miR-211-5p/FOXO3 axis accelerates osteogenic differentiation and fracture healing by mediating Wnt/β-catenin pathway

Huifei Zhai; Mianlong Lin; Changchun Lu; Jixia Liang

doi:10.1186/s13018-026-07003-y

. 2026 Jun 1;21:463. doi: 10.1186/s13018-026-07003-y

miR-211-5p/FOXO3 axis accelerates osteogenic differentiation and fracture healing by mediating Wnt/β-catenin pathway

Huifei Zhai ¹, Mianlong Lin ², Changchun Lu ³, Jixia Liang ^1,^✉

PMCID: PMC13435543 PMID: 42219493

Abstract

Background

As a disease closely related to aging, the prevalence and disability rate of osteoporotic fracture (OPF) are on the rise. MicroRNA (miRNA) expression abnormalities are closely linked to various skeletal disorders, yet the regulatory role of miR-211-5p in OPF remains unclear.

Objective

To investigate the expression patterns of miR-211-5p in OPF and its molecular mechanisms in the osteogenic differentiation and Wnt/β-catenin pathway of BMSCs and MC3T3-E1 cells.

Methods

Blood samples were collected from patients with osteoporosis (OP) and OPF who were treated in our hospital, as well as from healthy controls (HC). The levels of miR-211-5p and FOXO3 were quantified by RT-qPCR. The functional activity of the cells was evaluated using CCK-8 assay, flow cytometry, and Western blot techniques, respectively. The targeting relationship was verified by dual-luciferase activity reporter assay and RIP assay.

Results

Serum miR-211-5p was down-regulated in OPF patients, and holds diagnostic potential for identifying such patients. Following treatment, serum miR-211-5p levels increased over time in OPF patients, and osteogenic induction upregulated miR-211-5p level in BMSCs and MC3T3-E1 cells. miR-211-5p directly targeted FOXO3. Overexpression of FOXO3 partially reversed the activation of miR-211-5p mimic on cell viability, osteogenic markers and Wnt/β-catenin pathway effector markers.

Conclusion

miR-211-5p promotes bone formation by targeting and inhibiting FOXO3 to activate the Wnt/β-catenin pathway, thereby accelerating fracture healing.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13018-026-07003-y.

Keywords: miR-211-5p, FOXO3, Osteoporotic fracture, Osteogenic differentiation, Wnt/β-catenin pathway

Introduction

Osteoporotic fractures (OPF) are a classic complication of osteoporosis (OP) [1]. Their pathological basis involves reduced bone mass and disruption of the microscopic structure of bone tissue, leading to increased bone fragility and decreased strength, which in turn predisposes individuals to fractures [2, 3]. OPF predominantly affect the hip, vertebrae, and wrist, often leading to chronic pain, impaired fracture healing, and a marked elevation in disability rates [4]. Against the backdrop of a globally aging population, the prevalence of OPF continues to rise. Statistics indicate that over 9 million such fractures occur worldwide each year, imposing a heavy burden on public healthcare systems and patients' quality of life [5]. In current clinical practice, the diagnosis of OPF mainly relies on dual-energy X-ray absorptiometry (DXA) combined with imaging technology [6]. However, this gold standard mainly reflects the changes in bone mass or structural abnormalities after fracture, which is often lack of sensitive warning ability for the early degeneration of bone microstructure, and there is a certain risk of radiation exposure [7]. Therefore, identifying highly sensitive biomarkers for early warning has become a research focus in OPF treatment.

As a class of stable and conserved non-coding RNAs, microRNAs (miRNAs) offer new avenues for risk assessment and prognostic treatment in various diseases [8–10]. In the reports on bone diseases, miRNAs have been noted to mediate the process and may serve as candidate markers for intervention [11–16]. For instance, miRNA-6236 participated in and influenced angiogenesis in skeletal muscle following ischemia [17]. miR-126-5p promoted cell differentiation and skeletal muscle regeneration by enhancing MyoD expression through suppression of downstream FBXO32 levels [18]. Similarly, the role of miRNAs in fracture healing is an area of active research, with some miRNAs showing potential as diagnostic markers for delayed healing [19, 20]. miR-211-5p is located on human chromosome 15 with a length of 22 nucleotides [21]. Downregulation of miR-211-5p was revealed in postmenopausal osteoporosis (PMOP) rats, and its elevation may alleviate PMOP by mediating the JAK2/STAT3 pathway [22]. Additionally, miR-211-5p was recognized as being downregulated in osteoarthritis and identified as a downstream target of the lncRNA KCNQ1OT1. KCNQ1OT1 may suppress inflammatory responses and alleviate the progression of osteoarthritis by targeting the miR-211-5p/TCF4 axis [23]. Therefore, miR-211-5p’s function in fracture healing is worth further exploration.

Our study aims to analyze the level of miR-211-5p in serum samples from patients with OP and OPF, and to evaluate its diagnostic efficacy. Furthermore, we investigate the effects of abnormally expressed miR-211-5p on the activity and differentiation of BMSCs and MC3T3-E1 cells by in vitro assays. This aims to elucidate the functional mechanism of miR-211-5p in OPF, thereby providing novel insights and strategies for OPF intervention.

Materials and methods

Inclusion of patients

This study included 88 patients with OPF treated at Shantou Central Hospital with the approval of the Ethics Committee. Inclusion criteria: (1) BMD measured by DXA with lumbar spine or hip T-scores ≤ − 2.5; (2) History of traumatic fracture; (3) Undergoing surgical treatment at our hospital. Exclusion criteria: (1) Patients with concomitant malignancy, severe hepatic or renal disease, or other metabolic bone disorders; (2) Patients on long-term corticosteroids or other medications significantly affecting bone metabolism. Concurrently, 88 age-matched OP patients and healthy volunteers were enrolled.

Acquisition of serum samples

Fasting peripheral blood of 5 mL was collected from healthy controls and all patients within 24 h after admission. For OPF patients, additional blood samples are collected at 24 h, 1 week, 2 weeks, and 4 weeks post-treatment. The collected blood samples were centrifuged to separate the upper serum layer and stored uniformly in an ultra-low temperature refrigerator at − 80 °C.

Cell culture and osteogenic differentiation induction

Bone marrow mesenchymal stem cells (BMSC, Cat#7500, Lot#21,058) and mouse osteoblasts (MC3T3-E1, Cat#CRL-2593, Lot#70,045,219) were purchased from ScienCell (Carlsbad, USA). They were cultured in DMEM medium (Cat#C11330500BT, Lot#2458634CP; with 10% FBS, Cat#10,099-141C, Lot#2458634CP; Gibco, USA) at 37 °C and 5% CO₂ for incubation.

