MiR-328-5p/BCL7A axis is involved in fracture healing by modulating osteoblast function.

Introduction

Fractures are very common musculoskeletal injuries, affecting individuals across all age groups. These structural discontinuities in bone tissue typically result from traumatic incidents, though pathological fractures may occur secondary to underlying medical conditions [ 1 ]. Osteoporosis is a major risk factor affecting fracture healing, with susceptibility influenced by genetic factors such as polymorphisms in the glucagon-like peptide-1 receptor gene [ 2 ]. Additionally, several simple clinical predictors (body weight, body mass index, and age) have been identified to aid in guiding decisions during osteoporosis screening in women, thereby improving risk stratification [ 3 ]. The natural course of bone healing evolves through inflammatory, reparative, and remodeling phases in a coordinated manner. This cascade can be impaired by various disturbances such as ischemic conditions, infection, significant soft tissue damage, or suboptimal fixation integrity [ 4 – 6 ]. Modern fracture management relies heavily on internal fixation techniques, yet delayed healing complications persist [ 7 ]. This condition, diagnostically confirmed when radiographic evidence shows inadequate callus formation and non-union at 6 months post-injury, significantly impacts patient mobility and daily activities [ 8 , 9 ]. An early radiological assessment is used to identify fractures with a poor prognosis. At 4 months post-injury, radiographic features such as absent or minimal callus with a visible fracture line are used to screen for high-risk fractures that may progress to delayed or nonunion. Furthermore, the 4-month assessment serves as a critical predictive milestone, enabling the evaluation of relevant biomarkers for forecasting delayed healing [ 10 , 11 ]. Consequently, the discovery of novel biomolecules holds significant potential for enhancing fracture repair and improving patient care strategies.

Recent studies have highlighted the emerging roles of non-coding RNAs in musculoskeletal disorders, with research spanning tendon injury and homeostasis (e.g., miRNA and siRNA) [ 12 , 13 ], to osteoarthritis [ 14 ], rheumatoid arthritis [ 15 ], and osteoporosis [ 16 , 17 ] (involving miRNAs, siRNAs, and circRNAs). Research indicates that lncRNAs such as CRNDE and HCG18 regulate fracture healing and spinal tuberculosis progression by influencing cellular viability and modulating the TGF-β1/SMADs pathway, respectively [ 18 , 19 ]. Emerging evidence has revealed the crucial involvement of microRNAs (miRNAs) in modulating the development and progression of various skeletal disorders [ 20 ]. For instance, research using osteoporotic mouse models has identified miR-24-2-5p as a significant modulator of osteogenic differentiation and bone formation processes [ 21 ]. In the context of osteoarthritis pathogenesis, elevated expression of miR-653-5p has been shown to suppress cellular senescence in chondrocytes while mitigating degeneration of articular cartilage [ 22 ]. Microarray profiling revealed distinct miR-328-5p expression patterns between infected tibial nonunion cases and closed tibial fractures, supporting its potential as a diagnostic biomarker for infectious nonunion [ 23 ]. Previous investigations utilizing circRNA-miRNA network modeling identified miR-328-5p as a potential regulator in the osteogenic differentiation pathway of rat bone marrow-derived mesenchymal stem cells [ 24 ]. Research has identified increased miR-328-5p abundance in degenerative disc tissues, with functional studies indicating its contribution to disease progression [ 25 ]. The potential involvement of miR-328-5p in bone fracture repair and its regulatory mechanisms during osteoblast differentiation remain unclear. Therefore, this study specifically focuses on the unexplored role of miR-328-5p in osteogenic differentiation and fracture healing, with the aim of evaluating its potential as a therapeutic target and biomarker for delayed fracture healing.

The present study evaluated the clinical significance of serum miR-328-5p in patients with differing fracture healing progression. Moreover, in vitro studies were conducted to investigate the molecular mechanisms underlying miR-328-5p-mediated osteoblast differentiation, with the goal of offering novel ideas to accelerate fracture healing.

