LncRNA CASC15 serves as a diagnostic biomarker for postmenopausal osteoporotic fractures and its knockdown enhances HBMSC osteogenic differentiation via miR-152-3p.

Introduction

Postmenopausal osteoporosis (PMOP) is a common systemic condition disrupting bone metabolism. Epidemiological studies have indicated that the prevalence of osteoporosis among postmenopausal women in China (32.1%) is significantly higher than that in men aged 50 years and above (6.9%) [ 1 ]. A drop in estrogen levels upsets the equilibrium between bone breakdown and bone regeneration, leading to reduced bone density, impaired bone microarchitecture, and a significantly increased risk of fragility fractures [ 2 ]. Fractures, as the most severe complication of PMOP, severely impair patients’ quality of life and serve as a key indicator for assessing treatment efficacy [ 3 , 4 ]. Although bisphosphonates and similar drugs have been shown to reduce fracture risk [ 5 ] by activating the Nrf2 pathway [ 6 ] and increasing bone mineral density (BMD) [ 7 , 8 ] in postmenopausal women, long-term use is associated with an increased risk of secondary osteoporosis [ 9 ]. Currently, bone mineral density assessment primarily relies on dual-energy X-ray absorptiometry (DXA) scans [ 10 ]; however, current treatments cannot reverse established bone loss, and reliable biomarkers for the early prediction of fracture risk are lacking [ 11 ]. Therefore, the development of new diagnostic indicators and targeted therapies is urgently required to optimize the prognosis of PMOP and fracture risks.

Long non-coding RNAs (lncRNAs), as key participants in epigenetic regulation, have been shown in recent years to play important roles in bone metabolism [ 12 ]. For example, KCNQ1OT1 alleviates PMOP symptoms through the miR-421-3p/mTOR pathway [ 13 ]. HBMSCs possess multipotent differentiation potential, and their osteogenic differentiation capacity directly influences bone formation. The regulation of HBMSCs’ osteogenic differentiation by lncRNAs has emerged as a key therapeutic approach for intervening in bone metabolic diseases [ 14 ]. Cancer susceptibility-associated gene 15 (CASC15) is situated in the 22.3 region of chromosome 6’s short arm and was first identified as a lncRNA with high transcriptional activity [ 15 ]. Recent studies have shown that lncRNA CASC15 is significantly overexpressed in osteosarcoma patients, and downregulating its expression levels can influence the miR-338-3p/RAB14 and the Wnt/β-catenin signaling transduction pathway, effectively delaying the progression of osteosarcoma [ 16 , 17 ]. Additionally, this molecule exhibits abnormal expression in bone marrow samples from acute myeloid leukemia (AML) patients [ 18 ]and cases of multifocal bone lesions [ 19 ]. Further studies indicate that abnormal CASC15 expression in PMOP patients [ 20 ] also participates in regulating irisin-mediated chondrocyte differentiation [ 21 ]. However, its biological function in the pathogenesis of PMOP remains unclear. Therefore, we selected CASC15 as our research subject. Notably, online databases successfully predicted the binding regions of CASC15 and miR-152-3p. Previous findings support the pivotal function of miR-152-3p in driving HBMSC osteogenic differentiation [ 22 ]. However, the specific mechanisms of the CASC15 and miR-152-3p interaction in PMOP require further investigation.

Given this, we hypothesize that CASC15 regulates miR-152-3p through a competitive endogenous RNA (ceRNA) mechanism, thereby contributing to PMOP progression. This study focuses on CASC15, assessing its potential as a diagnostic fracture biomarker by measuring its expression levels in PMOP patients. We further used cell experiments to validate whether CASC15 knockout promotes BMSC osteogenic differentiation via miR-152-3p targeting, aiming to provide a theoretical basis for early diagnosis and targeted therapy of PMOP-related fractures.

Materials and methods

Participants

The study sample consisted of 141 female patients with PMOP who were treated at Rizhao Central Hospital between August 2021 and June 2023. A control group of 137 healthy postmenopausal women of similar age who did not exhibit symptoms of osteoporosis was also selected. PMOP patients were recruited based on the following criteria: (1) being 55–75 years old and more than 12 months postmenopausal; (2) clinically diagnosed with osteoporosis; (3) no previous medical or non-medical treatment for PMOP. In the healthy control group, participants with conditions affecting bone metabolism (e.g., hyperparathyroidism) or currently taking medications that impair bone metabolism were excluded. The study grouped PMOP participants into fracture ( n = 73) and non-fracture ( n = 68) cohorts according to fracture status. During the study period, various clinical indicators such as age, menopause, smoking habits, drinking habits, BMI, BMD (femoral neck), T-score, and Z-score were systematically collected. After fasting overnight, participants donated 5 mL of venous blood at the time of enrollment. These samples were immediately processed using a 10-minute centrifugation at 3,000 rpm. The separated serum was immediately frozen in liquid nitrogen and stored in a − 80 °C freezer.

