Comprehensive analysis of circRNA expression profiles in postmenopausal women differing in bone mineral density.

Postmenopausal osteoporosis (PMOP) seriously endangers the bone health of older women. Although there are currently indicators to diagnose PMOP, early diagnostic biomarkers are lacking. Circular ribonucleic acid (circRNA) has a stable structure, regulates gene expression, participates in the pathological process of disease, and has the potential to become a biomarker. The purpose of this study was to investigate circRNAs that could be used to predict patients with early PMOP. Ribonucleic acid (RNA) sequencing was performed on peripheral blood leukocytes from 15 female patients to identify differential circRNAs between different groups. Using bioinformatics analysis, enrichment analysis was performed to discover relevant functions and pathways. CircRNA-micro ribonucleic acid (miRNA) interaction analysis and messenger ribonucleic acid (mRNA) prediction and network construction help us to understand the relationship between circRNA, miRNA, and mRNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the gene expression of candidate circRNAs. We screened out 2 co-expressed differential circRNAs, namely hsa_circ_0060849 and hsa_circ_0001394. By analyzing the regulatory network, a total of 54 miRNAs and 57 osteoporosis-related mRNAs were identified, which, as potential downstream target genes of hsa_circ_0060849 and hsa_circ_0001394, may play a key role in the occurrence and development of PMOP. The occurrence and development of PMOP is regulated by circRNAs, and hsa_circ_0060849 and hsa_circ_0001394 can be used as new diagnostic markers and therapeutic targets for early PMOP.

Interferon Regulatory Factor 4 (IRF4) Plays a Key Role in Osteoblast Differentiation of Postmenopausal Osteoporosis.

BACKGROUND: Postmenopausal osteoporosis (PMOP) is a prevalent disease, which features decreased bone mass, bone weakness and deteriorated bone microstructure in postmenopausal women. Although many factors have been revealed to contribute to the occurrence of PMOP, its mechanism remains undefined. This work aimed to identify significant changes in gene expression during PMOP formation and to examine the most valuable differential genes in postmenopausal osteoporosis versus the control group. METHODS: The GSE68303 dataset that contains 12 ovariectomize (OVX) experimental and 11 sham groups was downloaded and analyzed. The results indicated that interferon regulatory factor 4 (IRF4) might be a hub gene in the development of postmenopausal osteoporosis. Western blot and immunohistochemistry were carried out to evaluate IRF4 levels in thoracic vertebra extracts from OVX and Sham mice. To assess IRF4's impact on osteogenic differentiation in postmenopausal bone marrow mesenchymal stem cells (BM-MSCs), IRF4 overexpression (OV-IRF4) and knockdown (Sh-IRF4) plasmids were constructed. RESULTS: The results showed that comparing with the sham group, bone samples from the OVX group showed higher IRF4 expression. Alkaline phosphatase (ALP) staining revealed that IRF4 overexpression significantly inhibited ALP activity, while IRF4 knockdown promoted ALP activity in BM-MSCs. Simvastatin-treated OVX mice showed increased total bone volume/total tissue volume (BV/TV) and elevated Runx2 expression by immunohistochemical staining compared with the OVX group. CONCLUSIONS: This study demonstrated that IRF4 is associated with OVX induced osteoporosis, it can regulate bone stability by inhibiting the osteogenic differentiation BM-MSCs. This study may help enhance our understanding of the molecular mechanism of PMOP formation, providing new insights into estrogen defiance induced osteoporosis.

Heterogeneous osteoimmune profiles via single-cell transcriptomics in osteoporotic patients who fail bisphosphonate treatment.

Postmenopausal osteoporosis arises from imbalanced osteoclast and osteoblast activity, and mounting evidence suggests a role for the osteoimmune system in bone homeostasis. Bisphosphonate (BP) is an antiresorptive agent, but its treatment failure rate can be as high as 40%. Here, we performed single-cell RNA sequencing on peripheral immune cells from carefully selected postmenopausal women: non-osteoporotic, osteoporosis improved after BP treatment, and BP-failed cases. We found an increase in myeloid cells in patients with osteoporosis (specifically, T cell receptor+ macrophages). Furthermore, lymphoid lineage cells varied significantly, notably elevated natural killer cells (NKs) in the BP-failed group. Moreover, we provide fruitful lists of biomarkers within the immune cells that exhibit condition-dependent differences. The existence of osteoporotic- and BP-failure-specific cellular information flows was revealed by cell-cell interaction analysis. These findings deepen our insight of the osteoporosis pathology enhancing comprehension of the role of immune heterogeneity in postmenopausal osteoporosis and BP treatment failure.