Materials and methods

Study population

We conducted a retrospective analysis of 125 tibial fracture cases treated with internal fixation at Jiangsu Province Hospital of Chinese Medicine , The Affiliated Hospital of Nanjing University of Chinese Medicine between 2023 and 2024. The cohort comprised 66 patients (52.8%) with proximal tibial fractures, 32 (25.6%) with tibial shaft fractures, and 27 (21.6%) with distal tibial fractures. The study was approved by the institutional review board, and informed consent was obtained from all participants.

Inclusion criteria: (1) radiologically confirmed tibial fracture (X-ray/CT), (2) adult patients (≥ 18 years); and (3) availability of detailed clinical data. Exclusion criteria: (1) significant organ dysfunction, (2) malignancy, (3) prior orthopedic surgical interventions, (4) metabolic bone disorders, (5) pregnancy or lactation status, and (6) infections or autoimmune conditions.

Fracture healing was assessed using anteroposterior and lateral radiographs of the affected site. Radiographic diagnostic criteria were established by integrating recognized diagnostic standards [ 26 ] with clinical practice. To ensure consistent and objective application of the radiographic criteria, all X-ray images were independently assessed by two experienced radiologists who were blinded to the group assignments. Key evaluation indicators included callus formation, fracture line clarity, and evidence of bone resorption. Fracture healing outcomes were categorized as follows: Normal healing group ( n = 69): evidence of bridging callus formation within the 4-month postoperative period. Delayed healing group ( n = 56): absent or minimal callus formation at 4-month follow-up, with persistent fracture lines and sclerotic bone edges visible on radiographic examination. The normal healing group consisted of 36 proximal (52.2%), 19 diaphyseal (27.5%), and 14 distal (20.3%) tibial fractures. The delayed healing group contained 30 proximal (53.6%), 13 diaphyseal (23.2%), and 13 distal (23.2%) fractures.

Peripheral venous blood (5 mL) was obtained from each participant before the surgical procedure. Following 30 min of coagulation at room temperature, samples were centrifuged at 3000 × g for 5 min to separate serum, which was subsequently cryopreserved at -80 °C for long-term storage.

Osteogenic differentiation induction

The hFOB 1.19 human osteoblast cell line (Procell, China) was maintained in DMEM/F12 supplemented with 10% FBS at 37 °C. To induce osteogenic differentiation, cells were treated with the following components (100 nM dexamethasone, 10 mM β-glycerophosphate, and 50 µg/mL ascorbic acid) for 14 days, with medium replacement every 2–3 days.

Cell transfection

Gain- and loss-of-function studies were performed using miR-328-5p mimics, inhibitors, and corresponding negative controls (NC, RiboBio, China), as well as siRNA targeting BCL7A (si-BCL7A) and si-NC (Genepharma, China). The hFOB 1.19 cells differentiated under osteogenic conditions were seeded in 24-well plates. At 70–80% confluence, transfections were carried out using Lipofectamine RNAiMAX (Thermo, USA) according to the manufacturer’s protocol.

RT-qPCR assay

RNA isolation was performed using Trizol reagent (Simgen, China) following the manufacturer’s instructions. Reverse transcription was carried out with the miRNA cDNA Synthesis Kit (Cwbio, China) and TIANScript II RT Kit (TIANGEN, China). Quantitative PCR analysis was conducted on the StepOnePlus Real-Time PCR System (Thermo, USA) using TB Green Premix Ex Taq (TaKaRa, Japan). U6 and GAPDH served as endogenous controls for miRNA and mRNA normalization, respectively. The thermal cycling conditions were: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Primer sequences are provided in Supplementary Materials (Table S1 ). Relative expression levels were calculated using the 2 −ΔΔCt method.

