The functional mechanism and clinical significance of miR-3651 in the healing process of Pilon fractures

Abstract

Background

Pilon fractures are high-energy injuries often associated with delayed healing after treatment. MiR-3651 is a short non-coding RNA, yet its expression level, therapeutic effect, and specific mechanism in the healing process of Pilon fractures remain unclear.

Aim

This study investigates the association between miR-3651 expression and Pilon fracture healing, assesses its diagnostic utility, and elucidates its regulatory mechanism.

Methods

The expression levels of miR-3651, KRAS, and osteoclast differentiation markers were detected using RT-qPCR. The diagnostic efficacy of miR-3651 for delayed healing of Pilon fractures was assessed via ROC curve analysis, and logistic regression was employed to identify risk factors influencing delayed healing. Cell viability and apoptosis were measured using the CCK-8 assay and flow cytometry. The targeting relationship between miR-3651 and KRAS was verified through the dual-luciferase reporter assay.

Results

MiR-3651 expression was significantly upregulated in the delayed healing group, with concomitant downregulation of its downstream target KRAS. MiR-3651 effectively distinguished between normal healing and delayed healing and was identified as a risk factor influencing the occurrence of delayed healing. Inhibition of miR-3651 expression reduced osteoclast activity and differentiation while promoting apoptosis; conversely, overexpression of miR-3651 produced the opposite effects. Knockdown of KRAS expression reversed the impact of miR-3651 inhibition on osteoclast activity, apoptosis, and differentiation.

Conclusions

Downregulation of miR-3651 expression inhibits osteoclast formation, and knockdown of KRAS restores this inhibitory effect.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13018-025-06569-3.

Introduction

In 1911, French radiologist Étienne Destot first introduced the term “Pilon fracture” to describe fractures of the distal tibia involving the ankle joint surface [1]. This type of fracture occurs in the distal third of the tibia and extends into the tibiotalar articular surface, typically resulting from high-energy trauma such as falls from height, jumps, or motor vehicle accidents [2]. Patients often present with compression of distal cancellous bone, bone defects, depression or comminution of the articular surface, and frequently concomitant fractures of the distal fibula. It is recognized as a common intra-articular fracture in clinical practice [3, 4]. As a complex intra-articular injury, the management of Pilon fractures poses a major challenge in orthopedics. Open reduction and internal fixation (ORIF) is currently the mainstay treatment modality; however, improper management can lead to complications such as surgical site infection and post-traumatic arthritis, significantly compromising postoperative functional recovery [5]. Therefore, investigating effective strategies to promote fracture healing and prevent delayed healing has become a key objective in orthopedic research. The identification of predictive biomarkers for delayed healing in Pilon fractures is of great importance for improving patient prognosis [6, 7]. Developing novel, effective, and safe therapeutic approaches to enhance healing quality and reduce the risk of delayed healing represents an urgent clinical need and a valuable research direction [8].

MicroRNAs (miRNAs) are endogenously produced, single-stranded non-coding RNA molecules that typically range from 22 to 24 nucleotides [9]. These mature miRNAs interact with the 3′-untranslated regions (3′-UTRs) of target mRNAs via full or partial base pairing, which can result in mRNA degradation, inhibition of translation, or other modes of post-transcriptional control [10, 11]. MiRNAs play a significant role in the progression and occurrence of various diseases [12, 13]. Consequently, miRNAs act as negative regulators of gene expression and participate broadly in leading to mRNA degradation, translational repression, or other forms of post-transcriptional regulation [14, 15]. As a result, miRNAs negatively regulate the expression of target genes and play extensive roles in biological processes such as development, proliferation, apoptosis, and differentiation [16, 17]. Studies have demonstrated that miRNAs are critically involved in skeletal development and homeostasis maintenance, particularly by coordinating the coupling process between osteoblasts and osteoclasts to regulate bone metabolism [18, 19].

Retain, but ensure citation [20] aligns with the miR-106a-5p example that follows. For instance, miR-106a-5p demonstrates diagnostic value in postmenopausal osteoporosis (PMOP) and correlates with serum ferritin levels; it exerts a protective effect under iron accumulation conditions by regulating PTEN [13]. The long non-coding RNA MYLK-AS1 has been proposed as a potential diagnostic marker for delayed fracture healing (DFH). Its overexpression suppresses miR-146a-5p, enhances osteoblast activity, reduces apoptosis, and thereby facilitates fracture healing [21]. In the context of Pilon fractures, miR-1271-5p contributes to the healing process by targeting ZBTB7A [22], while miR-122-5p promotes healing through inhibition of PDCD4 and represents a potential prognostic indicator for predicting nonunion [23].

