- Email: [email protected]
MicroRNA expression profiling of articular cartilage from a mouse model of post-traumatic OA
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
Purpose: To identify prognostic factors for the clinical outcome following arthroscopic partial meniscectomy Methods: The authors searched the Cochrane Library, Medline, Embase, Web of Science, Sportdiscus, Pubmed Publisher, Google Scholar and conference proceedings, checked reference lists and contacted authors. Main search terms included meniscal tear, treatment and meniscectomy. Two authors independently screened the search results. The main inclusion criteria were a minimum follow-up of 1 year after surgery and the use of a validated clinical questionnaire. The authors extracted data and assessed the Risk of Bias in duplicate, using the assessment form of the Evidence Based Guideline (Chochrane Group). Results: 24 of 4200 studies met the inclusion critera and were included. The authors found a risk of selection bias in 75 % (18 publications). None of the included articles showed a risk of information bias. In 92 % (22 publications) a risk of confounding bias was found. Strong evidence was found that the amount of resected tissue, the presence of radiological knee-osteoarthritis at baseline and the duration of complaints are prognostic factors for the clinical outcome of arthroscopic partial meniscectomy. Strong evidence was found that there is no correlation between pre-operative sport level or location of tear and clinical outcome. Moderate evidence was found that there is no correlation between gender, type of tear or pathophysiology (acute/chronic) and long term clinical outcome of arthroscopic partial meniscectomy. Conflicting evidence was found for the prognostic value of age, perioperative chondromalacie, BMI and leg alignment. Conclusions: Resecting more tissue, the presence of radiological kneeosteoarthritis and a long duration of complaints seem to result in a worse long-term clinical outcome following arthroscopic partial meniscectomy. There seems to be no correlation between location of tear, sport level, gender, type of tear or pathophysiology and clinical outcome.
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a 3-fold and 2-fold increase in the atrogenes MuRF-1 (p¼0.0283) and atrogin-1 (p¼0.0130), respectively. Conversely, tibialis anterior and gastrocnemius muscles of CA rabbits expressed 78% (p¼0.0095) and 70% (p¼0.0097) less myostatin with no alteration in TNF mRNA. Correlation analyses revealed that tibialis anterior whole-muscle CSD and type II myofibre CSD were inversely associated with the expression of pro-inflammatory, muscle-derived cytokines. Conclusions: Our model induced a severe sarcopenia characterized by reduced animal weight and muscle size, an up-regulation of atrogene expression, indicating muscle protein breakdown, and a decrease in muscle myostatin expression. The arthritic insult appeared to activate an inflammatory phenotype in skeletal muscle. We propose that inflamed muscle may contribute to the process of sarcopenia via a mechanism of autocrine atrophy triggered by increased secretion of muscle-derived, pro-inflammatory mediators. While our model reflects a severe inflammatory burden, the chronic low-grade inflammation seen in OA may activate similar catabolic pathways.
566 MUSCLE ALTERATIONS IN AN EXPERIMENTAL MODEL OF CHRONIC ARTHRITIS R.D. Little y, z, I. Prieto-Potin z, S. Perez-Baos z, A. Villalvilla z, P. Gratal z, G. Herrero-Beaumont z, R. Largo z. y Sch. of Publ. Hlth.and Preventive Med., Alfred Hosp., Monash Univ., Melbourne, Australia; z IIS-Fundacion Jimenez Diaz UAM, Madrid, Spain Purpose: Skeletal muscle mass is a consequence of the balance between anabolic and catabolic processes. Sarcopenia is a common comorbidity of osteoarthritis (OA) and is primarily attributed to age and inactivity, with a subsequent reduction in the anabolic pathway. However, the catabolic impact of chronic, low-grade inflammation is less known. Our aim was to evaluate the pathogenic mechanisms of sarcopenia secondary to systemic inflammation in a chronic arthritis frame. For this purpose, we characterise alterations to muscle structure and gene expression in an animal model of chronic inflammatory arthritis. Methods: Nineteen male, New Zealand white