- Email: [email protected]
Association of Circulating Micrornas With Osteoarthritis
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
mice. Raw data was parsed to remove undetectable miRNA across all samples and modified global means normalization applied prior to miRNA expression analyses. Results: A total of 256 miRNA were expressed with 100 miRNAs demonstrating a 3-fold change compared to day 0, unoperated (control) mice at any one time point in the OA tissue and were clustered into several different expression patterns during the time course (Figure 1). Subgroups of these RNAs were linked to functional activities through analyses of their targets that formed networks related to angiogenesis, growth factor signaling (Tgfb, Igf, Pdgf, Fgf, Wnt), inflammation (cytokines and chemokines), apoptosis and kinase pathways (Figure 2). The functional activities of miRNAs with the highest fold changes that have not been previously reported in OA, are being further examined for their biological effects in chondrocytes. Many miRNAs we find exhibiting stage -dependent expression in the DMM mouse model, had been reported in human OA tissue (e.g., miR-155, miR-34a, miR-140, miR-27). Hence, these miRNAs have the potential for diagnostic value and intervention of OA. Conclusions: Our whole-genome miRNA analysis of joint tissues (articular cartilage and subchondral bone) revealed that the deregulated miRNAs clustered into 4 distinct expression pattern groups (Figure 1). Bioinformatic profiling revealed a common theme that the main targets of miRNA deregulation during OA progression are growth factor pathways (Figure 2). Additionally, the miRNA expression profile and the subsequent pathway analysis revealed EGF and PDGF as novel OAaffected signaling pathways not previously shown to be perturbed in the natural progression of OA. In addition, miRNA deregulation suggests that the Bmp (Smad1/5/8) signaling is favored over Tgfb signaling in the DMM knee joint. Together, these data suggest that the function of miRNA involvement in OA may be to target and downregulate key signaling molecules in growth factor pathways that promote cartilage anabolism (Egf & Pdgf) and also skew signaling of the Tgfb superfamily such that BMP signaling is enhanced over Tgfb signaling that may enhance cartilage degeneration. The data presented here represent OA progression through days 0 - 28 in mice, which likely represent early and moderate stages of OA development in humans. In summary, we propose that one mechanism through which OA progression occurs may be through direct miRNA inhibition of key elements of the EGF, PDGF, TGFb and BMP signaling pathways. We also propose that early medical interventions following knee injury can be made that involve antimiRs against growth factor-targeting miRNAs.
Figure 1: Analysis of 202 miRNAs differentially expressed 3-fold in OA vs. non-OA knee osteochondral samples. A). Principal component analysis (PCA) reveals 4 distinct clusters of expression patterns. B0. Average expression pattem of miRNAs within each cluster (IeIV) fordays 0, 4, 7, 14, 21 & 28, post-OA-inducing surgery. C). Hierarchical clustering: analysis of each PCA cluster according to post-operative day.
