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Global gene expression of Oscar Knock-out (KO) mice with Wild Type (WT) mice.

ABSTRACT: Purpose: RNA sequencing (RNA-Seq) analyses of articular cartilage obtained after DMM surgery in Oscar-/- and WT mice. The purpose of this experiment was to demonstrate how the deficiency of Oscar gene affects downstream signal transduction in articular cartilage. Methods: Articular cartilage mRNA profiles of 10-weeks-old wild-type (WT) and Oscar knockout (Oscar−/−) mice were performed for the destabilization of the medial meniscus (DMM) and each cartilage tissue sample was harvested from two time-points (2- and 4-weeks after surgery). Sequencing libraries were prepared according to the manufacturer’s instructions (TruSeq Stranded mRNA Library Prep Kit; Illumina, San Diego, CA, USA). Paired-end sequencing of 101-mer read length was performed using a HISEQ 2500 sequencing system (Illumina). The sequencing quality of raw FASTQ files was assessed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Low-quality reads and adapter sequences in reads were eliminated using BBDuk (http://jgi.doe.gov/data-and-tools/bb-tools/). Results: Usinig RNA-Seq data in articular cartilage tissues from WT and Oscar–/– mice subjected to sham or DMM surgery and each cartilage sample obtained from 2- and 4-week time points after surgery, respectively. And we found that significantly enriched pathways in the 1270 common genes. Conclusions: Our study represents the Oscar gene deficiency analysis of articular cartilage transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

ORGANISM(S): Mus musculus

PROVIDER: GSE147529 | GEO | 2020/03/26

REPOSITORIES: GEO

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Project description:Mechanical Stimuli are arguably the most important aetiolgical factors in osteoarthritis (OA) development. Not only do we see disease arising from joints where the cartilage has sustained direct (e.g. intraarticular fracture) or indirect (e.g. meniscal injury) trauma, but mechanical factors are considered, at least partly, to explain the disease associations with aging and obesity. It is now well established that OA is not simply due to repeated wear and tear, leading to attrition of the articular surfaces, but that it requires activation of a number of inflammatory genes, which drive catabolic protease activity in the joint. These enzymes lead to breakdown of the major extracellular matrix components of cartilage, namely type II collagen, and the proteoglycan, aggrecan. Although it is unclear precisely which enzymes are responsible for matrix breakdown in human OA, Glasson et al showed that deletion of the aggrecan degrading enzyme, ADAMTS5 substantially protected the joint from surgically induced murine OA suggesting that it is a major aggrecanase in the mouse. This study was part of a wider study using the surgical model of murine OA, induced by destabilising the medial meniscus (DMM), to confirm the dependence of joint loading on disease progression and to reveal mechanosensitive genes within the joint which may be involved in the development of OA. Using this model we, and others, have shown that there is a robust degradation of articular cartilage over 4-12wks in male C57B/6 mice. We identified the gene response in joints early following induction of OA, prior to cartilage degradation, by extracting RNA from whole joints (after skin and muscle had been removed) 6hrs, 3 days and 7 days following sham and DMM surgery. An Affymetrix ST1 gene array was performed. Significantly regulated genes (>1.4 fold above naive and sham samples at any timepoint), or those considered to have a putative role in OA pathogenesis were selected for quantitative validation using Taqman low density microfluidic card arrays (TLDA). Gene expression in whole knee joints at 3 early time points post DMM surgery (before cartilage loss), 6hrs, 3days and 7days was examined. Two controls were identified, the first control consisted of age matched naïve mice that had undergone 15 minutes of anaesthesia but were not operated on (0hrs post surgery). The second control included mice which had undergone sham surgery (capsulotomy alone). For this RNA was taken at the same time points post surgery as DMM samples (6hrs, 3days and 7days). There were three biological replicates per condition (total of 21 samples). The right (ipsilateral) knee joint was taken and RNA extracted and processed for microarray analysis using the Affymetrix Mouse Gene 1.0 st v1 platform.

