miR-326 promotes osteogenic differentiation of bone marrow mesenchymal stem cells by targeting NAT10

Abstract

Objectives

Abnormal osteogenic differentiation of BMSCs is a core factor in osteoporosis (OP). This study investigates how miR-326 regulates osteogenic differentiation of hBMSCs by targeting NAT10, thereby influencing OP pathogenesis.

Method

Serum miR-326 levels were measured using RT-qPCR. Its diagnostic value for OP was assessed by ROC analysis, and its correlation with bone mineral density (BMD) was examined. Using a dexamethasone (DEX)-induced hBMSC OP model, we evaluated the effects of miR-326 on osteogenesis (RT-qPCR), cell viability (CCK-8 assay), and inflammation (ELISA). The miR-326–NAT10 interaction was confirmed by dual-luciferase assay. A rescue experiment with co-transfection verified their functional relationship.

Results

MiR-326 levels in OP patient serum were significantly decreased, and the area under the ROC curve demonstrated excellent diagnostic performance The OP group exhibited significantly lower BMD, which was positively correlated with reduced miR-326 expression. In DEX-induced hBMSCs, miR-326 overexpression promoted osteogenesis by upregulating key markers, enhanced cell viability, and attenuated inflammation. Dual-luciferase assays confirmed NAT10 as a direct target of miR-326. NAT10 was upregulated in OP patients and negatively correlated with miR-326, and oe-NAT10 rescued the pro-osteogenic and anti-inflammatory effects of miR-326.

Conclusions

miR-326 is downregulated in the OP and can serve as a potential diagnostic biomarker for OP. miR-326 promotes osteogenic differentiation of hBMSCs and inhibits inflammatory responses by targeting NAT10, exerting a protective role in the pathogenesis of OP.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13018-026-06702-w.

Introduction

The proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) are fundamental to both the pathogenesis and recovery of osteoporosis (OP) [1]. OP is characterized by a disturbance in the homeostasis between osteoblastic bone formation and osteoclast-mediated bone resorption [2]. These changes were accompanied by the depletion of hBMSCs and alterations in cell signaling, leading to the suppression of osteogenesis and the excessive expression of adipogenic and osteoclastogenic factors [3]. OP can cause fractures in the hip, vertebrae, and distal forearm, leading to significant disability and mortality [4]. Currently, the primary treatment for OP involves medications that promote bone formation or inhibit bone resorption [5]. Bisphosphonates inhibit bone resorption by inducing osteoclast apoptosis. Anti-resorptive medications such as denosumab and calcitonin, along with non-pharmacological measures like vitamin D and calcium supplementation, can be used to delay and treat OP [6]. However, none of these drugs can reverse existing bone loss and may cause other serious side effects [7]. Therefore, understanding OP pathogenesis is crucial for identifying effective intervention targets and improving disease diagnosis and prognosis. Research has established that bone metabolism is precisely regulated by a multi-layered network involving numerous factors. In addition to classical growth factor signaling pathways, such as the epidermal growth factor pathway which plays a critical role in skeletal development and homeostasis [8], investigators have also focused on developing diverse strategies to enhance the osteogenic differentiation of hBMSCs for bone regeneration [9, 10]. Concurrently, non-pharmacological interventions have been demonstrated to modulate bone metabolism by stimulating osteoblast proliferation and functional activity [11].

Non-coding RNAs (including miRNAs, siRNAs, circRNAs, etc.) have been shown to play important regulatory roles in various musculoskeletal disorders, such as tendon repair [12, 13], osteoarthritis [14], rheumatoid arthritis [15], and osteoporosis [16, 17]. Among them, miRNAs can exert regulatory effects by targeting key genes [18], and this targeting action may produce either promoting or inhibitory effects on osteoblast differentiation [19]. For example, miR-483-3p promotes osteogenic differentiation of BMSCs by inhibiting STAT1, reversing senile bone loss [20], while miR-9-5p blocks DDX17 under high glucose conditions but induces diabetic OP [21]. MiR-326 serves as a key regulatory factor in BMSC osteogenic differentiation. It can also be delivered via BMSC exosomes to target and inhibit HDAC3, thereby blocking chondrocyte pyroptosis and ameliorating osteoarthritis [22]. miR-326 is closely associated with bone metabolism markers in lung cancer bone metastasis and is a potential marker for monitoring disease progression [23]. In intervertebral disc degeneration, miR-326 is suppressed by SNHG1, thereby promoting the proliferation of nucleus pulposus cells [24]. Based on the existing evidence, this study speculates that miR-326 may be a key miRNA regulatory factor driving BMSC osteogenic differentiation.

