Identification of suitable reference gene and biomarkers of serum miRNAs for osteoporosis.
ABSTRACT: Our objective was to identify suitable reference genes in serum miRNA for normalization and screen potential new biomarkers for osteoporosis diagnosis by a systematic study. Two types of osteoporosis models were used like as mechanical unloading and estrogen deficiency. Through a large-scale screening using microarray, qPCR validation and statistical algorithms, we first identified miR-25-3p as a suitable reference gene for both type of osteoporosis, which also showed stability during the differentiation processes of osteoblast and osteoclast. Then 15 serum miRNAs with differential expression in OVX rats were identified by microarray and qPCR validation. We further detected these 15 miRNAs in postmenopausal women and bedrest rhesus monkeys and evaluated their diagnostic value by ROC analysis. Among these miRNAs, miR-30b-5p was significantly down-regulated in postmenopausal women with osteopenia or osteoporosis; miR-103-3p, miR-142-3p, miR-328-3p were only significantly decreased in osteoporosis. They all showed positive correlations with BMD. Except miR328-3p, the other three miRNAs were also declined in the rhesus monkeys after long-duration bedrest. Their AUC values (all >0.75) proved the diagnostic potential. Our results provided a reliable normalization reference gene and verified a group of circulating miRNAs as non-invasive biomarkers in the detection of postmenopausal- and mechanical unloading- osteoporosis.
Project description:The incidence of osteoporosis is high in postmenopausal women due to altered estrogen levels and continuous calcium loss that occurs with aging. Recent studies have shown that microRNAs (miRNAs) are involved in the development of osteoporosis. These miRNAs may be used as potential biomarkers to identify women at a high risk for developing the disease. In this study, whole blood samples were collected from 48 postmenopausal Chinese women with osteopenia or osteoporosis and pooled into six groups according to individual T-scores. A miRNA microarray analysis was performed on pooled blood samples to identify potential miRNA biomarkers for postmenopausal osteoporosis. Five miRNAs (miR-130b-3p, -151a-3p, -151b, -194-5p, and -590-5p) were identified in the microarray analysis. These dysregulated miRNAs were subjected to a pathway analysis investigating whether they were involved in regulating osteoporosis-related pathways. Among them, only miR-194-5p was enriched in multiple osteoporosis-related pathways. Enhanced miR-194-5p expression in women with osteoporosis was confirmed by quantitative reverse transcription-polymerase chain reaction analysis. For external validation, a significant correlation between the expression of miR-194-5p and T-scores was found in an independent patient collection comprised of 24 postmenopausal women with normal bone mineral density, 30 postmenopausal women with osteopenia, and 32 postmenopausal women with osteoporosis (p < 0.05). Taken together, the present findings suggest that miR-194-5p may be a viable miRNA biomarker for postmenopausal osteoporosis.
Project description:The study concerns effects of 21 days of sustained bedrest and hypoxia, alone and in combination, on skeletal muscle microRNA (miRNA) expression. It is expected that astronauts undertaking long-duration missions will be exposed not only to microgravity but also to a hypoxic environment. The molecular machinery underlying microgravity-induced alterations in skeletal muscle structure and function is still largely unknown. One possible regulatory mechanism is altered expression of miRNAs, a group of noncoding RNAs which down-regulate many different target genes through increased degradation or translation of their messenger RNA Thirteen healthy men underwent three 21-day interventions, interspersed by 4-month washout periods: horizontal bedrest in normoxia, bedrest in hypoxia, ambulation in hypoxia. The level of hypoxia corresponded to 4000 m altitude. miRNAs from v. lateralis muscle biopsies were analyzed using a microarray covering ?4000 human miRNAs. Sixteen mature miRNAs were up-regulated and three down-regulated after bedrest. The magnitudes of these changes were small and a large portion of the miRNAs affected by bedrest was also differentially expressed after washout periods. In fact, the number of differentially expressed probe sets over time was substantially larger than what could be detected after bedrest. Still, the majority of the miRNAs (let-7, miR-15, miR-25, miR-199, miR-133) that were differentially expressed following bedrest, belong to miRNA families previously reported in the context of muscle physiology, in particular to respond to changes in mechanical loading. Since only minor changes in miRNA expression could be detected after bedrest, our data indicate miRNA to play only a minor role in the substantial change in muscle phenotype seen with unloading.
Project description:This study aimed to identify specific microRNAs (miRNAs) related to postmenopausal osteoporosis (OP) in human. A total of 67 conserved miRNAs, including 50 miRNAs significantly up-regulated and 17 miRNAs significantly downregulated, showed differential expression between OP group and control group. 180 hairpin structures were predicted and 199 potential novel miRNA candidates with 18 to 25 nt in length, which will greatly enrich the human miRBase. 4 miRNAs (miR-518b, miR-582-3p, miR-148a-3p and miRNA-223-3p) had upregulated expression and 4 (miR-7d-5p, miR-210-3p, miR-324-5p and miR-654-3p) showed down-regulated expression. Target genes of these miRNAs were involved in bone development, cell proliferation in bone marrow, osteoblast development, negative regulation of osteoblast differentiation, and negative regulation of osteoclast development, as well as several osteogenesis related pathways. Canonical Wnt signaling pathway was selected for verification and function analysis. The expression of Wnt1, FZD10, LRP5, DVL2 and LEF1 was down-regulated significantly, while that of SFRP1, DKK1, and CHD8 was up-regulated markedly. In conclusion, these genes play important roles in OP, which improves our understanding of pathogenesis of OP.
