Hypomethylation of the RUNX2 Gene Is a New Potential Biomarker of Primary Osteoporosis in Men and Women.

The search for the molecular markers of osteoporosis (OP), based on the analysis of differential deoxyribonucleic acid (DNA) methylation in bone cells and peripheral blood cells, is promising for developments in the field of the early diagnosis and targeted therapy of the disease. The Runt-related transcription factor 2 (RUNX2) gene is one of the key genes of bone metabolism, which is of interest in the search for epigenetic signatures and aberrations associated with the risk of developing OP. Based on pyrosequencing, the analysis of the RUNX2 methylation profile from a pool of peripheral blood cells in men and women over 50 years of age of Russian ethnicity from the Volga-Ural region of Russia was carried out. The level of DNA methylation in three CpG sites of the RUNX2 gene was assessed and statistically significant hypomethylation was revealed in all three studied CpG sites in men (U = 746.5, p = 0.004; U = 784, p = 0.01; U = 788.5, p = 0.01, respectively) and in one CpG site in women (U = 537, p = 0.03) with primary OP compared with control. In the general sample, associations were preserved for the first CpG site (U = 2561, p = 0.0001766). The results were obtained for the first time and indicate the existence of potentially new epigenetic signatures of RUNX2 in individuals with OP.

Differential but complementary roles of HIF-1α and HIF-2α in the regulation of bone homeostasis.

Bone is a highly dynamic tissue undergoing continuous formation and resorption. Here, we investigated differential but complementary roles of hypoxia-inducible factor (HIF)-1α and HIF-2α in regulating bone remodeling. Using RNA-seq analysis, we identified that specific genes involved in regulating osteoblast differentiation were similarly but slightly differently governed by HIF-1α and HIF-2α. We found that increased HIF-1α expression inhibited osteoblast differentiation via inhibiting RUNX2 function by upregulation of Twist2, confirmed using Hif1a conditional knockout (KO) mouse. Ectopic expression of HIF-1α via adenovirus transduction resulted in the increased expression and activity of RANKL, while knockdown of Hif1a expression via siRNA or osteoblast-specific depletion of Hif1a in conditional KO mice had no discernible effect on osteoblast-mediated osteoclast activation. The unexpected outcome was elucidated by the upregulation of HIF-2α upon Hif1a overexpression, providing evidence that Hif2a is a transcriptional target of HIF-1α in regulating RANKL expression, verified through an experiment of HIF-2α knockdown after HIF-1α overexpression. The above results were validated in an ovariectomized- and aging-induced osteoporosis model using Hif1a conditional KO mice. Our findings conclude that HIF-1α plays an important role in regulating bone homeostasis by controlling osteoblast differentiation, and in influencing osteoclast formation through the regulation of RANKL secretion via HIF-2α modulation.

New mechanistic understanding of osteoclast differentiation and bone resorption mediated by P2X7 receptors and PI3K-Akt-GSK3ß signaling.