Western blot

Cells were lysed in RIPA buffer (Thermo, USA) for protein extraction. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk for 2 h at room temperature, membranes were incubated overnight with primary antibodies (Sigma-Aldrich, Germany) against runt-related transcription factor 2 (RUNX2, cat# SAB2106220-100UL, 1:1000), osteocalcin (OCN, cat# SAB5702281-100UL, 1:1000), osteopontin (OPN, cat# SAB1306579-400UL, 1:1000), BAF chromatin remodeling complex subunit BCL7A (BCL7A, cat# HPA019762-100UL, 1:1000), and GAPDH (cat# G9545-100UL, 1:5000). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, cat# ab6759, Abcam, UK) for 1 h. Protein bands were visualized using BeyoECL Plus (Beyotime, China) and quantified using iBright software (Thermo, USA). Target proteins were normalized to GAPDH and expressed as fold change relative to the control group.

Alkaline phosphatase (ALP) activity detection

Cells were washed with cold PBS and resuspended in assay buffer from the Alkaline Phosphatase Assay Kit (Abcam, UK). After homogenization and low-temperature centrifugation, the supernatant was collected. Alkaline phosphatase (ALP) activity was measured according to the manufacturer’s instructions by reading absorbance at 405 nm using the Synergy LX microplate reader (Agilent, USA).

Bioinformatics methods

Potential target genes for miR-328-5p were identified using the databases miRDB ( https://mirdb.org/mirdb/index.html ), miRWalk ( http://mirwalk.umm.uni-heidelberg.de/ ), and mirDIP ( https://ophid.utoronto.ca/mirDIP/ ). The Wei Sheng Xin website ( https://www.bioinformatics.com.cn/ ) was used to generate the Venn diagram. The binding sequence between miR-328-5p and BCL7A was predicted utilizing the miRDB database.

Dual-luciferase reporter gene assay

Wild-type (BCL7A-WT) and mutant (BCL7A-MT) luciferase reporter plasmids were synthesized by RiboBio (China). hFOB 1.19 cells were seeded in 24-well plates and co-transfected with these vectors alongside miR-328-5p mimics or inhibitors. After 48 h, luciferase activity was measured using the Luciferase Assay System (Promega, USA) and a microplate reader.

CCK-8 assay

Cells from each group were inoculated into 96-well plates. CCK-8 reagent (10 µL/well, Yeasen, China) was added at 0, 24, 48, and 72 h. After 1.5 h of incubation, optical density (OD) values were measured using a microplate reader.

Apoptosis assay

Transfected hFOB 1.19 cells were digested, and washed with pre-cooled PBS. Cells were resuspended in binding buffer from the Annexin V-FITC Apoptosis Detection Kit (Beyotime, China), stained with Annexin V-FITC/PI, and incubated at 37 °C for 15 min in the dark. Apoptosis rates were analyzed using CytoFLEX SRT flow cytometry (Beckman Coulter, USA).

Caspase-3 activity was measured using the Caspase 3 Activity Assay Kit (Beyotime, China). Absorbance was read at 405 nm using a microplate reader.

Statistical analysis

Data analysis was performed using GraphPad Prism 8.0 (USA) and IBM SPSS 22.0 (USA). Results are presented as mean ± standard deviation (SD). Group comparisons were conducted using Student’s t-test or one-way analysis of variance (ANOVA), with a significance threshold of p < 0.05. If ANOVA was significant, post hoc comparisons were carried out using Tukey’s test. Each experiment included at least three biological replicates. Clinical parameters were analyzed utilizing Chi-square tests and multivariate logistic regression. Diagnostic performance was evaluated by receiver operating characteristic (ROC) curve analysis, and correlations were examined using Pearson’s test.

Results

Clinical correlation between miR-328-5p and delayed healing

The investigation encompassed 125 patients with tibial fractures who were demographically matched and categorized based on healing status into a normal healing group (69 cases) and a delayed healing group (56 cases). Comparison of clinical characteristics revealed a higher proportion of diabetic patients in the delayed healing group, with no other notable differences observed between the groups (Table 1 ). Quantitative analysis disclosed significantly lower expression of miR-328-5p in the delayed healing cohort (Fig. 1 A).