Approval of the study was secured from the Ethics Committee at Rizhao Central Hospital. Before formal participation in the study, all individuals were given a thorough overview of the research and signed informed consent forms. Ethical compliance, as defined by the Declaration of Helsinki, was fully upheld during this study.

Cell culture and transfection

Human bone marrow mesenchymal stem cells (HBMSCs) were provided by the National Certified Cell Culture Collection Center in Shanghai, China. During cell culture, the complete growth medium consisted of DMEM/F12 (Gibco, USA) enriched with 10% fetal bovine serum (FBS) and a 1% antibiotic solution containing penicillin-streptomycin (Gibco, USA). All cells were maintained at standard conditions (37 °C, 5% CO₂ concentration, saturated humidity). When the cells reached 80–90% confluence, they were passage into new flasks. All experimental data were obtained from cells cultured in the 2nd to 6th generations.

Lentiviral vectors expressing short hairpin RNA targeting CASC15 (sh-CASC15) and a negative control vector (sh-NC) were constructed and transfected into HBMSCs. All transfections were performed using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. The miR-152-3p inhibitor and its control group (NC inhibitor) were introduced into HBMSCs from the second to third generation. HBMSCs were cultured at a cell density of 1 × 10⁵ cells per 6-well culture plate and harvested after entering the exponential growth phase. The control group comprised untreated HBMSCs that were cultured under normal conditions.

Cell viability assay

HBMSCs were plated in 96-well plates at an initial concentration of 5 × 10 3 cells per well and maintained under standard culture conditions (37 °C, 5% CO₂) for 0, 24, 48, and 72 h, respectively. Then, 10 µL of CCK-8 reagent (Beyotime, Shanghai) was introduced into each well, and the plates were further incubated for 2 h. Subsequently, the optical density (OD) at 450 nm was determined for each sample using a microplate reader (BioTek, USA) to assess cell viability.

Cell apoptosis assay

Apoptosis in HBMSCs 48 h post-transfection was assessed using the Membrane-Bound Protein-V/Propidium Iodide (PI) Apoptosis Detection Kit (Beyotime, Beijing). The procedure is as follows: Digest the cells with EDTA-free trypsin, then collect the cells by centrifugation at 4 °C for 5 min. Wash the cells with the binding buffer provided in the kit. Resuspend the cells in 10 µL Annexin V-FITC and 5 µL propidium iodide and incubate in the dark for 30 min. Finally, cell apoptosis was analyzed using flow cytometry (Becton, Dickinson and Company, USA).

Osteoblast differentiation

HBMSCs were seeded into culture flasks and cultured under normal conditions in an incubator set at a constant condition (37 °C, 5% CO₂). The cells were then transferred to osteogenic differentiation medium (DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 10 mM β-glycerophosphate, 100 µM L-ascorbic acid 2-phosphate, and 10 nM dexamethasone) and cultured again. During the differentiation process, freshly made medium was replaced with the old medium every 48 h to maintain cell growth.

RNA extraction and real-time quantitative reverse transcription PCR (RT-qPCR)

Total RNA extraction from serum and cells was performed using TRIzol reagent (Invitrogen, USA). After extraction, the concentration and purity of the RNA samples were analyzed using a NanoDrop 2000 spectrophotometer. Reverse transcription was performed according to the kit instructions to convert total RNA into cDNA (TaKaRa, Japan). Gene expression quantification of CASC15, miR-152-3p, and molecular markers related to bone formation, including runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), collagen I type (Col I), and osteocalcin (OCN), was conducted via RT-qPCR with SYBR Premix Ex Taq II (TaKaRa, Japan). For data normalization, GAPDH (lncRNA/mRNA) and U6 (miRNA) were selected as reference genes, and the2 −ΔΔCt method was used to calculate relative expression levels.

Western blotting (WB)

Protein levels of RUNX2, ALP, OCN, and Col I in HBMSCs following different transfection treatments were determined using WB. Protein concentration was measured using the BCA Protein Assay Kit (Thermo Fisher, USA). Equivalent amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, USA). PVDF membranes were incubated overnight at 4 °C with primary antibodies against anti-RUNX2 (1:1000, CST, USA), anti-ALP (1:1000, Abcam, Shanghai), anti-OCN (1:1000, Abcam, Shanghai), anti-Col I (1:1000, Abcam, Shanghai), and GAPDH (1:1000, Abcam, Shanghai) overnight at 4 °C. Membranes were washed with TBST and incubated with the corresponding secondary antibody (1:5000, Abcam, Shanghai) for 2 h. Expression levels of each target protein were normalized to GAPDH. All uncut original films are included in the supplementary materials.