S289
455 ASSOCIATION OF CIRCULATING MICRORNAS WITH OSTEOARTHRITIS J.-C. Rousseau y, E. Sornay-Rendu y, O. Borel y, z, R. Chapurlat y, x. y INSERM 1033, Lyon, France; z Ho ^pital E. Herriot, Hospices Civils de ^pital E. Herriot, Hospices Civils de Lyon, Lyon, Lyon, France; x Ho Universit e de Lyon, Lyon, France Purpose: Sensitive and specific blood biomarkers to detect the initial stages of osteoarthritis (OA) and to predict the future development of the disease are not available in clinical routine. Consequently, there is a considerable interest in the identification of new markers. In OA, studies investigating the altered expression of regulatory RNA that may be used clinically are scarce. MicroRNAs (miRs) are small non-coding RNAs of approximately 22 nucleotides in length that can silence gene expression by binding to complementary sequence on target messenger RNA transcripts resulting in translational repression or target degradation. They are easily accessible and stable. So, we studied the differential expression of circulating miRs in subjects with and without OA in the OFELY cohort. Methods: The study group included French women belonging to the population-based cohort OFELY (Os des FEmmes de LYon). Expression levels of serum miR were measured in 10 healthy women without OA at any site (knee, lumbar spine, hip and hand) and in 10 women with a Kellgren & Lawrence score of 2 and 3 (early and intermediate knee OA) and OA at others sites. The evaluation of the OA disease was performed by radiography for spine disc degeneration and knee OA, by clinical examination for hand OA and by questionnaire for hip OA. These evaluations have been performed at the same visit, 8 years after recruitment of the cohort. Both groups were matched for age (healthy: 61.9 ± 3.03 and OA: 63.9 ± 3.4 p¼0.17) and menopausal status. According to the manufacturer's protocol (EXIQON, Denmark) for the Next Generation Sequencing (NGS) method, RNA isolation was performed from 400 ml of serum followed by the library preparation and amplification and the mRNA sequencing (Illumina platform). Measurements were expressed as Tags per million (TPM), which is a unit used to measure expression in NGS experiments. The number of reads for a particular miR is divided by the total number of mapped reads and multiplied by 1 million (Tags Per Million, TPM). Results: We identified 421 miRs with an expression level 1 TPM and 241 with an expression level 10 TPM. When we compared the two groups, 22 miRs showed differential expression (p<0.05) between controls and OA patients. After Benjamini-Hochberg False Discovery Rate (FDR) correction has-miR-139-5p, has-miR-1299 and has-miR200a-3p remained significantly different between OA patients and controls (p<0.05, FDR at 5%) (Table).
S290
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
Names
Log fold change
P-value
FDR
Healthy average TPM
OA average TPM
has-miR-139-5p has-miR-1299 has-miR-200a-3p
0.734682 3.38328 1.881
0.000126 0.000201 0.000328
0.043432 0.043432 0.047335
90.1 12 77.2
143.3 0.8 29.4
Conclusions: With a NGS screening approach, we identified 3 miRs that are differentially expressed in women suffering from OA compared to healthy women. The next step will be the measurement of these specific miRs in the entire cohort to determine the clinical utility of these markers. 456 THE ROLE OF MICRORNAS IN OSTEOARTHRITIS AND AGEINGRELATED FUNCTIONAL DECLINE IN JOINT TISSUE HOMEOSTASIS R. McCormick, L. House, B. Poulet, M. Roebuck, S. Frostick, G. Bou-Gharious, K. Goljanek-Whysall. Univ. of Liverpool, Liverpool, United Kingdom Purpose: Osteoarthritis is a degenerative disease associated with changes in the articular cartilage and bone, severely affecting patients' mobility and quality of life. A similar, but less acute decline is also seen during ageing. microRNAs (miRs) are novel post-transcriptional regulators of gene expression. microRNAs have been implicated in different aspects of musculoskeletal ageing through their differential expression, however the functional consequences of this are still not fully understood. Methods: Wild type C57BL6/J adult (3e6 months old) mice and human cartilage explants from knee replacement surgeries were used. To study microRNA function in vivo, modified microRNA antagomiR was delivered via intraarticular injection into knee joints of mice or via transfection into the human explants. Knee joints were dissected for histological assessment or RNA isolation. Human cartilage explants were cultured with IL-1b, miR mimic and/or antagomiR. Fixed joints and explants were subsequently decalcified and processed for wax