OBJECTIVE: Osteoporosis is a global health issue characterized by decreased bone mass and microstructural degradation, leading to an increased risk of fractures. This study aims to explore the molecular mechanism by which P2X7 receptors influence osteoclast formation and bone resorption through the PI3K-Akt-GSK3ß signaling pathway. METHODS: An osteoporosis mouse model was generated through ovariectomy (OVX) in normal C57BL/6 and P2X7f/f; LysM-cre mice. Osteoclasts were isolated for transcriptomic analysis, and differentially expressed genes were selected for functional enrichment analysis. Metabolite analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and multivariate statistical analysis and pattern recognition were used to identify differential lipid metabolism markers and their distribution. Bioinformatics analyses were conducted using the Encyclopedia of Genes and Genomes database and the MetaboAnalyst database to assess potential biomarkers and create a metabolic pathway map. Osteoclast precursor cells were used for in vitro cell experiments, evaluating cell viability and proliferation using the Cell Counting Kit 8 (CCK-8) assay. Osteoclast precursor cells were induced to differentiate into osteoclasts using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-beta ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) staining was performed to compare differentiation morphology, size, and quantity between different groups. Western blot analysis was used to assess the expression of differentiation markers, fusion gene markers, and bone resorption ability markers in osteoclasts. Immunofluorescence staining was employed to examine the spatial distribution and quantity of osteoclast cell skeletons, P2X7 protein, and cell nuclei, while pit assay was used to evaluate osteoclast bone resorption ability. Finally, in vivo animal experiments, including micro computed tomography (micro-CT), hematoxylin and eosin (HE) staining, TRAP staining, and immunohistochemistry, were conducted to observe bone tissue morphology, osteoclast differentiation, and the phosphorylation level of the PI3K-Akt-GSK3ß signaling pathway. RESULTS: Transcriptomic and metabolomic data collectively reveal that the P2X7 receptor can impact the pathogenesis of osteoporosis through the PI3K-Akt-GSK3ß signaling pathway. Subsequent in vitro experiments showed that cells in the Sh-P2X7 + Recilisib group exhibited increased proliferative activity (1.15 versus 0.59), higher absorbance levels (0.68 versus 0.34), and a significant increase in resorption pit area (13.94 versus 3.50). Expression levels of osteoclast differentiation-related proteins MMP-9, CK, and NFATc1 were markedly elevated (MMP-9: 1.72 versus 0.96; CK: 2.54 versus 0.95; NFATc1: 3.05 versus 0.95), along with increased fluorescent intensity of F-actin rings. In contrast, the OE-P2X7 + LY294002 group showed decreased proliferative activity (0.64 versus 1.29), reduced absorbance (0.34 versus 0.82), and a significant decrease in resorption pit area (5.01 versus 14.96), accompanied by weakened expression of MMP-9, CK, and NFATc1 (MMP-9: 1.14 versus 1.79; CK: 1.26 versus 2.75; NFATc1: 1.17 versus 2.90) and decreased F-actin fluorescent intensity. Furthermore, in vivo animal experiments demonstrated that compared with the wild type (WT) + Sham group, mice in the WT + OVX group exhibited significantly increased levels of CTX and NTX in serum (CTX: 587.17 versus 129.33; NTX: 386.00 versus 98.83), a notable decrease in calcium deposition (19.67 versus 53.83), significant reduction in bone density, increased trabecular separation, and lowered bone mineral density (BMD). When compared with the KO + OVX group, mice in the KO + OVX + recilisib group showed a substantial increase in CTX and NTX levels in serum (CTX: 503.50 versus 209.83; NTX: 339.83 versus 127.00), further reduction in calcium deposition (29.67 versus 45.33), as well as decreased bone density, increased trabecular separation, and reduced BMD. CONCLUSION: P2X7 receptors positively regulate osteoclast formation and bone resorption by activating the PI3K-Akt-GSK3ß signaling pathway.

SP1 regulates BMSC osteogenic differentiation through the miR-133a-3p/MAPK3 axis : SP1 regulates osteogenic differentiation of BMSCs.

BACKGROUND: The progression of osteoporosis (OP) can dramatically increase the risk of fractures, which seriously disturb the life of elderly individuals. Specific protein 1 (SP1) is involved in OP progression. However, the mechanism by which SP1 regulates OP progression remains unclear. OBJECTIVE: This study investigated the mechanism underlying the function of SP1 in OP. METHODS: SAMP6 mice were used to establish an in vivo model of age-dependent OP, and BALB/c mice were used as controls. BMSCs were extracted from two subtypes of mice. Hematoxylin and eosin staining were performed to mark the intramedullary trabecular bone structure to evaluate histological changes. ChIP assay was used to assess the targeted regulation between SP1 and miR-133a-3p. The binding sites between MAPK3 and miR-133a-3p were verified using a dual-luciferase reporter assay. The mRNA levels of miR-133a-3p and MAPK3 were detected using quantitative reverse transcription polymerase chain reaction (RT-qPCR). The protein expression of SP1, MAPK3, Colla1, OCN, and Runx2 was examined using Western blotting. Alkaline phosphatase (ALP) kit and Alizarin Red S staining were used to investigate ALP activity and mineralized nodules, respectively. RESULTS: The levels of SP1 and miR-133a-3p were upregulated, whereas the expression of MAPK3 was downregulated in BMSCs from SAMP6 mice, and miR-133a-3p inhibitor accelerated osteogenic differentiation in BMSCs. SP1 directly targeted miR-133a-3p, and MAPK3 was the downstream mRNA of miR-133a-3p. Mechanically, SP1 accelerated osteogenic differentiation in BMSCs via transcriptional mediation of the miR-133a-3p/MAPK3 axis. CONCLUSION: SP1 regulates osteogenic differentiation by mediating the miR-133a-3p/MAPK3 axis, which would shed new light on strategies for treating senile OP.