rabbits were randomly assigned to two groups: control (n¼12) and chronic arthritis (CA, n¼7). CA was induced over six weeks by intra-articular injections of a heterologous antigen in previously immunized rabbits. Animals received intra-dermal ovalbumin sensitization and four subsequent intra-articular injections. Nineteen gastrocnemius muscles were collected for gene expression studies and 10 tibialis anterior muscles (control n¼6; CA n¼4) were collected for both gene expression and histological studies. IL-1b, IL-6, TNF, CCL-2 (MCP-1), myostatin and two atrogenes, MuRF-1 and atrogin-1, were quantified using real-time PCR. Tibialis anterior mid-belly cross-sections were stained with haematoxylin & eosin and for ATPase activity to measure whole muscle cross-sectional diameter (CSD) and type II fibre CSD, respectively. Rabbits were weighed weekly and serum C-Reactive Protein (CRP) was measured at week 6. Group comparisons were performed using Mann-Whitney tests and associations were tested using Spearman correlation analyses. Results: CA rabbits exhibited significantly less weight gain than controls, with a maximum cumulative difference of -160g vs. þ570g (p<0.0001), and a 74-fold increase in serum CRP (p¼0.0003). Tibialis anterior of CA rabbits showed a 16% reduction in type II myofibre CSD (p¼0.0159) and a 27% reduction in whole muscle CSD (p¼0.0095) alongside an 11-fold increase in IL-1b (p¼0.0095), a 21-fold increase in IL-6 (p¼0.0381) and a 14-fold increase in CCL-2 mRNA (p¼0.0095). Gastrocnemii of CA rabbits demonstrated similar, yet less pronounced, inflammatory activity with a 3-fold increase in IL-1b (p¼0.0012), and a 5-fold increase in both IL-6 (p¼0.0221) and CCL-2 (p¼0.0018) alongside
MiRNA 567 MICRORNA EXPRESSION PROFILING OF ARTICULAR CARTILAGE FROM A MOUSE MODEL OF POST-TRAUMATIC OA L.H. Kung y, L. Rowley y, V. Ravi z, K. Bell y, C.B. Little z, J.F. Bateman y. y Murdoch Childrens Res. Inst., Melbourne, Australia; z Raymond Purves Bone and Joint Res. Lab., Univ. of Sydney, Sydney, Australia Purpose: Osteoarthritis (OA) is the most common form of degenerative joint disease and it results in considerable disability and health care expenditure worldwide. OA is a disease that impacts on all tissues of the joint, however, it is primarily characterized by the progressive breakdown of articular cartilage (AC). Whilst many disease targets and pathological pathways are being continuously associated with OA, there are still no disease modifying OA drugs that have demonstrated dramatic success. Clearly, there is still more work needed to fully understand the complex mechanisms responsible and thus improve on the development of more effective therapeutic approaches. Intriguingly, an increasing number of microRNAs (miRNAs) are being identified as novel regulators of OA disease initiation and progression, making them exciting candidates for therapeutic targets and diagnostic biomarkers. The purpose of this study is to identify and characterize the expression profile of miRNAs in cartilage of normal, early and progressive stages of OA in a mouse model of post-traumatic OA. Methods: OA was induced in 10-12 week old male wild type mice by bilateral surgical destabilization of the medial meniscus (DMM). RNA from the medial tibial AC of DMM and sham-operated mice was isolated by laser microdissection at 1 and 6 weeks post-surgery. Four mice per time point per group were used for this experiment. miRNA expression profiling was performed using Agilent miRNA microarrays. The severity of chondrocyte hypertrophy, proteoglycan (PG) loss, and structural damage in the tibial AC of the contralateral medial femoro-tibial joint was scored by one observer blinded to surgical intervention and postoperative time. Results: Chondrocyte hypertrophy was considerably more extensive in the tibial AC of DMM compared with sham-operated joints at both 1
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Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