Although existing studies suggest that miR-326 is associated with bone metabolism, there is still a lack of conclusive evidence regarding its direct regulatory role in the osteogenic differentiation of hBMSCs. Therefore, this study aims to investigate whether miR-326 promotes osteogenic differentiation through NAT10, which could provide new insights for targeted therapy of OP.

Materials and methods

This study enrolled 103 OP patients who visited Hangzhou Fuyang Hospital of Orthopedics of Traditional Chinese Medicine from March 2021 to December 2023, along with 96 healthy individuals from physical examinations during the same period (control group). Inclusion criteria for the OP group were: age ≥ 50 years, with lumbar spine (LS) and/or hip bone mineral density (BMD) T-score ≤ − 2.5 SD as measured by dual-energy X-ray absorptiometry. Exclusion criteria were: secondary OP, malignant tumors, acute or chronic infections, hepatic or renal insufficiency, and recent use of medications affecting bone metabolism. The control group consisted of healthy individuals undergoing physical examinations. Age and BMI were recorded, and BMD was measured at the LS, femoral neck (FN), and total hip (TH). This study has been approved by the hospital ethics committee, and all participants have signed informed consent forms.

Culture of hBMSCs

Human bone marrow mesenchymal stem cells (hBMSCs) were purchased from Cyagen Bioscience (China). The cells were cultured in complete α-MEM medium (containing 10% FBS and 1% penicillin-streptomycin). After 48 h of inoculation, the medium was replaced to remove non-adherent cells, followed by routine medium changes every 3 days. When cell confluence reached 80–90%, cells were digested and subcultured using 0.25% trypsin-EDTA. Fourth-generation cells were selected for this experiment.

miR-326 overexpression transfection

hBMSCs were seeded in 6-well plates (5 × 10⁵ cells/well) and cultured until 70% confluency. Transfection was performed using Lipofectamine 3000 (Invitrogen), and the experiment was divided into control, miR-NC, and miR-326 mimic group. A 250 µL volume of transfection complex was added to each well. The medium was replaced with complete medium after 6 h of incubation, and subsequent assays were carried out 48 h later.

Establish an osteogenic differentiation Inhibition model using dexamethasone (Dex)

To simulate the osteoporotic pathological microenvironment and establish an in vitro model of impaired osteogenic differentiation, we treated hBMSCs with Dex. The cells were continuously cultured in complete medium supplemented with a high concentration of Dex (10⁻⁶ mol/L), with the medium being replaced every 2–3 days. The control group cells were cultured in parallel under the same conditions using complete medium without Dex supplementation.

CCK-8

The transfected hBMSCs were seeded in 96-well plates (3 × 10³ cells/well) with 6 replicates per group. At 24, 48, and 72 h of culture, 10 µL of CCK-8 reagent (Dojindo, Japan) was added to each well. Plates were then incubated at 37℃ for 2 h, and absorbance at 450 nm was measured using a microplate reader.

Inflammatory factor testing

Cell culture supernatants were collected, and the concentrations of IL-6, TNF-α, and IL-1β were measured using ELISA kits (R&D Systems, USA) according to the manufacturer’s instructions. Absorbance at 450 nm was read on a microplate reader, and concentrations were calculated from the standard curve.

Bioinformatic prediction of miR-326 targets

Potential target genes of hsa-miR-326 were predicted using the TargetScanHuman (version 8.0) and miRWalk 3.0 databases. The overlapping prediction results from both databases were integrated to form a candidate gene list for subsequent screening. Among the shared genes identified, NAT10 was selected for further investigation. This decision was based on the presence of a highly conserved 7mer-m8 binding site for miR-326 within its 3′ UTR, which is associated with a high predicted probability of conserved targeting (0.85) and favorable binding free energy (ΔG = − 24.50 kcal/mol), indicating a strong and specific potential for interaction. Furthermore, literature review reveals that NAT10 plays a critical role in bone metabolism [25, 26]. Therefore, NAT10 was chosen as the candidate gene for this study.

Dual-luciferase reporter gene assay

NAT10 3’UTR wild-type (WT) and binding site mutant (MUT) plasmids were constructed (Shanghai GenePharma Co., Ltd.). hBMSCs were seeded in 24-well plates (1 × 10⁵ cells/well) and co-transfected with miR-326 mimic (50 nM) and plasmid (100 ng/well) using Lipofectamine transfection reagent. After 48 h, luciferase activity was measured using the dual-luciferase reporter assay system (Promega, USA).