Recently, numerous studies have confirmed that multiple miRNAs play crucial regulatory roles in skeletal diseases and bone-related cellular biological processes [24]. Among them, miR-3651—a miRNA previously reported to be functionally active in various malignancies such as colorectal cancer—has not yet been fully elucidated in terms of its role and molecular mechanisms in fracture occurrence and healing, particularly in the context of Pilon fractures [25]. This study focuses on the function of miR-3651 in the healing process of Pilon fractures and its regulatory mechanism governing osteoclast activity and differentiation. The findings are expected to provide a theoretical foundation for predicting and preventing DFH, as well as to offer valuable insights for developing novel therapeutic strategies targeting Pilon fractures and other bone-related disorders. To elucidate this, we first analyzed miR-3651 expression in a cohort of Pilon fracture patients, followed by functional validation in an osteoclast differentiation model. To our knowledge, this is the first study to implicate miR-3651 in Pilon fracture osteoclast regulation via KRAS.

Materials and methods

Patient enrollment and sample collection

This study included 146 patients diagnosed with Pilon fractures and treated at Beijing Jishuitan Hospital from January to November 2024. Based on fracture healing status at the 4-month follow-up, they were classified into a normal healing group (n = 70) and a delayed healing group (n = 76). Before inclusion in the study, written informed consent was obtained from all participants or their direct relatives, and the experimental procedures were reviewed and approved by the Ethics Committee of Beijing Jishuitan Hospital.

Inclusion criteria were: (1) Radiologically confirmed Pilon fracture; (2) Initial fracture management performed at our hospital. (3) Presence of visible callus formation in the normal healing group; (4) Absence of persistent callus, clear radiographic fracture line, or evidence of bone resorption at the fracture site in the delayed healing group.

Exclusion criteria included: (1) Comorbid malignant tumors or pre-existing orthopedic conditions;

(2) Diagnosed osteoporosis; (3) Use of medications known to affect bone metabolism before enrollment.

Peripheral venous blood was collected from both groups. Samples were centrifuged (1500 × g, 10 min) within half an hour after collection. The serum supernatant was aliquoted into sterile cryovials labeled with sample identifiers and collection dates, and stored at − 80 °C.

Cell culture and induction

The THP-1 human monocyte cell line (ATCC, USA) was cultured in RPMI-1640 medium containing 10% FBS at 37 °C in a 5% CO₂ atmosphere.

Upon reaching 80–90% confluency, cells were stimulated with 200 ng/mL phorbol 12-myristate 13-acetate (PMA; MedChemExpress, USA) for 48 h to induce macrophage differentiation. Subsequently, to further induce osteoclast differentiation, cells were treated with a combination of 25 ng/mL macrophage colony-stimulating factor (M-CSF; PeproTech, USA) and 30 ng/mL receptor activator of nuclear factor kappa-B ligand (RANKL; R&D Systems, USA). The medium is changed every two days for 7 days to induce the formation of osteoclasts.

Cell transfection

MiR-3651 mimic and negative control (mimic-NC), miR-3651 inhibitor and its control (inhibitor-NC), KRAS siRNA (si-KRAS) and its control (si-NC), KRAS overexpression vector (OE-KRAS) and control (OE-NC) were synthesized by Qiagen (Germany). Transfection into osteoclasts was performed using Entranster™-H4000 reagent (Beijing, China).

RNA extraction and RT‑qPCR analysis

Total RNA was isolated from both serum samples and cultured cells with Trizol reagent. Following extraction, cDNA was reverse transcribed using the HiScript® II Q RT SuperMix for qPCR kit (Vazyme, Nanjing). SYBR® Green PCR Master Mix (Qiagen, Beijing) was employed for quantitative real-time PCR in accordance with the manufacturer’s protocol. The 2^-ΔΔCt method was applied to determine relative expression levels, with U6 and GAPDH serving as endogenous controls. The sequences of the primers used are shown in Supplementary Table 1.

CCK-8 assay

Cells were plated overnight in 96-well plates at a density of 5 × 10³ cells per milliliter. After grouping with quadruplicate wells per condition, the medium was replaced with 100 µL fresh culture medium per well. After incubation for 24, 48, or 72 h, 10 µL of CCK-8 reagent was added to every well. After 4 h incubation, absorbance was measured at 450 nm to calculate cell viability.