Table 1 Clinical information of the study subjects Characteristics Normal healing ( n = 69) Delayed healing ( n = 56) p value Age, years 44.20 ± 12.25 46.30 ± 12.22 0.342 Sex, n (%) 0.847 Male 37 (53.62) 31 (55.36) Female 32 (46.38) 25 (44.64) Smoking, n (%) 0.300 No 48 (69.57) 34 (60.71) Yes 21 (30.43) 22 (39.29) Drinking, n (%) 0.511 No 47 (68.12) 35 (62.50) Yes 22 (31.88) 21 (37.50) Diabetes, n (%) 0.001 No 60 (86.96) 34 (60.71) Yes 9 (13.04) 22 (39.29) Fracture side, n (%) 0.809 Left 36 (52.17) 28 (50.00) Right 33 (47.83) 28 (50.00) Fracture classification, n (%) 0.836 Proximal 36 (52.20) 30 (53.60) Diaphyseal 19 (27.50) 13 (23.20) Distal 14 (20.30) 13 (23.20) Osteosynthesis method, n (%) 0.186 Open reduction 39 (56.52) 25 (44.64) Closed reduction 30 (43.48) 31 (55.36) Internal fixation method, n (%) 0.360 Plate 35 (50.72) 33 (58.93) Intramedullary nail 34 (49.28) 23 (41.07) Data are presented as n (%) for categorical variables and mean ± standard deviation for continuous variables. p -Values were calculated using Chi-square test for categorical variables and independent samples t-test for age, representing comparisons between the Normal healing and Delayed healing groups

Fig. 1 Correlation of serum miR-328-5p expression with fracture healing. A Serum levels of miR-328-5p in patients with normal fracture healing ( n = 69) and delayed healing ( n = 56), as determined by RT-qPCR. Data are presented as scatter plots with mean ± SD. B ROC curve analysis evaluating the diagnostic potential of serum miR-328-5p levels to distinguish delayed healing patients from normal healing controls

ROC curve analysis identified serum miR-328-5p as a promising biomarker for delayed fracture healing, with an area under the curve (AUC) of 0.873 [95% confidence interval (CI): 0.810–0.936]. At the optimal cut-off value of 0.7275, the sensitivity and specificity were 0.696 and 0.942, respectively (Fig. 1 B). Logistic regression analysis was performed to identify factors associated with fracture healing. The model was adjusted for age, gender, smoking status, drinking status, fracture side, osteosynthesis method, and internal fixation method. Multivariate analysis demonstrated a close association between miR-328-5p expression and delayed healing, with an odds ratio (OR) of 0.078 (95% CI 0.031–0.197; p < 0.001). Diabetes was also identified as a risk factor for delayed healing (OR = 3.427, 95% CI 1.157–10.148; p = 0.026). The model explained 46.0% of the variance in the outcome (Nagelkerke’s R 2 = 0.460, Table 2 ).

Table 2 Logistic regression analysis of delayed fracture healing-related clinical indicators Variables Odds ratio (95% confidence interval) p value Age 1.936 (0.739–5.070) 0.179 Sex 1.333 (0.499–3.558) 0.566 Smoking 1.866 (0.695–5.013) 0.216 Drinking 1.689 (0.641–4.456) 0.289 Diabetes 3.427 (1.157–10.148) 0.026 Fracture side 1.149 (0.456–2.895) 0.768 Fracture classification 1.408 (0.464–4.270) 0.546 Osteosynthesis method 1.378 (0.532–3.573) 0.509 Internal fixation method 0.748 (0.295–1.899) 0.541 MiR-328-5p 0.078 (0.031–0.197) < 0.001 Nagelkerke’s R 2 = 0.460

Expression of miR-328-5p and key markers during osteogenic differentiation

During osteogenic differentiation of hFOB 1.19 cells, miR-328-5p expression increased in a time-dependent manner (Fig. 2 A). Concurrently, both mRNA (Fig. 2 B) and protein (Fig. 2 C) levels of RUNX2, a key transcription factor regulating osteoblast differentiation, were significantly elevated. ALP activity, a critical marker of osteogenesis, was also markedly enhanced (Fig. 2 D). Furthermore, the expression of bone-related genes OCN and OPN was significantly upregulated at both the mRNA (Fig. 2 E) and protein (Fig. 2 F) levels.