Investigating the impact of Wnt pathway-related genes on biomarker and diagnostic model development for osteoporosis in postmenopausal females.

The Wnt signaling pathway is essential for bone development and maintaining skeletal homeostasis, making it particularly relevant in osteoporosis patients. Our study aimed to identify distinct molecular clusters associated with the Wnt pathway and develop a diagnostic model for osteoporosis in postmenopausal Caucasian women. We downloaded three datasets (GSE56814, GSE56815 and GSE2208) related to osteoporosis from the GEO database. Our analysis identified a total of 371 differentially expressed genes (DEGs) between low and high bone mineral density (BMD) groups, with 12 genes associated with the Wnt signaling pathway, referred to as osteoporosis-associated Wnt pathway-related genes. Employing four independent machine learning models, we established a diagnostic model using the 12 osteoporosis-associated Wnt pathway-related genes in the training set. The XGB model showed the most promising discriminative potential. We further validate the predictive capability of our diagnostic model by applying it to three external datasets specifically related to osteoporosis. Subsequently, we constructed a diagnostic nomogram based on the five crucial genes identified from the XGB model. In addition, through the utilization of DGIdb, we identified a total of 30 molecular compounds or medications that exhibit potential as promising therapeutic targets for osteoporosis. In summary, our comprehensive analysis provides valuable insights into the relationship between the osteoporosis and Wnt signaling pathway.

Relationship between polymorphisms and mutations at rs7125942 and rs3736228 of LRP5 gene and bone metabolism in postmenopausal women.

OBJECTIVE: To analyze the relationship between the polymorphism and mutation of rs7125942 and rs3736228 locus in the low-density lipoprotein receptor-related protein 5 (LRP5) genotype and bone mineral density (BMD) in postmenopausal women in Xinjiang, China, to provide a basis for prevention and treatment of the disease. METHODS: According to the results of dual-energy X-ray (DEXA) determination of BMD, the 136 subjects were divided into three groups: Group A: normal bone mass, Group B: osteopenia, Group C: osteoporosis. 1. Age, body, mass index (BMI), and menopause of all subjects were recorded. 2. Fasting blood glucose (FBG), glycosylated hemoglobin (HbA1c), calcium (Ca), phosphorus (P), alkaline phosphatase (ALP), and clinical biochemical data were determined. 3. LRP5 locus polymorphisms were determined by time-of-flight mass spectrometry. RESULTS: 1. Compared with group A, the age, ALP, Cr, and BUN levels in group B and group C were increased, but UA levels were lower (P < 0.05), and Serum P was higher in the group C (P < 0.05). 2. There was no statistically significant difference in the prevalence of diabetes between the three groups (P > 0.05). 3. The ROC curves for different BMD sites such as L1, L2, L3, L4, L total, and femoral neck were 0.929, 0.955, 0.901, 0.914, 0.885, and 0.873 (P < 0.01). 4. At rs7125942 locus, there was statistically significant difference in the distribution of wild-type (CC) and mutant (CG) with the normal bone mass (NBM) group and the abnormal bone mass (ABM) group (P < 0.05). 5. At rs7125942 locus, compared with wild-type (CC), mutant (CG) had lower LDL and FPG in NBM group (P < 0.05), and lower serum ALP in the ABM group (P < 0.05). At rs3736228 locus, the BMD (Femoral neck) of mutant (CT/TT) was lower than that of wild-type (CC) in the NBM group (P < 0.05). 6. Age and menopausal years were negatively correlated with BMD of the femoral neck and L1-4 (P < 0.05), and BMI and TG were positively (P < 0.05), and the results of multiple linear regression analysis showed that age, BMI, and TG were both independent factors affecting BMD (P < 0.05).

Identifying the effective combination of acupuncture and traditional Chinese medicinal herbs for postmenopausal osteoporosis therapy through studies of their molecular regulation of bone homeostasis.