embedding. Serial coronal sections were stained with Safranin O at regular intervals across the joint, and scored for articular cartilage degradation severity, using the OARSI grading system (from grades 0e6). RNA was used for microRNA and mRNA profiling using highthroughput sequencing and subsequently qPCR. Results: Our data shows dysregulation of miR-199 in human and mouse models of OA. Using qPCR and luciferase assays, we validated a selection of the microRNAs target genes differentially expressed in mouse femorotibial joints and human cartilage explants models of OA. miR-199 was further analysed using in vitro and in vivo gain- and loss-of-function approaches. Conclusions: In summary, we have shown the regulation of OA-associated target genes by miR-199 in the in vitro and in vivo models of OA and have shown that dysregulation of miR-199 expression may contribute to the pathogenic phenotype in the OA models. The authors would like to acknowledge financial support from the MRC-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA) and BBSRC. 457 MICRORNA-34A: ROLE IN THE DEVELOPMENT OF OSTEOARTHRITIS DURING OBESITY H. Endisha y, z, P. Datta y, A. Sharma y, G. Tavallaee y, z, M. Kapoor y, z. y Krembil Res. Inst., Toronto, ON, Canada; z Univ. of Toronto, Toronto, ON, Canada Purpose: The prevalence of obesity is increasing at an alarming rate and is one of the major risk factors of osteoarthritis (OA). Previous studies conducted in our lab show that mice fed a high-fat diet (HFD) exhibited an accelerated surgically-induced OA phenotype compared to lean diet (LD) mice. In humans, the synovial fluid of end-stage knee OA patients expressed significantly higher levels of microRNA-34a (miR-34a) than early-stage OA patients. MicroRNAs are endogenous short non-coding RNA segments that are negative regulators of gene expression and involved in many biological processes. Recent studies have shown that
miR-34a levels are elevated in obesity; however, the functional role of elevated miR-34a in obesity is unknown. Despite the strong association between obesity and OA pathogenesis, no studies have examined the role of miR-34a in the development of OA during obesity. We hypothesize that during obesity expression of miR-34a is elevated and contributes to OA pathophysiology. Methods: Mouse blood was collected via saphenous vein at 9 weeks of age and at the end of a HFD (34.3% fat by weight, 5.1 kcal/g) or LD (4.2% fat content, 3.7 kcal/g) course, which spans 18 weeks. Human plasma was taken at preadmission from end-stage OA patients undergoing total knee replacement (TKR) surgery. Patients with no co-morbidities were segregated according to body mass index (BMI) into normal weight (BMI¼ 18.5e24.9 kg/m2) and overweight groups (BMI>¼ 25 kg/m2). Following RNA isolation, miRNA and gene expression from plasma and cultured cells, respectively were determined by qRT-PCR. Chondrocytes and synovial fibroblasts were plated in replicates and transfected with 100nM miR-34a mimic for 24h. Results: Mir-34a was detectable in plasma of human and mice. Plasma miR-34a levels were significantly up-regulated in HFD mice and correlated with percent body fat, body weight, and fasting blood glucose compared to baseline mice and LD controls. Similarly, human plasma miR-34a levels were up-regulated in overweight end-stage OA patients compared to normal weight end-stage OA patients. In-situ hybridization showed HFD mouse knee joints expressed higher levels of miR34a than LD mouse knees and localized to cartilage and synovial membrane. Chondrocytes treated with a miR-34a mimic express a reduction of SIRT1 (a direct target of miR-34a), anabolic (aggrecan) and autophagy markers (ATG5, ULK1), as well as, elevated catabolic markers (MMP13), suggesting that miR-34a contributes to cartilage degeneration. Synovial fibroblasts treated with miR-34a mimic express significantly elevated inflammatory (TNF-a, IL-6), fibrotic (TGF-b, Type 1 Collagen), and autophagy markers (ULK1, ATG3, ATG5), suggesting that miR-34a is involved in mediating synovial inflammation and fibrosis. Conclusions: This is the first report to show that mir-34a is up-regulated in the plasma of obese individuals with end-stage OA compared to non-obese OA individuals, as well as, in the plasma, cartilage and synovium of obese mice compared to lean mice. Next, our data shows that miR-34a is involved in cartilage degeneration by promoting chondrocyte loss and catabolic activity. Furthermore, our data shows that miR-34a is involved in promoting synovial inflammation and fibrosis. Thus targeting miR-34a may provide therapeutic benefit in delaying or preventing the progression of OA. In this context, we are currently investigating the exact role and therapeutic potential of miR34a in OA pathogenesis using miR-34a knock-out mice and miR-34a anti-sense inhibitors. 