and 6 weeks (p ¼ 0.002 and 0.004, respectively), but did not differ with time in either surgical group. There was no AC PG loss in either surgical group at 1 week, but significant PG loss was observed in the DMM group with time (p < 0.001) and in DMM versus sham at 6 weeks (p < 0.001). Similarly maximal and total AC structural damage scores did not differ between DMM and sham at 1 week, but both scores progressed with time in DMM (p < 0.003) and in DMM versus sham at 6 weeks (p < 0.001). miRNA expression profiling identified 122 and 74 miRNAs in AC that were differentially expressed (adj.p.value < 0.05) in DMM versus sham-operated mice at 1 and 6 weeks post-surgery, respectively. Moreover, 57 dysregulated miRNAs were found to be common to both 1 and 6 week time points. Amongst the 57, several miRNAs were identified with a FC > 2.0 and adj.p.value < 0.05, including miR-6931-5p, miR3082-5p, miR-1187, miR-669n, miR-468-3p, miR-669l-5p, miR-669e-5p and miR-672-5p. In addition, differentially expressed miRNAs were also found to be exclusively associated with either 1 or 6 weeks post-surgery (65 miRNAs at 1 week and 17 miRNAs at 6 weeks). Conclusions: We demonstrated typical OA pathological features in the AC of DMM-operated mice compared with sham, some of which were time dependent (PG loss and structural damage). Our microarray data revealed dynamic changes in miRNA expression profiles between normal (Sham) and OA cartilage (DMM). Interestingly, we observed changes in miRNA expression that mirrored the temporal changes in PG loss and structural damage. Similarly, we also demonstrated changes in miRNA expression which were independent of time, reflecting the chondrocyte hypertrophy scores which were also static with time. In conclusion, we have identified potential miRNA regulators of OA initiation and progression in AC which have not been previously associated with OA. Future studies are now focused on determining their role in OA disease progression and identifying miRNA gene targets. 568 MICRORNA-455 TARGETS MULTIPLE GENES IN THE WNT SIGNALLING PATHWAY T.E. Swingler y, L.T. Le y, S. Gardner y, M.J. Barter z, D.A. Young z, T. Dalmay y, I.M. Clark y. y Univ. of East Anglia, Norwich, United Kingdom; z Newcastle Univ., Newcastle, United Kingdom Purpose: There is increasingly compelling evidence linking miRNAs to both normal cartilage physiology and pathology. MiRNAs are 20-24 nucleotide non-coding RNA molecules that post-transcriptionally regulate gene expression. A single miRNA can regulate multiple mRNAs via binding to sequences in the 3’UTR either promoting mRNA decay or repressing translation. MicroRNAs are known to simultaneously target many transcripts, possibly to fine tune complex signalling pathways. We have previously identified several microRNAs that are regulated in chondrogenesis and also osteoarthritis, including miRNA-455, a key Sox9-responsive microRNA. We now focus on pathways through which miR-455 functions in cartilage. Methods: Primary chondrocytes were transfected with either 50nM miR-455-3p mimic or inhibitor or non-targeting controls. Cells were incubated for 48 hours and harvested for mRNA using Trizol (Invitrogen). Whole genome micro-array analysis (Illumina Human HT-12 v4 Expression BeadChip) was performed to identify genes regulated at the mRNA level. We further analysed the UTRs of the regulated genes for miR-455 binding sites. Pathway analysis was performed. The 3’UTR of potential targets were cloned and luciferase assays performed. Western blots were performed to confirm the potential targets change at the protein level. The TopFlash-luc reporter plasmid was used to examine the effect of miR-455 on WNT signalling. Results: Whole genome analysis measured genes that were repressed by miR-455 mimic and induced by miR-455 inhibitor. Further bioinformatics analysis identified those genes with potential miR-455 binding sites in their 3’UTR. Direct targets of miR-455 are most likely to be within this pool. These data revealed that miR-455 regulates at least 13 genes in the canonical and non-canonical WNT signalling pathway. Regulated genes include Dickkopf-related protein 1 (DKK1), Beta-catenin, Glycogen synthase kinase 3 beta (GSK-3b), Wingless-type MMTV integration site family, member A and B (WNT5A/B) and Calcium/ calmodulin dependent protein kinase II association domain (CaMKII). Luciferase assays examining response of the 3’UTR to miR-455 mimics confirmed the genes to be direct targets of miR-455. Target expression was confirmed at the protein level using western blot. Using the TCF responsive TOPFlash-luc reporter showed that miR455 inhibits canonical WNT signalling.