Project description:Objective: To Identify gene changes in the medial tibial plateau of articular cartilage at 2, 4 and 8 weeks after destabilisation of the medial meniscus (DMM) in mice and compare our data set with previously published sets to ascertain dysregulated pathways and genes in osteoarthritis. Materials and methods: RNA was extracted from the ipsilateral and contralateral medial tibial plateaus, amplified, labelled and hybridized on Illumina WG_v2 microarrays. Results were confirmed by RT-PCR for selected genes. Results: Transcriptional analysis and network reconstruction revealed changes in extracellular matrix (ECM) and cytoskeletal genes induced by DMM. TGFalpha signalling pathway and complement and coagulation cascade genes were regulated at 2 weeks. Fibronectin (FN1) is a hub in a reconstructed network of potential protein-protein interactions at 2 weeks. Regulated genes decrease over time and by eight weeks fibromodulin (FMOD) and tenascin N (TNN) are the only dysregulated genes present in the DMM operated knees. Comparison with human and rodent published gene sets discovered genes overlapping between our array and 6 others. Conclusions: Cartilage contributes a minute percentage to the RNA extracted from the whole joint (>0.2%), yet is sensitive to changes in gene expression post-DMM. The post-DMM transcriptional reprogramming wanes over time and dissipates by 8 weeks. Common pathways between published gene sets include focal adhesion, regulation of actin cytoskeleton and TGFalpha. Common genes include Jagged 1 (Jag1), Tetraspanin 2 (Tspan2), neuroblastoma, suppression of tumourigenicity 1 (Nbl1) and N-myc downstream regulated gene 2 (Ndrg2). The concomitant genes and pathways we identify are novel and warrant further investigation as biomarkers or modulators of osteoarthritis. In total 18 samples. Each sample was a pool of the medial tibial plateau of 8 mice. Two categories (Treated and untreated) with three biological replicates each and three time points (2, 4 and 8 weeks post-surgery).

Project description:As the most common degenerative joint disease, osteoarthritis (OA) contributes significantly to pain and disability during aging. Several genes of interest involved in articular cartilage damage in OA have been identified. However, the direct causes of OA are poorly understood. Evaluating the public human RNA-seq dataset showed that Cbfβ, (subunit of a heterodimeric Cbfβ/Runx1,Runx2, or Runx3 complex) expression is decreased in the cartilage of patients with OA. Here, we found that the chondrocyte-specific deletion of Cbfβ in tamoxifen-induced Cbfβf/fCol2α1-CreERT mice caused a spontaneous OA phenotype, worn articular cartilage, increased inflammation, and osteophytes. RNA-sequencing analysis showed that Cbfβ deficiency in articular cartilage resulted in reduced cartilage regeneration, increased canonical Wnt signaling and inflammatory response, and decreased Hippo/YAP signaling and TGF-β signaling. Immunostaining and western blot validated these RNA-seq analysis results. ACLT surgery-induced OA decreased Cbfβ and Yap expression and increased active β-catenin expression in articular cartilage, while local AAV-mediated Cbfβ overexpression promoted Yap expression and diminished active β-catenin expression in OA lesions. Remarkably, AAV-mediated Cbfβ overexpression in knee joints of mice with OA showed the significant protective effect of Cbfβ on articular cartilage in the ACLT OA mouse model. Overall, this study, using loss-of-function and gain-of-function approaches, uncovered that low expression of Cbfβ may be the cause of OA. Moreover, Local admission of Cbfβ may rescue and protect OA through decreasing Wnt/β-catenin signaling, and increasing Hippo/Yap signaling and TGFβ/Smad2/3 signaling in OA articular cartilage, indicating that local Cbfβ overexpression could be an effective strategy for treatment of OA. Using unbiased genome-wide RNA-seq data from Cbfβf/f;Col2α1-Cre hip joint articular cartilage and Cbfβf/f;Aggrecan-cre knee joint articular cartilage and their controls, we examined Cbfβ-mediated transcriptional targets for articular cartilage regeneration in OA.

Project description:Background: Meniscus tears are the most common injury in the knee and are associated with an increased risk of osteoarthritis (OA). The molecular profile of knees with meniscus tears is not well-studied. Therefore, to advance our understanding of the early response of the knee to injury, we compared the gene expression profile of meniscus and articular cartilage within the same knees following meniscus injury. Hypothesis/Purpose: To identify differences between the molecular signatures of meniscus and articular cartilage from knees with intact articular cartilage undergoing arthroscopic partial meniscectomy. Study Design: Descriptive laboratory study Methods: Patients (n=12) with a known isolated medial meniscus tear without any knee chondrosis or radiographic OA were consented prior to surgery. During arthroscopic partial meniscectomy, a sample of their injured meniscus and a sample of their articular cartilage off the medial femoral condyle were procured. The transcriptome signatures, as measured through Affymetrix microarray, were compared between the two tissues and underlying biological processes were explored computationally. Results: 3566 gene transcripts were differentially expressed between meniscus and articular cartilage. Gene transcripts down-regulated in articular cartilage were associated with extracellular matrix organization, wound healing, cell adhesion, and chemotaxis. Gene transcripts up-regulated in articular cartilage were associated with blood vessels morphogenesis and angiogenesis. Examples of individual genes with significant differences in expression between the two tissues include IBSP (23.76 fold; P < 0.001), upregulated in meniscus, and TREM1 (3.23 fold; P = 0.006), upregulated in meniscus. Conclusion: The meniscus and articular cartilage have distinct gene expression profiles in knees with meniscus tears and intact articular cartilage. Total RNA obtained from injured meniscus and normal articular cartilage from patients undergoing partial meniscectomy.

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Global gene expression of Oscar Knock-out (KO) mice with Wild Type (WT) mice.

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