Delivery of miR-15b-5p via magnetic nanoparticle-enhanced bone marrow mesenchymal stem cell-derived extracellular vesicles mitigates diabetic osteoporosis by targeting GFAP.

Diabetic osteoporosis (DO) presents significant clinical challenges. This study aimed to investigate the potential of magnetic nanoparticle-enhanced extracellular vesicles (GMNPE-EVs) derived from bone marrow mesenchymal stem cells (BMSCs) to deliver miR-15b-5p, thereby targeting and downregulating glial fibrillary acidic protein (GFAP) expression in rat DO models. Data was sourced from DO-related RNA-seq datasets combined with GEO and GeneCards databases. Rat primary BMSCs, bone marrow-derived macrophages (BMMs), and osteoclasts were isolated and cultured. EVs were separated, and GMNPE targeting EVs were synthesized. Bioinformatic analysis revealed a high GFAP expression in DO-related RNA-seq and GSE26168 datasets for disease models. Experimental results confirmed elevated GFAP in rat DO bone tissues, promoting osteoclast differentiation. miR-15b-5p was identified as a GFAP inhibitor, but was significantly downregulated in DO and enriched in BMSC-derived EVs. In vitro experiments showed that GMNPE-EVs could transfer miR-15b-5p to osteoclasts, downregulating GFAP and inhibiting osteoclast differentiation. In vivo tests confirmed the therapeutic potential of this approach in alleviating rat DO. Collectively, GMNPE-EVs can effectively deliver miR-15b-5p to osteoclasts, downregulating GFAP expression, and hence, offering a therapeutic strategy for rat DO.

PCSK9 inhibitors and osteoporosis: mendelian randomization and meta-analysis.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors represent an effective strategy for reducing cardiovascular disease risk. Yet, PCSK9's impact on osteoporosis remains unclear. Hence, we employed Mendelian randomization (MR) analysis for examining PCSK9 inhibitor effects on osteoporosis. METHODS: Single nucleotide polymorphisms (SNPs) for 3-hydroxy-3-methylglutaryl cofactor A reductase (HMGCR) and PCSK9 were gathered from available online databases for European pedigrees. Four osteoporosis-related genome-wide association studies (GWAS) data served as the main outcomes, and coronary artery disease (CAD) as a positive control for drug-targeted MR analyses. The results of MR analyses examined by sensitivity analyses were incorporated into a meta-analysis for examining causality between PCSK9 and HMGCR inhibitors and osteoporosis. RESULTS: The meta-analysis involving a total of 1,263,102 subjects, showed that PCSK9 inhibitors can increase osteoporosis risk (P < 0.05, I2, 39%). However, HMGCR inhibitors are not associated with osteoporosis risk. Additionally, a replication of the analysis was conducted with another exposure-related GWAS dataset, which led to similar conclusions. CONCLUSION: PCSK9 inhibitors increase osteoporosis risk. However, HMGCR inhibitors are unremarkably linked to osteoporosis.

The clinical characteristics and pathogenic variants of primary pigmented nodular adrenocortical disease in 210 patients: a systematic review.

Aims: Primary pigmented nodular adrenocortical disease (PPNAD), as a rare kind of Cushing's syndrome, is frequently misdiagnosed. To get a better understanding of the disease, we analyzed the clinical characteristics and pathogenic variants of PPNAD. Methods: Databases were searched, and the pathogenic variants and clinical manifestations of patients were summarized from the relevant articles. Results: A total of 210 patients in 86 articles were enrolled with a median age of 22 and a female-to-male ratio of 2:1. Sixty-six (31.43%) patients were combined with Carney complex (CNC) and 94.29% were combined with osteoporosis/osteopenia. Among 151 patients who underwent genetic testing, 87.42% (132/151) had pathogenic variants. Six gene mutations (PRKAR1A, PDE11A, PRKACA, CTNNB1, PDE8B, and ARMC5) were detected in the patients. The most common mutation was PKAR1A, accounting for 79.47% (120/151). There was a significant correlation between PRKAR1A pathogenic variant and spotty skin pigmentation in CNC concurrent with PPNAD (p < 0.05). Among pregnant patients with PPNAD, those without surgical treatment and with bilateral adrenalectomy suffered from a high-risk perinatal period. However, patients with unilateral adrenalectomy presented a safe perinatal period. Conclusions: For young patients with Cushing's syndrome, especially female patients with spotty skin pigmentation and osteoporosis/osteopenia, PPNAD should be considered. Unilateral adrenal resection may be considered as an option for women with fertility needs. In view of the difficulty of PPNAD diagnosis, genetic testing before surgery might be a reasonable option. Patients with PPNAD with spotty skin pigmentation should consider the PRKAR1A pathogenic variant and pay attention to CNC. Systematic review registration: https://www.crd.york.ac.uk/prospero, identifier CRD42023416988.