Conclusions: In this study we show that miRNA-455 inhibits Wnt signalling in chondrocytes. The Wnt signalling pathway is complex and central to skeletal development/ homeostasis and also implicated in osteoarthritis. We have shown that miRNA-455 inhibits the expression of at least 13 genes in the Wnt pathway, and we have validated several of these as new direct targets of this miRNA. 569 INFLUENCE OF OSTEOARTHRITIC CARTILAGE EXPLANTS ON THE EXPRESSION OF MIRNAS AND PREDICTED TARGETS IN COCULTURED CHONDROCYTES AND MESENCHYMAL STEM CELLS €ssel y. y Experimental Orthopedics, U. Mayer y, H.-R. Springorum z, S. Gra Ctr. of BioMed. Technology, Regensburg, Germany; z Dept. of Orthopedic Surgery, Univ. of Regensburg, Asklepiosklinikum, Bad Abbach, Germany Purpose: Stem cell-based strategies are promising in regenerative medicine but not much is known about the capacity of adult mesenchymal stem cells (MSC) in cartilage regeneration under osteoarthritic (OA) conditions. Contact to OA cartilage alters gene expression of cocultured chondrocytes and chondrogenically differentiating MSC regarding genes as collagen I, II, III and X, essential for chondrogenic, hypertrophic and mesenchymal phenotypes. Using an established in vitro coculture model, the influence of OA cartilage on cocultured chondrocytes and MSC is investigated concerning the expression of miR-124a, miR-675 and miR-29b described to be associated with the regulation of Sox9, collagen II and collagen I/III, respectively. Methods: Human chondrocytes are isolated from post-surgery discarded OA cartilage by collagenase II digestion and MSC by density gradient centrifugation of bone marrow cells derived from femoral heads of patients undergoing total hip replacement due to OA. Chondrocytes/MSC are embedded in fibrin gels and cultured either on OA cartilage explants (coculture) or without explants (monoculture) for 7 and 28 days in chondrogenic medium. Expression of miR-124a, miR-675 and miR-29b presumably associated with SOX9, COL2A1 and COL1A1/COL3A1 is analyzed by quantitative real-time PCR (qPCR). Expression levels of miRs and predicted targets are normalized to U6 snRNA and GAPDH, respectively and calibrated to correspondent monocultures. To verify if miR-29b inhibits collagen I/ III expression, miR-29b is overexpressed in MSC by transfection with a miR-29b mimic and subsequently gene expression of miR-29b and its targets COL1A1/COL3A1 is analyzed by qPCR and protein expression by western blot. To check if soluble factors derived from OA cartilage are responsible for the upregulation of miR-29b, MSC/ chondrocytes kept in micromass pellets are cultured in chondrogenic medium conditioned with OA cartilage explants or in unconditioned medium and miR-29b and COL1A1/COL3A1 expression is analyzed. As IL-6 occurs in higher concentration in supernatants of cocultures with OA cartilage which is a possible factor for upregulating miR-29b expression via activation of STAT3, stimulation experiments with IL-6 are in progress. Results: Coculture with OA cartilage leads to an increased expression of miR-124a, miR-675 and miR-29b in chondrocytes and MSC compared to monocultured cells. SOX9 expression is unaltered in cocultures whereas COL2A1, COL1A1 and COL3A1 expression is reduced. Transfection of MSC with a miR-29b mimic results in reduced collagen I and III expression. Chondrocytes and MSC cultured in chondrogenic medium conditioned with OA cartilage explants show higher miR-29b and lower COL1A1 and COL3A1 expression levels compared to chondrocytes and MSC cultured in unconditioned chondrogenic medium. Conclusions: Coculture with OA cartilage increases the expression of all investigated miRs and reduces collagen I, II and III expression in MSC and chondrocytes. miR-675, described as a cartilage-specific, Sox9-dependent positive regulator of collagen II, is supposed to target a collagen II transcriptional repressor. Therefore, reduced collagen II expression should be associated with reduced miR-675 and Sox9 expression. However, coculture with OA cartilage leads to increased miR-675 expression while Sox9 expression is unaltered although expression of miR-124a which targets Sox9 is increased. Thus miR-675 and miR-124a seem to target other or additional mRNAs suggesting that reduced collagen II levels are caused by other factors or miRs in our system. Increased miR-29b expression induced by soluble factors, i.e. cytokines or MMPs, released from OA cartilage into cell culture supernatant is responsible for the reduced collagen I and III expression.