RT-PCR

Total RNA was isolated from 200 µL serum with TRIzol LS reagent, and its concentration and purity were determined by NanoDrop 2000. Quantitative real-time PCR (Applied Biosystems) was then conducted using the miScript SYBR Green PCR Kit. Gene levels of ALP (alkaline phosphatase), RUNX2 (Runt-related transcription factor 2), OCN (osteocalcin), OPN (osteopontin), and NAT10 were normalized to GAPDH, while miR-326 level was normalized to U6, with both calculated via the 2^−ΔΔCt method.

ALP activity assay

After the completion of osteogenic induction, cells were collected and lysed on ice using lysis buffer. An equal volume of the clarified lysate supernatant was incubated with pNPP substrate working solution at 37℃ in the dark for an appropriate duration. The reaction was terminated by adding 2 M NaOH, and the absorbance was measured at a wavelength of 405 nm. The total protein concentration of the same lysate was determined using the BCA assay for subsequent normalization.

Western blot analysis

Total cellular proteins were collected, and their concentrations were determined using the BCA assay. Equal amounts of protein samples were separated by SDS-PAGE and subsequently transferred onto a PVDF membrane. The membrane was blocked with 5% skimmed milk and then incubated with the corresponding primary antibody at 4℃ overnight. After washing with TBST, the membrane was incubated with an HRP-conjugated secondary antibody at room temperature for 1 h. Signals were detected using an ECL chemiluminescence reagent and captured with an imaging system. GAPDH served as the loading control, and band intensities were quantified using ImageJ software.

Statistical analysis

Statistical analysis was performed using SPSS 25.0 and GraphPad Prism 9. Data are expressed as mean ± SD, and intergroup comparisons were assessed by independent samples t-test or one-way ANOVA with Tukey’s test, respectively. The diagnostic efficacy of miR-326 was analyzed using ROC curves. Correlation analysis was performed using Pearson’s test. Statistical significance was set at p < 0.05.

Results

Baseline clinical characteristics

According to the collected subject data, BMD was significantly lower in the OP group compared with controls at all sites measured. Specifically, the OP group exhibited significantly lower BMD at the LS (0.80 ± 0.03 vs. 0.91 ± 0.03 g/cm²), FN (0.62 ± 0.02 vs. 0.73 ± 0.05 g/cm²), and TH (0.68 ± 0.03 vs. 0.76 ± 0.03 g/cm²) compared to the control group (p < 0.001, Table 1).

Table 1.

Basic information of the research subjects

Clinical characteristics	Control group (n = 96)	Osteoporosis group (n = 103)	p value
Age (years)	62.46 ± 4.00	62.3 ± 2.75	0.746
BMI (kg/m2)	23.11 ± 3.41	23.20 ± 3.40	0.854
LS BMD (g/cm2)	0.91 ± 0.03	0.8 ± 0.03	< 0.001
FN BMD (g/cm2)	0.73 ± 0.05	0.62 ± 0.02	< 0.001
TH BMD (g/cm2)	0.76 ± 0.03	0.68 ± 0.03	< 0.001

BMI, body mass index; LS BMD, lumbar spine bone mineral density; FN BMD femoral neck bone mineral density; TH BMD, total hip bone mineral density. n represents the sample size, and the data are presented as mean ± standard deviation

Study on the expression and correlation of miR-326 in OP

Compared with healthy controls, the relative level of serum miR-326 in OP patients was significantly decreased (p < 0.0001, Fig. 1A). As shown in Fig. 1B, miR-326 had an AUC of 0.836 (95% CI 0.780–0.891) in distinguishing OP, with a sensitivity of 0.850 and a specificity of 0.719. Correlation analysis revealed that serum miR-326 levels were significantly positively correlated with LS (r = 0.630, Fig. 1C), FN (r = 0.663, Fig. 1D), and TH (r = 0.765, Fig. 1E) density (all p < 0.001).

Fig. 1.