Impdh2 deficiency suppresses osteoclastogenesis through mitochondrial oxidative phosphorylation and alleviates ovariectomy-induced osteoporosis.

Abnormalities in osteoclastic generation or activity disrupt bone homeostasis and are highly involved in many pathologic bone-related diseases, including rheumatoid arthritis, osteopetrosis, and osteoporosis. Control of osteoclast-mediated bone resorption is crucial for treating these bone diseases. However, the mechanisms of control of osteoclastogenesis are incompletely understood. In this study, we identified that inosine 5'-monophosphate dehydrogenase type II (Impdh2) positively regulates bone resorption. By histomorphometric analysis, Impdh2 deletion in mouse myeloid lineage cells (Impdh2LysM-/- mice) showed a high bone mass due to the reduced osteoclast number. qPCR and western blotting results demonstrated that the expression of osteoclast marker genes, including Nfatc1, Ctsk, Calcr, Acp5, Dcstamp, and Atp6v0d2, was significantly decreased in the Impdh2LysM-/- mice. Furthermore, the Impdh inhibitor MPA treatment inhibited osteoclast differentiation and induced Impdh2-cytoophidia formation. The ability of osteoclast differentiation was recovered after MPA deprivation. Interestingly, genome-wide analysis revealed that the osteoclastic mitochondrial biogenesis and functions, such as oxidative phosphorylation, were impaired in the Impdh2LysM-/- mice. Moreover, the deletion of Impdh2 alleviated ovariectomy-induced bone loss. In conclusion, our findings revealed a previously unrecognized function of Impdh2, suggesting that Impdh2-mediated mechanisms represent therapeutic targets for osteolytic diseases.

Navigating the gut-bone axis: The pivotal role of Coprococcus3 in osteoporosis prevention through Mendelian randomization.

Osteoporosis (OP) constitutes a notable public health concern that significantly impacts the skeletal health of the global aging population. Its prevalence is steadily escalating, yet the intricacies of its diagnosis and treatment remain challenging. Recent investigations have illuminated a profound interlink between gut microbiota (GM) and bone metabolism, thereby opening new avenues for probing the causal relationship between GM and OP. Employing Mendelian randomization (MR) as the investigative tool, this study delves into the causal rapport between 211 varieties of GM and OP. The data are culled from genome-wide association studies (GWAS) conducted by the MiBioGen consortium, in tandem with OP genetic data gleaned from the UK Biobank, BioBank Japan Project, and the FinnGen database. A comprehensive repertoire of statistical methodologies, encompassing inverse-variance weighting, weighted median, Simple mode, Weighted mode, and MR-Egger regression techniques, was adroitly harnessed for meticulous analysis. The discernment emerged that the genus Coprococcus3 is inversely associated with OP, potentially serving as a deterrent against its onset. Additionally, 21 other gut microbial species exhibited a positive correlation with OP, potentially accentuating its proclivity and progression. Subsequent to rigorous scrutiny via heterogeneity and sensitivity analyses, these findings corroborate the causal nexus between GM and OP. Facilitated by MR, this study successfully elucidates the causal underpinning binding GM and OP, thereby endowing invaluable insights for deeper exploration into the pivotal role of GM in the pathogenesis of OP.

The association between osteoporosis and frailty: a cross-sectional observational study and mendelian randomization analysis.