Flow cytometry assay

Cell suspensions were prepared in 100 µL of binding buffer and co-stained with 5 µL Annexin V-FITC and 100 µL propidium iodide (PI). Following a 30-minute incubation period in darkness, sample analysis was conducted using flow cytometry.

Western blot assay

Total protein was extracted using RIPA buffer containing protease and phosphatase inhibitors (SolarBio, China). After electrophoresis and transfer, the PVDF membrane was incubated with primary antibodies (Abcam, UK) against β-actin, p-Akt, Akt, p-Erk, Erk, and KRAS at 4 °C overnight, followed by corresponding secondary antibodies for 2 h at room temperature. Protein bands were visualized with a chemiluminescence system (Bio-Rad, USA).

Dual-luciferase reporter assays

Putative interaction sites between miR-3651 and KRAS were predicted using miRDB and miRWalk databases. Wild-type (KRAS-WT) and mutant (KRAS-MUT) luciferase reporter plasmids were generated (Qiagen, Germany). Cells underwent co-transfection with miR-3651 mimic or inhibitor along with the corresponding reporter constructs. Luciferase activity was quantified 24 h post-transfection employing a dual-luciferase reporter assay system (Promega, USA). Data were normalized against Renilla luciferase activity and presented as relative fold changes.

Statistical analysis

Statistical analysis was conducted with GraphPad Prism 9 and SPSS 22.0 software. Data following a normal distribution are expressed as mean ± SD. Intergroup differences were assessed by Student’s t-test, while one-way ANOVA was used for multi-group comparisons. A P-value below 0.05 was deemed statistically significant. Using the receiver operating characteristic (ROC) curve analysis, the diagnostic value of miR-3651 for DFH was evaluated. Using Logistic regression analysis, the risk factors for the occurrence of DFH were identified.

Results

MiR-3651 has potential diagnostic value for Pilon fractures

Bioinformatics analysis of the GSE125289 dataset revealed miR-3651 as a differentially expressed miRNA in fracture healing (Fig. 1A). Subsequent validation in serum samples from enrolled patients confirmed that miR-3651 level was upregulated in the delayed healing group (Fig. 1B). The comparative analysis of the baseline characteristics of the two groups revealed that there were no significant differences in clinical features between the two groups, such as age and gender (Table 1). The ROC area under the curve (AUC) was 0.9206 (95% CI: 0.8665–0.9747; P < 0.001), accompanied by a sensitivity of 92.65% and a specificity of 91.30% (Fig. 1C). Logistic regression analysis identified miR-3651 as a risk factor influencing the occurrence of delayed healing (OR = 3.873, P < 0.001) (Table 2). These findings indicate that miR-3651 is a reliable biomarker for DFH.

Fig. 1.

Expression level of miR-3651 and diagnostic capability. A The volcano plot identified miR-3651 as the differentially expressed gene in fractures. B The expression levels of miR-3651 in the serum of patients with normal fracture healing and DFH. C ROC curve for miR-3651 in diagnosing DFH

Table 1.

Basic data and clinical characteristics of Pilon fracture patients

Parameters	Normal healing (n = 70)	Delayed healing (n = 76)	P value
Age (years)	42.10 ± 10.58	42.09 ± 13.09	0.134
Gender/male (%)	36 (51.43)	40 (52.63)	0.508
BMI (kg/m2)	22.71 ± 1.28	22.30 ± 1.43	0.448
Smoking (%)	23 (32.86)	25 (32.89)	0.568
Drinking (%)	26 (37.14)	28 (36.84)	0.553
Fracture side/left (%)	36 (51.43)	35 (46.05)	0.314

BMI, body mass index

Table 2.

Logistic regression analysis was conducted to explore the association between patient characteristics and delayed healing of pilonidal fractures

Parameters	OR	95% CI	P value
Age	1.225	0.6047–2.471	0.572
Gender	1.134	0.537–2.932	0.406
BMI	1.353	0.663–2.761	0.742
Smoking	1.108	0.511–2.404	0.795
Drinking	1.070	0.506–2.264	0.859
Fracture side	1.213	0.591–2.488	0.599
MiR-3651	3.837	1.914–7.692	< 0.001