Fig. 2 Expression changes of miR-328-5p and osteogenic markers during in vitro osteogenic differentiation. Human osteoblasts were subjected to osteogenic induction for the indicated time periods (0, 7, 14 days). A Expression levels of miR-328-5p measured by RT-qPCR. B and C mRNA ( B ) and protein ( C ) expression of the osteogenic transcription factor RUNX2, analyzed by RT-qPCR and western blot, respectively. D Alkaline phosphatase (ALP) activity measured using a colorimetric assay kit. E and F mRNA ( E ) and protein ( F ) levels of osteocalcin (OCN) and osteopontin (OPN). GAPDH was used as a loading control. Data are representative of five biological replicates (mean ± SD)

MiR-328-5p targeted BCL7A in osteoblasts

Bioinformatic analysis identified three candidate targets of miR-328-5p. Subsequent experimental validation showed that only BCL7A was transcriptionally responsive to miR-328-5p overexpression (Fig. 3 A). The predicted binding sites of miR-328-5p within the 3’-untranslated region (UTR) of BCL7A is illustrated in Fig. 3 B. Dual-luciferase reporter assays confirmed this interaction. MiR-328-5p overexpression suppressed, while its inhibition enhanced, the luciferase activity of BCL7A-WT. No significant effects were observed on BCL7A-MT (Fig. 3 C).

Fig. 3 Targeting relationship of miR-328-5p with BCL7. A Relative mRNA levels of several bioinformatically predicted target genes in cells transfected with a miR-328-5p mimic or negative control (mimic NC), as measured by RT-qPCR. B Schematic diagram of the predicted binding site between miR-328-5p and the 3’ untranslated region (3’UTR) of BCL7A mRNA. C Luciferase reporter assay validating the direct interaction. Firefly luciferase activity was normalized to Renilla luciferase activity (mean ± SD, n = 3). D Serum BCL7A mRNA levels in patients with normal and delayed healing, quantified by RT-qPCR. E Pearson correlation analysis between serum miR-328-5p and BCL7A levels in the delayed healing group

Comparative analysis showed that BCL7A expression was significantly higher in the delayed healing group than in the normal healing group (Fig. 3 D). Moreover, a negative association was observed between miR-328-5p and BCL7A expression in the delayed healing cohort (Pearson’s r = -0.7865, p < 0.001; Fig. 3 E).

MiR-328-5p affected osteogenic differentiation through BCL7A

Transfection with miR-328-5p inhibitors significantly reduced miR-328-5p expression in osteogenic differentiated cells (Fig. 4 A). Suppression of miR-328-5p led to a marked elevation in BCL7A expression at both the mRNA (Fig. 4 B) and protein (Fig. 4 C) levels, which was effectively reversed by co-transfection with BCL7A siRNA (Fig. 4 B and C).

Fig. 4 Effect of miR-328-5p on BCL7A expression. Osteoblasts were transfected with the following constructs: inhibitor NC, miR-328-5p inhibitor, miR-328-5p inhibitor + si NC, or miR-328-5p inhibitor + si-BCL7A. A Transfection efficiency was confirmed by measuring miR-328-5p levels via RT-qPCR. B and C The mRNA ( B ) and protein ( C ) expression of BCL7A was analyzed by RT-qPCR and western blot, respectively. GAPDH served as the loading control. Data are from five biological replicates (mean ± SD)

Further experiments demonstrated that miR-328-5p downregulation considerably reduced RUNX2 expression at the mRNA (Fig. 5 A) and protein (Fig. 5 B) levels, and suppressed ALP activity (Fig. 5 C). In addition, inhibition of miR-328-5p decreased the mRNA (Fig. 5 D) and protein (Fig. 5 E) expression of OCN and OPN.