458 ELEVATED OSTEOCLASTIC MIR-214-3P TARGETS TIMP2 TO PROMOTE SUBCHONDRAL BONE REMODELING IN EARLY OSTEOARTHRITIS L. Jin, X. Wu, A. Lu, G. Zhang. Hong Kong Baptist Univ., Hong Kong, Hong Kong Purpose: Emerging evidence demonstrates that the active osteoclastmediated subchondral bone (SCB) remodeling precedes the articular cartilage degradation to play a critical role in the initiation and progression of osteoarthritis (OA). Several recent studies have shown that the microRNAs were differentiated expressed in SCB between OA individuals and non-OA controls, suggesting a potential role of microRNAs in OA pathogenesis. In our previous studies, we have documented the pro-osteoclastogenic role of miR-214-3p. Moreover, we have demonstrated that osteoclast could release exosomal miR-214-3p into circulation. In this study, we study the role of miR-214-3p in osteoclastmediated SCB remodeling in early OA development. Methods: First, the SCB miR-214-3p expression in femoral head samples from OA patients and individuals with femoral neck fracture (nonOA control) were examined by QPCR. Next, a mouse model with surgery-induced OA, i.e. anterior cruciate ligament transection (ACLT) model, was established for investigating the miR-214-3p expression in serum and SCB osteoclasts by QPCR, the cartilage degradation by safranin O-staining histology and the circulating bone remodeling marker (CTX-I) and cartilage degradation marker (CTX-II) by ELISA. Thereafter, a series of functional experiments, including luciferase reporter assay and gain-and-loss of function assay, were performed to identified the potential targets of miR-214-3p in regulating OA osteoclast formation and their resorptive activity in cartilage explant in vitro.
mice. Raw data was parsed to remove undetectable miRNA across all samples and modified global means normalization applied prior to miRNA expression analyses. Results: A total of 256 miRNA were expressed with 100 miRNAs demonstrating a 3-fold change compared to day 0, unoperated (control) mice at any one time point in the OA tissue and were clustered into several different expression patterns during the time course (Figure 1). Subgroups of these RNAs were linked to functional activities through analyses of their targets that formed networks related to angiogenesis, growth factor signaling (Tgfb, Igf, Pdgf, Fgf, Wnt), inflammation (cytokines and chemokines), apoptosis and kinase pathways (Figure 2). The functional activities of miRNAs with the highest fold changes that have not been previously reported in OA, are being further examined for their biological effects in chondrocytes. Many miRNAs we find exhibiting stage -dependent expression in the DMM mouse model, had been reported in human OA tissue (e.g., miR-155, miR-34a, miR-140, miR-27). Hence, these miRNAs have the potential for diagnostic value and intervention of OA. Conclusions: Our whole-genome miRNA analysis of joint tissues (articular cartilage and subchondral bone) revealed that the deregulated miRNAs clustered into 4 distinct expression pattern groups (Figure 1). Bioinformatic profiling revealed a common theme that the main targets of miRNA deregulation during OA progression are growth factor pathways (Figure 2). Additionally, the miRNA expression profile and the subsequent pathway analysis revealed EGF and PDGF as novel OAaffected signaling pathways not previously shown to be perturbed in the natural progression of OA. In addition, miRNA deregulation suggests that the Bmp (Smad1/5/8) signaling is favored over Tgfb signaling in the DMM knee joint. Together, these data suggest that the function of miRNA involvement in OA may be to target and downregulate key signaling molecules in growth factor pathways that promote cartilage anabolism (Egf & Pdgf) and also skew signaling of the Tgfb superfamily such that BMP signaling is enhanced over Tgfb signaling that may enhance cartilage degeneration. The data presented here represent OA progression through days 0 - 28 in mice, which likely represent early and moderate stages of OA development in humans. In summary, we propose that one mechanism through which OA progression occurs may be through direct miRNA inhibition of key elements of the EGF, PDGF, TGFb and BMP signaling pathways. We also propose that early medical interventions following knee injury can be made that involve antimiRs against growth factor-targeting miRNAs.