The bold value indicates statistical significance

BMI, body mass index

The effect of miR-3651 on osteoclast differentiation of THP-1 cells

We next used an M-CSF and RANKL-induced THP-1 cell model to examine the role of miR-3651 in osteoclast differentiation. The results showed that miR-3651 expression was increased in the induced group, transfection with an inhibitor or mimic effectively decreased or increased miR-3651 levels, respectively (Fig. 2A). Functionally, M-CSF and RANKL stimulation notably increased osteoclast activity and decreased apoptosis in THP-1 cells. Blocking miR-3651 reversed these effects, bringing cell activity back to baseline levels and significantly increasing apoptosis. Conversely, overexpressing miR-3651 further boosted cell activity and suppressed apoptosis (Fig. 2B and C). MiR-3651 can reduce the activity of osteoclasts, and by inducing their programmed cell death, it further weakens the stability of this cell population. At the molecular level, the expression of key osteoclastogenic markers (NFATC1, TRAP, and c-FOS) was significantly elevated following induction. This upregulation was markedly suppressed by miR-3651 inhibition and further potentiated by its overexpression (Fig. 2D–F). Collectively, these results demonstrate that miR-3651 serves as a key regulator during osteoclast differentiation.

Fig. 2.

Overexpression and inhibition of miR-3651 on osteoclasts. A Changes in expression levels after the dysregulation of miR-3651. B The changes in osteoclast activity after the dysregulation of miR-3651. C The changes in osteoclast apoptosis after the dysregulation of miR-3651. D-F The changes in the levels of osteoclast differentiation markers (NFATC1, TRAP, and c-FOS) after the dysregulation of miR-3651

The interaction between miR-3651 and KRAS

By integrating the prediction results from the miRDB and miRWalk databases, we identified three candidate target genes of miR-3651 (KRAS, ADAM17, and ZSCAN30) (Fig. 3A). Subsequently, these genes were verified in the patient serum, and it was found that the expression difference of KRAS was the most significant (Fig. 3B). To confirm the direct targeting relationship between miR-3651 and KRAS, a dual-luciferase reporter assay was conducted. Bioinformatics prediction indicated a binding site between miR-3651 and KRAS (Fig. 3C). The luciferase activity assay demonstrated that inhibition of miR-3651 increased the luciferase activity of the wild-type KRAS vector (KRAS-WT), while overexpression of miR-3651 markedly suppressed it. In contrast, no significant change was observed in the luciferase activity of the mutant vector (KRAS-MUT) (Fig. 3D). Finally, Pearson correlation analysis supported a negative regulatory relationship between miR-3651 and KRAS (Fig. 3E).

Fig. 3.

KRAS served as the downstream target of miR-3651. A Venn diagram of the target genes of miR-3651 combined with prediction. B The expression level of three candidate target genes in the serum of patients with Pilon fractures. C Predict the complementary sites of miR-3651 and KRAS. D The luciferase activity validated the relationship between miR-3651 and KRAS. E The level of miR-3651 was negatively correlated with that of KRAS

KRAS can reverse the effect of miR-3651 on osteoclasts.

This research sought to examine the role of miR-3651 on pro-osteoclastogenic effects via regulation of KRAS. In the osteoclast model induced by M-CSF and RANKL, inhibiting miR-3651 in this model markedly upregulated KRAS expression, an effect that was completely reversed by concurrent knockdown of KRAS (Fig. 4A). In terms of cellular function, the suppression of cell viability (Fig. 4B) and promotion of apoptosis (Fig. 4C) caused by miR-3651 inhibition were also rescued by KRAS knockdown, restoring phenotypes to baseline levels. Similarly, the downregulation of key osteoclastogenic markers (NFATC1, TRAP, c-FOS) resulting from miR-3651 inhibition was reversed when KRAS was simultaneously knocked down (Fig. 4D and E).

Fig. 4.

Knockdown of KRAS restored the promoting effect of the miR-3651 inhibitor on fracture healing. A MiR-3651 regulated the changes in the expression level of KRAS. B MiR-3651 regulated the effect of KRAS on cell activity. C MiR-3651 regulated the effect of KRAS on cell apoptosis. D-F MiR-3651 regulated the effect of KRAS on osteoclast differentiation markers (NFATC1, TRAP, and c-FOS)

In this model, overexpression of miR-3651 significantly reduces the expression of KRAS (Fig. 5A), while simultaneous overexpression of KRAS can reverse this effect. In terms of cellular functions, the increase in cell viability (Fig. 5B) and the inhibition of cell apoptosis (Fig. 5C) caused by miR-3651 overexpression can also be restored to baseline levels by co-overexpression of KRAS. Moreover, the upregulation of osteoclast markers (NFATC1, TRAP, c-FOS) induced by miR-3651 was reversed by KRAS co-overexpression, restoring their expression to baseline (Fig. 5D–F). In summary, these findings indicate that miR-3651 significantly influences M-CSF and RANKL-induced osteoclast proliferation, apoptosis, and differentiation by targeting and regulating KRAS expression.