Diagnostic value of serum miR-326 for osteoporosis and its correlation analysis with bone mineral density. A ROC curve of miR-326 for distinguishing OP patients from controls; B Relative expression of miR-326 in the control and OP groups; C The correlation between miR-326 and LS BMD; D The correlation between miR-326 and FN BMD; E The correlation between miR-326 expression and TH BMD; ****p < 0.0001

Functional validation of miR-326 in Dex-inhibited osteogenic differentiation of hBMSCs

To investigate the intrinsic function of miR-326 under physiological conditions, we analyzed its role in normally cultured hBMSCs. As shown in Supplementary Figure S1, transfection with miR-326 mimic successfully increased intracellular miR-326 levels (Supplementary Figure S1 A). Correspondingly, overexpression of miR-326 significantly elevated the mRNA expression of key osteogenic markers (ALP, RUNX2, OCN, OPN) (Supplementary Figure S1 B-E), and enhanced ALP enzymatic activity and cell viability (Supplementary Figure S1 F-G). Furthermore, the secretion of inflammatory cytokines was notably reduced following miR-326 overexpression (Supplementary Figure S1 H-J). The Dex-induced hBMSC model revealed that Dex treatment significantly decreased miR-326 expression (p < 0.0001, Fig. 2A), while the Dex-miR-mimic group markedly increased its expression (p < 0.001). Notably, Dex treatment markedly suppressed the expression of osteogenic differentiation markers at both mRNA and protein levels, whereas overexpression of miR-326 effectively counteracted this suppression in both dimensions (Fig. 2B–I). Furthermore, overexpression of miR-326 also reversed the inhibitory effect of Dex on ALP enzyme activity (Fig. 2J). The CCK-8 results showed that miR-326 overexpression could enhance cell viability and effectively reverse the effect of Dex on cell viability (p < 0.0001, Fig. 3A). In addition, Dex treatment significantly increased the secretion of inflammatory factors TNF-α, IL-6, and IL-1β (p < 0.0001), while miR-326 overexpression markedly suppressed these inflammatory levels (p < 0.0001, Fig. 3B–D).

Fig. 2.

Effect of miR-326 overexpression on osteogenic differentiation markers and ALP enzymatic activity in the Dex-induced hBMSC model. A The transfection experiment successfully regulated miR-326; The effects of miR-326 overexpression on osteogenic differentiation markers B, C ALP, D, E RUNX2, F, G OCN, and H–I) OPN under Dex induction; J Measurement of ALP enzymatic activity; ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05

Fig. 3.

The effect of miR-326 overexpression on cell viability and inflammatory factors in the Dex-induced hBMSC model. A Effect of miR-326 overexpression on Dex-induced viability of hBMSCs; The effect of miR-326 overexpression on Dex-induced inflammation in hBMSCs B TNF-α, C IL-6, D IL-1β; ****p < 0.0001; ****p < 0.0001

The targeting relationship between miR-326 and NAT10

Bioinformatics analysis predicted that hsa-miR-326 could directly target and regulate NAT10 by complementary binding to the 3781–3787 site of the NAT10 3’ UTR (Fig. 4A). The dual-luciferase assay demonstrated that miR-326 mimic significantly inhibited luciferase activity in WT-NAT10 (p < 0.0001), while this inhibitory effect was abolished in MT-NAT10 (Fig. 4B), confirming that miR-326 directly targets specific sites on the NAT10 3’ UTR. In cellular assays, Dex treatment upregulated the mRNA expression of NAT10, and this upregulation was reversed by miR-326 overexpression. Conversely, inhibition of miR-326 further enhanced NAT10 expression (p < 0.0001, Fig. 4C). The observed trends in mRNA expression were further corroborated by Western blot analysis (p < 0.0001, Fig. 4D).Further analysis of clinical samples revealed that the mRNA expression of NAT10 was significantly elevated in OP patient tissues compared to the control group (p < 0.0001, Fig. 4E), showing a significant negative correlation with miR-326 (r = − 0.730, p < 0.0001, Fig. 4F).

Fig. 4.

Verification of the relationship between miR-326 and NAT10. A Bioinformatics analysis predicted the targeting binding between NAT10 and miR-326; B Dual-luciferase reporter verified that miR-326 mimic significantly inhibits WT-NAT10 luciferase activity; C, D miR-326 overexpression/inhibition on NAT10 mRNA and protein levels in Dex-treated cells; E The relative levels of NAT10 in the osteoporosis group and the healthy group; F Correlation analysis between miR-326 and NAT10; ****p < 0.0001. ****p < 0.0001, ***p < 0.001, **p < 0.01

NAT10 reversed the effect of miR-326 on osteogenic differentiation of hBMSCs

By investigating the effect of NAT10 on Dex-miR-mimic, it was found that NAT10 overexpression inhibited the enhanced cell viability induced by miR-326 overexpression (p < 0.0001, Fig. 5A). Furthermore, in terms of osteogenic differentiation, overexpression of NAT10 reversed the miR-326-induced upregulation in the mRNA (p < 0.0001, Fig. 5B) and protein (p < 0.0001, Fig. 5C) expression of key osteogenic markers, and also inhibited the recovery of ALP enzymatic activity (p < 0.0001, Fig. 5D). Analysis of inflammatory factors revealed that oe-NAT10 could significantly reverse the inhibitory effect of miR-326 overexpression on inflammatory secretion (p < 0.0001, p < 0.001, Fig. 5E).