Worldwide, as the population age, osteoporosis is becoming increasingly common, and osteoporotic fractures have a significant economic burden. Postmenopausal women are the most susceptible to developing osteoporosis and the most critical time to prevent it is during the perimenopausal and early menopausal years. In this regard, we hypothesize rational combination of acupuncture and Traditional Chinese Medicine (TCM) in the form of herbal extract could prevent osteoporosis in women. Estrogen deficiency during menopause causes low-level inflammation that stimulates the formation of osteoclasts, the bone-resorbing cells, and simultaneously inhibits the viability and function of osteoblasts, the bone-forming cells. The most potent inflammatory cytokine in skeletal homeostasis is the receptor activator of nuclear factor kappa B ligand (RANKL) that stimulates osteoclast function. Conversely, the canonical Wnt pathway is essential for osteoblastogenesis and bone formation, and estrogen deficiency leads to diminished functioning of this pathway. TCM and acupuncture could target the RANKL and the Wnt pathway in favorable ways to prevent the accelerated bone loss experienced during the early menopausal stage and promote the gain in bone mass in postmenopausal women. In this review, we propose a rational combination of specific TCM and acupuncture targeting those signaling molecules/pathways by the drugs that are in clinical use for the treatment of postmenopausal osteoporosis. Our rational approach revealed that Danshen (Radix Salviae Miltiorrhizae) could exert a synergistic effect with acupuncture. We then propose a translational path for developing the putative combination in women with postmenopausal osteoporosis to curtail the risk of osteoporotic fractures.

Glycolithocholic acid increases the frequency of circulating Tregs through constitutive androstane receptor to alleviate postmenopausal osteoporosis.

BACKGROUND AND PURPOSE: Gut microbiota and their metabolic activity are important regulators of host immunity. However, the role of gut microbiota and their metabolic activity-mediated osteoimmunity in postmenopausal osteoporosis (PMO) remains unknown. This study aimed to explore the role of gut microbiota and their metabolic activity in PMO. EXPERIMENTAL APPROACH: 16S rDNA sequencing was used for analyzing the gut microbiota diversity of patients with PMO and rat models, and a targeted metabolism study was performed for analyzing metabolite levels. Flow cytometry was used for analyzing the frequency of immune cells. Micro-CT was used for analyzing bone damage in rat models. Fecal microbiota transplantation was performed for exploring the therapeutic effect of the gut microbiota on PMO. CD4+ T cells were co-cultured with bone marrow mesenchymal stem cells for evaluating their molecular mechanisms. KEY RESULTS: Patients with PMO exhibited reduced gut microbiota diversity, and fecal glycolithocholic acid (GLCA) levels correlated with the degree of osteoporosis. GLCA levels in the gut were positively correlated with the frequency of circulating Tregs in ovariectomized rats. Restoration of the gut microbiota alleviated osteoporosis in ovariectomized rats. Circulating GLCA augmented CD4+ T cell differentiation into Tregs via constitutive androstane receptors. The increased frequency of Tregs further promoted the osteogenic differentiation of bone marrow mesenchymal stem cells to alleviate osteoporosis. CONCLUSION AND IMPLICATIONS: GLCA alleviated PMO by increasing the frequency of circulating Tregs, acting via the constitutive androstane receptor. This study reveals a new strategy for the treatment of PMO, with GLCA as a potential drug candidate.

Identification of a potential signature to predict the risk of postmenopausal osteoporosis.

BACKGROUND: Postmenopausal osteoporosis (PMOP) is related to the elevated risk of fracture in postmenopausal women. Thus, to effectively predict the occurrence of PMOP, we explored a novel gene signature for the prediction of PMOP risk. METHODS: The WGCNA analysis was conducted to identify the PMOP-related gene modules based on the data from GEO database (GSE56116 and GSE100609). The "limma" R package was applied for screening differentially expressed genes (DEGs) based on the data from GSE100609 dataset. Next, LASSO Cox algorithm were applied to identify valuable PMOP-related risk genes and construct a risk score model. GSEA was then conducted to analyze potential signaling pathways between high-risk (HR) score and low-risk (LR) score groups. RESULTS: A novel risk model with five PMOP-related risk genes (SCUBE3, TNNC1, SPON1, SEPT12 and ULBP1) was developed for predicting PMOP risk status. RT-qPCR and western blot assays validated that compared to postmenopausal non-osteoporosis (non-PMOP) patients, SCUBE3, ULBP1, SEPT12 levels were obviously elevated, and TNNC1 and SPON1 levels were reduced in blood samples from PMOP patients. Additionally, PMOP-related pathways such as MAPK signaling pathway, PI3K-Akt signaling pathway and HIF-1 signaling pathway were significantly activated in the HR-score group compared to the LR-score group. The circRNA-gene-miRNA and gene-transcription factor networks showed that 533 miRNAs, 13 circRNAs and 40 TFs might be involved in regulating the expression level of these five PMOP-related genes. CONCLUSION: Collectively, we developed a PMOP-related gene signature based on SCUBE3, TNNC1, SPON1, SEPT12 and ULBP1 genes, and higher risk score indicated higher risk suffering from PMOP.