Fig. 5.

Overexpression of KRAS restored the inhibitory effect of miR-3651 on fracture healing. A The change in KRAS expression level after overexpression of miR-3651. B-C The effect of miR-3651 and KRAS overexpression on cell activity and apoptosis. D-F RT-qPCR revealed the changes in the markers of osteoclast differentiation (NFATC1, TRAP, and c-FOS)

MiR-3651 potentially regulated ERK and AKT signaling via KRAS.

To elucidate the downstream mechanism by which miR-3651 regulates osteoclast differentiation, we examined the activation status of the KRAS signaling pathway (ERK and AKT). Western Blot results showed that overexpression of miR-3651 significantly suppressed KRAS protein levels, and its inhibition enhanced KRAS expression (Fig. 6A and B). While miR-3651 overexpression promoted the phosphorylation of ERK and AKT, this effect was reversed by concurrent KRAS overexpression. Conversely, miR-3651 inhibition suppressed phosphorylation of both proteins, and this suppression was rescued by KRAS knockdown (Fig. 6C–F). These results indicated that miR-3651 potentially regulates the ERK and AKT signaling pathways by targeting KRAS, thereby influencing the differentiation and function of osteoclasts.

Fig. 6.

MiR-3651 targeted KRAS with potential regulation of ERK/AKT signaling. A-B Changes in KRAS protein levels. C-D Changes in the protein levels of phosphorylated ERK and total ERK. E-F Changes in the levels of phosphorylated AKT and total AKT protein

Discussion

Pilon fractures refer to intra-articular fractures of the distal tibia, often accompanied by articular surface impaction and fractures of the distal fibula [26]. These injuries primarily affect males aged 41–45 and are typically caused by high-energy trauma such as falls from height or traffic accidents, accounting for approximately 3%-10% of all tibial fractures. Compared to low-energy rotational ankle fractures, Pilon fractures demonstrate more severe injury mechanisms, fracture patterns, and soft tissue damage [27, 28]. They are frequently comminuted and associated with bone defects and significant soft tissue compromise, leading to a high rate of postoperative complications, including delayed healing, infection, and joint stiffness [29, 30]. Consequently, treatment and rehabilitation remain clinically challenging. Given these complexities, identifying factors that predict and prevent delayed healing is of considerable clinical importance. Current preventive and therapeutic measures include pharmacological interventions (e.g., calcitonin), low-intensity pulsed ultrasound (LIPUS), and intermittent pneumatic compression (IPC) [31, 32]. However, the efficacy of these approaches is often inconsistent, and some involve complex implementation protocols.

MiRNAs, a class of short non-coding RNAs, precisely modulate post-transcriptional gene expression [33, 34]. Through sequence-specific interactions with target mRNAs, miRNAs facilitate transcript degradation or inhibit translation, including events, thereby playing a crucial role in processes such as proliferation, differentiation, and programmed cell death [35, 36]. Accumulating studies have further established miRNAs as key regulatory molecules in the pathogenesis and advancement of diverse human diseases. Within the skeletal system, multiple studies have identified miRNAs, revealing the regulatory roles of these molecules [37, 38]. For example, miR-216a has been shown to promote osteoblast differentiation and bone matrix deposition by inhibiting the expression of the proto-oncogene Cbl, thereby blocking the PI3K/Akt signaling pathway [39]. In another study, miR-98-5p was found to target HMGA2, thereby suppressing osteogenic differentiation and cell proliferation, which ultimately impairs bone regeneration [40]. Despite these advances, the functions of numerous miRNAs with potential regulatory importance in bone biology and related disorders remain largely unexplored. Further investigation is needed to elucidate their mechanisms of action and therapeutic relevance.

Our study focuses on miR-3651, which has been previously identified as a differentially expressed microRNA in fracture tissues. Previous studies have demonstrated its functional significance in hepatocellular carcinoma, wherein it enhances tumor growth and metastatic potential through activation of the PI3K/AKT/mTOR signaling cascade, suggesting its potential as a diagnostic and therapeutic target [41]. Furthermore, miR-3651 shows promise as a prognostic factor for early-stage breast cancer by targeting FRMD3 [42]. Given the multi-pathway regulatory capacity of miRNAs, these findings provide a rationale for developing personalized treatment strategies in oncology. Building on this background, we investigated its role in fracture healing. Increased circulating miR-3651 levels were observed in individuals experiencing delayed Pilon fracture healing. Building on our ROC analysis, which demonstrated that miR-3651 exhibited strong discriminatory power for delayed healing. Notably, despite comparable baseline characteristics such as age and gender between the two groups, logistic regression analysis confirmed miR-3651 as an independent risk factor for DFH.