Project description:Secreted frizzled-related protein-1 (SFRP1) is a negative regulatory molecule of the WNT signaling pathway and serves as a therapeutic target for bone formation in osteoporosis. In this study, we first established an ovariectomized (OVX) rat model to simulate postmenopausal osteoporosis and found significant changes in miR-542-3p and sFRP1 expression by RNA sequencing and qRT-PCR. In addition, there was a significant negative correlation between miR-542-3p and sFRP1 mRNA levels in postmenopausal women with osteoporosis. We found that miR-542-3p inhibited the expression of sFRP1 mRNA by luciferase reporter assay. When the miR-542-3p binding site in sFRP1 3'UTR was deleted, it did not affect its expression. Western blot results showed that miR-542-3p inhibited the expression of SFRP1 protein. The expression of SFRP1 was significantly increased in osteoblast-induced mesenchymal stem cells (MSC), whereas the expression of miR-542-3p was significantly decreased. And miR-542-3p transfected MSCs showed a significant increase in osteoblast-specific marker expression, indicating that miR-542-3p is necessary for MSC differentiation. Inhibition of miR-542-3p reduced bone formation, confirmed miR-542-3p play a role in bone formation in vivo. In general, these data suggest that miR-542-3p play an important role in bone formation via inhibiting SFRP1 expression and inducing osteoblast differentiation.
Project description:Emerging evidence indicates that many microRNAs (miRNAs) are indispensable regulators of osteoblast differentiation and bone formation. However, the role of miRNAs in mechanotransduction of osteoblasts remains to be elucidated. This study aimed to identify a mechanosensitive miRNA that regulates Activin A receptor type I (ACVR1)-induced osteogenic differentiation. After 4 weeks of hindlimb unloading (HLU) suspension of 6-month-old male C57BL/6J mice, femurs and tibias were harvested to extract total bone RNAs. Elevated levels of miR-208a-3p correlated with a lower degree of bone formation in whole-bone samples of HLU mice. However, in vitro overexpression of miR-208a-3p inhibited osteoblast differentiation, whereas silencing of miR-208a-3p by antagomiR-208a-3p promoted expression of osteoblast activity, bone formation marker genes, and matrix mineralization under mechanical unloading condition. Bioinformatics analysis and a luciferase assay revealed that ACVR1 is a target gene of miR-208a-3p that negatively regulates osteoblast differentiation under mechanical unloading environment. Further, this study also demonstrates that in vivo pre-treatment with antagomiR-208a-3p led to an increase in bone formation and trabecular microarchitecture and partly rescued the bone loss caused by mechanical unloading. Collectively, these results suggest that in vivo, inhibition of miRNA-208a-3p by antagomiR-208a-3p may be a potential therapeutic strategy for ameliorating bone loss.
Project description:MicroRNAs (miRNAs or miRs) are a class of short non-coding RNAs that serve an important regulatory role in living organisms. These molecules are associated with multiple biological processes and are potential biomarkers in multiple diseases. The present study aimed to further identify miRNAs that are differentially expressed in circulating monocytes (CMCs) from postmenopausal Mexican-Mestizo women. Microarray analyses of monocytes using Affymetrix miRNA 4.0 and Human Genome U133 Plus 2.0 arrays were performed in 6 normal and 6 osteoporotic women, followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) validation. The overexpression of miR-1270, miR-548×-3p and miR-8084 were detected in the osteoporosis compared with the normal group according to the microarray analysis; miR-1270, a miRNA with several target genes associated with bone remodeling, was validated by RT-qPCR. Bioinformatics analysis identified that interferon regulatory factor 8 (IRF8) is the most likely target gene of miR-1270, which is associated with osteoclastogenesis. Furthermore, the findings of the present study demonstrate that an upregulation of miR-1270 may reduce the gene expression of IRF8 in CMCs (osteoclast precursors), implicating its potential role in leading to low bone mineral density and contributing to osteoporosis development in postmenopausal women.