Eupatilin ameliorates postmenopausal osteoporosis via elevating microRNA-211-5p and repressing JAK2/STAT3 pathway.

Postmenopausal osteoporosis (PMOP) poses a significant threat to women's health worldwide. Eupatilin is a key bioactive component of the Chinese herbal medicine Artemisia asiatica Nakai. Recent research reports have proved the inhibitory function of Eupatilin in many diseases. MicroRNAs (miRNAs) are 21-23 nucleotide-long, single-stranded, noncoding RNA molecules generated endogenously, and many studies have indicated that miRNAs are involved in the development of osteoporosis. This study explored the role and potential mechanism of Eupatilin underlying PMOP. First, rats were given intragastric administration of Eupatilin every day and subcutaneous injections of oligonucleotides or plasmids that interfered with miR-211-5p or janus kinase 2 (JAK2) once a week. After 4 weeks, the PMOP rat model was established. Then, serum alkaline phosphatase, calcium, and phosphorus levels, as well as femur bone mineral density and biomechanical parameters, were detected. Hematoxylin-eosin staining and Masson staining were applied for detecting the pathological condition of femur, and immunohistochemical staining was for detecting osteocalcin. MC3T3-E1 cells were transfected with plasmid vectors interfering with miR-211-5p or JAK2; and cell viability, lactate dehydrogenase cytotoxicity, and cell mineralization were subsequently examined. The relationship between miR-211-5p and JAK2/signal transducer and activator of transcription 3 (STAT3) pathway was analyzed. The targeting relation between miR-211-5p and JAK2 was also verified. The experimental results revealed that Eupatilin improved the pathological conditions of PMOP rats by promoting the proliferation and mineralization of osteoblasts. MiR-211-5p was down-regulated and JAK2/STAT3 was upregulated in PMOP rats. Upregulation of miR-211-5p further improved the pathological conditions of PMOP rats based on Eupatilin treatment. MiR-211-5p inhibited the JAK2/STAT3 pathway. JAK2 offset the effects of elevated miR-211-5p on PMOP rats. Overall, Eupatilin attenuates PMOP through elevating miR-211-5p and repressing JAK2/STAT3 pathway, which suggests the utility of Eupatilin as a potential drug for POMP treatment.

Systems genetics and bioinformatics analyses using ESR1-correlated genes identify potential candidates underlying female bone development.

Estrogen receptor α (ESR1) is involved in E2 signaling and plays a major role in postmenopausal bone loss. However, the molecular network underlying ESR1 has not been explored. We used systems genetics and bioinformatics to identify important genes associated with Esr1 in postmenopausal bone loss. We identified ~2300 Esr1-coexpressed genes in female BXD bone femur, functional analysis of which revealed 'osteoblast signaling' as the most enriched pathway. PPI network led to the identification of 25 'female bone candidates'. The gene-regulatory analysis revealed RUNX2 as a key TF. ANKRD1 and RUNX2 were significantly different between osteoporosis patients and healthy controls. Sp7, Col1a1 and Pth1r correlated with multiple femur bone phenotypes in BXD mice. miR-3121-3p targeted Csf1, Ankrd1, Sp7 and Runx2. ß-estradiol treatment markedly increased the expression of these candidates in mouse osteoblast. Our study revealed that Esr1-correlated genes Ankrd1, Runx2, Csf1 and Sp7 may play important roles in female bone development.

Polymorphisms in the glucagon-like peptide-1 receptor gene and their interactions on the risk of osteoporosis in postmenopausal Chinese women.

