Project description:The aim of this study is to identify, for the first time, the genome-wide DNA methylation profiles of human articular chondrocytes from OA and healtly cartilage samples. Genome wide DNA methylation profiling of normal and osteoarthritic samples. The Illumina Infinium 27k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,000 CpGs in cartilage knee samples. Samples included 18 healthy controls and 23 OA patients.
Project description:The aim of this study is to identify, for the first time, the genome-wide DNA methylation profiles of human articular chondrocytes from OA and healtly cartilage samples. Genome wide DNA methylation profiling of normal and osteoarthritic samples. The Illumina Infinium 27k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,000 CpGs in cartilage knee samples. Samples included 18 healthy controls and 23 OA patients. Bisulphite converted DNA from the 31 samples were hybridised to the Illumina Infinium 27k Human Methylation Beadchip v1.2
Project description:Osteoarthritis (OA) is a chronic disease of the joint characterized by a progressive degradation of articular cartilage and subchondral bone. In healthy tissue, specialized cells called chondrocytes are regulating a balanced cartilage catabolism and anabolism. By contrast osteoarthritic joints are characterized by a dramatic increase of cartilage catabolism, due to changes of gene expression patterns within chondrocytes. To identify potential epigenetic differences regulating this process a genome-wide methylation screening of paired unaffected and osteoarthritic knee cartilage samples was performed. Therefore samples of macroscopic arthritic and non-arthritic cartilage areas of the femoral condyle of five female patients were collected and DNA isolation was performed. For being able to investigate methylation changes on a genome-wide scale using only limited amounts of DNA a specific amplification protocol for mainly methylated DNA has been established, based on combinations of different methylation-sensitive and M-bM-^@M-^Sindependent restriction digestions. The amplified DNA was then labeled and hybridized onto Agilent M-bM-^@M-^\Human Promoter Whole GenomeM-bM-^@M-^] microarrays. A random variance t-test for paired (per patient) samples was performed, identifying 1214 differentially methylated genetic targets between arthritic and non-arthritic samples. The biological relevance of these genes was then further investigated via Gene Ontology (GO) and KEGG pathway analysis. DNA isolated of paired arthritic and non-arthritic knee cartilage samples of five different female osteoarthritis patients (10 samples) was methylation-specifically amplified using combinations of methylation-sensitive and -insensitive restriction enzymes. Amplicons were dye labeled (Cy3) and hybridized onto 2x244k Agilent Human Promoter microarrays.
Project description:Osteoarthritis (OA) is a chronic disease of the joint characterized by a progressive degradation of articular cartilage and subchondral bone. In healthy tissue, specialized cells called chondrocytes are regulating a balanced cartilage catabolism and anabolism. By contrast osteoarthritic joints are characterized by a dramatic increase of cartilage catabolism, due to changes of gene expression patterns within chondrocytes. To identify potential epigenetic differences regulating this process a genome-wide methylation screening of paired unaffected and osteoarthritic knee cartilage samples was performed. Therefore samples of macroscopic arthritic and non-arthritic cartilage areas of the femoral condyle of five female patients were collected and DNA isolation was performed. For being able to investigate methylation changes on a genome-wide scale using only limited amounts of DNA a specific amplification protocol for mainly methylated DNA has been established, based on combinations of different methylation-sensitive and –independent restriction digestions. The amplified DNA was then labeled and hybridized onto Agilent “Human Promoter Whole Genome” microarrays. A random variance t-test for paired (per patient) samples was performed, identifying 1214 differentially methylated genetic targets between arthritic and non-arthritic samples. The biological relevance of these genes was then further investigated via Gene Ontology (GO) and KEGG pathway analysis.
Project description:Genome wide DNA methylation profiling of different passages of human articular chondrocytes treated with 5-azacytidine. The Illumina Infinium 450k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,578 CpGs.Samples included 6 different passages of human articular chondrocytes with 5-azacytidine treatment, 6 different passages without 5-azacytidine treatment,
Project description:Genome wide DNA methylation profiling of different passages of human articular chondrocytes treated with 5-azacytidine. The Illumina Infinium 450k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,578 CpGs.Samples included 6 different passages of human articular chondrocytes with 5-azacytidine treatment, 6 different passages without 5-azacytidine treatment, Bisulphite converted DNA from the 12 samples were hybridised to the Illumina Infinium 450k Human Methylation Beadchip v1.2
Project description:Effect of stimulation with IL-1beta and p38 MAPK inhibition with SB203580 or Birb 796 on human articular osteoarthritic chondrocytes
Project description:Osteoarthritis (OA) is the most common form of arthritis worldwide. It is a complex disease affecting the whole joint but is generally characterized by progressive degradation of articular cartilage. Recent genome-wide association screens have implicated distinct DNA methylation signatures in OA patients. We show that the de novo DNA methyltransferase (Dnmt) 3b, but not Dnmt3a, is present in healthy murine and human articular chondrocytes and expression decreases in OA mouse models and in chondrocytes from human OA patients. Targeted deletion of Dnmt3b in murine articular chondrocytes results in an early onset and progressive post-natal OA-like pathology. RNA-seq and MethylC-seq analyses of Dnmt3b loss-of-function chondrocytes shows that cellular metabolic processes are affected. Specifically, TCA metabolites and mitochondrial respiration are elevated. Importantly, a chondroprotective effect was found following Dnmt3b gain-of-function in murine articular chondrocytes in vitro and in vivo. This study shows that Dnmt3b plays a significant role in regulating post-natal articular cartilage homeostasis. Cellular pathways regulated by Dnmt3b in chondrocytes may provide novel targets for therapeutic approaches to treat OA.