Fig. 5.

NAT10 overexpression reversed the rescuing effect of miR-326 on Dex-impaired osteogenic differentiation capacity of hBMSCs. A Effect of oe-NAT10 on cell viability; B, C Effect of oe-NAT10 on the relative expression levels of key osteogenic markers at mRNA and protein levels; D Effect of oe-NAT10 on ALP enzymatic activity; E The effect of oe-NAT10 on inflammatory response; ****p < 0.0001, ***p < 0.001

Discussion

OP is characterized by reduced bone mass and the destruction of bone microstructure, resulting from impaired osteogenic differentiation of BMSCs, which leads to bone formation disorders [27]. To model this pathological condition, we employed Dex, a well-established inhibitor of osteogenic differentiation [28]. This approach aimed to elucidate the underlying molecular mechanisms by investigating the role of miR-326 in hBMSCs. Our findings provide novel insights into the pathogenesis of OP and may offer an experimental foundation for developing new diagnostic markers and therapeutic strategies.

MicroRNAs are key regulators of bone homeostasis and metabolism, potentially affecting the functions of osteoblasts and osteoclasts, such as cell proliferation, inflammation, and more [29]. As an important microRNA, miR-326 plays a crucial regulatory role in the pathogenesis of OP. Xu et al. discovered aberrant expression of miR-326-3p in osteoporotic rat models through high-throughput sequencing, but neither its clinical significance nor functional mechanisms were investigated further [30]. Building upon this research, our study confirmed the significant downregulation of miR-326 in OP patients and validated its strong diagnostic value (AUC = 0.836) through clinical samples. Decreased BMD in the LS, FN, and TH is associated with an increased risk of osteoporotic fractures [31], while this study found that miR-326 levels were significantly positively correlated with these BMD measurements. This further confirms that miR-326 may serve as a diagnostic biomarker and potential therapeutic target for OP. This is consistent with the growing recognition of miRNAs as promising diagnostic biomarkers in musculoskeletal disorders [32, 33]. Dysregulation of specific miRNAs has been closely linked to the progression and diagnosis of osteoporosis [33]. High concentrations of Dex (10⁻⁶ mol/L) can promote apoptosis and inhibit proliferation of hBMSCs [28, 34]. In addition, ALP, OCN, OPN, and Runx2 are key markers in the osteogenic process [35]. Their expression and activity are affected during the pathological progression of OP, potentially influencing bone formation and maintenance [36]. Although Li et al. initially speculated that overexpression of miR-326-5p might promote osteogenic differentiation, its specific downstream targets and the complete regulatory pathway in OP remain unclear [37]. This study found that Dex treatment significantly reduced the expression of miR-326 and suppressed the expression of osteogenesis-related genes, while overexpression of miR-326 could promote the impaired osteogenic differentiation induced by Dex and enhance cell viability. IL-1β, IL-6 and TNF-α are important regulators of bone metabolism, and their elevated levels are negatively correlated with BMD [38]. We found that Dex treatment increased inflammatory factor concentrations, while miR-326 overexpression effectively inhibited their secretion. Collectively, these studies suggest that therapeutic targeting of specific miRNA-mediated inflammatory pathways holds potential for treating a spectrum of bone and joint disorders [39, 40]. Notably, this study not only verified the function of miR-326 in the pathological model but also directly confirmed its physiological role through complementary experiments. In normally cultured hBMSCs without Dex treatment, miR-326 overexpression similarly significantly upregulated the expression of key osteogenic genes (ALP, RUNX2, OCN, and OPN) and enhanced ALP enzymatic activity. These results indicate that miR-326 itself acts as an intrinsic initiator and promoter of the osteogenic differentiation program in hBMSCs, and its bone-promoting effects extend beyond merely antagonizing Dex‑induced inhibition. These results collectively suggest that miR-326 may play a positive role in osteogenic differentiation. The regulatory function of miR-326 operates within the intricate miRNA network associated with bone metabolism. For instance, miR-217 has been shown to influence postmenopausal osteoporosis by modulating the OPG/RANKL/RANK pathway [41], while ceRNA networks are also widely involved in the regulation of bone metabolism [42]. This study places miR-326 within this regulatory framework, highlighting its unique role through interaction with specific molecular targets. NAT10 is an enzyme that catalyzes RNA N4-acetylcytidine (ac4C) modification in eukaryotes [43]. Currently, the functions of NAT10 have been primarily revealed in the field of oncology, and its expression is regulated by different miRNAs. In colorectal cancer, it exerts tumor-suppressive effects by being inhibited by miR-6716-5p [44], whereas in gastric cancer, it is upregulated through the DARS-AS1/miR-330-3p axis, promoting tumor progression [45]. Moreover, NAT10 also exhibits pleiotropic functions in non-oncological fields. For instance, NAT10 is involved in osteogenic differentiation and OP [46]. Research has found that NAT10 promotes osteoclast differentiation and postmenopausal OP [25], while also playing a critical role in inflammatory bone loss [26]. However, which specific miRNA regulates NAT10 expression in OP remains a critical question to be investigated. This study, through TargetScan and miRWalk prediction combined with dual-luciferase reporter gene assay, has for the first time discovered and confirmed that miR-326 can directly target the 3’-untranslated region of NAT10 and bind to it. NAT10 is a high-confidence predicted target of miR-326 and has established functional relevance in bone homeostasis, as reported in the literature [25, 26]. Unlike its previously known role in tumor cells, this study focuses on the field of OP and confirms the existence of a novel regulatory axis, miR-326/NAT10. We observed a significant upregulation of NAT10 in OP serum, which showed a negative correlation with miR-326 expression. Functional experiments further demonstrated that overexpression of NAT10 could reverse the promoting effects of miR-326 on cell viability and osteogenic differentiation, while also reversing miR-326’s inhibitory effect on cellular inflammation.