BACKGROUND: Osteoporosis and frailty are two common features in the elderly population. Despite many review articles mentioning the association between osteoporosis and frailty, there is a lack of original research directly investigating their relationship. Therefore, this study was conducted to examine the correlation between osteoporosis and frailty. METHODS: We conducted a cross-sectional study using data from the National Health and Nutrition Examination Survey (NHANES), using logistic regression analysis to assess the association of osteoporosis with the frailty index. In addition, we further explored the causal relationship between them using Mendelian randomization (MR) study. RESULTS: In the cross-sectional study, 19,091 non-frailty participants and 5878 frailty participants were included in this study. We observed a significant positive association between osteoporosis and frailty after adjusting for demographic characteristics, body mass index (BMI), smoking, and alcohol use (OR = 1.454, 95% CI [1.142,1.851], P = 0.003). Moreover, the MR study showed a bidirectional causal relationship between osteoporosis and frailty. When osteoporosis was used as an exposure factor, the frailty pooled OR value calculated utilizing the inverse variance weighted (IVW) method was 2.81 (95% CI [1.69, 4.68], P = 6.82 × 10- 5). When frailty was used as an exposure factor, the OR value calculated using the IVW method was 1.01 (95% CI [1.00,1.01], P = 3.65 × 10- 7). CONCLUSIONS: Osteoporosis was positively correlated with frailty, and the results remained robust after adjusting for covariates. Further, MR studies have shown a bidirectional causal relationship between osteoporosis and frailty.

Associations of sleep behaviors and genetic risk with risk of incident osteoporosis: A prospective cohort study of 293,164 participants.

BACKGROUND: Unhealthy sleep behaviors are associated with higher risks of osteoporosis (OP), while prospective evidence is limited. This study aimed to prospectively investigate this association, quantify the attributable burden of OP incidence reduction due to unhealthy sleep behaviors, and explore potential modifications by genetic risk factors. METHODS: This longitudinal cohort study was conducted utilizing data from the UK Biobank, comprising 293,164 participants initially free of OP and with requisite sleep behaviors data at baseline. We followed the participants after recruitment until November 30, 2022, to ascertain incident OP. We assessed the associations of five sleep behaviors including sleep duration, chronotype, insomnia, daytime napping, and morning wake-up difficulties, as well as sleep behavior patterns identified based on the above sleep behaviors, with the risk of OP, using Cox models adjusted for multiple confounders. The analyses were then performed separately among individuals with different OP susceptibility, indexed by standard polygenetic risk scores(PRS) for OP. Our secondary outcome was OP with pathologic fracture. Subgroup and sensitivity analyses were performed. Additionally, attributable risk percent in the exposed population (AR%) and population attributable fraction (PAF) of sleep behaviors were calculated. RESULTS: Over a median follow-up of 13.7 years, 8253 new-onset OP cases were documented. Unhealthy sleep behaviors, such as long or short sleep duration, insomnia, daytime napping, morning wake-up difficulties, and unhealthy sleep patterns, were associated with elevated risks of OP (HRs ranging from 1.14 to 1.46, all P-value <0.001) compared to healthy sleep behaviors. Similar associations were observed for OP with pathologic fractures. Insomnia exhibited the largest AR% of 39.98 % (95%CI: 36.46, 43.31) and PAF of 33.25 % (95%CI: 30.00, 36.34) among healthy sleep patterns and components. A statistically significant multiplicative interaction was noted between sleep behaviors and OP PRS on OP risk (all P-interaction <0.001). CONCLUSIONS: Four unhealthy sleep behaviors and sleep behavior patterns were associated to increased OP risk, with insomnia contributing the most to OP incidence, while genetic risk for OP modified this association. These findings underscore the crucial role of adhering to healthy sleep behaviors for effective OP prevention.

Two-sample Mendelian randomization studies revealed a causal relationship between insulin use and osteoporosis: An observational study.

OBJECTIVE: To investigate causal associations between diabetes, insulin treatment and osteoporosis using LDSC analysis with a 2-way Mendelian randomization study. METHODS: LDSC analysis was used to estimate the likelihood-scale heritability of the genome-wide association study used with genetic correlation between the 2 genome-wide association study used. Then a 2-sample Mendelian randomization study was performed using 3 methods including inverse variance weighted, MR Egger, and weighted median. RESULTS: The genetic correlation between diabetes, insulin treatment (h2_Z = 3.70, P = 2.16e-4), osteoporosis (h2_Z = 4.93, h2_p = 8.13e-7) and genes was significant. There was a significant genetic correlation (rg = 0.122, P = 0.0211). There was a causal association between diabetes, insulin treatment and osteoporosis [P = 0.003754, OR (95%CI) = 0.998876 (0.998116-0.999636)], while no causal association existed between osteoporosis and insulin use (P = 0.998116-0.999636) causal association existed (P = 0.333244). CONCLUSION: There was a strong genetic correlation between diabetes, insulin treatment and osteoporosis, a causal association between diabetes, insulin treatment and osteoporosis, and no causal association between osteoporosis and diabetes, insulin treatment.