Fig. 5 Effect of miR-328-5p on osteogenic differentiation mediated by BCL7A. Osteoblasts were transfected with the following constructs: inhibitor NC, miR-328-5p inhibitor, miR-328-5p inhibitor + si NC, or miR-328-5p inhibitor + si-BCL7A. A and B The mRNA ( A ) and protein ( B ) expression of RUNX2, analyzed by RT-qPCR and western blot, respectively. C ALP activity measured using a colorimetric assay kit. D and E The mRNA ( D ) and protein ( E ) levels of OCN and OPN. GAPDH was used as a loading control. All data are from five biological replicates (mean ± SD)

Functional analysis revealed that suppression of miR-328-5p significantly inhibited cell proliferation (Fig. 6 A), promoted apoptosis (Fig. 6 B), and increased caspase-3 activity (Fig. 6 C). Importantly, these effects were largely rescued by BCL7A knockdown, which restored proliferation and reduced apoptosis (Fig. 6 A–C).

Fig. 6 Effect of miR-328-5p on cell proliferation and apoptosis mediated by BCL7A. Osteoblasts were transfected with the following constructs: inhibitor NC, miR-328-5p inhibitor, miR-328-5p inhibitor + si NC, or miR-328-5p inhibitor + si-BCL7A. A Cell viability was assessed using the CCK-8 assay at 24, 48, and 72 h post-transfection. B Cell apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI double staining. C Caspase-3 activity was measured using a commercial activity assay kit. Data are presented as mean ± SD of three or five replicates

Discussion

Recent clinical and preclinical research has explored various strategies to accelerate healing, including pharmacological agents [ 27 ], intermittent pneumatic compression [ 28 ], and low-intensity pulsed ultrasound [ 29 ]. For osteoporotic vertebral compression fractures, surgical interventions such as vertebroplasty and kyphoplasty are commonly employed to stabilize the spine and alleviate pain [ 30 , 31 ]. Despite considerable progress in fracture management, approximately10-20% of cases still experience impaired healing [ 8 ]. The tibia, particularly its middle and distal segments, has a relatively limited blood supply. Fractures in this region can further compromise vascular integrity, leading to inadequate perfusion at the fracture site [ 32 ]. Such impaired blood flow adversely affects bone regeneration and increases the risk of delayed healing in tibial fractures [ 33 ]. Natural compounds such as asperuloside have been demonstrated to alleviate osteoporosis by promoting osteocyte autophagy and modulating the Nrf2 pathway, offering a potential therapeutic approach for enhancing bone regeneration and fracture healing [ 34 ]. In order to improve the current landscape of fracture treatment, extensive research has been conducted to elucidate the molecular mechanisms underlying fracture healing. The diagnostic and therapeutic potential of miRNAs in skeletal diseases is increasingly being unraveled. Systematic reviews confirm the diagnostic relevance of miRNAs in osteoarthritis [ 35 ]. Recent studies have highlighted the regulatory roles of miRNAs across various stages of bone repair [ 36 , 37 ]. For instance, Cheng et al. demonstrated that miR-21 facilitates tibial fracture healing in rats by modulating the TGF-β1/Smad signaling pathway [ 38 ]. A previous study also reported abnormal expression of miR-328-5p in patients with infected tibial nonunion [ 23 ]. Therefore, this study aimed to investigate the clinical relevance of miR-328-5p in delayed healing of tibial fractures and to explore its potential mechanisms.