The cells that reached 90% confluence were seeded in 6-well plates, and osteogenic medium containing vitamin C (50 μg/ml, Sigma-Aldrich, Cat#A4544, Lot#0000526693), β-glycerophosphate (10 Mm, Sigma-Aldrich, Cat#G9422, Lot#0000543566), and dexamethasone (10 nM, Sigma-Aldrich, Cat#D4902, Lot#BCCP4649) was added to induce osteoblast differentiation. The medium was changed every three days, with testing performed on days 0, 7, and 14.

Cell transfection

Negative control mimic NC, inhibitor NC, ov-NC and miR-211-5p mimic/inhibitor, ov-FOXO3 were synthesized and provided by RiboBio (Guangzhou, China). They were transfected into BMSC and MC3T3-E1 cells for 48 h by lipofectamine 2000 (Invitrogen, USA, Cat#11,668,019, Lot#1,467,572) reagent. The sequences of the RNA oligonucleotides are shown in Supplementary Table 1.

RT-qPCR

Total RNA was extracted from serum and cells according to Trizol reagent protocols (Invitrogen, USA, Cat#15,596,026, Lot#424,311). Briefly, TRIzol was used for full lysis followed by the addition of chloroform reagent (Sigma-Aldrich, USA, Cat#151,858, Lot#LRAC0011). The aqueous phase containing RNA was collected and the RNA was precipitated with an equal volume of isopropanol (Sigma-Aldrich, USA, Cat#I9516, Lot#SHBT5354). Then, RNA concentration and purity were validated using NanoDrop 2000. Subsequently, cDNA templates were obtained by reverse transcription of miRNA and mRNA using Hairpin-it™ (GenePharma, Shanghai, China, Cat#E22001, Lot#20250312E22001) and HiFiScript kits (Kangwei, Beijing, China, Cat#CW2582, Lot#20250214CW2582), respectively. Finally, RT-qPCR amplification was performed in a 20 μL reaction system containing TB Green reagent (TaKaRa, Japan, Cat#RR820A, Lot#AN41007N), specific primers, and 2 μL of cDNA template, using CFX96 Real-Time PCR system (Bio-Rad, USA). The PCR reaction was performed with the following cycling profile: initial denaturation at 95 °C for 30 s, and then 40 cycles consisting of denaturation at 95 °C for 5 s and annealing at 60 °C for 30 s. U6 and GAPDH served as internal controls, and the relative expression of miR-211-5p and FOXO3 were calculated using the 2^{^(−ΔΔCt)} method. Primer sequences are as follows: miR-211-5p, forward 5'-GATGCTGTAATGGATGATATGA-3' and reverse 5'-ATTGGAACGATACAGAGAAGATT-3'; FOXO3, forward 5'-CGGCTAGCTGCGCCTTGGCTTTATAACT-3' and reverse 5'-GGCTCGAGCC

CTCCTTCACTGCTACTGG-3'; U6, forward 5'-CGCTTCGGCAGCACATATAC-3' and reverse 5'- TTCACGAATTTGCGTGTCAT-3'; GAPDH, forward 5'-GAAGGTGAAGGTCGGAGTC-3' and reverse 5'-GAAGATGGTGATGGGATTTC-3'.

Cell proliferation

At the indicated time points (24 h and 48 h), CCK-8 solution (10 μL; Dojindo, Japan, Cat#CK04-13, Lot#D2408) were supplemented to each well. After 2 h incubation at 37 °C, the absorbance at 450 nm was detected using a microplate reader (Bio Tek, USA, ELx808TM).

Cell apoptosis

After trypsin digestion and pre-cooled PBS washing, the cells were sequentially incubated with Annexin V-FITC and PI staining solutions (5 μL; Abcam; Annexin V-FITC/PI Apoptosis Detection Kit, Cat#ab14085, Lot#GR256892-1). Incubate at room temperature in the dark for 15 min, then stop the reaction by placing the sample on ice. Collect data using flow cytometer (BD Biosciences, FacsCANTO II) and calculate the proportion of each cell population.

Detection of osteogenic factors

The levels of osteogenesis-related factors Runx2 and OCN in cells were measured using ELISA kits, which were purchased from Elabscience Biotechnology Co (Wuhan, China, Cat#E-EL-H0925, Cat#E-EL-M0864).

ALP activity was measured through Alkaline Phosphatase Assay Kit (Beyotime, China, Cat#P0321, Lot#20250318P0321).

Dual-luciferase reporter assay

Predict the complementary sites for miR-211-5p and FOXO3 via ENCORI (https://rnasysu.com/encori/). FOXO3 3’UTR fragments containing wild-type (WT) and mutant (MUT) binding sites were cloned into pmiGLO reporter plasmid (Promega, Cat#E1330; FJ376737.1; Plasmid map is shown in Supplementary Fig. 1, Sequences are listed in Supplementary Table 2). The above plasmids were co-transfected with miR-211-5p mimic/inhibitor or mimic/inhibitor NC into BMSC and MC3T3-E1 cells. Luciferase activity was measured using dual-luciferase assay system (Promega, USA, Cat#E1910, Lot# 00000461) after 48 h.

RIP assay

Cells were resuspended using precooled RIP lysates (Magna RIP Kit; Millipore, USA, Cat#17–700, Lot#3,678,915). The cell lysates were incubated on ice for 5 min and then centrifuged to collect the supernatant. Subsequently, either Ago2 antibody or IgG control antibody were coupled to the magnetic beads overnight at 4 °C with rotation. The next day, the magnetic beads were washed twice with cold RIP wash buffer. After proteinase K (Magna RIP Kit; Millipore, USA; Cat#17–700, Lot#3,678,915) treatment of precipitated complexes at 55 °C for 30 min to digest proteins, RNA was extracted and quantified.

Western blot

Total proteins were extracted with RIPA buffer (Beyotime, China; Cat#P0013B, Lot#B250302) and separated by SDS-PAGE, then transferred to PVDF membranes. After blocking with 5% nonfat milk (Beyotime, China; Cat#P0216, Lot#20,250,218), followed by overnight incubation at 4 °C with primary antibodies (1:1000): FOXO3 (Cat#2497), non-phospho (active) β-catenin (Cat#8814), AXIN2 (Cat#2151), and GAPDH (Cat#2118; Cell Signaling Technology, USA). Followed by HRP-conjugated secondary antibodies (1:5000; Abcam, UK; Cat#ab6721) at room temperature for 1 h. Signals were visualized with ECL substrate (Thermo Fisher, USA; Cat# 32,106, Lot#NB168871B) and quantified using ImageJ software (Version 1.53c).