Role of miRNA-regulated type H vessel formation in osteoporosis.

Osteoporosis (OP) is a chronic systemic bone metabolism disease characterized by decreased bone mass, microarchitectural deterioration, and fragility fractures. With the demographic change caused by long lifespans and population aging, OP is a growing health problem. The role of miRNA in the pathogenesis of OP has also attracted widespread attention from scholars in recent years. Type H vessels are unique microvessels of the bone and have become a new focus in the pathogenesis of OP because they play an essential role in osteogenesis-angiogenesis coupling. Previous studies found some miRNAs regulate type H vessel formation through the regulatory factors, including platelet-derived growth factor-BB (PDGF-BB), hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), and so on. These findings help us gain a more in-depth understanding of the relationship among miRNAs, type H vessels, and OP to find a new perspective on treating OP. In the present mini-review, we will introduce the role of type H vessels in the pathogenesis of OP and the regulation of miRNAs on type H vessel formation by affecting regulatory factors to provide some valuable insights for future studies of OP treatment.

GREM2 inactivation increases trabecular bone mass in mice.

Osteoporosis is a common skeletal disease affecting millions of individuals world-wide, with an increased risk of fracture, and a decreased quality of life. Despite its well-known consequences, the etiology of osteoporosis and optimal treatment methods are not fully understood. Human genetic studies have identified genetic variants within the FMN2/GREM2 locus to be associated with trabecular volumetric bone mineral density (vBMD) and vertebral and forearm fractures, but not with cortical bone parameters. GREM2 is a bone morphogenetic protein (BMP) antagonist. In this study, we employed Grem2-deficient mice to investigate whether GREM2 serves as the plausible causal gene for the fracture signal at the FMN2/GREM2 locus. We observed that Grem2 is moderately expressed in bone tissue and particularly in osteoblasts. Complete Grem2 gene deletion impacted mouse survival and body growth. Partial Grem2 inactivation in Grem2+/- female mice led to increased trabecular BMD of femur and increased trabecular bone mass in tibia due to increased trabecular thickness, with an unchanged cortical thickness, as compared with wildtype littermates. Furthermore, Grem2 inactivation stimulated osteoblast differentiation, as evidenced by higher alkaline phosphatase (Alp), osteocalcin (Bglap), and osterix (Sp7) mRNA expression after BMP-2 stimulation in calvarial osteoblasts and osteoblasts from the long bones of Grem2-/- mice compared to wildtype littermates. These findings suggest that GREM2 is a possible target for novel osteoporotic treatments, to increase trabecular bone mass and prevent osteoporotic fractures.

miRNA-seq analysis of high glucose induced osteoblasts provides insight into the mechanism underlying diabetic osteoporosis.

The present study aims to explore the etiology of Diabetic osteoporosis (DOP), a chronic complication associated with diabetes mellitus. Specifically, the research seeks to identify potential miRNA biomarkers of DOP and investigated role in regulating osteoblasts. To achieve this, an animal model of DOP was established through the administration of a high-sugar and high-fat diet, and then injection of streptozotocin. Bone microarchitecture and histopathology analysis were analyzed. Rat calvarial osteoblasts (ROBs) were stimulated with high glucose (HG). MiRNA profiles of the stimulated osteoblasts were compared to control osteoblasts using sequencing. Proliferation and mineralization abilities were assessed using MTT assay, alkaline phosphatase, and alizarin red staining. Expression levels of OGN, Runx2, and ALP were determined through qRT-PCR and Western blot. MiRNA-sequencing results revealed increased miRNA-702-5p levels. Luciferase reporter gene was utilized to study the correlation between miR-702-5p and OGN. High glucose impaired cell proliferation and mineralization in vitro by inhibiting OGN, Runx2, and ALP expressions. Interference with miR-702-5p decreased OGN, Runx2, and ALP levels, which were restored by OGN overexpression. Additionally, downregulation of OGN and Runx2 in DOP rat femurs was confirmed. Therefore, the miRNA-702-5p/OGN/Runx2 signaling axis may play a role in DOP, and could be diagnostic biomarker and therapeutic target for not only DOP but also other forms of osteoporosis.