Postmenopausal osteoporosis (PMOP) is a prevalent form of primary osteoporosis, affecting over 40% of postmenopausal women. Previous studies have suggested a potential association between single nucleotide polymorphisms (SNPs) in glucagon-like peptide-1 receptor (GLP-1R) and PMOP in postmenopausal Chinese women. However, available evidence remains inconclusive. Therefore, this study aimed to investigate the possible association between GLP-1R SNPs and PMOP in Han Chinese women. Thus, we conducted a case-control study with 152 postmenopausal Han Chinese women aged 45-80 years, including 76 women with osteoporosis and 76 without osteoporosis. Seven SNPs of the GLP-1R were obtained from the National Center of Biotechnology Information and Genome Variation Server. We employed three genetic models to assess the association between GLP-1R genetic variants and osteoporosis in postmenopausal women, while also investigating SNP-SNP and SNP-environment interactions with the risk of PMOP. In this study, we selected seven GLP-1R SNPs (rs1042044, rs2268641, rs10305492, rs6923761, rs1126476, rs2268657, and rs2295006). Of these, the minor allele A of rs1042044 was significantly associated with an increased risk of PMOP. Genetic model analysis revealed that individuals carrying the A allele of rs1042044 had a higher risk of developing osteoporosis in the dominant model (P = 0.029, OR = 2.76, 95%CI: 1.09-6.99). Furthermore, a multiplicative interaction was found between rs1042044 and rs2268641 that was associated with osteoporosis in postmenopausal women (Pinteraction = 0.034). Importantly, this association remained independent of age, menopausal duration, family history of osteoporosis, and body mass index. However, no significant relationship was observed between GLP-1R haplotypes and PMOP. In conclusion, this study suggests a close association between the A allele on the GLP-1R rs1042044 and an increased risk of PMOP. Furthermore, this risk was significantly augmented by an SNP-SNP interaction with rs2268641. These results provide new scientific insights into the development of personalized prevention strategies and treatment approaches for PMOP.

Based on network pharmacology and molecular docking to explore the molecular mechanism of Ginseng and Astragalus decoction against postmenopausal osteoporosis.

Traditional Chinese medicine suggests that Ginseng and Astragalus Decoction (GAD) may effectively treat postmenopausal osteoporosis (PMO). However, the exact mechanism of action for GAD remains unclear. This study aims to utilize network pharmacology and molecular docking technology to explore the potential mechanism of GAD in treating PMO. The main chemical components of GAD were identified by consulting literature and traditional Chinese medicine systems pharmacology database. GeneCards and online mendelian inheritance in man were used to identify PMO disease targets, and Cytoscape 3.8.2 software was used to construct a herb-disease-gene-target network. The intersection of drug targets and disease targets was introduced into the search tool for the retrieval of interacting genes platform to construct a protein-protein interaction network. Additionally, we further conducted gene ontology and Kyoto encyclopedia of genes and genomes enrichment analyses, followed by molecular docking between active ingredients and core protein targets. We have identified 59 potential targets related to the treatment of PMO by GAD, along with 33 effective components. Quercetin and kaempferol are the compounds with higher degree. In the protein-protein interaction network, IL6, AKT1, and IL1B are proteins with high degree. The enrichment analysis of gene ontology and KEEG revealed that biological processes involved in treating PMO with GAD mainly include response to hormones, positive regulation of phosphorylation, and regulation of protein homodimerization activity. The signal pathways primarily include Pathways in cancer, PI3K-Akt signaling pathway, and AGE-RAGE signaling pathway. Molecular docking results indicate that kaempferol and quercetin have a high affinity for IL6, AKT1, and IL1B. Our research predicts that IL6, AKT1, and IL1B are highly likely to be potential targets for treating PMO with GAD. PI3K/AKT pathway and AGE-ARGE pathway may play an important role in PMO.

MiR-210 promotes bone formation in ovariectomized rats by regulating osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells through downregulation of EPHA2.

PURPOSE: In osteoporosis, the balance between osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs) is disrupted. The osteogenic differentiation of bone marrow MSCs (BMSCs) is important for improving osteoporosis. The aim of this study was to explore the role and molecular mechanism of miR-210 in the balance of osteogenic/adipogenic differentiation of BMSCs in postmenopausal osteoporosis. METHODS: Postmenopausal osteoporosis rat models were constructed by ovariectomy (OVX). BMSCs were isolated from the femur in rats of Sham and OVX groups. MiR-210 was overexpressed and suppressed by miR-210 mimics and inhibitor, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative mRNA expression of miR-210, ephrin type-A receptor 2 (EPHA2), alkaline phosphatase (ALP), osterix (OSX), osteocalcin (Bglap), Runt-related transcription factor 2 (Runx2), peroxisome proliferator activated receptor gamma, and fatty acid binding protein 4 (FABP4) in each group of rat femoral tissues or BMSCs. Western blot was applied to detect the protein expression level of EPHA2 in rat femoral tissues and cells. Alizarin red S staining and oil red O staining were performed to assess the osteogenic and adipogenic differentiation of BMSCs, respectively. In addition, the targeting relationship between miR-210 and EPHA2 was verified by a dual luciferase gene reporter assay. RESULTS: The expression of miR-210 was significantly reduced in femoral tissues and BMSCs of OVX rats, and its low expression was associated with reduced bone formation. The osteogenic differentiation was enhanced in OVX rats treated with miR-210 mimic. Overexpression of miR-210 in transfected BMSCs was also found to significantly promote osteogenic differentiation and even inhibit adipogenic differentiation in BMSCs, while knockdown of miR-210 did the opposite. Further mechanistic studies showed that miR-210 could target and inhibit the expression of EPHA2 in BMSCs, thus promoting osteogenic differentiation and inhibiting adipogenic differentiation of BMSCs. CONCLUSION: MiR-210 promotes osteogenic differentiation and inhibits adipogenic differentiation of BMSCs by down-regulating EPHA2 expression. As it plays an important role in the osteogenic/adipogenic differentiation of osteoporosis, miR-210 can serve as a potential miRNA biomarker for osteoporosis.