Statistical analysis

Quantitative data are expressed as mean ± standard deviation (SD), while count data are presented as n. Intergroup differences were assessed using one-way analysis of variance (ANOVA). ROC curve analysis was performed to evaluate the diagnostic performance of miR-211-5p, and the 95% confidence interval (CI) for the area under the curve (AUC) was calculated using GraphPad Prism software (version 9.0; Boston, MA, USA). Pearson correlation analysis determined the relationship between miR-211-5p and key indicators. Each set of experiments was performed at least three times to ensure the referability of the results. P < 0.05 was considered as statistically significant cutoff.

Results

Analysis of subjects’ clinical indicators

The clinical characteristics were recorded in Table 1. Each group included 88 participants. The HC group comprised 38 males and 50 females, with an average age of 65.30 ± 5.98 years; OP group consisted of 31 males and 57 females, averaging 66.04 ± 4.97 years; OPF group included 26 males and 62 females, also averaging 66.04 ± 4.97 years. Analysis revealed no significant differences among the three groups in terms of age, BMI, gender, or the presence of diabetes and hypertension (P > 0.05). However, substantial differences were observed in BMD and 25-(OH) vitamin D levels (P < 0.001).

Table 1.

Comparison of clinical information of recruited volunteers

Items	HC (n = 88)	OP (n = 88)	OPF (n = 88)	P value
Age (year)	65.30 ± 5.98	66.04 ± 4.97	66.83 ± 6.23	0.209
BMI (kg/m²)	23.66 ± 3.35	22.84 ± 2.09	22.75 ± 3.35	0.087
Gender (male/female)	38/50	31/57	26/62	0.168
Diabetes (yes/no)	10/78	14/74	17/71	0.346
Hypertension (yes/no)	32/56	37/51	35/53	0.742
Bone mineral density (T score)	−0.02 ± 0.48	-2.75 ± 0.46	-3.53 ± 0.31	< 0.001
25-(OH) vitamin D (ng/mL)	39.92 ± 6.49	28.98 ± 3.93	20.46 ± 3.63	< 0.001
Location of fracture (n/%)				–
Hip	–	–	51/57.95
Vertebral	–	–	18/20.45
Distal radius	–	–	13/14.77
Other	–	–	6/6.82

Open in a new tab

HC healthy controls, OP osteoporosis, OPF osteoporotic fractures, BMI body mass index

Expression and clinical significance of miR-211-5p in OPF

Compared with the HC group, serum miR-211-5p levels decreased in both the OP and OPF groups, with a more pronounced decrease observed in the latter (Fig. 1A). ROC analysis indicated that the AUC for distinguishing OP from HC using miR-211-5p was 0.751 (95% CI: 0.680–0.822; Fig. 1B), while the AUC for identifying OPF patients within the OP group was 0.809 (95% CI: 0.745–0.874; Fig. 1C). Furthermore, correlation analysis showed that miR-211-5p was positively correlated with BMD (r = 0.672, P < 0.001; Fig. 1D) and 25-(OH) vitamin D (r = 0.638, P < 0.001; Fig. 1E). Serum miR-211-5p levels gradually increased over time following treatment (Fig. 1F). Collectively, these findings suggest that serum miR-211-5p serves as a promising non-invasive biomarker for the screening of OPF.

Fig. 1 — Serum miR-211-5p expression was downregulated in OPF patients. A miR-211-5p was decreased in the serum of OP and OPF patients. B Diagnosis of miR-211-5p in OP patients. C Diagnosis of miR-211-5p in OPF patients. D, E miR-211-5p was positively correlated with BMD and 25-(OH) vitamin D. F The level of miR-211-5p in serum gradually increased with the recovery of patients. (n = 3 biological replicates, P < 0.001)

Osteogenic differentiation upregulated the miR-211-5p level

Following osteogenic differentiation induction in BMSC and MC3T3-E1 cells, miR-211-5p levels progressively increased (Fig. 2A). Concurrently, the levels of related osteogenic markers Runx2, ALP, and OCN also rose at 14 days compared to 0 day (Fig. 2B–D). This suggests that miR-211-5p is likely a positive regulatory factor promoting osteogenic differentiation in BMSC/MC3T3-E1 cells.

Abnormal expression of miR-211-5p regulated cell function

Transfection assay revealed that miR-211-5p mimic increased the miR-211-5p level in BMSC and MC3T3-E1 cells, while miR-211-5p inhibitor suppressed it (Fig. 3A). Upregulation of miR-211-5p accelerated cell growth and suppressed cell apoptosis, while downregulation of miR-211-5p resulted in the opposite effect (Fig. 3B, C). Additionally, Runx2, ALP, and OCN levels significantly enhanced when miR-211-5p was elevated, while osteogenic differentiation was suppressed when miR-211-5p was silenced (Fig. 3D–F). These in vitro results indicate that miR-211-5p acts as a key positive regulator of osteogenic potential.

Fig. 3 — Effect of dysregulation of miR-211-5p on cell function. A Transfection efficiency of miR-211-5p mimic and miR-211-5p inhibitor. B, C Changes in cell proliferation and apoptosis after transfection. D, E, F Changes in Runx2, ALP, and OCN levels in BMSC and MC3T3-E1 cells after transfection. (n = 3 biological replicates, P < 0.05)

FOXO3 was negatively regulated by miR-211-5p

Bioinformatics websites predicted specific binding sites between miR-211-5p and FOXO3 (Fig. 4A). miR-211-5p mimic/inhibitor in BMSCs markedly suppressed/promoted luciferase activity of the WT-FOXO3 reporter plasmid. The same effect was observed in MC3T3-E1 cells (Fig. 4B). RIP assays revealed that miR-211-5p and FOXO3 are enriched in cells, indicating their interaction (Fig. 4C).

Moreover, serum FOXO3 expression was higher in OPF patients than in the OP and HC groups (Fig. 4D). This elevated expression was negatively correlated with the level of miR-211-5p (r = − 0.664, P < 0.001; Fig. 4E). Serum FOXO3 level was gradually downregulated over time (Fig. 4F). This confirms that FOXO3 is a direct downstream target of miR-211-5p, and the inverse clinical association between them suggests the potential existence of a regulatory axis in the pathogenesis of OPF.

