ABSTRACT: We studied microRNA expression levels from serum of postmenopausal women with osteoporosis by investigating the anti-osteoporotic treatment denosumab for a period of 2 years in a longitudinal study. Serum RNA was extracted and subject to small RNA sequencing on an Illumina HiSeqV4 SR50 using 50bp, single end reads. After mapping against GRCh38.p12 provided by Ensembl and miRBase v22.1 differential expression analysis was undertaken with edgeRv3.28 using a quasi-likelihood negative binomial generalized log-linear model. We identified a panel of altered small non-coding RNAs between 3 different time points for all patients.
Project description:We studied microRNA expression levels from serum and ulna bone in an in vivo model of type II diabetes (ZDF rats) by investigating different anti-osteoporotic treatments (anti-sclerostin, PTH and insulin). Serum and ulna bone RNA were extracted and subject to small RNA sequencing on an Illumina NextSeq550 SR75 High using 75bp, single end reads. After mapping against Rnor.6.0 provided by Ensembl and miRBase v22.1 differential expression analysis was undertaken with edgeRv3.32 using a quasi-likelihood negative binomial generalized log-linear model. We identified a panel of altered small non-coding RNAs.
Project description:We studied extracellular vesicles (EVs) from synovial fibroblast (SF). EVs were isolated from the secretome of non-senescent and irradiation-induced senescent SFs using size exclusion chromatography. EV RNA was extracted and subject to small RNA sequencing on an Illumina NovaSeq SP using 100bp, single end reads. After mapping against GRCh38.p12 and miRBase v22.1 differential expression analysis was undertaken with edgeRv3.28 using a quasi-likelihood negative binomial generalized log-linear model. We identified a panel of differentially expressed small non-coding RNAs.
Project description:We studied extracellular vesicles (EVs) from plasma and synovial fluid in an in vivo model of equine osteoarthritis by investigating longitudinal samples. EVs were isolated using size exclusion chromatography from plasma and synovial fluid of four horses subjected to an osteochondral fragment model of osteoarthritis at 0, 10, 35, 42, 49, 56, 63 days with relevant controls.EV RNA was extracted and subject to small RNA sequencing on an Illumina NovaSeq SP using 100bp, single end reads. After mapping against EquCab.3.0 and miRBase v22.1 differential expression analysis was undertaken with edgeRv3.28 using a quasi-likelihood negative binomial generalized log-linear model. We identified a panel of altered small non-coding RNAs.
Project description:The objective of this study was the identification of serum microRNAs that can differentiate osteoporotic fracture patients with and without type-2 diabetes from healthy control subjects. For that purpose circulating microRNAs were profiled by real-time quantitative PCR using a custom 384-well panel in 200 µl serum samples. Univariate and multivariate statistical tools were used in order to identify single as well as combinations of circulating microRNas that were characteristic of patients with prevalent osteoporotic fractures: a qRT-PCR-based classifier consisting of miR-550a-5p, miR-96-5p, miR-32-3p and miR-486-5p can distinguish T2D women with (DMFx) and without fragility fractures (DM) with high specifitiy and sensitivity (AUC = 0.93). A classifier consisting of miR-188-3p, miR-382-3p, miR-942 and miR-155-5p was capable of differentiating between postmenopausal women with osteoporotic fractures and fracture-free controls with an AUC of 0.98.
Project description:RNA was purified from serum of osteoporotic and healthy postmenopausal mexican women using miRNeasy Serum/Plasma kit (QIAGEN). For microRNA expression analysis we used the Human MicroRNA A+B Cards Set v3.0 TaqMan Low Density Array platform (Applied Biosystems). Analysis was performed in the Expression Suite v.1.1.3 software (Applied Biosystems).
Project description:Purpose: Identification of osteoclast derived coupling factors using Denosumab as a biological probe Method: harvested RNA from centrifuged bone biopsies (heterogeneous population containing bone lining cells, osteoblasts, osteoclasts and osteocytes, without in vitro culture Results: Identification of genes differentially regulated in denosumab treated participants that correlate with bone remodeling indices Conclusions: Using this approach we identified denosumab regulated genes that correlate with bone remodeling in the untreated participants and may represent novel osteoclast derived coupling factors. Of interest, we identified DPP4 to be suppressed in denosumab treated participants, providing a novel link between bone remodeling and energy metabolism.