Long non-coding RNA MIAT serves as a biomarker of fragility fracture and promotes fracture healing.

BACKGROUND: Fragility fracture is common in the elderly. Osteoblast differentiation is essential for bone healing and regeneration. Expression pattern of long non-coding RNA MIAT during fracture healing was examined, and its role in osteoblast differentiation was investigated. METHODS: 90 women with simple osteoporosis and 90 women with fragility fractures were included. Another 90 age-matched women were set as the control group. mRNA levels were tested using RT-qPCR. Cell viability was detected via CCK-8, and osteoblastic biomarkers, including ALP, OCN, Collagen I, and RUNX2 were tested via ELISA. The downstream miRNAs and genes targeted by MIAT were predicted by bioinformatics analysis, whose functions and pathways were annotated via GO and KEGG analysis. RESULTS: Serum MIAT was upregulated in osteoporosis women with high accuracy of diagnostic efficacy. Serum MIAT was even elevated in the fragility fracture group, but decreased in a time manner after operation. MIAT knockdown promoted osteogenic proliferation and differentiation of MC3T3-E1, but the influences were reversed by miR-181a-5p inhibitor. A total of 137 overlapping target genes of miR-181a-5p were predicted based on the miRDB, TargetScan and microT datasets, which were mainly enriched for terms related to signaling pathways regulating pluripotency of stem cells, cellular senescence, and osteoclast differentiation. CONCLUSIONS: LncRNA MIAT serves as a promising biomarker for osteoporosis, and promotes osteogenic differentiation via targeting miR-181a-5p.

CircZfp644-205 inhibits osteoblast differentiation and induces apoptosis of pre-osteoblasts via sponging miR-455-3p and promoting SMAD2 expression.

BACKGROUND: Circular RNAs (circRNAs) are involved in the progression of osteoporosis; however, their impact on osteogenic differentiation has yet to be fully elucidated. In this study, we identified a novel circRNA known as circZfp644-205 and investigated its effect on osteogenic differentiation and apoptosis in osteoporosis. METHODS: CircZfp644-205, miR-445-3p, and SMAD2 levels were measured using quantitative real-time polymerase chain reaction (qRT-PCR). MC3T3-E1 cells were subjected to microgravity (MG) to establish a cell model. Osteogenic differentiation was assessed using qRT-PCR, Alizarin Red S staining, alkaline phosphatase staining, and western blot. The apoptosis was evaluated using flow cytometry. The relationship between miR-445-3p and circZfp644-205 or SMAD2 was determined using bioinformatics, RNA pull-down, and luciferase reporter assay. Moreover, a hindlimb unloading mouse model was generated to investigate the role of circZfp644-205 in vivo using Micro-CT. RESULTS: CircZfp644-205 expression was up-regulated significantly in HG-treated MC3T3-E1 cells. Further in vitro studies confirmed that circZfp644-205 knockdown inhibited the osteogenic differentiation and induced apoptosis of pre-osteoblasts. CircZfp644-205 acted as a sponge for miR-455-3p, which reversed the effects of circZfp644-205 on pre-osteoblasts. Moreover, miR-455-3p directly targeted SMAD2, thus inhibiting the expression of SMAD2 to regulate cellular behaviors. Moreover, circZfp644-205 alleviated the progression of osteoporosis in mice. CONCLUSIONS: This study provides a novel circRNA that may serve as a potential therapeutic target for osteoporosis and expands our understanding of the molecular mechanism underlying the progression of osteoporosis.

Muscle-origin creatinine-cystatin C ratio is an osteoporosis marker in individuals with normal renal function: evidence from observational and Mendelian randomization analysis.