Effect of miR‑211‑5p/FOXO3 axis on cell viability and Wnt/β-catenin pathway

Transfection of miR‑211‑5p mimic downregulated the level of FOXO3 in BMSC and MC3T3-E1 cells, while high levels of FOXO3 counteracted this inhibitory effect (Fig. 5A). Meanwhile, co-transfection of miR‑211‑5p mimic and ov-FOXO3 reversed the promotion of cell proliferation and the inhibition of cell apoptosis induced by miR‑211‑5p (Fig. 5B, C). Following co-transfection with ov-FOXO3, the Runx2, ALP, and OCN levels were also suppressed, indicating that osteogenic differentiation was inhibited (Fig. 6A–C). Additionally, high levels of miR-211-5p increased cellular active β-catenin and AXIN2. However, in the miR-211-5p mimic + ov-FOXO3 group, overexpression of FOXO3 partially rescued the Wnt/β-catenin pathway activation effect of miR-211-5p (Fig. 6D, E). These results demonstrate that miR-211-5p promotes osteogenic potential by directly targeting FOXO3, thereby activating the Wnt/β-catenin signaling pathway.

Fig. 5 — Regulation of cell activity by miR-211-5p and FOXO3 co-transfection. A Upregulation of miR-211-5p and FOXO3 regulated FOXO3 expression in BMSC and MC3T3-E1 cells. B, C miR-211-5p and FOXO3 mediated cell viability and apoptosis. (n = 3 biological replicates, P < 0.05)

Fig. 6 — Osteoblast differentiation and the Wnt/β-catenin pathway. A, B, C Effect of miR-211-5p and FOXO3 on osteogenic markers, and measured these markers using the ELISA method. D, E Active β-catenin and AXIN2 levels were regulated by the miR-211-5p/FOXO3 axis, which were determined by Western blot. (n = 3 biological replicates, P < 0.05)

Discussion

OPF is an important risk factor leading to disability and death in the elderly, which seriously impairs the quality of life of patients [3, 24]. In view of this, it is particularly urgent and of great scientific value to screen new biomarkers with high specificity to provide solid theoretical support for the clinical diagnosis and treatment of OPF. In addition to the extensive involvement of non-coding RNAs such as small interfering RNAs in tendon homeostasis and inflammation related to rheumatoid arthritis [25, 26], recent studies have also confirmed that a variety of pharmacological, physical and biological methods are involved in the regulation of fracture repair [27–31].

In numerous studies, the role of miRNAs in fracture occurrence and progression has garnered extensive attention [32, 33]. For example, upregulating miR-1224-5p accelerated fracture healing by inducing osteoclast differentiation and activating the ADCY2/Rap1 pathway [34]. Long and colleagues revealed that miR-181a-5p mediated BMP3 to promote osteogenic differentiation, thereby reducing the risk of fractures that may be caused by osteoporosis [35]. Additionally, miR-193a-3p was believed to accelerate fracture healing by regulating PTEN and was considered a potential target for fracture repair [36]. Recent studies have shown that fracture healing is a complex process regulated by multiple molecular and microenvironmental factors. Specifically, several long non-coding RNA/microRNA regulatory axes, such as the CRNDE/miR-29a-3p and HCG18/miR-146a-5p pathways have been shown to be closely associated with delayed fracture healing [37, 38]. Furthermore, miR-204-5p participates in disease regulation by inhibiting apoptosis, while stress-induced hyperglycemia is also an important predictor of delayed healing following tibial fractures[39, 40]. The present study found that miR-211-5p expression was reduced in OP and OPF patients, especially in OPF, and its expression tended to increase with the progression of treatment. miR‑211‑5p has shown a good application potential in the differential diagnosis of healthy population and OP, as well as OP and OPF. These results suggest that miR-211-5p may reflect the improvement of fracture healing and may be used as a potential indicator for therapeutic efficacy monitoring.

The pathophysiological process of fracture healing is complex, and its core lies in the effective proliferation and differentiation of osteoblasts [41]. Given that BMSC is a precursor cell of osteoblasts and plays a crucial regulatory role in bone remodeling, we selected BMSC and MC3T3-E1 as in vitro study subjects for molecular mechanism study. During osteogenic differentiation induction, intracellular miR-211-5p levels were upregulated, accompanied by elevated expression of osteogenic markers, indicating that miR-211-5p exerts a positive regulatory role in osteogenic differentiation. In addition, miR-211-5p mimic increased the miR-211-5p level, cell viability and osteogenic markers in BMSC and MC3T3-E1 cells, while miR-211-5p inhibitor suppressed their expression. Similarly, miR-211-5p was confirmed to be upregulated in differentiated MC3T3-E1 cells, and ALP, OSX, OCN and Runx2 contents were elevated in previous studies [42]. The above findings indicate that miR-211-5p may serve as a regulatory factor mediating osteoblast proliferation, apoptosis, and osteogenic differentiation.

Previous studies have confirmed that miRNAs participate in the process of diseases by targeting downstream factors [43, 44]. Bioinformatics prediction and experimental verification showed that FOXO3 was the downstream action site of miR-211-5p. Their targeting relationship was also identified in past studies of premature ovarian failure [45]. FOXO3 was significantly increased in the serum of OPF patients and was negatively regulated by miR‑211‑5p. This trend was also observed in fatty liver disease, acute adrenal syndrome, and liver cancer [46, 47]. Interestingly, FOXO3 was upregulated in mouse models of muscle atrophy [48]. Recent studies have also revealed that FOXO3 is highly expressed in OP patients, and it has good diagnostic efficacy as a key oxidative stress indicator of OP [49]. Further in vitro assays demonstrated that overexpression of FOXO3 counteracted the effects of miR-211-5p, inhibiting the activity of BMSC and MC3T3-E1 cells, suppressing osteogenic differentiation markers, and accelerating apoptosis. Moreover, Wnt/β-catenin signaling pathway is recognized as the decisive cascade regulating bone formation and osteoblast differentiation [50]. During osteogenesis, dephosphorylation of β-catenin leads to its nuclear translocation and initiation of transcription [51]. As a direct target of this pathway, AXIN2 expression levels often sensitively reflect the signaling output of the Wnt/β-catenin pathway [52]. We noted that the transfection of the miR-211-5p/FOXO3 axis increased the levels of active β-catenin and AXIN2, suggesting that the Wnt/β-catenin pathway was activated. These findings reveal that miR-211-5p activates the Wnt/β-catenin signaling pathway by targeting FOXO3, thereby promoting osteogenic differentiation and bone formation. Regrettably, the research is confined to in vitro cell studies and lacks evidence in vivo animal models, indicating that the therapeutic potential of miR-211-5p warrants further evaluation.

In summary, miR-211-5p was negatively expressed in OPF, which accelerated fracture healing and promotes bone formation via the FOXO3/Wnt/β-catenin pathway. This finding holds significant implications for optimizing diagnostic strategies and refining treatment plans for OPF patients.