Subcellular fractionation

Nuclear-cytoplasmic fractionation of HBMSCs was performed using the PARIS kit (Invitrogen, USA) according to the manufacturer’s recommended procedure. Total RNA was then extracted from the nucleolar fraction and cytoplasmic fraction using the TRIzol method. To investigate the intracellular localization of CASC15, RNA samples were extracted and subjected to RT-qPCR analysis. U6 and GAPDH were used as internal references for nuclear and cytoplasmic RNA normalization, respectively.

Dual luciferase (DLR)assay

Potential binding sites between CASC15 and miR-152-3p were predicted using the ENCORI database. Based on this predicted interaction, wild-type CASC15 (CASC15-WT) and mutant CASC15 (CASC15-MUT) 3’-UTR luciferase reporter gene vectors were constructed, followed by co-transfection with miR-152-3p mimics and negative control mimics (mimic NC). After 48 h of culture, the luciferase activity of the vectors was quantitatively analyzed using the DLR Report Gene Assay Kit (Promega, USA).

RNA Immunoprecipitation (RIP) assay

Following the manufacturer’s protocol, immunoprecipitation of RNA-binding proteins was achieved using the EZ-Magna RIPTM Kit (Millipore, USA). The specific steps are as follows: First, collect the transfected cells, add RIP lysis buffer to lyse the cells, and then incubate the obtained cell lysate with magnetic beads pre-incubated with anti-Ago2 or IgG antibodies in RIP buffer for 4 h. The magnetic beads, after being incubated, were cleaned three times, followed by digestion with proteinase K. RNA was then extracted and purified using TRIzol reagent. Finally, the relative enrichment of CASC15 and miR-152-3p in the precipitated RNA was analyzed using RT-qPCR technology.

Statistical analysis

All data in this study were obtained from at least three independent replicate experiments and are expressed as the mean ± standard deviation (SD) after normalization (mean normalization) to mitigate systematic variations. Statistical analysis was performed using GraphPad Prism 9.0 software. For comparisons between groups, statistical significance between two groups was evaluated using a two-tailed Student’s t-test, whereas one-way analysis of variance (ANOVA) was performed for multiple-group comparisons. Results were considered statistically significant if the P -value was less than 0.05.

Results

Expression levels of CASC15 in PMOP patients and its clinical diagnostic efficacy

Through a comparative analysis of the basic demographic characteristics of the subjects, the results showed that compared with the healthy control group, PMOP patients had significantly lower BMD (femoral neck), T-score, and Z-score ( P < 0.05). Nevertheless, the two groups showed no notable variations regarding age, age at menopause, BMI, smoking history, and drinking habits ( P > 0.05, Table 1 ). Further detection of CASC15 expression levels in serum from both groups revealed that CASC15 was significantly overexpressed in PMOP patients ( P < 0.0001, Fig. 1 A). Evaluation using the ROC curve indicated that CASC15 has excellent diagnostic value as a biomarker for distinguishing PMOP patients from healthy individuals, with an AUC value of 0.9874, a diagnostic sensitivity of 95.04%, and a specificity of 90.51% (Fig. 1 B). Additionally, among PMOP patients, those who experienced fractures ( n = 73) exhibited significantly higher CASC15 expression levels than those who did not ( n = 68) ( P < 0.0001, Fig. 1 C). Further ROC analysis demonstrated that CASC15 effectively distinguished between patients with and without fractures (AUC = 0.8069; sensitivity = 69.86%; specificity = 86.76%) (Fig. 1 D).

Analysis of independent risk factors for fractures in PMOP patients

Pearson correlation analysis was utilized for assessing the association between CASC15 and clinical indicators in PMOP patients. CASC15 levels were significantly negatively correlated with T-score, Z-score, and BMD ( P < 0.0001, Table 2 ). To further identify independent risk factors for fracture in PMOP patients, a binary logistic regression model was used for analysis. The regression results indicated that T-score ( P = 0.025), Z-score ( P = 0.033), bone mineral density ( P = 0.012), and CASC15 expression levels ( P < 0.0001) were all significantly associated with fracture risk ( P < 0.05, Table 3 ), and these indicators can all serve as independent predictive factors for fracture risk in PMOP patients.