Figure 1: Analysis of 202 miRNAs differentially expressed 3-fold in OA vs. non-OA knee osteochondral samples. A). Principal component analysis (PCA) reveals 4 distinct clusters of expression patterns. B0. Average expression pattem of miRNAs within each cluster (IeIV) fordays 0, 4, 7, 14, 21 & 28, post-OA-inducing surgery. C). Hierarchical clustering: analysis of each PCA cluster according to post-operative day.
S289
455 ASSOCIATION OF CIRCULATING MICRORNAS WITH OSTEOARTHRITIS J.-C. Rousseau y, E. Sornay-Rendu y, O. Borel y, z, R. Chapurlat y, x. y INSERM 1033, Lyon, France; z Ho ^pital E. Herriot, Hospices Civils de ^pital E. Herriot, Hospices Civils de Lyon, Lyon, Lyon, France; x Ho Universit e de Lyon, Lyon, France Purpose: Sensitive and specific blood biomarkers to detect the initial stages of osteoarthritis (OA) and to predict the future development of the disease are not available in clinical routine. Consequently, there is a considerable interest in the identification of new markers. In OA, studies investigating the altered expression of regulatory RNA that may be used clinically are scarce. MicroRNAs (miRs) are small non-coding RNAs of approximately 22 nucleotides in length that can silence gene expression by binding to complementary sequence on target messenger RNA transcripts resulting in translational repression or target degradation. They are easily accessible and stable. So, we studied the differential expression of circulating miRs in subjects with and without OA in the OFELY cohort. Methods: The study group included French women belonging to the population-based cohort OFELY (Os des FEmmes de LYon). Expression levels of serum miR were measured in 10 healthy women without OA at any site (knee, lumbar spine, hip and hand) and in 10 women with a Kellgren & Lawrence score of 2 and 3 (early and intermediate knee OA) and OA at others sites. The evaluation of the OA disease was performed by radiography for spine disc degeneration and knee OA, by clinical examination for hand OA and by questionnaire for hip OA. These evaluations have been performed at the same visit, 8 years after recruitment of the cohort. Both groups were matched for age (healthy: 61.9 ± 3.03 and OA: 63.9 ± 3.4 p¼0.17) and menopausal status. According to the manufacturer's protocol (EXIQON, Denmark) for the Next Generation Sequencing (NGS) method, RNA isolation was performed from 400 ml of serum followed by the library preparation and amplification and the mRNA sequencing (Illumina platform). Measurements were expressed as Tags per million (TPM), which is a unit used to measure expression in NGS experiments. The number of reads for a particular miR is divided by the total number of mapped reads and multiplied by 1 million (Tags Per Million, TPM). Results: We identified 421 miRs with an expression level 1 TPM and 241 with an expression level 10 TPM. When we compared the two groups, 22 miRs showed differential expression (p<0.05) between controls and OA patients. After Benjamini-Hochberg False Discovery Rate (FDR) correction has-miR-139-5p, has-miR-1299 and has-miR200a-3p remained significantly different between OA patients and controls (p<0.05, FDR at 5%) (Table).