Supplementary Information

Below is the link to the electronic supplementary material.

13018_2026_7003_MOESM1_ESM.tif^{(1.7MB, tif)}

Supplementary Material 1: Fig. 1. The pmiGLO plasmid map.

Supplementary Material 2.^{(596.2KB, pdf)}

Supplementary Material 3.^{(17.4KB, docx)}

Supplementary Material 4.^{(24.5KB, docx)}

Acknowledgements

Not applicable.

Author contributions

Conceptualization, H.Z., J.L., M.L., C.L.; Data curation, H.Z., J.L., M.L., C.L.; Formal analysis, H.Z., J.L., M.L., C.L.; Funding acquisition, C.L.; Investigation, M.L.; Methodology, H.Z., J.L., M.L., C.L.; Project administration, C.L.; Resources, M.L.; Software, M.L., C.L.; Supervision, C.L.; Validation, H.Z., J.L., M.L.; Visualization, M.L., M.L.; Roles/Writing—original draft, M.L.; Writing—review & editing, H.Z., J.L., C.L.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability

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

Declarations

Ethics approval and consent to participate

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Shantou Central Hospital. Informed consent was obtained from all individual participants included in the study.

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.

References

1.Han H, Li R, Fu D, et al. Correlation between bone density, bone metabolism markers with lipid metabolism markers and body mass index. BMC Musculoskelet Disord. 2024;25(1):162. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Chen Y, Wu X, Li J, Jiang Y, Xu K, Su J. Bone-targeted nanoparticle drug delivery system: an emerging strategy for bone-related disease. Front Pharmacol. 2022;13:909408. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Xue H, Liu W, Li R, et al. Advantages of robot-assisted PKP under local anesthesia in the treatment of OVCF: a retrospective, non-randomized, controlled, clinical study. Front Surg. 2024;11:1445461. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Kang X, Tian B, Zhao ZD, Zhang BF, Zhang M. Evaluation of the association between low-density lipoprotein (LDL) and all-cause mortality in geriatric patients with hip fractures: a prospective cohort study of 339 patients. J Pers Med. 2023. 10.3390/jpm13020345. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Wiedl A, Förch S, Fenwick A, Mayr E. Fractures’ associated mortality risk in orthogeriatric inpatients: a prospective 2-year survey. Eur Geriatr Med. 2021;12(1):61–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Zheng K, Bai J, Yang H, et al. Nanomaterial-assisted theranosis of bone diseases. Bioact Mater. 2023;24:263–312. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Azimi Manavi B, Mohebbi M, Stuart AL, et al. Antipsychotic medication use in association with quantitative heel ultrasound (QUS). Bone Rep. 2023;18:101694. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Giordano L, Porta GD, Peretti GM, Maffulli N. Therapeutic potential of microRNA in tendon injuries. Br Med Bull. 2020;133(1):79–94. [DOI] [PubMed] [Google Scholar]
9.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. [DOI] [PubMed] [Google Scholar]
10.Yang X, Yin P, Yao X, Zhang J. MicroRNAs in the diagnosis of osteoarthritis: a systematic review and meta-analysis of observational studies. J Orthop Surg Res. 2025;20(1):654. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Seeliger C, Balmayor ER, van Griensven M. miRNAs related to skeletal diseases. Stem Cells Dev. 2016;25(17):1261–81. [DOI] [PubMed] [Google Scholar]
12.Srivastava S, Rathor R, Singh SN, Suryakumar G. Emerging role of MyomiRs as biomarkers and therapeutic targets in skeletal muscle diseases. Am J Physiol Cell Physiol. 2021;321(5):C859–75. [DOI] [PubMed] [Google Scholar]
13.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. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Gargano G, Pagano SM, Maffulli N. Circular RNAs in the management of human osteoporosis. British Med Bullet. 2025;153(1). [DOI] [PubMed]
15.Yang X, Jin Q, Guo L. MiR-217 participates in the progression of postmenopausal osteoporosis by regulating the OPG/RANKL/RANK pathway. J Orthop Surg Res. 2025;20(1):600. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Liu X, Zhang X, Cen M. Dysregulation of miR-106a-5p/PTEN axis associated with progression and diagnostic of postmenopausal osteoporosis. J Orthop Surg Res. 2025;20(1):456. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Mani AM, Lamin V, Peach RC, et al. miRNA-6236 regulation of postischemic skeletal muscle angiogenesis. J Am Heart Assoc. 2024;13(23):e035923. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Yan Y, Zheng M, Wang X, et al. MiR-126-5p derived from bone marrow mesenchymal stem cell exosomes promotes skeletal muscle regeneration by regulating FBXO32/MyoD signaling. Acta Physiol (Oxf). 2025;241(11):e70114. [DOI] [PubMed] [Google Scholar]
19.Wu X, Shen T, Ji W, et al. lncRNA CASC11 regulates the progress of delayed fracture healing via sponging miR-150-3p. J Orthop Surg Res. 2024;19(1):757. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Li Y, Sun Y, Ma K, Wang S, Wang Z, Huang L. Functional mechanism and clinical implications of LINC00339 in delayed fracture healing. J Orthop Surg Res. 2024;19(1):511. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Zhang Y, Wang H, Xia Y. The expression of miR-211-5p in atherosclerosis and its influence on diagnosis and prognosis. BMC Cardiovasc Disord. 2021;21(1):371. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Hong L, Yang C. Eupatilin ameliorates postmenopausal osteoporosis via elevating microRNA-211-5p and repressing JAK2/STAT3 pathway. Environ Toxicol. 2024;39(4):2218–28. [DOI] [PubMed] [Google Scholar]
23.Aili D, Wu T, Gu Y, Chen Z, Wang W. Knockdown of long non-coding RNA KCNQ1OT1 suppresses the progression of osteoarthritis by mediating the miR-211-5p/TCF4 axis in vitro. Exp Ther Med. 2021;21(5):455. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Lin S, Wu J, Chen B, Li S, Huang H. Identification of a potential MiRNA-mRNA regulatory network for osteoporosis by using bioinformatics methods: a retrospective study based on the Gene Expression Omnibus Database. Front Endocrinol. 2022;13:844218. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Gargano G, Oliviero A, Oliva F, Maffulli N. Small interfering RNAs in tendon homeostasis. Br Med Bull. 2021;138(1):58–67. [DOI] [PubMed] [Google Scholar]