The influence of CASC15 on osteoblast activity

To investigate the effect of CASC15 on the differentiation of HBMSCs into osteoblasts, RNA interference was used for gene knockdown. First, RT-qPCR was used to confirm that sh-CASC15 transfection was highly efficient, successfully reducing the expression level of this gene in HBMSCs ( P < 0.0001, Fig. 2 A). The experimental results showed that when the CASC15 level was suppressed, the vitality of HBMSCs was significantly improved ( P < 0.0001, Fig. 2 B), while the apoptosis rate showed a decreasing trend ( P < 0.0001, Fig. 2 C). After one week of osteogenic induction culture, detection of osteogenic-related markers revealed that the levels of RUNX2, ALP, OCN, and Col I in the absence of CASC15 resulted in a significant upregulation relative to the untreated group ( P < 0.0001, Fig. 2 D), consistent with enhanced cell viability.

miR-152-3p serves as a downstream target of CASC15

To elucidate the molecular mechanism by which CASC15 regulates osteoblast function, we first analyzed the intracellular distribution characteristics of CASC15. The results showed that CASC15 is primarily distributed in the cytoplasm (Fig. 3 A). A bioinformatics analysis performed using the ECORI database revealed the presence of complementary binding sequences between CASC15 and miR-152-3p (Fig. 3 B). DLR assays confirmed that after transfection with a miR-152-3p mimic significantly reduced the fluorescent signal of the WT-CASC15 reporter vector, while no notable change was observed with the mutant vector ( P < 0.0001, Fig. 3 C). RIP experiments further showed that, compared with the IgG control, the RNA complexes enriched by Ago2 antibodies contained significantly higher levels of CASC15 and miR-152-3p ( P < 0.0001, Fig. 3 D). Compared with healthy controls, PMOP patients had significantly reduced expression levels of miR-152-3p in serum ( P < 0.0001, Fig. 3 E). Additionally, the expression of miR-152-3p was measured in PMOP patients with and without fractures, and findings revealed that miR-152-3p expression was markedly lower in PMOP patients with fractures compared to those without fractures ( P < 0.0001, Fig. 3 F). These results indicate a target relationship wherein CASC15 potentially targets miR-152-3p.

CASC15 regulates osteoblast function by targeting miR-152-3p

To determine whether the molecular mechanism through which CASC15 mediates osteogenic differentiation of BMSCs involves miR-152-3p, we first examined the effect of silencing CASC15 on miR-152-3p expression. The results showed that knocking down CASC15 led to a significant increase in miR-152-3p expression; however, this upregulation effect could be significantly reversed upon treatment with a miR-152-3p inhibitor ( P < 0.0001, Fig. 4 A). Downregulation of CASC15 significantly promoted the proliferation activity of HBMSCs and inhibited cell apoptosis, but this change could be offset by the miR-152-3p inhibitor ( P < 0.0001, Fig. 4 B, C). Additionally, the protein and mRNA expression of key osteogenic differentiation markers (including RUNX2, ALP, OCN, and Col I) was promoted by CASC15 downregulation, but the introduction of a miR-152-3p inhibitor effectively eliminated this regulatory effect ( P < 0.0001, Fig. 4 D, E).

Discussion

Amid socioeconomic advancements and an aging demographic, OP has become a major disease affecting human health, and PMOP is the most common type [ 23 ]. The key mechanism is that insufficient osteogenic differentiation of BMSCs cannot offset bone resorption [ 24 ]. The effectiveness of surgical treatment for fractures remains limited at present [ 25 ]. The persistence of low back pain in the short term after surgery is associated with factors such as reduced bone density [ 26 ]. Therefore, investigating the diagnostic role of lncRNAs in PMOP and the mechanisms regulating BMSC osteogenic differentiation is of great significance.

LncRNAs are molecules longer than 200 nucleotides with tissue specificity, participating in cellular homeostasis and immune regulation. Their abnormal expression is associated with various diseases and can serve as diagnostic markers and therapeutic targets [ 27 , 28 ]. Increasing evidence suggests that lncRNAs regulate osteoblast differentiation, proliferation, and apoptosis, thereby influencing bone formation and bone homeostasis. For example, lnc-ob1 has been found to promote bone formation by upregulating Osterix in osteoblasts [ 29 ]. ANCR exerts its inhibitory effect on osteoblast differentiation in PMOP by specifically targeting EZH2 and RUNX2 [ 30 ]. In this study, we focused on the lncRNA CASC15, which was found to be significantly overexpressed in PMOP patients and demonstrated strong diagnostic potential for PMOP and fracture risk assessment.