MiR-328-5p ha been implicated in a variety of diseases, including acute kidney injury, intervertebral disc degeneration, and diabetic nephropathy [ 25 , 39 , 40 ]. It has been shown to suppress adipogenesis in human bone marrow mesenchymal stem cells (BMSCs) by targeting FASN [ 41 ]. Additionally, miR-328-5p exhibits anti-inflammatory effects on endothelial cells and may suppress atherosclerosis progression [ 42 ]. This study enrolled 125 patients with tibial fractures, among whom 56 cases (44.8%) exhibited delayed healing. Reduced miR-328-5p expression was observed in these patients, indicating its strong diagnostic potential for delayed fracture healing. Further analysis confirmed a significant association between miR-328-5p downregulation and delayed healing, suggesting its involvement in bone repair mechanisms. Diabetes was also identified as a significant contributing factor to delayed fracture healing. The delayed healing rate observed in this study (44.8%) exceeded the commonly reported range of 10–20% in general fracture populations [ 43 ]. This discrepancy may be attributed to specific features of the study design and the patient cohort. Unlike broad epidemiological studies, our investigation focused specifically on tibial fractures, which are known to carry a higher risk of healing complications due to their tenuous blood supply, particularly in the middle and distal segments [ 44 ]. Furthermore, the cohort included a substantial number of patients with high-risk factors for impaired healing, such as high-energy trauma mechanisms, smoking, and diabetes. The consistent and rigorous application of radiographic and clinical diagnostic criteria for delayed healing may have also contributed to the relatively high observed rate. It is worth noting that previous studies focusing on similar high-risk populations have also reported delayed healing rates exceeding 30–40% [ 10 , 45 ], which aligns with the findings of the present study.

Osteoblasts orchestrate fracture repair through bone matrix synthesis, secretion, and mineralization, which are fundamental processes in bone formation and remodeling [ 46 ]. The osteogenic differentiation cascade is tightly regulated by key anabolic pathways, with RUNX2 serving as a master transcriptional regulator of bone formation processes [ 47 ]. RUNX2 activates the expression of osteogenesis-related genes (ALP, OPN, OCN), which mediate critical phases of skeletal development and mineralization [ 48 ]. Elevated ALP activity is strongly correlated with osteogenic differentiation status, making it a reliable functional marker for osteoblast development [ 49 ]. OCN represents an osteoblast-specific secreted protein that serves as a marker of terminal osteogenic differentiation [ 50 ]. As a secreted phosphoprotein, OPN contributes to skeletal homeostasis by coordinating extracellular matrix deposition and influencing osteoblast activity via integrin-mediated signaling [ 51 ]. Previous studies have revealed that osteogenic differentiation is not only modulated by specific pathways such as SPI1/SMAD5, but also correlates with osteoblast autophagy [ 34 , 52 , 53 ]. In this study, an osteogenic differentiation model was constructed to explore the role of miR-328-5p in osteoblasts. The expression of miR-328-5p was markedly augmented during osteogenic differentiation. Subsequent analyses confirmed that miR-328-5p silencing suppressed the expression of critical bone formation markers described above. MiR-21 is widely regarded as a potent promoter of osteogenesis, enhancing the osteogenic differentiation of BMSCs by upregulating the expression of ALP and Runx2 genes [ 54 ]. Additionally, the miR-29 family has also been shown to modulate osteoblast differentiation, primarily through mechanisms involving Wnt/β-Catenin signaling and mitophagy [ 55 , 56 ]. Our study is the first to establish a functional link between miR-328-5p and osteoblast differentiation in the specific context of tibial fracture healing, highlighting its unique and previously unexplored role in this pathological process. Furthermore, the current study revealed that miR-328-5p suppression impaired osteoblast viability and enhanced apoptosis. These results suggest that miR-328-5p-mediated regulation of osteoblast function may represent a novel pathway influencing fracture repair processes.