To explore the potential cellular mechanism underlying this clinical association, we utilized the M-CSF and RANKL-induced THP-1 cell model. In this model, overexpression of miR-3651 enhanced cell viability, suppressed apoptosis, and up-regulated osteoclast differentiation markers, whereas inhibition of miR-3651 produced the opposite effects. Osteoclasts with inhibited apoptosis may survive for a longer time and maintain their activity for a longer period, thereby causing excessive bone resorption, disrupting the coupling balance between osteogenesis and osteolysis, and ultimately leading to DFH. Osteoclasts with promoted apoptosis, on the other hand, will inhibit DFH and promote healing [43]. To bridge these functional findings with a molecular mechanism, we predicted and validated KRAS as a downstream target of miR-3651. KRAS is an important oncogene in the human body. MiR-210-3p inhibits the normal osteogenic differentiation of BMSCs by targeting KRAS and inhibiting the MAPK signaling pathway; overexpression of KRAS can reverse the inhibitory effect of miR-210-3p [44]. Its expression was downregulated in the serum of patients with Pilon fracture delayed healing. A direct targeting relationship between miR-3651 and KRAS was validated using a dual-luciferase reporter system, indicating a negative regulatory relationship. Most importantly, subsequent functional rescue experiments demonstrated that miR-3651 significantly influences the activity, apoptosis, and differentiation of M-CSF and RANKL-induced osteoclasts by targeting KRAS expression. Further experiments have confirmed that miR-3651 regulates the differentiation of osteoclasts by modulating the activities of ERK and AKT in the downstream pathway of KRAS. The miR-3651/KRAS axis plays a crucial role in coordinating signal transduction through the ERK and AKT pathways, and these two pathways are known to play a vital regulatory role in the survival, proliferation, and differentiation of osteoclasts.

From the perspective of clinical prospects, our findings illuminate a potential new strategy for mitigating delayed healing. The serum upregulation of miR-3651 could serve as an early warning biomarker. More importantly, therapeutic inhibition of miR-3651 could restore normal KRAS signaling and rebalance bone remodeling, offering a targeted approach for patients at high risk for delayed union. However, this study has limitations. First, validation with primary cells and animal models is crucial for confirming physiological relevance and translational potential [45]. But due to difficulties in obtaining primary cells and time constraints of animal studies, our current investigation primarily used the THP-1 cell line for mechanistic exploration. Future studies should validate these findings in primary human osteoclasts and animal models to confirm translational potential. Second, bone resorption capacity represents a core metric for evaluating osteoclast function and is a key phenotype in skeletal disease research [46]. Although we examined how signaling pathways affect osteoclast differentiation, functional bone resorption assays were not performed under current experimental constraints. Subsequent work will prioritize these functional assessments to comprehensively evaluate osteoclast activity.

Conclusion

In conclusion, upregulated miR-3651 in delayed Pilon fracture healing serves as a diagnostic biomarker and risk factor, promoting osteoclast activity via KRAS suppression. These results suggest miR-3651 inhibition as a novel adjunct to ORIF, warranting prospective trials.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization, S.L., Z.S., T.D., X.C., B.H., Y.W., R.W., J.W.; Data curation, S.L., Z.S., T.D., X.C., B.H., Y.W., R.W., J.W.; Formal analysis, S.L., Z.S., T.D., X.C., B.H., Y.W., R.W., J.W.; Funding acquisition, J.W.; Investigation, T.D., X.C., B.H.; Methodology, S.L., Z.S., T.D., X.C., B.H., Y.W., R.W., J.W.; Project administration, J.W.; Resources, Y.W., R.W.; Software, S.L., Z.S., T.D.; Supervision, J.W.; Validation, Y.W., R.W.; Visualization, S.L., Z.S.; Roles/Writing - original draft, S.L., Z.S.; Writing - review & editing, J.W.

Funding

This work was funded by Beijing Natural Science Foundation (L254086, L246006).

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 study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Beijing Jishuitan Hospital before the study began. The written informed consent has been obtained from the participants involved.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.