As a type of short RNA molecule, microRNAs (miRNAs) do not participate in the protein-coding process. Earlier investigations have reported that various miRNAs play key roles in tendon injury repair [ 31 ], osteoarthritis [ 32 ], and osteoporosis [ 33 , 34 ]. Furthermore, small interfering RNA (siRNA) and circular RNA (circRNA) are involved in bone metabolism and associated inflammatory processes by modulating gene expression and growth factor activity [ 35 – 38 ]. miR-152-3p is significantly reduced during the osteogenic induction of HBMSCs [ 39 ]and in multiple myeloma (MM) patients [ 40 ]. It is also significantly downregulated in Parkinson’s disease (PD) models [ 41 ]. In the database, lncRNA CASC15 has binding sites with miR-152-3p. However, the potential regulatory mechanism of lncRNA CASC15 in PMOP patients remains unclear. Subcellular localization analysis of CASC15 revealed that it may act as a sponge molecule in the cytoplasm to bind miR-152-3p and exert its function in PMOP. DLR assays and RIP experiments confirmed that miR-152-3p acts as a downstream effector of CASC15. HBMSCs are key cells in the bone formation process, playing a crucial role in bone tissue development, repair, and maintenance of bone homeostasis [ 42 ]. The presence of GLP-1R on the surface of HBMSCs has been shown to regulate bone metabolism [ 43 ]. Consequently, they are commonly used as cellular models for studying the pathogenesis and therapeutic approaches of bone-related diseases. Through functional knockout experiments, we found that CASC15 regulates osteogenic differentiation of HBMSCs by binding to miR-152-3p. Inhibiting CASC15 expression enhances the proliferation capacity of HBMSCs, reduces apoptosis rates, and upregulates the expression levels of osteogenic-related markers such as RUNX2, ALP, OCN, and Col I. RUNX2, as a key regulatory factor in osteoblast differentiation, plays a crucial role in promoting osteoblast-specific gene expression, regulating osteoblast maturation, and bone formation [ 44 ]. Studies have shown that RUNX2 gene deficiency leads to impaired osteoblast differentiation and severe bone mineralization abnormalities in mice [ 45 ]. RUNX2 influences bone formation by regulating osteoblast-related gene expression and signaling pathways (Wnt/β-catenin, BMP/Smad). Abnormal expression levels of RUNX2 are closely associated with skeletal system diseases such as osteoporosis and osteoarthritis [ 46 – 48 ]. ALP is an enzyme closely associated with osteoblast function, participating in phospholipid hydrolysis and bone matrix mineralization processes [ 49 ]. Increased activity of this enzyme is considered an early marker of osteoblast precursor and osteoblast stem cell differentiation [ 50 ]. Research indicates that elevated ALP levels are inversely associated with femoral BMD [ 51 ]and elevated levels have been associated with an increased risk of fractures [ 52 ]. OCN is a bone-specific non-collagenous matrix protein that not only regulates the mineralization process but also bidirectionally modulates the biological functions of osteoblasts and osteoclasts, making it an important marker for assessing bone formation [ 53 , 54 ]. Col I, as the primary structural component of the bone organic matrix, provides a foundational scaffold for maintaining skeletal mechanical properties and microstructural integrity [ 55 ]. The upregulation of these osteogenic markers following CASC15 knockout in this study suggests that CASC15 may influence osteoblast differentiation and function through a negative regulatory mechanism.

Nevertheless, this study retains certain limitations. Firstly, the sample size was relatively small; subsequent studies should expand the sample cohort to further validate the reliability of CASC15 as a diagnostic biomarker. Secondly, we will conduct longitudinal follow-up studies to track the expression dynamics of CASC15 in PMOP patients, analyzing its correlation with fracture occurrence timing and severity to clarify its prognostic assessment value. Regarding mechanisms, the present study only preliminarily explored the functional role of the CASC15/miR-152-3p pathway at the cellular level. An animal model of PMOP has yet to be established to validate its in vivo effects, and the downstream target genes of miR-152-3p remain to be thoroughly characterized. To address these limitations, subsequent research will explore the signaling pathways through which CASC15 targets miR-152-3p to influence fracture healing using GO and KEGG databases, followed by experimental validation.

In summary, this study reveals the important role of CASC15 in PMOP, providing new insights into the diagnosis, fracture risk prediction, and molecular mechanisms of PMOP. The CASC15/miR-152-3p interaction may serve as a critical target in PMOP therapy.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1

Acknowledgements

Not applicable.