Purpose: To identify prognostic factors for the clinical outcome following arthroscopic partial meniscectomy Methods: The authors searched the Cochrane Library, Medline, Embase, Web of Science, Sportdiscus, Pubmed Publisher, Google Scholar and conference proceedings, checked reference lists and contacted authors. Main search terms included meniscal tear, treatment and meniscectomy. Two authors independently screened the search results. The main inclusion criteria were a minimum follow-up of 1 year after surgery and the use of a validated clinical questionnaire. The authors extracted data and assessed the Risk of Bias in duplicate, using the assessment form of the Evidence Based Guideline (Chochrane Group). Results: 24 of 4200 studies met the inclusion critera and were included. The authors found a risk of selection bias in 75 % (18 publications). None of the included articles showed a risk of information bias. In 92 % (22 publications) a risk of confounding bias was found. Strong evidence was found that the amount of resected tissue, the presence of radiological knee-osteoarthritis at baseline and the duration of complaints are prognostic factors for the clinical outcome of arthroscopic partial meniscectomy. Strong evidence was found that there is no correlation between pre-operative sport level or location of tear and clinical outcome. Moderate evidence was found that there is no correlation between gender, type of tear or pathophysiology (acute/chronic) and long term clinical outcome of arthroscopic partial meniscectomy. Conflicting evidence was found for the prognostic value of age, perioperative chondromalacie, BMI and leg alignment. Conclusions: Resecting more tissue, the presence of radiological kneeosteoarthritis and a long duration of complaints seem to result in a worse long-term clinical outcome following arthroscopic partial meniscectomy. There seems to be no correlation between location of tear, sport level, gender, type of tear or pathophysiology and clinical outcome.
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a 3-fold and 2-fold increase in the atrogenes MuRF-1 (p¼0.0283) and atrogin-1 (p¼0.0130), respectively. Conversely, tibialis anterior and gastrocnemius muscles of CA rabbits expressed 78% (p¼0.0095) and 70% (p¼0.0097) less myostatin with no alteration in TNF mRNA. Correlation analyses revealed that tibialis anterior whole-muscle CSD and type II myofibre CSD were inversely associated with the expression of pro-inflammatory, muscle-derived cytokines. Conclusions: Our model induced a severe sarcopenia characterized by reduced animal weight and muscle size, an up-regulation of atrogene expression, indicating muscle protein breakdown, and a decrease in muscle myostatin expression. The arthritic insult appeared to activate an inflammatory phenotype in skeletal muscle. We propose that inflamed muscle may contribute to the process of sarcopenia via a mechanism of autocrine atrophy triggered by increased secretion of muscle-derived, pro-inflammatory mediators. While our model reflects a severe inflammatory burden, the chronic low-grade inflammation seen in OA may activate similar catabolic pathways.
566 MUSCLE ALTERATIONS IN AN EXPERIMENTAL MODEL OF CHRONIC ARTHRITIS R.D. Little y, z, I. Prieto-Potin z, S. Perez-Baos z, A. Villalvilla z, P. Gratal z, G. Herrero-Beaumont z, R. Largo z. y Sch. of Publ. Hlth.and Preventive Med., Alfred Hosp., Monash Univ., Melbourne, Australia; z IIS-Fundacion Jimenez Diaz UAM, Madrid, Spain Purpose: Skeletal muscle mass is a consequence of the balance between anabolic and catabolic processes. Sarcopenia is a common comorbidity of osteoarthritis (OA) and is primarily attributed to age and inactivity, with a subsequent reduction in the anabolic pathway. However, the catabolic impact of chronic, low-grade inflammation is less known. Our aim was to evaluate the pathogenic mechanisms of sarcopenia secondary to systemic inflammation in a chronic arthritis frame. For this purpose, we characterise alterations to muscle structure and gene expression in an animal model of chronic inflammatory arthritis. Methods: Nineteen male, New Zealand white rabbits were randomly assigned to two groups: control (n¼12) and chronic arthritis (CA, n¼7). CA was induced over six