26.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. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Migliorini F, Cocconi F, Vecchio G, Schäefer L, Koettnitz J, Maffulli N. Pharmacological agents for bone fracture healing: talking points from recent clinical trials. Expert Opin Investig Drugs. 2023;32(9):855–65. [DOI] [PubMed] [Google Scholar]
28.Khanna A, Gougoulias N, Maffulli N. Intermittent pneumatic compression in fracture and soft-tissue injuries healing. Br Med Bull. 2008;88(1):147–56. [DOI] [PubMed] [Google Scholar]
29.Martinez de Albornoz P, Khanna A, Longo UG, Forriol F, Maffulli N. The evidence of low-intensity pulsed ultrasound for in vitro, animal and human fracture healing. Br Med Bull. 2011;100:39–57. [DOI] [PubMed] [Google Scholar]
30.Kuzuca BC, Doral MN, Mangiavini L, Sapmaz E, Maffulli N, Koken M. Investigation into the effect of epidermal growth factor and hyaluronic acid on fracture healing in a rat femoral fracture model. Acta Orthop Traumatol Turc. 2023;57(5):229–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Liu J, Liu TT, Zhang HC, Li C, Wei W, Chao AJ. Effects of Jintiange on the healing of osteoporotic fractures in aged rats. J Orthop Surg Res. 2024;19(1):828. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Komatsu DE, Duque E, Hadjiargyrou M. MicroRNAs and fracture healing: pre-clinical studies. Bone. 2021;143:115758. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Zhang Z, Wang L, Zhang F, Jing S, Cen M. Functional mechanism and clinical implications of mir-1271-5p in pilon fracture healing processes. J Orthop Surg Res. 2024;19(1):782. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Hu L, Xie X, Xue H, et al. MiR-1224-5p modulates osteogenesis by coordinating osteoblast/osteoclast differentiation via the Rap1 signaling target ADCY2. Exp Mol Med. 2022;54(7):961–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Long Z, Dou P, Cai W, Mao M, Wu R. MiR-181a-5p promotes osteogenesis by targeting BMP3. Aging. 2023;15(3):734–47. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Zheng K, Wang Y. MiR-193a-3p promotes fracture healing via targeting PTEN gene. Mol Biotechnol. 2021;63(7):605–12. [DOI] [PubMed] [Google Scholar]
37.Li Y, Ye S, Han Z, Wei C, Huang Y. LncRNA CRNDE ameliorates bone fracture by regulating cell viability and apoptosis of osteoblasts. J Orthop Surg Res. 2025;20(1):521. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Li F, Tan H, Zhang X, Zhao X, Li X, Chen G. LncRNA HCG18 regulates the progression of spinal tuberculosis by modulating the hsa-miR-146a-5p/TGF-β1/SMADs pathway. J Orthop Surg Res. 2025;20(1):484. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Zhao C, Li Q, Shen C. Therapeutic potential of miR-204-5p in intervertebral disc degeneration: targeting the SSRP1/NF-κB pathway to inhibit apoptosis. J Orthop Surg Res. 2025;20(1):586. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Ji X, Zhao D, Xin Z, Feng H, Huang Z. The predictive value of stress-induced hyperglycemia parameters for delayed healing after tibial fracture post-surgery. J Orthop Surg Res. 2024;19(1):666. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Zhu L, Xie L, Wang Z, Li KL, Cai W. Mass spectrometry-based metabolomics reveal the effects and potential mechanism of isochlorogenic acid A in MC3T3-E1 cells. Front Mol Biosci. 2025;12:1518873. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Ju W, Zhang G, Zhang X, Wang J, Wu T, Li H. Involvement of MiRNA-211-5p and Arhgap11a interaction during osteogenic differentiation of MC3T3-E1 cells. Front Surg. 2022;9:857170. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Meng K, Li Z, Cui X. Three LHPP gene-targeting co-expressed microRNAs (microRNA-765, microRNA-21, and microRNA-144) promote proliferation, epithelial-mesenchymal transition, invasion, and are independent prognostic biomarkers in renal cell carcinomas patients. J Clin Lab Anal. 2021;35(12):e24077. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Zhou W, Chen L, Wu H, et al. Altered microRNAs in C3H10T1/2 cells induced by p.E95K mutant IHH signaling. Hereditas. 2021;158(1):48. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Zhang M, Xing J, Zhao S, et al. Exosomal YB-1 facilitates ovarian restoration by MALAT1/miR-211-5p/FOXO(3) axis. Cell Biol Toxicol. 2024;40(1):29. [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Wan Z, Liu X, Yang X, et al. MicroRNA-411-5p alleviates lipid deposition in metabolic dysfunction-associated steatotic liver disease by targeting the EIF4G2/FOXO3 axis. Cellular Mol Life Sci CMLS. 2024;81(1):398. [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Zhao P, Wang Z, Shi M, et al. Serum miR-27a reduction and FOXO3 elevation in elderly patients with severe pneumonia complicated with ARDS: association with disease severity and prognosis. J Inflamm Res. 2025;18:14465–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Ma H, Jing Y, Zeng J, et al. Human umbilical cord mesenchymal stem cell-derived exosomes ameliorate muscle atrophy via the miR-132-3p/FoxO3 axis. J Orthop Transl. 2024;49:23–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Zheng S, Luo X, Chen G, Wang P. Multi-omics integration and machine learning unveil FOXO3/SIRT1 axis as oxidative stress biomarkers and therapeutic targets in osteoporosis. Medicine Baltimore. 2026;105(5):e47356. [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Liu G, Lin J, Chen X, Liu R. Gingival fibroblast suppress the osteogenesis process mediated by bone substitute materials via WNT/β-catenin signaling pathway in vitro and in vivo. Front Bioeng Biotechnol. 2025;13:1521134. [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Bian Y, Wang G, Zhou J, et al. Astragalus membranaceus (Huangqi) and Rhizoma curcumae (Ezhu) decoction suppresses colorectal cancer via downregulation of Wnt5/β-catenin signal. Chin Med. 2022;17(1):11. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Su G, Wang D, Yang Q, et al. Cepharanthine suppresses APC-mutant colorectal cancers by down-regulating the expression of β-catenin. Nat Prod Bioprospect. 2024;14(1):18. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

13018_2026_7003_MOESM1_ESM.tif^{(1.7MB, tif)}

Supplementary Material 1: Fig. 1. The pmiGLO plasmid map.