Identification of ROCK1 as a novel biomarker for postmenopausal osteoporosis and pan-cancer analysis.

BACKGROUND: Postmenopausal osteoporosis (PMOP) is a prevalent bone disorder with significant global impact. The elevated risk of osteoporotic fracture in elderly women poses a substantial burden on individuals and society. Unfortunately, the current lack of dependable diagnostic markers and precise therapeutic targets for PMOP remains a major challenge. METHODS: PMOP-related datasets GSE7429, GSE56814, GSE56815, and GSE147287, were downloaded from the GEO database. The DEGs were identified by "limma" packages. WGCNA and Machine Learning were used to choose key module genes highly related to PMOP. GSEA, DO, GO, and KEGG enrichment analysis was performed on all DEGs and the selected key hub genes. The PPI network was constructed through the GeneMANIA database. ROC curves and AUC values validated the diagnostic values of the hub genes in both training and validation datasets. xCell immune infiltration and single-cell analysis identified the hub genes' function on immune reaction in PMOP. Pan-cancer analysis revealed the role of the hub genes in cancers. RESULTS: A total of 1278 DEGs were identified between PMOP patients and the healthy controls. The purple module and cyan module were selected as the key modules and 112 common genes were selected after combining the DEGs and module genes. Five Machine Learning algorithms screened three hub genes (KCNJ2, HIPK1, and ROCK1), and a PPI network was constructed for the hub genes. ROC curves validate the diagnostic values of ROCK1 in both the training (AUC = 0.73) and validation datasets of PMOP (AUC = 0.81). GSEA was performed for the low-ROCK1 patients, and the top enriched field included protein binding and immune reaction. DCs and NKT cells were highly expressed in PMOP. Pan-cancer analysis showed a correlation between low ROCK1 expression and SKCM as well as renal tumors (KIRP, KICH, and KIRC). CONCLUSIONS: ROCK1 was significantly associated with the pathogenesis and immune infiltration of PMOP, and influenced cancer development, progression, and prognosis, which provided a potential therapy target for PMOP and tumors. However, further laboratory and clinical evidence is required before the clinical application of ROCK1 as a therapeutic target.

Anti-osteoporosis mechanism of resistance exercise in ovariectomized rats based on transcriptome analysis: a pilot study.

Postmenopausal osteoporosis is the main cause of fractures in women. Resistance exercise has a positive effect on bone mineral density in postmenopausal osteoporosis patients, but its mechanism is unclear. The purpose of this study was to explore the mechanism of resistance exercise in improving ovariectomized osteoporotic rats based on the transcriptome sequencing technique. Eighteen female Sprague-Dawley rats were randomly divided into the sham-operated group, the non-exercise group, and the resistance exercise group. The rat model of postmenopausal osteoporosis was established by bilateral ovariectomy. Ten weeks after the operation, the resistance exercise group received 2 weeks of adaptive training, and 12 weeks of resistance exercise began in the 13th week. The rats were trained 5 days per week, in 4 sets of 3 repetitions per day. After the intervention, all rats were sacrificed, and the body weight, bone mineral density, trabecular bone microarchitecture, and bone biomechanics were examined. At the same time, RNA-seq and enrichment analysis of gene ontology and Kyoto Encyclopedia of Genes and Genomes were performed on the left tibias, followed by Elisa and RT-qPCR verification. It had been found that resistance exercise can effectively counteract the weight gain of ovariectomized osteoporotic rats, and has a good effect on bone mineral density and trabecular bone microarchitecture. Enrichment analysis showed that regulation of gene expression and osteoclast differentiation is the most closely related biological process and signaling pathway shared by RE/Ovx and NE/Ovx groups. Our results revealed that resistance exercise can play a role in inhibiting osteoclast activation and preventing the enhancement of osteoclast bone resorption function in ovariectomized osteoporotic rats by inhibiting Fos/Fosb-regulated TRAP activation and relieving Calcr inhibition, which has important application value in preventing bone loss caused by estrogen deficiency.