weeks by intra-articular injections of a heterologous antigen in previously immunized rabbits. Animals received intra-dermal ovalbumin sensitization and four subsequent intra-articular injections. Nineteen gastrocnemius muscles were collected for gene expression studies and 10 tibialis anterior muscles (control n¼6; CA n¼4) were collected for both gene expression and histological studies. IL-1b, IL-6, TNF, CCL-2 (MCP-1), myostatin and two atrogenes, MuRF-1 and atrogin-1, were quantified using real-time PCR. Tibialis anterior mid-belly cross-sections were stained with haematoxylin & eosin and for ATPase activity to measure whole muscle cross-sectional diameter (CSD) and type II fibre CSD, respectively. Rabbits were weighed weekly and serum C-Reactive Protein (CRP) was measured at week 6. Group comparisons were performed using Mann-Whitney tests and associations were tested using Spearman correlation analyses. Results: CA rabbits exhibited significantly less weight gain than controls, with a maximum cumulative difference of -160g vs. þ570g (p<0.0001), and a 74-fold increase in serum CRP (p¼0.0003). Tibialis anterior of CA rabbits showed a 16% reduction in type II myofibre CSD (p¼0.0159) and a 27% reduction in whole muscle CSD (p¼0.0095) alongside an 11-fold increase in IL-1b (p¼0.0095), a 21-fold increase in IL-6 (p¼0.0381) and a 14-fold increase in CCL-2 mRNA (p¼0.0095). Gastrocnemii of CA rabbits demonstrated similar, yet less pronounced, inflammatory activity with a 3-fold increase in IL-1b (p¼0.0012), and a 5-fold increase in both IL-6 (p¼0.0221) and CCL-2 (p¼0.0018) alongside
MiRNA 567 MICRORNA EXPRESSION PROFILING OF ARTICULAR CARTILAGE FROM A MOUSE MODEL OF POST-TRAUMATIC OA L.H. Kung y, L. Rowley y, V. Ravi z, K. Bell y, C.B. Little z, J.F. Bateman y. y Murdoch Childrens Res. Inst., Melbourne, Australia; z Raymond Purves Bone and Joint Res. Lab., Univ. of Sydney, Sydney, Australia Purpose: Osteoarthritis (OA) is the most common form of degenerative joint disease and it results in considerable disability and health care expenditure worldwide. OA is a disease that impacts on all tissues of the joint, however, it is primarily characterized by the progressive breakdown of articular cartilage (AC). Whilst many disease targets and pathological pathways are being continuously associated with OA, there are still no disease modifying OA drugs that have demonstrated dramatic success. Clearly, there is still more work needed to fully understand the complex mechanisms responsible and thus improve on the development of more effective therapeutic approaches. Intriguingly, an increasing number of microRNAs (miRNAs) are being identified as novel regulators of OA disease initiation and progression, making them exciting candidates for therapeutic targets and diagnostic biomarkers. The purpose of this study is to identify and characterize the expression profile of miRNAs in cartilage of normal, early and progressive stages of OA in a mouse model of post-traumatic OA. Methods: OA was induced in 10-12 week old male wild type mice by bilateral surgical destabilization of the medial meniscus (DMM). RNA from the medial tibial AC of DMM and sham-operated mice was isolated by laser microdissection at 1 and 6 weeks post-surgery. Four mice per time point per group were used for this experiment. miRNA expression profiling was performed using Agilent miRNA microarrays. The severity of chondrocyte hypertrophy, proteoglycan (PG) loss, and structural damage in the tibial AC of the contralateral medial femoro-tibial joint was scored by one observer blinded to surgical intervention and postoperative time. Results: Chondrocyte hypertrophy was considerably more extensive in the tibial AC of DMM compared with sham-operated joints at both 1
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Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