This study primarily validated NAT10 as a functional downstream target of miR-326, though the precise mechanisms by which NAT10 promotes osteogenesis remain incompletely understood. NAT10 is the principal mRNA acetyltransferase and enhances target mRNA stability and translation efficiency through ac4C modification [47–49]. We hypothesize that under osteoporotic conditions or in Dex-treated bone marrow mesenchymal stem cells, upregulated NAT10 may selectively stabilize the transcripts of one or more “osteogenesis inhibitors” or “pro-inflammatory factors” via ac4C modification. By targeting and inhibiting NAT10, miR-326 may reduce the stability of these detrimental transcripts, thereby alleviating their suppressive effects on osteogenesis. Recent studies provide supporting evidence, NAT10 can promote osteoclastogenesis by activating the MAPK pathway through ac4C modification of Fos mRNA [26]. This suggests that NAT10 may dynamically regulate key signaling pathways influencing bone homeostasis, such as MAPK and NF-κB, through similar RNA modification mechanisms. Thus, the bone-promoting effect of the miR-326/NAT10 axis may partly result from its interference with dysregulated RNA epitranscriptional networks that are abnormally activated in OP. Future research should employ approaches such as ac4C sequencing to further elucidate the precise mechanisms by which NAT10 regulates osteogenic differentiation.

Although this study has achieved certain progress and findings, the following limitations still exist: First, the experimental design was limited, such as the lack of comparative analysis among patients of different age groups and disease stages. Future studies require more comprehensive designs to further explore the role of miR-326 in OP. Second, this study did not establish appropriate animal models to observe the expression pattern of miR-326 during hBMSCs osteogenic differentiation in vivo, evaluate its effectiveness in OP, or further validate the in vitro experimental results.

Conclusion

This study found that miR-326 was reduced in OP and positively correlated with LS, FN, and TH BMD. In vitro experiments revealed a novel mechanism: miR-326 promotes osteogenic differentiation of hBMSCs and inhibits inflammatory responses by directly targeting NAT10, ultimately providing a bone-protective effect. This discovery deepens our understanding of OP pathogenesis and provides a theoretical basis and experimental support for developing novel diagnostic and therapeutic strategies targeting the miR-326/NAT10 axis.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

LQ Z designed this study. GX C and JJ L conducted the experiment and analyzed the data. YB Z wrote the manuscript. XZ H revised the manuscript. All authors reviewed and approved for publication.

Funding

No funds, grants, or other support was received.

Data availability

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

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 Hangzhou Fuyang Hospital of Orthopedics of Traditional Chinese Medicine before the study began. The participants’ right to be informed about the study was ensured and agreed to participate in the study.

Consent for publication

All patients provided written informed consent.

Competing interests

The authors declare no competing interests.