Background: Creatinine-cystatin C ratio (CCR) has been demonstrated as an objective marker of sarcopenia in clinical conditions but has not been evaluated as an osteoporosis marker in individuals with normal renal function. Methods: We selected 271,831 participants with normal renal function from UK Biobank cohort. Multivariable linear/logistic regression and Cox proportional hazards model were used to investigate the phenotypic relationship between CCR and osteoporosis in total subjects and gender-stratified subjects. Based on the genome-wide association study (GWAS) data, linkage disequilibrium regression (LDSC) and Mendelian randomization (MR) analysis were performed to reveal the shared genetic correlations and infer the causal effects, respectively. Results: Amongst total subjects and gender-stratified subjects, serum CCR was positively associated with eBMD after adjusting for potential risk factors (all P<0.05). The multivariable logistic regression model showed that the decrease in CCR was associated with a higher risk of osteoporosis/fracture in all models (all P<0.05). In the multivariable Cox regression analysis with adjustment for potential confounders, reduced CCR is associated with the incidence of osteoporosis and fracture in both total subjects and gender-stratified subjects (all P<0.05). A significant non-linear dose-response was observed between CCR and osteoporosis/fracture risk (P non-linearity < 0.05). LDSC found no significant shared genetic effects by them, but PLACO identified 42 pleiotropic SNPs shared by CCR and fracture (P<5×10-8). MR analyses indicated the causal effect from CCR to osteoporosis/fracture. Conclusions: Reduced CCR predicted increased risks of osteoporosis/fracture, and significant causal effects support their associations. These findings indicated that the muscle-origin serum CCR was a potential biomarker to assess the risks of osteoporosis and fracture.

Farnesyl Diphosphate Synthase Gene Associated with Loss of Bone Mass Density and Alendronate Treatment Failure in Patients with Primary Osteoporosis.

Aminobisphosphonates (NBPs) are the first-choice medication for osteoporosis (OP); NBP treatment aims at increasing bone mineral density (BMD) by inhibiting the activity of farnesyl diphosphate synthase (FDPS) enzyme in osteoclasts. Despite its efficacy, inadequate response to the drug and side effects have been reported. The A allele of the rs2297480 (A > C) SNP, found in the regulatory region of the FDPS gene, is associated with reduced gene transcription. This study evaluates the FDPS variant rs2297480 (A > C) association with OP patients' response to alendronate sodium treatment. A total of 304 OP patients and 112 controls were enrolled; patients treated with alendronate sodium for two years were classified, according to BMD variations at specific regions (lumbar spine (L1-L4), femoral neck (FN) and total hip (TH), as responders (OP-R) (n = 20) and non-responders (OP-NR) (n = 40). We observed an association of CC genotype with treatment failure (p = 0.045), followed by a BMD decrease in the regions L1-L4 (CC = -2.21% ± 2.56; p = 0.026) and TH (CC = -2.06% ± 1.84; p = 0.015) after two years of alendronate sodium treatment. Relative expression of the FDPS gene was also evaluated in OP-R and OP-NR patients. Higher expression of the FDPS gene was also observed in OP-NR group (FC = 1.84 ± 0.77; p = 0.006) when compared to OP-R. In conclusion, the influence observed of FDPS expression and the rs2897480 variant on alendronate treatment highlights the importance of a genetic approach to improve the efficacy of treatment for primary osteoporosis.

CRIP1 regulates osteogenic differentiation of bone marrow stromal cells and pre-osteoblasts via the Wnt signaling pathway.

With the aging of the global demographic, the prevention and treatment of osteoporosis are becoming crucial issues. The gradual loss of self-renewal and osteogenic differentiation capabilities in bone marrow stromal cells (BMSCs) is one of the key factors contributing to osteoporosis. To explore the regulatory mechanisms of BMSCs differentiation, we collected bone marrow cells of femoral heads from patients undergoing total hip arthroplasty for single-cell RNA sequencing analysis. Single-cell RNA sequencing revealed significantly reduced CRIP1 (Cysteine-Rich Intestinal Protein 1) expression and osteogenic capacity in the BMSCs of osteoporosis patients compared to non-osteoporosis group. CRIP1 is a gene that encodes a member of the LIM/double zinc finger protein family, which is involved in the regulation of various cellular processes including cell growth, development, and differentiation. CRIP1 knockdown resulted in decreased alkaline phosphatase activity, mineralization and expression of osteogenic markers, indicating impaired osteogenic differentiation. Conversely, CRIP1 overexpression, both in vitro and in vivo, enhanced osteogenic differentiation and rescued bone mass reduction in ovariectomy-induced osteoporosis mice model. The study further established CRIP1's modulation of osteogenesis through the Wnt signaling pathway, suggesting that targeting CRIP1 could offer a novel approach for osteoporosis treatment by promoting bone formation and preventing bone loss.