Project description:Purpose: Identification of osteoclast derived coupling factors using Denosumab as a biological probe Method: harvested RNA from centrifuged bone biopsies (heterogeneous population containing bone lining cells, osteoblasts, osteoclasts and osteocytes, without in vitro culture Results: Identification of genes differentially regulated in denosumab treated participants that correlate with bone remodeling indices Conclusions: Using this approach we identified denosumab regulated genes that correlate with bone remodeling in the untreated participants and may represent novel osteoclast derived coupling factors. Of interest, we identified DPP4 to be suppressed in denosumab treated participants, providing a novel link between bone remodeling and energy metabolism.
Project description:Osteoporosis is the consequence of altered bone metabolism resulting in the systemic reduction of bone strength and increased risk of fragility fractures. MicroRNAs (miRNAs) regulate gene expression on a post-transcriptional level are known to take part in the control of bone formation and bone resorption. Recently, targeted secretion of miRNAs from cells originating from various tissues has been described, which allows for their minimal-invasive detection in serum/plasma and use as biomarkers for presence and progression of pathological conditions. One pilot study has reported circulating miRNAs in serum and tissue of fracture patients. However, further studies are required to explore whether a dysbalance in bone homeostasis of fracture patients can reliably be reflected by specific circulating miRNAs, and whether these miRNAs might serve as drugable targets. Here, we report results from a comprehensive multiplex study of 175 miRNAs in serum samples obtained from 7 patients with osteoporotic fractures at the femoral neck, and 7 age-matched controls. Following elaborate quality control statistical analysis of this exploratory dataset identified 9 microRNAs with altered serum levels in response to fracture (adjusted p-value < 0.1). Of these, hsa-miR-10a/b gave excellent discrimination of both groups (AUC = 1.0), and clustering of samples based on the top10 miRNAs confirmed the high discriminatory power of circulating microRNAs for osteoporotic fractures. In the next step 3 miRNAs with unknown roles in osteogenic differentiation and 4 miRNA from a previous study were tested for their effects on osteogenic differentiation. Of these, 3 miRNAs showed robust effects on osteogenic differentiation. Overall, these data provide important insights into changes in serum miRNA in post-traumatic patients. Future studies will show, whether this knowledge can be used to improve current diagnostic methodologies to predict fracture risk and design novel treatment strategies for osteoporosis patients. Two groups with n=7 per group; one groups represents cases with osteoporotic fractures, the control group is age-matched without fractures
Project description:Denosumab is a fully human monoclonal antibody that binds receptor activator of nuclear factor-κB ligand (RANKL) and is routinely administered to cancer patients to reduce the incidence of new bone metastasis. RANK-RANKL regulates bone turnover by controlling osteoclast recruitment, development, and activity. However, this interaction also can regulate a broad range of immune cells including dendritic cells and medullary thymic epithelial cells (mTECs). Inhibition of the latter results in reduced thymic negative selection of T cells and could enhance the generation of tumor-specific T cells. We examined whether administering denosumab could modify modulate circulating immune cells in cancer patients. Blood was collected from 23 prostate cancer patients and 3 renal cell carcinoma patients, who provided written informed consent, with advanced disease who were receiving denosumab, prior to and during denosumab treatment. Using high-dimensional mass cytometry, we found that denosumab treatment by itself induced modest effects on circulating immune cell frequency and activation. We also found minimal changes in the circulating T cell repertoire and the frequency of new thymic emigrants with denosumab treatment. However, when we stratified patients by whether they were receiving chemotherapy and/or steroids, patients receiving these concomitant treatments showed significantly greater immune modulation, including an increase in the frequency of NK cells early and classical monocytes later. We also saw broad induction of CTLA-4 and TIM3 expression in circulating lymphocytes and some monocyte populations. These findings suggest that denosumab treatment by itself has modest immunomodulatory effects, but when combined with conventional cancer treatment, can lead to the induction of immunologic checkpoints.