Author contributions

Conceptualization, X.W., W.Z., J.L., K.M. and Z.W., X.D.; Data curation, X.W., W.Z., J.L., K.M. and S.W., X.D.; Formal analysis, K.M. and S.W.; Funding acquisition, Z.W.; Investigation, K.M. and S.W.; Methodology, X.W., J.L., W.Z., K.M. and S.W., X.D.; Project administration, Z.W., X.D.; Resources, K.M. and S.W.; Software, X.W., J.L., K.M. and S.W.; Supervision, Z.W., X.D.; Validation, K.M. and S.W.; Visualization, Z.W.; Roles/Writing - original draft, K.M.; Writing–review & editing, X.W., W.Z., J.L., Z.W., X.D.

Funding

Not applicable.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Rizhao Central Hospital before the study began.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Xuri Wu and Weiyang Zhang have contributed equally to this work.

References

1. Wang L, Yu W, Yin X, Cui L, Tang S, Jiang N, et al. Prevalence of osteoporosis and fracture in china: the China osteoporosis prevalence study. JAMA Netw Open. 2021;4(8):e2121106.

2. Li J, Chen X, Lu L, Yu X. The relationship between bone marrow adipose tissue and bone metabolism in postmenopausal osteoporosis. Cytokine Growth Factor Rev. 2020;52:5288–98.

3. Cai XP, Zhao Q, Guo ZD, Lin SJ, Chen ZX, Chen MY, et al. Potential diagnostic value of PD-1 in peripheral blood mononuclear cells of postmenopausal osteoporosis patients. J Clin Lab Anal. 2020;34(6):e23223.

4. Pfeifer M, Kohlwey L, Begerow B, Minne HW. Effects of two newly developed spinal orthoses on trunk muscle strength, posture, and quality-of-life in women with postmenopausal osteoporosis: a randomized trial. Am J Phys Med Rehabil. 2011;90(10):805–15.

5. Migliorini F, Colarossi G, Baroncini A, Eschweiler J, Tingart M, Maffulli N. Pharmacological management of postmenopausal osteoporosis: a level I evidence based–expert opinion. Expert Rev Clin Pharmacol. 2021;14(1):105–19.

6. Huang F, Wang Y, Liu J, Cheng Y, Zhang X, Jiang H. Asperuloside alleviates osteoporosis by promoting autophagy and regulating Nrf2 activation. J Orthop Surg Res. 2024;19(1):855.

7. Conti V, Russomanno G, Corbi G, Toro G, Simeon V, Filippelli W, et al. A polymorphism at the translation start site of the vitamin D receptor gene is associated with the response to anti-osteoporotic therapy in postmenopausal women from Southern Italy. Int J Mol Sci. 2015;16(3):5452–66.

8. Migliorini F, Maffulli N, Colarossi G, Eschweiler J, Tingart M, Betsch M. Effect of drugs on bone mineral density in postmenopausal osteoporosis: a bayesian network meta-analysis. J Orthop Surg Res. 2021;16(1):533.

9. Migliorini F, Colarossi G, Eschweiler J, Oliva F, Driessen A, Maffulli N. Antiresorptive treatments for corticosteroid-induced osteoporosis: a bayesian network meta-analysis. Br Med Bull. 2022;143(1):46–56.

10. Leeyaphan J, Rojjananukulpong K, Intarasompun P, Peerakul Y. Simple clinical predictors for making directive decisions in osteoporosis screening for women: a cross-sectional study. J Orthop Surg Res. 2024;19(1):789.

11. Ho-Pham LT, Nguyen ND, Nguyen TV. Quantification of the relative contribution of Estrogen to bone mineral density in men and women. BMC Musculoskelet Disord. 2013;14(1):14366.

12. Jiménez-Ortega RF, Aparicio-Bautista DI, Becerra-Cervera A, Ortega-Meléndez AI, Patiño N, Rivera-Paredez B et al. The regulatory role of long Non-Coding RNAs in the development and progression of osteoporosis. Int J Mol Sci. 2025;26(9):4273.

13. Wang Z, Zhang H, Li Q, Zhang L, Chen L, Wang H, et al. Long non-coding RNA KCNQ1OT1 alleviates postmenopausal osteoporosis by modulating miR-421-3p/mTOR axis. Sci Rep. 2023;13(1):2333.

14. An F, Wang X, Wang C, Liu Y, Sun B, Zhang J et al. Research progress on the role of lncRNA-miRNA networks in regulating adipogenic and osteogenic differentiation of bone marrow mesenchymal stem cells in osteoporosis. Front Endocrinol (Lausanne). 2023;14:141210627.

15. Zhao M, Zhu N, Hao F, Song Y, Wang Z, Ni Y et al. The regulatory role of Non-coding RNAs on programmed cell death four in inflammation and cancer. Front Oncol. 2019;9:9919.

16. Zhang H, Wang J, Ren T, Huang Y, Yu Y, Chen C et al. LncRNA CASC15 is upregulated in osteosarcoma plasma exosomes and CASC15 knockdown inhibits osteosarcoma progression by regulating miR-338-3p/RAB14 axis. Onco Targets Ther. 2020;13:12055–12066.

17. Wang H, Zhang P. lncRNA–CASC15 promotes osteosarcoma proliferation and metastasis by regulating epithelial–mesenchymal transition via the Wnt/β–catenin signaling pathway. Oncol Rep. 2021; 45(5):76.

18. Xia PH, Xu ZJ, Jin Y, Ma JC, Wen XM, Yuan Q, et al. [Clinical significance of low expression of LncRNA CASC15 in acute myeloid leukemia with NPM1 Mutations]. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2022;30(3):659–70.

19. Li WC, Liu L, Wang ZD, Chen H, Liu G, Feng ZC. Cystic angiomatosis in children: clinical experience and review of literature. World J Surg Oncol. 2022;20(1):390.

20. Wang S. Investigation of long non-coding RNA expression profiles in patients with post-menopausal osteoporosis by RNA sequencing. Exp Ther Med. 2020;20(2):1487–97.

21. Chen Y, Sha W, Zhang Y, Kou W, Yang L, Guo R, et al. Irisin-regulated LncRNAs and their potential regulatory functions in chondrogenic differentiation of human mesenchymal stem cells. Open Med (Wars). 2024;19(1):20241073.

22. Wang X, Liu Y, Lei P. LncRNA HOTAIRM1 promotes osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting miR-152-3p/ETS1 axis. Mol Biol Rep. 2023;50(7):5597–608.

23. Wong SK. Glycogen synthase Kinase-3 beta (GSK3β) as a potential drug target in regulating osteoclastogenesis: an updated review on current evidence. Biomolecules. 2024; 14(4):502.

24. Bateman ME, Strong AL, McLachlan JA, Burow ME, Bunnell BA. The effects of endocrine disruptors on adipogenesis and osteogenesis in mesenchymal stem cells: a review. Front Endocrinol (Lausanne). 2016;7:171.

25. Andersen M, Andresen AK, Hartvigsen J, Hermann AP, Sørensen J, Carreon LY. Vertebroplasty for painful osteoporotic vertebral compression fractures: a protocol for a single-center doubled-blind randomized sham-controlled clinical trial. VOPE2. J Orthop Surg Res. 2024;19(1):813.

26. Shen L, Yang H, Zhou F, Jiang T, Jiang Z. Risk factors of short-term residual low back pain after PKP for the first thoracolumbar osteoporotic vertebral compression fracture. J Orthop Surg Res. 2024;19(1):792.

27. Lin W, Zhou Q, Wang CQ, Zhu L, Bi C, Zhang S, et al. LncRNAs regulate metabolism in cancer. Int J Biol Sci. 2020;16(7):1194–206.

28. He T, Liu W, Cao L, Liu Y, Zou Z, Zhong Y et al. CircRNAs and LncRNAs in osteoporosis. Differentiation. 2020;116:16–25.

29. Sun Y, Cai M, Zhong J, Yang L, Xiao J, Jin F, et al. The long noncoding RNA lnc-ob1 facilitates bone formation by upregulating Osterix in osteoblasts. Nat Metab. 2019;1(4):485–96.

30. Cai N, Li C, Wang F. Silencing of LncRNA-ANCR promotes the osteogenesis of osteoblast cells in postmenopausal osteoporosis via targeting EZH2 and RUNX2. Yonsei Med J. 2019;60(8):751–9.

31. Giordano L, Porta GD, Peretti GM, Maffulli N. Therapeutic potential of MicroRNA in tendon injuries. Br Med Bull. 2020;133(1):79–94.

32. Oliviero A, Della Porta G, Peretti GM, Maffulli N. MicroRNA in osteoarthritis: physiopathology, diagnosis and therapeutic challenge. Br Med Bull. 2019;130(1):137–47.

33. Xu R, Shen X, Si Y, Fu Y, Zhu W, Xiao T, et al. MicroRNA-31a-5p from aging BMSCs links bone formation and resorption in the aged bone marrow microenvironment. Aging Cell. 2018;17(4):e12794.

34. Huang Y, Yang Y, Wang J, Yao S, Yao T, Xu Y et al. miR-21-5p targets SKP2 to reduce osteoclastogenesis in a mouse model of osteoporosis. J Biol Chem. 2021;296:100617.

35. Gargano G, Oliviero A, Oliva F, Maffulli N. Small interfering RNAs in tendon homeostasis. Br Med Bull. 2021;138(1):58–67.