S290
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
Names
Log fold change
P-value
FDR
Healthy average TPM
OA average TPM
has-miR-139-5p has-miR-1299 has-miR-200a-3p
0.734682 3.38328 1.881
0.000126 0.000201 0.000328
0.043432 0.043432 0.047335
90.1 12 77.2
143.3 0.8 29.4
Conclusions: With a NGS screening approach, we identified 3 miRs that are differentially expressed in women suffering from OA compared to healthy women. The next step will be the measurement of these specific miRs in the entire cohort to determine the clinical utility of these markers. 456 THE ROLE OF MICRORNAS IN OSTEOARTHRITIS AND AGEINGRELATED FUNCTIONAL DECLINE IN JOINT TISSUE HOMEOSTASIS R. McCormick, L. House, B. Poulet, M. Roebuck, S. Frostick, G. Bou-Gharious, K. Goljanek-Whysall. Univ. of Liverpool, Liverpool, United Kingdom Purpose: Osteoarthritis is a degenerative disease associated with changes in the articular cartilage and bone, severely affecting patients' mobility and quality of life. A similar, but less acute decline is also seen during ageing. microRNAs (miRs) are novel post-transcriptional regulators of gene expression. microRNAs have been implicated in different aspects of musculoskeletal ageing through their differential expression, however the functional consequences of this are still not fully understood. Methods: Wild type C57BL6/J adult (3e6 months old) mice and human cartilage explants from knee replacement surgeries were used. To study microRNA function in vivo, modified microRNA antagomiR was delivered via intraarticular injection into knee joints of mice or via transfection into the human explants. Knee joints were dissected for histological assessment or RNA isolation. Human cartilage explants were cultured with IL-1b, miR mimic and/or antagomiR. Fixed joints and explants were subsequently decalcified and processed for wax embedding. Serial coronal sections were stained with Safranin O at regular intervals across the joint, and scored for articular cartilage degradation severity, using the OARSI grading system (from grades 0e6). RNA was used for microRNA and mRNA profiling using highthroughput sequencing and subsequently qPCR. Results: Our data shows dysregulation of miR-199 in human and mouse models of OA. Using qPCR and luciferase assays, we validated a selection of the microRNAs target genes differentially expressed in mouse femorotibial joints and human cartilage explants models of OA. miR-199 was further analysed using in vitro and in vivo gain- and loss-of-function approaches. Conclusions: In summary, we have shown the regulation of OA-associated target genes by miR-199 in the in vitro and in vivo models of OA and have shown that dysregulation of miR-199 expression may contribute to the pathogenic phenotype in the OA models. The authors would like to acknowledge financial support from the MRC-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA) and BBSRC. 457 MICRORNA-34A: ROLE IN THE DEVELOPMENT OF OSTEOARTHRITIS DURING OBESITY H. Endisha y, z, P. Datta y, A. Sharma y, G. Tavallaee y, z, M. Kapoor y, z. y Krembil Res. Inst., Toronto, ON, Canada; z Univ. of Toronto, Toronto, ON, Canada Purpose: The prevalence of obesity is increasing at an alarming rate and is one of the major risk factors of osteoarthritis (OA). Previous studies conducted in our lab show that mice fed a high-fat diet (HFD) exhibited an accelerated surgically-induced OA phenotype compared to lean diet (LD) mice. In humans, the synovial fluid of end-stage knee OA patients expressed significantly higher levels of microRNA-34a (miR-34a) than early-stage OA patients. MicroRNAs are endogenous short non-coding RNA segments that are negative regulators of gene expression and involved in many biological processes. Recent studies have shown that