Long non-coding RNA Homeobox D gene cluster antisense growth-associated long noncoding RNA/microRNA-182-5p/Homeobox protein A10 alleviates postmenopausal osteoporosis via accelerating osteoblast differentiation of bone marrow mesenchymal stem cells.

BACKGROUND: Studies have illuminated that long non-coding RNA (lncRNA) influences bone cell differentiation and formation. Nevertheless, whether lncRNA Homeobox D gene cluster antisense growth-associated long noncoding RNA (HAGLR) was implicated in postmenopausal osteoporosis (PMOP) was yet uncertain. PURPOSE: The research was to explore HAGLR's role in the osteogenic differentiation (OD) process of bone marrow mesenchymal stem cells (BMSCs). METHODS: BMSCs were isolated from mouse bone marrow tissues and identified by electron microscope and flow cytometry. HAGLR, microRNA (miR)-182-5p, and homeobox protein A10 (Hoxa10) levels in BMSCs were detected. Mouse BMSC OD process was induced, and calcium deposition and alkaline phosphatase content were analyzed, as well as expressions of runt-related transcription factor 2, osteopontin, and osteocalcin, and cell apoptosis. Bilateral ovaries were resected from mice to construct the ovariectomized model and bone mineral density, maximum bending stress, maximum load, and elastic modulus of the femur were tested, and the femur was histopathologically evaluated. Chondrocyte apoptosis in the articular cartilage of mice was analyzed. Analysis of the interaction of HAGLR, miR-182-5p with Hoxa10 was conducted. RESULTS: HAGLR and Hoxa10 were down-regulated and miR-182-5p was elevated in PMOP patients. During the BMSC OD process, HAGLR and Hoxa10 levels were suppressed, while miR-182-5p was elevated. Promotion of HAGLR or suppression of miR-182-5p accelerated OD of BMSCs. Inhibition of miR-182-5p reversed the inhibitory effect of HAGLR on BMSC OD. In in vivo experiments, up-regulating HAGLR alleviated PMOP, while silencing Hoxa10 reversed the effects of upregulating HAGLR. HAGLR performed as a sponge for miR-182-5p, while miR-182-5p targeted Hoxa10. CONCLUSION: In general, HAGLR boosted the OD process of BMSCs and relieved PMOP via the miR-182-5p/Hoxa10 axis. These data preliminarily reveal the key role of HAGLR in PMOP, and the research results have a certain reference for the treatment of PMOP.

Identification of biomarkers associated with oxidative stress-related genes in postmenopausal osteoporosis.

Osteoporosis (OP) is a prevalent metabolic disease, with aging and menopause being the major risk factors. Studies have shown that nearly one-third of postmenopausal women suffer from osteoporosis. However, there is a scarcity of research on antioxidant systems for the prevention and treatment of postmenopausal osteoporosis (PM-OP). To address this gap, we performed differential analysis using Limma to identify differentially expressed genes (DEGs) in PM-OP samples. We employed weighted correlation network analysis (WGCNA) to identify oxidative stress (OS)-related genes (OSRGs) highly correlated with PM-OP. The intersection of key modular genes and DEGs yielded differentially expressed OSRGs (DE-OSRGs) specific to PM-OP. We conducted GO and KEGG functional enrichment analyses on these genes. Additionally, we constructed a protein-protein interaction (PPI) network and utilized support vector machine recursive feature elimination (SVM-RFE) and random forest (RF) algorithms to identify signature genes. The diagnostic value of the signature genes was evaluated and validated using ROC curves. GSEA enrichment analysis was employed to explore the potential mechanisms associated with the signature genes. Finally, we constructed a regulatory network involving TF-miRNA-mRNA interactions for the signature genes and verified the biological roles of FOXO3 and DDIT3 in PM-OP and healthy groups using quantitative real-time PCR (qRT-PCR). Our analysis revealed 20 DE-OSRGs specific to PM-OP, obtained by intersecting modular and differential genes. The PPI network identified central genes (DDIT3, MAPK8, CDK2, SIRT1, and FOXO3) with more than 3 nodes. Through integration with machine learning algorithms, we identified DDIT3 and FOXO3 as signature genes. The ROC curve analysis indicated that the AUC value was greater than 0.7, suggesting the potential diagnostic value of these signature genes. Furthermore, GSEA results revealed their involvement in pathways related to the regulation of neutrophil activation, oxidative phosphorylation, MAPK signaling, mitochondrial matrix, and phagocytosis. Lastly, we constructed a regulatory network comprising 27 nodes (22 TFs, 3 miRNAs, and 2 mRNAs) and 28 edges. Additionally, qRT-PCR confirmed the significant up-regulation of FOXO3 and DDIT3 expressions in the PM-OP group compared to the healthy control group. In summary, this study employed bioinformatics analysis to identify OS-related biomarkers (DDIT3 and FOXO3) in PM-OP, providing new biological targets for clinical treatment.