36. Gargano G, Oliva F, Oliviero A, Maffulli N. Small interfering RNAs in the management of human rheumatoid arthritis. Br Med Bull. 2022;142(1):34–43.

37. Gargano G, Asparago G, Spiezia F, Oliva F, Maffulli N. Small interfering RNAs in the management of human osteoporosis. Br Med Bull. 2023;148(1):58–69.

38. Gargano G, Pagano SM, Maffulli N. Circular RNAs in the management of human osteoporosis. Br Med Bull. 2025; 153(1):ldae024.

39. Zarecki P, Hackl M, Grillari J, Debono M, Eastell R. Serum MicroRNAs as novel biomarkers for osteoporotic vertebral fractures. Bone. 2020;130:115105.

40. Zheng JF, Guo NH, Zi FM, Cheng J. Long Noncoding RNA H19 promotes tumorigenesis of multiple myeloma by activating BRD4 signaling by targeting MicroRNA 152-3p. Mol Cell Biol. 2020;40(3):e00382-19.

41. Zhai K, Liu B, Gao L. Long-Noncoding RNA TUG1 promotes parkinson’s disease via modulating MiR-152-3p/PTEN pathway. Hum Gene Ther. 2020;31(23–24):1274–87.

42. Zhang FF, Hao Y, Zhang KX, Yang JJ, Zhao ZQ, Liu HJ, et al. Interplay between mesenchymal stem cells and macrophages: promoting bone tissue repair. World J Stem Cells. 2024;16(4):375–88.

43. Bao X, Liu C, Liu H, Wang Y, Xue P, Li Y. Association between polymorphisms of glucagon-like peptide-1 receptor gene and susceptibility to osteoporosis in Chinese postmenopausal women. J Orthop Surg Res. 2024;19(1):869.

44. Feng Y, Mao T, Yi J, Zhang N, Gu Y, Shen H et al. Runt-related transcription factors: from pathogenesis to therapeutic targets in multiple-organ fibrosis. Front Cell Dev Biol. 2025;13:1528645.

45. Plotkin LI, Bruzzaniti A. Molecular signaling in bone cells: regulation of cell differentiation and survival. Adv Protein Chem Struct Biol. 2019;116:237–81.

46. Xu F, Li W, Yang X, Na L, Chen L, Liu G. The roles of epigenetics regulation in bone metabolism and osteoporosis. Front Cell Dev Biol. 2020;8:619301.

47. Liang J, Chen J, Ye Z, Bao D. Cathelicidin LL-37 improves bone metabolic balance in rats with ovariectomy-induced osteoporosis via the Wnt/beta-catenin pathway. Physiol Res. 2022;71(3):369–77.

48. Han D, Wang W, Gong J, Ma Y, Li Y. Microbiota metabolites in bone: shaping health and confronting disease. Heliyon. 2024;10(7):e28435.

49. Gao X, Qin W, Chen L, Fan W, Ma T, Schneider A, et al. Effects of targeted delivery of Metformin and dental pulp stem cells on osteogenesis via demineralized dentin matrix under high glucose conditions. ACS Biomater Sci Eng. 2020;6(4):2346–56.

50. Rodríguez-Lozano FJ, Lozano A, López-García S, García-Bernal D, Sanz JL, Guerrero-Gironés J, et al. Biomineralization potential and biological properties of a new tantalum oxide (Ta( 2 )O( 5 ))-containing calcium silicate cement. Clin Oral Investig. 2022;26(2):1427–41.

51. Migliorini F, Maffulli N, Spiezia F, Tingart M, Maria PG, Riccardo G. Biomarkers as therapy monitoring for postmenopausal osteoporosis: a systematic review. J Orthop Surg Res. 2021;16(1):318.

52. Migliorini F, Maffulli N, Spiezia F, Peretti GM, Tingart M, Giorgino R. Potential of biomarkers during pharmacological therapy setting for postmenopausal osteoporosis: a systematic review. J Orthop Surg Res. 2021;16(1):351.

53. Lee NK, Sowa H, Hinoi E, Ferron M, Ahn JD, Confavreux C, et al. Endocrine regulation of energy metabolism by the skeleton. Cell. 2007;130(3):456–69.

54. Yao Z, Huang W, Yang Y, Zou L, Zhang Y, Zhang J, et al. Investigation of the osteogenic effects of ICA and ICSII on rat bone marrow mesenchymal stem cells. Sci Rep. 2025;15(1):3060.

55. Rossi V, Lee B, Marom R. Osteogenesis imperfecta: advancements in genetics and treatment. Curr Opin Pediatr. 2019;31(6):708–15.

Associated Data

Supplementary Materials

Supplementary Material 1

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.