Supplementary Material 2.^{(596.2KB, pdf)}

Supplementary Material 3.^{(17.4KB, docx)}

Supplementary Material 4.^{(24.5KB, docx)}

Data Availability Statement

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

[CR1] 1.Han H, Li R, Fu D, et al. Correlation between bone density, bone metabolism markers with lipid metabolism markers and body mass index. BMC Musculoskelet Disord. 2024;25(1):162. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR2] 2.Chen Y, Wu X, Li J, Jiang Y, Xu K, Su J. Bone-targeted nanoparticle drug delivery system: an emerging strategy for bone-related disease. Front Pharmacol. 2022;13:909408. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR3] 3.Xue H, Liu W, Li R, et al. Advantages of robot-assisted PKP under local anesthesia in the treatment of OVCF: a retrospective, non-randomized, controlled, clinical study. Front Surg. 2024;11:1445461. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR4] 4.Kang X, Tian B, Zhao ZD, Zhang BF, Zhang M. Evaluation of the association between low-density lipoprotein (LDL) and all-cause mortality in geriatric patients with hip fractures: a prospective cohort study of 339 patients. J Pers Med. 2023. 10.3390/jpm13020345. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR5] 5.Wiedl A, Förch S, Fenwick A, Mayr E. Fractures’ associated mortality risk in orthogeriatric inpatients: a prospective 2-year survey. Eur Geriatr Med. 2021;12(1):61–8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR6] 6.Zheng K, Bai J, Yang H, et al. Nanomaterial-assisted theranosis of bone diseases. Bioact Mater. 2023;24:263–312. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR7] 7.Azimi Manavi B, Mohebbi M, Stuart AL, et al. Antipsychotic medication use in association with quantitative heel ultrasound (QUS). Bone Rep. 2023;18:101694. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR8] 8.Giordano L, Porta GD, Peretti GM, Maffulli N. Therapeutic potential of microRNA in tendon injuries. Br Med Bull. 2020;133(1):79–94. [DOI] [PubMed] [Google Scholar]

[CR9] 9.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. [DOI] [PubMed] [Google Scholar]

[CR10] 10.Yang X, Yin P, Yao X, Zhang J. MicroRNAs in the diagnosis of osteoarthritis: a systematic review and meta-analysis of observational studies. J Orthop Surg Res. 2025;20(1):654. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR11] 11.Seeliger C, Balmayor ER, van Griensven M. miRNAs related to skeletal diseases. Stem Cells Dev. 2016;25(17):1261–81. [DOI] [PubMed] [Google Scholar]

[CR12] 12.Srivastava S, Rathor R, Singh SN, Suryakumar G. Emerging role of MyomiRs as biomarkers and therapeutic targets in skeletal muscle diseases. Am J Physiol Cell Physiol. 2021;321(5):C859–75. [DOI] [PubMed] [Google Scholar]

[CR13] 13.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. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR14] 14.Gargano G, Pagano SM, Maffulli N. Circular RNAs in the management of human osteoporosis. British Med Bullet. 2025;153(1). [DOI] [PubMed]

[CR15] 15.Yang X, Jin Q, Guo L. MiR-217 participates in the progression of postmenopausal osteoporosis by regulating the OPG/RANKL/RANK pathway. J Orthop Surg Res. 2025;20(1):600. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR16] 16.Liu X, Zhang X, Cen M. Dysregulation of miR-106a-5p/PTEN axis associated with progression and diagnostic of postmenopausal osteoporosis. J Orthop Surg Res. 2025;20(1):456. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR17] 17.Mani AM, Lamin V, Peach RC, et al. miRNA-6236 regulation of postischemic skeletal muscle angiogenesis. J Am Heart Assoc. 2024;13(23):e035923. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR18] 18.Yan Y, Zheng M, Wang X, et al. MiR-126-5p derived from bone marrow mesenchymal stem cell exosomes promotes skeletal muscle regeneration by regulating FBXO32/MyoD signaling. Acta Physiol (Oxf). 2025;241(11):e70114. [DOI] [PubMed] [Google Scholar]

[CR19] 19.Wu X, Shen T, Ji W, et al. lncRNA CASC11 regulates the progress of delayed fracture healing via sponging miR-150-3p. J Orthop Surg Res. 2024;19(1):757. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR20] 20.Li Y, Sun Y, Ma K, Wang S, Wang Z, Huang L. Functional mechanism and clinical implications of LINC00339 in delayed fracture healing. J Orthop Surg Res. 2024;19(1):511. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR21] 21.Zhang Y, Wang H, Xia Y. The expression of miR-211-5p in atherosclerosis and its influence on diagnosis and prognosis. BMC Cardiovasc Disord. 2021;21(1):371. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR22] 22.Hong L, Yang C. Eupatilin ameliorates postmenopausal osteoporosis via elevating microRNA-211-5p and repressing JAK2/STAT3 pathway. Environ Toxicol. 2024;39(4):2218–28. [DOI] [PubMed] [Google Scholar]

[CR23] 23.Aili D, Wu T, Gu Y, Chen Z, Wang W. Knockdown of long non-coding RNA KCNQ1OT1 suppresses the progression of osteoarthritis by mediating the miR-211-5p/TCF4 axis in vitro. Exp Ther Med. 2021;21(5):455. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR24] 24.Lin S, Wu J, Chen B, Li S, Huang H. Identification of a potential MiRNA-mRNA regulatory network for osteoporosis by using bioinformatics methods: a retrospective study based on the Gene Expression Omnibus Database. Front Endocrinol. 2022;13:844218. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR25] 25.Gargano G, Oliviero A, Oliva F, Maffulli N. Small interfering RNAs in tendon homeostasis. Br Med Bull. 2021;138(1):58–67. [DOI] [PubMed] [Google Scholar]

[CR26] 26.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. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR27] 27.Migliorini F, Cocconi F, Vecchio G, Schäefer L, Koettnitz J, Maffulli N. Pharmacological agents for bone fracture healing: talking points from recent clinical trials. Expert Opin Investig Drugs. 2023;32(9):855–65. [DOI] [PubMed] [Google Scholar]

[CR28] 28.Khanna A, Gougoulias N, Maffulli N. Intermittent pneumatic compression in fracture and soft-tissue injuries healing. Br Med Bull. 2008;88(1):147–56. [DOI] [PubMed] [Google Scholar]

[CR29] 29.Martinez de Albornoz P, Khanna A, Longo UG, Forriol F, Maffulli N. The evidence of low-intensity pulsed ultrasound for in vitro, animal and human fracture healing. Br Med Bull. 2011;100:39–57. [DOI] [PubMed] [Google Scholar]

