparable to values from other validated targets of miR-140 (Supplementary dataset 3).Table 2Proteins that were downreguated by miR-140 and are predicted targets of miR-140GeneDIANAmTmiRandamiRWalkPITATargetscanSUMANKRD17111115ARFIP2111115FBLIM1111115FBN1111115IL1B000112IL6010102IMPACT010102ITGA2110114MCC011114OXR1111003SDC4110103SLC7A5111115TJP1110114TSC22D21111150, not predicted; 1, predicted. Open table in a new tab 0, not predicted; 1, predicted. OA is a serious disease that impacts on the life of a considerable proportion of the adult human population. The pathogenic factors contributing to this disorder are inflammation, ECM degradation and failure to rebuild the articular cartilage. miR-140 has been shown in animal models to protect against arthritis, in part through the downregulation of ADAMTS5. Using an in vitro model of OA we now extend this insight by showing that forced expression of miR-140 inhibits the NFKB signaling pathway and downstream inflammatory cytokines, possibly inhibits ECM degradation proteins and enhances levels of proteins essential for the regeneration of new hyaline cartilage. Recently it was shown that intra-articular injection of miR-140 ameliorated rheumatoid arthritis in mice.25Peng JS Chen SY Wu CL Chong HE Ding YC Shiau AL et al.Amelioration of experimental autoimmune arthritis through targeting of synovial fibroblasts by intra-articular delivery of microRNAs 140-3p and 140-5p.Arthritis Rheumatol. 2016; 68: 370-381Crossref PubMed Scopus (58) Google Scholar The current results support the notion that intra-articular injection of miR-140 in patients with asymptomatic or early stage OA may prevent the onset of full-blown disease. The in vitro model of adding rhIL1B to ACs is a well-established model. In this study, the concentration of rhIL1B added was 1/100 of the concentration frequently used by others. Where 0.1 ng/ml corresponds to the concentration of IL1B found in the synovial fluid of OA patients,26Vangsness Jr, CT Burke WS Narvy SJ MacPhee RD Fedenko AN Human knee synovial fluid cytokines correlated with grade of knee osteoarthritis–a pilot study.Bull NYU Hosp Jt Dis. 2011; 69: 122-127PubMed Google Scholar and it is therefore a pathophysiologically relevant concentration. It is also similar to the concentration of IL1B found to be secreted following sterile trauma to other tissues.27Sjögren F Anderson C Sterile trauma to normal human dermis invariably induces IL1beta, IL6 and IL8 in an innate response to “danger”.Acta Derm Venereol. 2009; 89: 459-465Crossref PubMed Scopus (35) Google Scholar To simulate the effect of intraarticular injection, we transfected miR-140 into ACs in the rhIL1B induced OA model. The rationale behind this was several fold: loss of miR-140 has been shown to induce OA in knockout mice, overexpression of miR-140 in transgenic mice has been shown to protect against collagen-induced arthritis and miR-140 expression has been found to be reduced in cartilage and synovial fluid of human OA patients.11Miyaki S Sato T Inoue A Otsuki S Ito Y Yokoyama S et al.MicroRNA-140 plays dual roles in both cartilage development and homeostasis.Genes Dev. 2010; 24: 1173-1185Crossref PubMed Scopus (458) Google Scholar,28Iliopoulos D Malizos KN Oikonomou P Tsezou A Integrative microRNA and proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks.PLoS One. 2008; 3: e3740Crossref PubMed Scopus (418) Google Scholar,29Miyaki S Nakasa T Otsuki S Grogan SP Higashiyama R Inoue A et al.MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses.Arthritis Rheum. 2009; 60: 2723-2730Cros" @default.
• W2530316795 created "2016-10-21" @default.
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• W2530316795 title "microRNA-140 Inhibits Inflammation and Stimulates Chondrogenesis in a Model of Interleukin 1β-induced Osteoarthritis" @default.
• W2530316795 cites W1491975479 @default.
• W2530316795 cites W1527682346 @default.
• W2530316795 cites W1537032233 @default.
• W2530316795 cites W1951304988 @default.
• W2530316795 cites W1967210326 @default.
• W2530316795 cites W1967342160 @default.
• W2530316795 cites W1969722892 @default.
• W2530316795 cites W1981186391 @default.
• W2530316795 cites W1985163729 @default.
• W2530316795 cites W1985982440 @default.
• W2530316795 cites W1999382756 @default.
• W2530316795 cites W2002388650 @default.
• W2530316795 cites W2007033032 @default.
• W2530316795 cites W2012276832 @default.
• W2530316795 cites W2014028055 @default.
• W2530316795 cites W2022687771 @default.
• W2530316795 cites W2036786326 @default.
• W2530316795 cites W2039799680 @default.
• W2530316795 cites W2043965285 @default.
• W2530316795 cites W2050066317 @default.
• W2530316795 cites W2060951121 @default.
• W2530316795 cites W2061265481 @default.
• W2530316795 cites W2074549189 @default.
• W2530316795 cites W2079836802 @default.
• W2530316795 cites W2080396135 @default.
• W2530316795 cites W2080752012 @default.
• W2530316795 cites W2085192138 @default.
• W2530316795 cites W2087958138 @default.
• W2530316795 cites W2088287778 @default.
• W2530316795 cites W2095415569 @default.
• W2530316795 cites W2108768458 @default.
• W2530316795 cites W2111222524 @default.
• W2530316795 cites W2114370145 @default.
• W2530316795 cites W2128818253 @default.
• W2530316795 cites W2138664009 @default.
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