Numerous studies have established that miRNAs modulate the pathological processes of skeletal diseases, mainly through post-transcriptional gene expression modulation [ 57 , 58 ]. Mechanistic studies reveal their role in postmenopausal osteoporosis via the miR-217/OPG/RANKL/RANK and miR-106a-5p/PTEN axes [ 59 , 60 ]. MiR-204-5p shows promise for disc degeneration by targeting SSRP1/NF-κB [ 61 ]. Li et al. suggested that miR-495 participates in osteoporotic fracture repair via SKP2-mediated regulation of osteoblast differentiation [ 62 ]. Furthermore, miR-22-3p has been shown to modulate osteogenic differentiation in BMSCs and promote bone healing via the SOSTDC1/PI3K/AKT signaling pathway [ 63 ]. To elucidate the mechanism by which miR-328-5p regulates osteogenic differentiation and osteoblast function, this study identified the downstream genes of miR-328-5p. Through comprehensive screening, we discovered that miR-328-5p directly targets and suppresses BCL7A expression. Moreover, clinical data unveiled an inverse relationship between BCL7A and miR-328-5p in individuals with delayed fracture healing. Research has demonstrated that deletion of the BCL7A gene contributes to the proliferation of multiple myeloma cells and accelerates disease development [ 64 ]. BCL7A also functions as a tumor suppressor in osteosarcoma and acute myelogenous leukemia, impeding disease progression [ 65 , 66 ]. Notably, our findings reveal for the first time that BCL7A is involved in the regulation of osteogenic differentiation and is directly targeted by miR-328-5p in the context of bone repair, distinguishing our work from previous research focused solely on its role in cancers. The experimental results demonstrated that BCL7A silencing partially reversed the consequences of miR-328-5p suppression on osteogenic gene expression, cellular proliferation, and apoptosis in osteoblasts. These findings imply that miR-328-5p may regulate fracture repair through BCL7A-dependent modulation of osteogenic differentiation processes. Collectively, our findings suggest the potential involvement of a miR-328-5p/BCL7A regulatory axis in osteoblast function and delayed fracture healing, offering additional mechanistic insights that may complement the current understanding of miRNA-mediated repair.

This study represents the first investigation into both the clinical significance and biological functions of miR-328-5p in bone fracture repair, offering new insights into the mechanistic study underlying fracture healing. However, several limitations should be acknowledged. First, the limited sample size in this study may constrain the statistical power of our findings. Future validation through larger, multicenter cohorts is needed to confirm these findings. Second, while this study demonstrates that miR-328-5p promotes osteogenic differentiation by directly targeting and inhibiting BCL7A, as evidenced by the successful rescue experiments, a standalone investigation into the gain- and loss-of-function effects of BCL7A itself was not conducted within the scope of this research. This represents a limitation of our current work. Therefore, directly elucidating the precise role of BCL7A in osteogenic differentiation through overexpression and knockdown approaches constitutes a key direction for our future investigations. A further limitation of this study is that the specific downstream signaling mechanisms through which BCL7A operates remain unclear. It is well established that the PI3K/AKT pathway plays a central role in promoting osteogenic differentiation in mesenchymal stem cells [ 67 – 69 ]. For instance, AKT activation phosphorylates GSK-3β, leading to the stabilization and nuclear translocation of β-catenin, which subsequently upregulates the expression of key osteogenic genes such as Runx2 [ 70 ]. Based on this evidence, further investigation is warranted to determine whether BCL7A modulates osteogenic differentiation via the PI3K/AKT signaling pathway. Elucidating this mechanism will constitute a central objective of our future research. In addition, the regulatory mechanism of the miR-328-5p/BCL7A axis in fracture healing requires further validation through animal models. For instance, employing fracture mouse models to administer miR-328-5p agomirs or antagomirs and directly observing their effects on the fracture healing process would provide more robust evidence for our conclusions. This constitutes a critical direction for future research.

In summary, the findings demonstrate that decreased miR-328-5p expression is associated with delayed fracture healing, highlighting its potential as a diagnostic biomarker. MiR-328-5p facilitates osteogenic differentiation by targeting BCL7A and modulates key osteoblast functions. These results identify a novel miR-328-5p/BCL7A regulatory axis, providing new insights for the diagnosis and treatment of delayed fracture healing. However, as this study is largely based on in vitro evidence and clinical correlations, further in vivo validation remains necessary.

Supplementary Information

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Supplementary Material 1

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Acknowledgements

Not applicable.

Author contributions

ZH designed the study, performed the experiment, and was a major contributor in writing the manuscript. CJW involved in interpretation of data, drafting the manuscript and revising it critically for important intellectual content. All authors read and approved of the final manuscript.

Funding

No funding was received to assist with the preparation of this manuscript.

Data availability

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

Declarations

Ethics approval and consent to participate

The Ethics Committee of Jiangsu Province Hospital of Chinese Medicine, The Affiliated Hospital of Nanjing University of Chinese Medicine approved this study, and all participants provided informed consent.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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