miR-34a levels are elevated in obesity; however, the functional role of elevated miR-34a in obesity is unknown. Despite the strong association between obesity and OA pathogenesis, no studies have examined the role of miR-34a in the development of OA during obesity. We hypothesize that during obesity expression of miR-34a is elevated and contributes to OA pathophysiology. Methods: Mouse blood was collected via saphenous vein at 9 weeks of age and at the end of a HFD (34.3% fat by weight, 5.1 kcal/g) or LD (4.2% fat content, 3.7 kcal/g) course, which spans 18 weeks. Human plasma was taken at preadmission from end-stage OA patients undergoing total knee replacement (TKR) surgery. Patients with no co-morbidities were segregated according to body mass index (BMI) into normal weight (BMI¼ 18.5e24.9 kg/m2) and overweight groups (BMI>¼ 25 kg/m2). Following RNA isolation, miRNA and gene expression from plasma and cultured cells, respectively were determined by qRT-PCR. Chondrocytes and synovial fibroblasts were plated in replicates and transfected with 100nM miR-34a mimic for 24h. Results: Mir-34a was detectable in plasma of human and mice. Plasma miR-34a levels were significantly up-regulated in HFD mice and correlated with percent body fat, body weight, and fasting blood glucose compared to baseline mice and LD controls. Similarly, human plasma miR-34a levels were up-regulated in overweight end-stage OA patients compared to normal weight end-stage OA patients. In-situ hybridization showed HFD mouse knee joints expressed higher levels of miR34a than LD mouse knees and localized to cartilage and synovial membrane. Chondrocytes treated with a miR-34a mimic express a reduction of SIRT1 (a direct target of miR-34a), anabolic (aggrecan) and autophagy markers (ATG5, ULK1), as well as, elevated catabolic markers (MMP13), suggesting that miR-34a contributes to cartilage degeneration. Synovial fibroblasts treated with miR-34a mimic express significantly elevated inflammatory (TNF-a, IL-6), fibrotic (TGF-b, Type 1 Collagen), and autophagy markers (ULK1, ATG3, ATG5), suggesting that miR-34a is involved in mediating synovial inflammation and fibrosis. Conclusions: This is the first report to show that mir-34a is up-regulated in the plasma of obese individuals with end-stage OA compared to non-obese OA individuals, as well as, in the plasma, cartilage and synovium of obese mice compared to lean mice. Next, our data shows that miR-34a is involved in cartilage degeneration by promoting chondrocyte loss and catabolic activity. Furthermore, our data shows that miR-34a is involved in promoting synovial inflammation and fibrosis. Thus targeting miR-34a may provide therapeutic benefit in delaying or preventing the progression of OA. In this context, we are currently investigating the exact role and therapeutic potential of miR34a in OA pathogenesis using miR-34a knock-out mice and miR-34a anti-sense inhibitors. 458 ELEVATED OSTEOCLASTIC MIR-214-3P TARGETS TIMP2 TO PROMOTE SUBCHONDRAL BONE REMODELING IN EARLY OSTEOARTHRITIS L. Jin, X. Wu, A. Lu, G. Zhang. Hong Kong Baptist Univ., Hong Kong, Hong Kong Purpose: Emerging evidence demonstrates that the active osteoclastmediated subchondral bone (SCB) remodeling precedes the articular cartilage degradation to play a critical role in the initiation and progression of osteoarthritis (OA). Several recent studies have shown that the microRNAs were differentiated expressed in SCB between OA individuals and non-OA controls, suggesting a potential role of microRNAs in OA pathogenesis. In our previous studies, we have documented the pro-osteoclastogenic role of miR-214-3p. Moreover, we have demonstrated that osteoclast could release exosomal miR-214-3p into circulation. In this study, we study the role of miR-214-3p in osteoclastmediated SCB remodeling in early OA development. Methods: First, the SCB miR-214-3p expression in femoral head samples from OA patients and individuals with femoral neck fracture (nonOA control) were examined by QPCR. Next, a mouse model with surgery-induced OA, i.e. anterior cruciate ligament transection (ACLT) model, was established for investigating the miR-214-3p expression in serum and SCB osteoclasts by QPCR, the cartilage degradation by safranin O-staining histology and the circulating bone remodeling marker (CTX-I) and cartilage degradation marker (CTX-II) by ELISA. Thereafter, a series of functional experiments, including luciferase reporter assay and gain-and-loss of function assay, were performed to identified the potential targets of miR-214-3p in regulating OA osteoclast formation and their resorptive activity in cartilage explant in vitro.