and 6 weeks (p ¼ 0.002 and 0.004, respectively), but did not differ with time in either surgical group. There was no AC PG loss in either surgical group at 1 week, but significant PG loss was observed in the DMM group with time (p < 0.001) and in DMM versus sham at 6 weeks (p < 0.001). Similarly maximal and total AC structural damage scores did not differ between DMM and sham at 1 week, but both scores progressed with time in DMM (p < 0.003) and in DMM versus sham at 6 weeks (p < 0.001). miRNA expression profiling identified 122 and 74 miRNAs in AC that were differentially expressed (adj.p.value < 0.05) in DMM versus sham-operated mice at 1 and 6 weeks post-surgery, respectively. Moreover, 57 dysregulated miRNAs were found to be common to both 1 and 6 week time points. Amongst the 57, several miRNAs were identified with a FC > 2.0 and adj.p.value < 0.05, including miR-6931-5p, miR3082-5p, miR-1187, miR-669n, miR-468-3p, miR-669l-5p, miR-669e-5p and miR-672-5p. In addition, differentially expressed miRNAs were also found to be exclusively associated with either 1 or 6 weeks post-surgery (65 miRNAs at 1 week and 17 miRNAs at 6 weeks). Conclusions: We demonstrated typical OA pathological features in the AC of DMM-operated mice compared with sham, some of which were time dependent (PG loss and structural damage). Our microarray data revealed dynamic changes in miRNA expression profiles between normal (Sham) and OA cartilage (DMM). Interestingly, we observed changes in miRNA expression that mirrored the temporal changes in PG loss and structural damage. Similarly, we also demonstrated changes in miRNA expression which were independent of time, reflecting the chondrocyte hypertrophy scores which were also static with time. In conclusion, we have identified potential miRNA regulators of OA initiation and progression in AC which have not been previously associated with OA. Future studies are now focused on determining their role in OA disease progression and identifying miRNA gene targets. 568 MICRORNA-455 TARGETS MULTIPLE GENES IN THE WNT SIGNALLING PATHWAY T.E. Swingler y, L.T. Le y, S. Gardner y, M.J. Barter z, D.A. Young z, T. Dalmay y, I.M. Clark y. y Univ. of East Anglia, Norwich, United Kingdom; z Newcastle Univ., Newcastle, United Kingdom Purpose: There is increasingly compelling evidence linking miRNAs to both normal cartilage physiology and pathology. MiRNAs are 20-24 nucleotide non-coding RNA molecules that post-transcriptionally regulate gene expression. A single miRNA can regulate multiple mRNAs via binding to sequences in the 3’UTR either promoting mRNA decay or repressing translation. MicroRNAs are known to simultaneously target many transcripts, possibly to fine tune complex signalling pathways. We have previously identified several microRNAs that are regulated in chondrogenesis and also osteoarthritis, including miRNA-455, a key Sox9-responsive microRNA. We now focus on pathways through which miR-455 functions in cartilage. Methods: Primary chondrocytes were transfected with either 50nM miR-455-3p mimic or inhibitor or non-targeting controls. Cells were incubated for 48 hours and harvested for mRNA using Trizol (Invitrogen). Whole genome micro-array analysis (Illumina Human HT-12 v4 Expression BeadChip) was performed to identify genes regulated at the mRNA level. We further analysed the UTRs of the regulated genes for miR-455 binding sites. Pathway analysis was performed. The 3’UTR of potential targets were cloned and luciferase assays performed. Western blots were performed to confirm the potential targets change at the protein level. The TopFlash-luc reporter plasmid was used to examine the effect of miR-455 on WNT signalling. Results: Whole genome analysis measured genes that were repressed by miR-455 mimic and induced by miR-455 inhibitor. Further bioinformatics analysis identified those genes with potential miR-455 binding sites in their 3’UTR. Direct targets of miR-455 are most likely to be within this pool. These data revealed that miR-455 regulates at least 13 genes in the canonical and non-canonical WNT signalling pathway. Regulated genes include Dickkopf-related protein 1 (DKK1), Beta-catenin, Glycogen synthase kinase 3 beta (GSK-3b), Wingless-type MMTV integration site family, member A and B (WNT5A/B) and Calcium/ calmodulin dependent protein kinase II association domain (CaMKII). Luciferase assays examining response of the 3’UTR to miR-455 mimics confirmed the genes to be direct targets of miR-455. Target expression was confirmed at the protein level using western blot. Using the TCF responsive TOPFlash-luc reporter showed that miR455 inhibits canonical WNT signalling.