Project description:MicroRNAs (miRNAs) are single-stranded RNA molecules that control gene expression in various processes, such as cancers, Alzheimer's disease, and bone metabolic diseases. However, the regulatory roles of miRNAs in osteoporosis have not been systematically analyzed. Here, we performed a comprehensive analysis to identify the differentially expressed miRNAs involved in osteoporosis. MiRNAs associated with osteoporosis were collected through literature retrieval and further screened based on specific inclusion and exclusion criteria. The osteoporosis therapeutic targets of miRNAs were obtained by the integration of miRWalk 3.0 database and five human disease therapeutic target databases. Then, the network analysis and functional enrichment analysis of miRNAs and their targets were performed. As a result, 11 eligible miRNAs were identified highly associated with osteoporosis. MiRNA-mRNA network demonstrated there were the complex mutual interactions between miRNAs and their targets. Besides, ADRB2, AR, ESR1, FGFR1, TRAF6, etc., were identified as the top hub genes in protein-protein interaction (PPI) network. Functional enrichment analysis revealed that miRNAs and their targets were mainly mapped on processes associated with bone and immune system, such as bone remolding, bone mineralization, PI3K/AKt, TNF signaling pathways and Th17 cell differentiation. RT-PCR results showed that the expression of miR-335-3p was significantly down-regulated in hind limb unloading (HLU) mice tibia samples compared with controls, the remaining 10 miRNAs were significantly up-regulated after HLU (P < 0.01). In summary, we identified 11 differentially expressed miRNAs and their hub target genes in osteoporosis, which may be novel diagnostic biomarkers for osteoporosis.
Project description:MicroRNAs (miRNAs) are the most abundant RNA species found in serum, and recently, several miRNAs have been found to be associated with osteoporosis. However, the development of such associated miRNAs into diagnostic and therapeutic targets remains unaddressed, mostly because of a lack of functional validation. Here, we identified circulating miR-338 associated with postmenopausal osteoporosis, and performed functional validation <i>in vivo</i> and <i>in vitro</i>. <b>Methods</b>: We collected the serum from postmenopausal osteoporosis patients (N=15) and female volunteers of the same age but with normal bone density (N=15) and examined the enrichment of miR-338 cluster. We also confirmed such enrichment using mice subjected to ovariectomy at different stages. We employed primary bone marrow stromal cells from mice and the MC-3T3 cell line along with CRISPR, RNA-seq and ChIP-qPCR to validate the biological function of secreted miR-338 cluster on osteoblastic differentiation and their upstream regulators. Moreover, we generated miR-338 knockout mice and OVX mice injected with an inhibitor against miR-338 cluster to confirm its biological function <i>in vivo</i>. <b>Results</b>: We observed a significant enrichment of miR-338 cluster in postmenopausal osteoporosis patients. Such enrichment was also prominent in serum from mice subjected to ovariectomy and was detected much earlier than bone density decreases revealed by micro-CT. We also confirmed the presence of an estrogen-dependent <i>Runx2</i>/<i>Sox4</i>/miR-338 positive feedback loop that modulated osteoblast differentiation, providing a possible explanation for our clinical findings. Moreover, deletion of the miR-338 cluster or direct intravenous injection of an miR-338 cluster inhibitor significantly prevented osteoporosis after ovariectomy. <b>Conclusion</b>: Circulating miR-338 cluster in the serum could serve as a promising diagnostic and therapeutic target for postmenopausal osteoporosis patients.
Project description:Recent studies have confirmed that microRNAs and lncRNAs can affect bone cell differentiation and bone formation. In this study, miR-139-3p was upregulated in the femurs of hindlimb unloading mice and MC3T3-E1 cells under simulated microgravity; this effect was related to osteoblast differentiation and apoptosis. Silencing miR-139-3p attenuated the suppression of differentiation and the promotion of MC3T3-E1 cell apoptosis induced by simulated microgravity. ELK1 is a target of miR-139-3p and is essential for miR-139-3p to regulate osteoblast differentiation and apoptosis. An osteoblast differentiation-related lncRNA that could interact with miR-139-3p (lncRNA ODSM) was identified in MC3T3-E1 cells under simulated microgravity. Further investigations demonstrated that lncRNA ODSM could promote MC3T3-E1 cell differentiation. Therefore, this research was the first to reveal the critical role of the lncRNA ODSM/miR-139-3p/ELK1 pathway in osteoblasts, and these findings suggest the potential value of miR-139-3p in osteoporosis diagnosis and therapy.
Project description:Unloading-induced bone loss is a threat to human health and can eventually result in osteoporotic fractures. Although the underlying molecular mechanism of unloading-induced bone loss has been broadly elucidated, the pathophysiological role of long noncoding RNAs (lncRNAs) in this process is unknown. Here, we identified a novel lncRNA, OGRU, a 1816-nucleotide transcript with significantly decreased levels in bone specimens from hindlimb-unloaded mice and in MC3T3-E1 cells under clinorotation-unloading conditions. OGRU overexpression promoted osteoblast activity and matrix mineralization under normal loading conditions, and attenuated the suppression of MC3T3-E1 cell differentiation induced by clinorotation unloading. Furthermore, this study found that supplementation of pcDNA3.1(+)-OGRU via (DSS)<sub>6</sub>-liposome delivery to the bone-formation surfaces of hindlimb-unloaded (HLU) mice partially alleviated unloading-induced bone loss. Mechanistic investigations demonstrated that OGRU functions as a competing endogenous RNA (ceRNA) to facilitate the protein expression of Hoxa10 by competitively binding miR-320-3p and subsequently promote osteoblast differentiation and bone formation. Taken together, the results of our study provide the first clarification of the role of lncRNA OGRU in unloading-induced bone loss through the miR-320-3p/Hoxa10 axis, suggesting an efficient anabolic strategy for osteoporosis treatment.