Hmga1-overexpressing lentivirus protects against osteoporosis by activating the Wnt/ß-catenin pathway in the osteogenic differentiation of BMSCs.

Postmenopausal osteoporosis is associated with bone formation inhibition mediated by the impaired osteogenic differentiation potential of bone marrow mesenchymal stem cells (BMSCs). However, identifying and confirming the essential genes in the osteogenic differentiation of BMSCs and osteoporosis remain challenging. The study aimed at revealing the key gene that regulated osteogenic differentiation of BMSCs and led to osteoporosis, thus exploring its therapeutic effect in osteoporosis. In the present study, six essential genes related to the osteogenic differentiation of BMSCs and osteoporosis were identified, namely, fibrillin 2 (Fbn2), leucine-rich repeat-containing 17 (Lrrc17), heat shock protein b7 (Hspb7), high mobility group AT-hook 1 (Hmga1), nexilin F-actin-binding protein (Nexn), and endothelial cell-specific molecule 1 (Esm1). Furthermore, the in vivo and in vitro experiments showed that Hmga1 expression was increased during the osteogenic differentiation of rat BMSCs, while Hmga1 expression was decreased in the bone tissue of ovariectomized (OVX) rats. Moreover, the expression of osteogenic differentiation-related genes, the activity of alkaline phosphatase (ALP), and the number of mineralized nodules were increased after Hmga1 overexpression, which was partially reversed by a Wnt signaling inhibitor (DKK1). In addition, after injecting Hmga1-overexpressing lentivirus into the bone marrow cavity of OVX rats, the bone loss, and osteogenic differentiation inhibition of BMSCs in OVX rats were partially reversed, while osteoclast differentiation promotion of BMSCs in OVX rats was unaffected. Taken together, the present study confirms that Hmga1 prevents OVX-induced bone loss by the Wnt signaling pathway and reveals that Hmga1 is a potential gene therapeutic target for postmenopausal osteoporosis.

Identification and evaluation of circulating exosomal miRNAs for the diagnosis of postmenopausal osteoporosis.

BACKGROUND: Postmenopausal osteoporosis (PMOP) is a common condition that leads to a loss of bone density and an increased risk of fractures in women. Recent evidence suggests that exosomal miRNAs are involved in regulating bone development and osteogenesis. However, exosomal miRNAs as biomarkers for PMOP diagnosis have not been systematically evaluated. In this study, we aim to identify PMOP-associated circulating exosomal miRNAs and evaluate their diagnostic performance. METHODS: We performed next-generation sequencing and bioinformatics analysis of plasma exosomal miRNAs from 12 PMOP patients and 12 non-osteoporosis controls to identify PMOP-associated exosomal miRNAs, and then validated them in an independent natural community cohort with 26 PMOP patients and 21 non-osteoporosis controls. Exosomes were isolated with the size exclusion chromatography method from the plasma of elder postmenopausal women. The plasma exosomal miRNA profiles were characterized in PMOP paired with controls with next-generation sequencing. Potential plasma exosomal miRNAs were validated by qRT-PCR in the validation cohort, and their performance in diagnosing PMOP was systematically evaluated with the receiver operating characteristic curve. RESULTS: Twenty-seven miRNAs were identified as differentially expressed in PMOP versus controls in sequencing data, of which six exosomal miRNAs (miR-196-5p, miR-224-5p, miR320d, miR-34a-5p, miR-9-5p, and miR-98-5p) were confirmed to be differentially expressed in PMOP patients by qRT-PCR in the validation cohort. The three miRNAs combination (miR-34a-5p + miR-9-5p + miR-98-5p) demonstrated the best diagnostic performance, with an AUC = 0.734. In addition, the number of pregnancies was found to be an independent risk factor that can improve the performance of exosomal miRNAs in diagnosing PMOP. CONCLUSIONS: These results suggested that the plasma exosomal miRNAs had the potential to serve as noninvasive diagnostic biomarkers for PMOP.