Conclusions: In this study we show that miRNA-455 inhibits Wnt signalling in chondrocytes. The Wnt signalling pathway is complex and central to skeletal development/ homeostasis and also implicated in osteoarthritis. We have shown that miRNA-455 inhibits the expression of at least 13 genes in the Wnt pathway, and we have validated several of these as new direct targets of this miRNA. 569 INFLUENCE OF OSTEOARTHRITIC CARTILAGE EXPLANTS ON THE EXPRESSION OF MIRNAS AND PREDICTED TARGETS IN COCULTURED CHONDROCYTES AND MESENCHYMAL STEM CELLS €ssel y. y Experimental Orthopedics, U. Mayer y, H.-R. Springorum z, S. Gra Ctr. of BioMed. Technology, Regensburg, Germany; z Dept. of Orthopedic Surgery, Univ. of Regensburg, Asklepiosklinikum, Bad Abbach, Germany Purpose: Stem cell-based strategies are promising in regenerative medicine but not much is known about the capacity of adult mesenchymal stem cells (MSC) in cartilage regeneration under osteoarthritic (OA) conditions. Contact to OA cartilage alters gene expression of cocultured chondrocytes and chondrogenically differentiating MSC regarding genes as collagen I, II, III and X, essential for chondrogenic, hypertrophic and mesenchymal phenotypes. Using an established in vitro coculture model, the influence of OA cartilage on cocultured chondrocytes and MSC is investigated concerning the expression of miR-124a, miR-675 and miR-29b described to be associated with the regulation of Sox9, collagen II and collagen I/III, respectively. Methods: Human chondrocytes are isolated from post-surgery discarded OA cartilage by collagenase II digestion and MSC by density gradient centrifugation of bone marrow cells derived from femoral heads of patients undergoing total hip replacement due to OA. Chondrocytes/MSC are embedded in fibrin gels and cultured either on OA cartilage explants (coculture) or without explants (monoculture) for 7 and 28 days in chondrogenic medium. Expression of miR-124a, miR-675 and miR-29b presumably associated with SOX9, COL2A1 and COL1A1/COL3A1 is analyzed by quantitative real-time PCR (qPCR). Expression levels of miRs and predicted targets are normalized to U6 snRNA and GAPDH, respectively and calibrated to correspondent monocultures. To verify if miR-29b inhibits collagen I/ III expression, miR-29b is overexpressed in MSC by transfection with a miR-29b mimic and subsequently gene expression of miR-29b and its targets COL1A1/COL3A1 is analyzed by qPCR and protein expression by western blot. To check if soluble factors derived from OA cartilage are responsible for the upregulation of miR-29b, MSC/ chondrocytes kept in micromass pellets are cultured in chondrogenic medium conditioned with OA cartilage explants or in unconditioned medium and miR-29b and COL1A1/COL3A1 expression is analyzed. As IL-6 occurs in higher concentration in supernatants of cocultures with OA cartilage which is a possible factor for upregulating miR-29b expression via activation of STAT3, stimulation experiments with IL-6 are in progress. Results: Coculture with OA cartilage leads to an increased expression of miR-124a, miR-675 and miR-29b in chondrocytes and MSC compared to monocultured cells. SOX9 expression is unaltered in cocultures whereas COL2A1, COL1A1 and COL3A1 expression is reduced. Transfection of MSC with a miR-29b mimic results in reduced collagen I and III expression. Chondrocytes and MSC cultured in chondrogenic medium conditioned with OA cartilage explants show higher miR-29b and lower COL1A1 and COL3A1 expression levels compared to chondrocytes and MSC cultured in unconditioned chondrogenic medium. Conclusions: Coculture with OA cartilage increases the expression of all investigated miRs and reduces collagen I, II and III expression in MSC and chondrocytes. miR-675, described as a cartilage-specific, Sox9-dependent positive regulator of collagen II, is supposed to target a collagen II transcriptional repressor. Therefore, reduced collagen II expression should be associated with reduced miR-675 and Sox9 expression. However, coculture with OA cartilage leads to increased miR-675 expression while Sox9 expression is unaltered although expression of miR-124a which targets Sox9 is increased. Thus miR-675 and miR-124a seem to target other or additional mRNAs suggesting that reduced collagen II levels are caused by other factors or miRs in our system. Increased miR-29b expression induced by soluble factors, i.e. cytokines or MMPs, released from OA cartilage into cell culture supernatant is responsible for the reduced collagen I and III expression.