[CR30] 30.Kuzuca BC, Doral MN, Mangiavini L, Sapmaz E, Maffulli N, Koken M. Investigation into the effect of epidermal growth factor and hyaluronic acid on fracture healing in a rat femoral fracture model. Acta Orthop Traumatol Turc. 2023;57(5):229–36. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR31] 31.Liu J, Liu TT, Zhang HC, Li C, Wei W, Chao AJ. Effects of Jintiange on the healing of osteoporotic fractures in aged rats. J Orthop Surg Res. 2024;19(1):828. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR32] 32.Komatsu DE, Duque E, Hadjiargyrou M. MicroRNAs and fracture healing: pre-clinical studies. Bone. 2021;143:115758. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR33] 33.Zhang Z, Wang L, Zhang F, Jing S, Cen M. Functional mechanism and clinical implications of mir-1271-5p in pilon fracture healing processes. J Orthop Surg Res. 2024;19(1):782. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR34] 34.Hu L, Xie X, Xue H, et al. MiR-1224-5p modulates osteogenesis by coordinating osteoblast/osteoclast differentiation via the Rap1 signaling target ADCY2. Exp Mol Med. 2022;54(7):961–72. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR35] 35.Long Z, Dou P, Cai W, Mao M, Wu R. MiR-181a-5p promotes osteogenesis by targeting BMP3. Aging. 2023;15(3):734–47. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR36] 36.Zheng K, Wang Y. MiR-193a-3p promotes fracture healing via targeting PTEN gene. Mol Biotechnol. 2021;63(7):605–12. [DOI] [PubMed] [Google Scholar]

[CR37] 37.Li Y, Ye S, Han Z, Wei C, Huang Y. LncRNA CRNDE ameliorates bone fracture by regulating cell viability and apoptosis of osteoblasts. J Orthop Surg Res. 2025;20(1):521. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR38] 38.Li F, Tan H, Zhang X, Zhao X, Li X, Chen G. LncRNA HCG18 regulates the progression of spinal tuberculosis by modulating the hsa-miR-146a-5p/TGF-β1/SMADs pathway. J Orthop Surg Res. 2025;20(1):484. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR39] 39.Zhao C, Li Q, Shen C. Therapeutic potential of miR-204-5p in intervertebral disc degeneration: targeting the SSRP1/NF-κB pathway to inhibit apoptosis. J Orthop Surg Res. 2025;20(1):586. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR40] 40.Ji X, Zhao D, Xin Z, Feng H, Huang Z. The predictive value of stress-induced hyperglycemia parameters for delayed healing after tibial fracture post-surgery. J Orthop Surg Res. 2024;19(1):666. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR41] 41.Zhu L, Xie L, Wang Z, Li KL, Cai W. Mass spectrometry-based metabolomics reveal the effects and potential mechanism of isochlorogenic acid A in MC3T3-E1 cells. Front Mol Biosci. 2025;12:1518873. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR42] 42.Ju W, Zhang G, Zhang X, Wang J, Wu T, Li H. Involvement of MiRNA-211-5p and Arhgap11a interaction during osteogenic differentiation of MC3T3-E1 cells. Front Surg. 2022;9:857170. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR43] 43.Meng K, Li Z, Cui X. Three LHPP gene-targeting co-expressed microRNAs (microRNA-765, microRNA-21, and microRNA-144) promote proliferation, epithelial-mesenchymal transition, invasion, and are independent prognostic biomarkers in renal cell carcinomas patients. J Clin Lab Anal. 2021;35(12):e24077. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR44] 44.Zhou W, Chen L, Wu H, et al. Altered microRNAs in C3H10T1/2 cells induced by p.E95K mutant IHH signaling. Hereditas. 2021;158(1):48. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR45] 45.Zhang M, Xing J, Zhao S, et al. Exosomal YB-1 facilitates ovarian restoration by MALAT1/miR-211-5p/FOXO(3) axis. Cell Biol Toxicol. 2024;40(1):29. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR46] 46.Wan Z, Liu X, Yang X, et al. MicroRNA-411-5p alleviates lipid deposition in metabolic dysfunction-associated steatotic liver disease by targeting the EIF4G2/FOXO3 axis. Cellular Mol Life Sci CMLS. 2024;81(1):398. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR47] 47.Zhao P, Wang Z, Shi M, et al. Serum miR-27a reduction and FOXO3 elevation in elderly patients with severe pneumonia complicated with ARDS: association with disease severity and prognosis. J Inflamm Res. 2025;18:14465–74. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR48] 48.Ma H, Jing Y, Zeng J, et al. Human umbilical cord mesenchymal stem cell-derived exosomes ameliorate muscle atrophy via the miR-132-3p/FoxO3 axis. J Orthop Transl. 2024;49:23–36. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR49] 49.Zheng S, Luo X, Chen G, Wang P. Multi-omics integration and machine learning unveil FOXO3/SIRT1 axis as oxidative stress biomarkers and therapeutic targets in osteoporosis. Medicine Baltimore. 2026;105(5):e47356. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR50] 50.Liu G, Lin J, Chen X, Liu R. Gingival fibroblast suppress the osteogenesis process mediated by bone substitute materials via WNT/β-catenin signaling pathway in vitro and in vivo. Front Bioeng Biotechnol. 2025;13:1521134. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR51] 51.Bian Y, Wang G, Zhou J, et al. Astragalus membranaceus (Huangqi) and Rhizoma curcumae (Ezhu) decoction suppresses colorectal cancer via downregulation of Wnt5/β-catenin signal. Chin Med. 2022;17(1):11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR52] 52.Su G, Wang D, Yang Q, et al. Cepharanthine suppresses APC-mutant colorectal cancers by down-regulating the expression of β-catenin. Nat Prod Bioprospect. 2024;14(1):18. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

miR-211-5p/FOXO3 axis accelerates osteogenic differentiation and fracture healing by mediating Wnt/β-catenin pathway

Huifei Zhai

Mianlong Lin

Changchun Lu

Jixia Liang

Abstract

Background

Objective

Methods

Results

Conclusion

Supplementary Information

Introduction

Materials and methods

Inclusion of patients

Acquisition of serum samples

Cell culture and osteogenic differentiation induction

Cell transfection

RT-qPCR

Cell proliferation

Cell apoptosis

Detection of osteogenic factors

Dual-luciferase reporter assay

RIP assay

Western blot

Statistical analysis

Results

Analysis of subjects’ clinical indicators

Table 1.

Expression and clinical significance of miR-211-5p in OPF

Fig. 1.

Osteogenic differentiation upregulated the miR-211-5p level

Fig. 2.

Abnormal expression of miR-211-5p regulated cell function

Fig. 3.

FOXO3 was negatively regulated by miR-211-5p

Fig. 4.

Effect of miR‑211‑5p/FOXO3 axis on cell viability and Wnt/β-catenin pathway

Fig. 5.

Fig. 6.

Discussion

Supplementary Information

Acknowledgements

Author contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases