Elevated miR-409-5p may promote the progression of osteoarthritis by targeting DLST as a potential biomarker function of miR-409-5p in osteoarthritis

Abstract

Background

Osteoarthritis (OA) significantly impairs the quality of life of middle-aged and elderly individuals. MicroRNAs (miRNAs) are known to play key regulatory roles in OA progression.

Aims

To investigate the potential role mechanisms of miR-409-5p in OA.

Methods

This study enrolled 93 OA patients and 75 non-OA controls. The expression levels of miR-409-5p, DLST, and OA-related markers were measured using RT-qPCR or Western Blot. The targeting relationship between miR-409-5p and DLST was verified by dual luciferase reporter assays. Cell viability was assessed with the CCK-8 assay, and inflammatory cytokine concentrations were measured via ELISA.

Results

Serum miR-409-5p expression was higher in OA patients than in controls and showed good diagnostic value for OA. Inhibiting miR-409-5p suppressed IL-1β-induced viability in CHON-001 cells, promoted aggrecan expression, suppressed MMP13 expression, and reduced secretion of IL-6 and IL-8. MiR-409-5p directly targeted DLST, and their expression levels were negatively correlated. Knockdown of DLST abolished the beneficial effects of miR-409-5p inhibition on cell viability, extracellular matrix degradation, and inflammatory responses.

Conclusions

MiR-409-5p was highly expressed in OA and may promote disease progression by targeting DLST.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13018-026-06687-6.

Introduction

Osteoarthritis (OA) is a degenerative joint disease commonly found in the elderly and can lead to disability [1]. According to the Global Burden of Disease (GBD) report, around 595 million people worldwide were affected by arthritis in 2020, and this number continues to increase [2]. Historically, OA was regarded as a simple “wear and tear” condition; however, advances in modern medicine have deepened our understanding of its complexity [3, 4]. The pathogenesis of OA involves a cascade of events, including progressive cartilage loss, chronic synovitis, and hardening of the underlying subchondral bone [5]. Inflammatory mediators interleukin-1β (IL-1β), IL-6, and IL-8 could promote OA progression by inducing chondrocyte apoptosis and extracellular matrix (ECM) degradation [6]. Clinically, OA typically presents as joint pain, swelling, stiffness, limited mobility, and joint deformity, with symptoms progressing gradually over time [7]. Current diagnostic approaches rely mainly on clinical symptoms and imaging, yet these methods often fail to detect early-stage OA [8]. Without timely diagnosis and intervention, patients with severe OA may require total joint replacement, which imposes significant challenges to daily living and substantial economic burdens [9]. Therefore, identifying biomarkers that could facilitate the diagnosis and monitoring of OA progression is an urgent priority.

Non-coding RNAs (ncRNAs) play a crucial regulatory role in the pathogenesis and progression of musculoskeletal disorders. Research indicates their broad potential in elucidating disease mechanisms, including tendon homeostasis, osteoporosis (OP), and rheumatoid arthritis (RA) [10–13]. Among these, microRNAs (miRNAs) exert key functions in multiple musculoskeletal disease processes [14]. For instance, miR-204-5p delays intervertebral disc degeneration by targeting the SSRP1/NF-κB pathway, while miR-217 and miR-106a-5p participate in osteoporosis progression by regulating OPG/RANKL/RANK or PTEN signalling pathways [15–17]. Notably, certain miRNAs have demonstrated potential therapeutic value in in vitro studies of tendon injuries [18]. Concurrently, long non-coding RNAs (lncRNAs) such as CRNDE and HCG18 influence fracture healing and the pathological progression of spinal tuberculosis through miRNA-binding mechanisms [19, 20].

In the context of OA, the role of miRNAs is gaining increasing recognition. Recent studies suggest miRNAs possess potential functions in alleviating OA [21, 22]. Meta-analyses further indicate that miRNAs demonstrate high accuracy in diagnosing OA, rendering them viable as non-invasive biomarkers [23]. Mechanistically, miRNAs primarily exert post-transcriptional negative regulation by binding to mRNAs, thereby participating in the pathogenesis of OA [24]. With advances in detection technologies, a growing number of miRNAs have been implicated in the regulation of OA progression [25]. For instance, miR-146a-5p is up-regulated in OA cartilage and may contribute to disease development by suppressing CXCR4 expression [26]. Similarly, miR-151a-3p is elevated in the OA patients and may regulate cell viability and apoptosis through targeting SOX9 [27]. Moreover, miR-4701-5p has been shown to alleviate inflammatory injury in OA cell models by down-regulating HMGA1, suggesting its potential as a therapeutic target [28]. Another miRNA, miR-409-5p, acts as an osteogenesis inhibitor and has been reported to be up-regulated in OA cartilage [29, 30]. Nevertheless, its specific function in OA remains poorly understood.

Therefore, this study focuses on miR-409-5p to evaluate its diagnostic value in OA and to explore its role in chondrocyte viability, ECM degradation, and inflammatory responses.

Materials and methods

Study subjects

This study enrolled 93 patients diagnosed with OA at West China Hospital, Sichuan University, between January 2022 and June 2024. In addition, 75 age-matched healthy controls without joint inflammation or other systemic diseases were recruited from the hospital’s health examination center. This study was approved by the Ethics Committee of West China Hospital, Sichuan University. All patients and their families were informed and provided written informed consent. The diagnosis of OA was established based on published criteria [31], which required the presence of recurrent knee pain in the past month plus at least two of the following: (1) joint space narrowing, capsular degeneration on standing radiographs, or subchondral sclerosis; (2) morning stiffness ≤ 30 min; (3) crepitus or friction sensation during movement. OA group exclusion criteria: (1) Kellgren-Lawrence grade (K-L grade) IV; (2) presence of bone resorption, bone metastases from any cancer, autoimmune diseases, or other inflammatory arthritis; (3) history of neurological or psychiatric disorders.

Baseline data collection

Fasting blood was collected from subjects, left to stand for 2 h, centrifuged, and the supernatant obtained and stored at -80℃. Baseline data for all subjects were collected and recorded, including age, BMI, gender, and smoking and drinking habits. For OA patients, disease duration was additionally recorded, including VAS score, WOMAC score, K-L grade, and disease site.

Cell culture

The present study utilized the CHON-001 human chondrocyte line, procured from the American Type Culture Collection (ATCC, USA). Cells were cultured in DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin-streptomycin mixture (Solarbio, China). Cultures were maintained in humidified air supplemented with 5% CO₂ at 37℃. Following preliminary experiments and reference to prior studies, an OA cell model was established by stimulating CHON-001 cells with 10 ng/mL IL-1β for 24 h [32, 33]. Cell transfection.

The miR-409-5p mimic, miR-409-5p inhibitor, and si-Dihydrolipoamide S-succinyltransferase (DLST) were purchased from Ribobio Biotechnology (Guangzhou, China). Lipofectamine 3000 reagent (ThermoFisher, USA) was used for transfection experiments. Medium was replaced 6 h post-transfection.

RT-qPCR

Total RNA was extracted from serum and cells with Trizol reagent (Takara Bio, Japan). Reverse transcription was performed using a commercial kit (ThermoFisher, USA), followed by RT-qPCR with SYBR Green (Beyotime, China). The small nuclear RNA U6 and GAPDH were employed as endogenous references for miRNA and mRNA assays, respectively. Relative expression levels were calculated based on the 2^–ΔΔCt algorithm. The primers for RT-qPCR were shown in Supplementary Table 1.

Cell viability assay

Cells were seeded in 96-well plates at 5 × 10³ cells/well. CCK-8 reagent (Solarbio, China) was added at 0, 24, 48, and 72 h, and absorbance was measured after 2 h of incubation.

Western blot (WB)

Processed CHON-001 cells were harvested and lysed using high-efficiency RIPA lysis buffer (R0010, SolarBio, China). Following centrifugation, samples underwent SDS-polyacrylamide gel electrophoresis and were transferred onto PVDF membranes. Protein expression levels of aggrecan, matrix metallopeptidase 13 (MMP13), and DLST were detected using WB technology.

ELISA

The concentrations of inflammatory cytokines IL-6 and IL-8 were detected using an ELISA kit (Beyotime, China).

Dual luciferase reporter assay

Binding sites of miR-409-5p on DLST were predicted using ENCORI and TargetScan. Luciferase reporter plasmids for DLST wild-type (DLST-WT) and mutant (DLST-MUT) were provided by Wuhan GeneCreate Biotechnology Co., Ltd. Reporter plasmids were co-transfected into CHON-001 cells with miR-409-5p mimics or inhibitors. Luciferase activity was measured using a dual luciferase assay kit (GeneCreate, China), with Renilla luciferase as the internal control.

Statistical analysis

The experimental data were processed using GraphPad 8.0 and SPSS 27.0. Continuous variable outcomes are reported as means ± standard deviation. For inter-group comparisons, the independent samples t-test was applied to compare two sets of data, and ANOVA was employed to assess differences among multiple groups. The chi-square test was used to analyze categorical variables. ROC curves were plotted to evaluate the diagnostic value of miR-409-5p for OA. Correlation analysis was performed using Pearson correlation analysis. Each experiment was conducted in triplicate as independent biological replicates. P < 0.05 was statistically significant.

Results

Baseline information of study subjects

No significant differences were observed between the control group and OA patients in terms of age, BMI, gender, smoking, and drinking habits. OA patients generally had a longer disease duration. Information regarding VAS scores, Lysholm scores, WOMAC scores, K-L grades, and affected joint locations was described in Table 1.

Table 1.

Comparison of basic information

	Healthy (n = 75)	Osteoarthritis patients (n = 93)	χ2/F	P value
Age (years)	56.89 ± 6.64	57.38 ± 7.11	0.203	0.653
BMI (kg/m²)	23.68 ± 3.11	24.46 ± 3.48	2.214	0.139
Gender, n (%)			1.275	0.259
male	38 (50.7)	39 (41.9)
female	37 (49.3)	54 (58.1)
Smoking, n (%)			0.898	0.343
Yes	41 (54.7)	44 (47.3)
No	34 (45.3)	49 (52.7)
Drinking, n (%)			1.110	0.292
Yes	35 (46.7)	51 (54.8)
No	40 (53.3)	42 (45.2)
Course of disease (months)	/	37.47 ± 14.46	/	/
VAS score	/	5.43 ± 1.11	/	/
Lysholm knee score	/	57.89 ± 10.32	/	/
WOMAC score	/	50.84 ± 9.76	/	/
K-L grade, n (%)
I	/	37 (39.8)	/	/
II	/	39 (41.9)	/	/
III	/	17 (18.3)	/	/
Disease site, n (%)
Left knee	/	42 (45.2)	/	/
Right knee	/	37 (39.8)	/	/
Both knees	/	14 (15.1)	/	/

BMI, body mass index; VAS score, Visual Analogue score; WOMAC score, Western Ontario and McMaster Universities Arthritis Index; K-L grade, Kellgren-Lawrence grade

Expression and diagnostic value of miR-409-5p in OA serum

Quantification via RT-qPCR revealed that miR-409-5p expression was significantly elevated in the serum of OA patients compared to the control group (Fig. 1A). ROC curve analysis indicated that miR-409-5p exhibits good diagnostic value for OA (Fig. 1B, area under the curve (AUC) = 0.844, sensitivity = 80.65%, specificity = 76.00%).

Fig. 1.

Elevated miR-409-5p served as a diagnostic marker for OA. A RT-qPCR detected the expression level of miR-409-5p in serum. B ROC curve evaluated the diagnostic role of miR-409-5p for OA. ***P < 0.001

Effects of miR-409-5p on chondrocyte viability, ECM degradation, and inflammatory response

Preliminary experiments revealed that the relative expression levels of miR-409-5p progressively increased with rising IL-1β concentrations and prolonged stimulation duration. Notably, the most pronounced change in miR-409-5p occurred when CHON-001 cells were stimulated with 10 ng/mL IL-1β for 24 h (Fig. 2A-B). Consequently, this condition was adopted to establish the OA cell model for subsequent investigations. Following the establishment of an OA cell model using IL-1β, miR-409-5p expression increased while cell viability markedly decreased. Co-incubation with a miR-409-5p inhibitor effectively suppressed miR-409-5p expression and restored cell viability (Fig. 2C-D). Moreover, IL-1β stimulation reduced Aggrecan expression while increasing MMP13 expression. Following transfection with miR-409-5p inhibitor, Aggrecan expression recovered, and MMP13 expression was suppressed (Fig. 2E-F). ELISA results indicated that IL-1β stimulation elevated IL-6 and IL-8 concentrations; following transfection with miR-409-5p inhibitor, IL-6 and IL-8 concentrations decreased (Fig. 2G).

Fig. 2.

Knockdown of miR-409-5p inhibited IL-1β-induced chondrocyte injury. A RT-qPCR detected the effect of different concentrations of IL-1β on the expression level of miR-409-5p. B RT-qPCR was conducted to examine the effects of different stimulation durations on miR-409-5p expression. Following IL-1β stimulation and transfection with miR-409-5p inhibitor, changes in miR-409-5p expression C, cell viabilityD, ECM degradation-related genes E-F, and inflammatory factors G were assessed. *P < 0.05, **P < 0.01, ***P < 0.001

MiR-409-5p may target DLST

ENCORI and TargetScan 8.0 predictions indicate that miR-409-5p binds to the DLST (Fig. 3A). Dual luciferase reporter assays revealed that transfection with miR-409-5p mimic or inhibitor altered luciferase activity in DLST-WT, while having no discernible effect on DLST-MUT (Fig. 3B). The quantification by RT-qPCR revealed that DLST expression in serum from OA patients was significantly reduced compared to the control group (Fig. 3C). Analysis of the correlation quantified by Pearson’s coefficient revealed a strong negative association linking miR-409-5p with DLST (Fig. 3D, r = -0.699). Furthermore, the relative expression level of DLST decreased with increasing IL-1β concentration and prolonged exposure time (Fig. 3E-F). Transfection with miR-409-5p inhibitor increased DLST expression under IL-1β stimulation (Fig. 3G-H).

Fig. 3.

MiR-409-5p may have a target relationship with DLST. A Predicted binding sites between miR-409-5p and DLST. B A dual luciferase assay was used to detect the targeting relationship between miR-409-5p and DLST. C RT-qPCR was used to detect the expression of DLST in serum. D Pearson correlation analysis was used to assess the correlation between miR-409-5p and DLST. E RT-qPCR detected the effect of different IL-1β concentrations on DLST expression. F RT-qPCR detected the effect of different stimulation times on DLST expression. G-H RT-qPCR and WB detected changes in DLST expression after IL-1β stimulation and transfection with miR-409-5p inhibitor. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant

MiR-409-5p may regulate chondrocyte function via DLST

Transfection with miR-409-5p inhibitor increased DLST expression, while subsequent transfection with si-DLST reduced DLST expression (Fig. 4A-B). CCK-8 assay revealed that miR-409-5p inhibitor inhibition suppressed IL-1β-stimulated cell viability, whereas si-DLST transfection partially restored viability (Fig. 4C). Moreover, transfection with miR-409-5p inhibitor increased Aggrecan expression and decreased MMP13 expression, while transfection with si-DLST reversed these changes (Fig. 4D). ELISA results showed that transfection with miR-409-5p inhibitor decreased IL-6 and IL-8 concentrations, while transfection with si-DLST reversed these changes (Fig. 4E-F).

Fig. 4.

MiR-409-5p may regulate chondrocyte function through DLST. IL-1β stimulation combined with miR-409-5p inhibitor and si-DLST transfection: changes in DLST expression A-B, cell viability C, ECM degradation-related genes D-E, and inflammatory factors F. *P < 0.05, **P < 0.01, ***P < 0.001

Discussion

OA is a prevalent degenerative joint disorder characterized by dysregulated chondrocyte metabolism and sustained inflammatory responses [34]. As a leading cause of disability worldwide, OA imposes substantial medical and socioeconomic burdens [35]. Inflammatory cytokines contribute to OA pathogenesis by inducing chondrocyte apoptosis and disrupting ECM metabolism [36]. MiRNAs, a class of small non-coding RNAs, have attracted increasing attention in OA research [14, 37]. Accumulating evidence indicates that multiple miRNAs participate in OA progression by modulating inflammatory responses and cartilage homeostasis [37]. For example, miR-335-5p is highly expressed in OA and may exacerbate disease severity by inhibiting VCAM1 expression [38]. In contrast, down-regulation of miR-23b-3p alleviates chondrocyte injury through up-regulation of COL11A2, thereby attenuating OA progression [32]. Similarly, studies have found that miR-296-5p inhibits chondrocyte apoptosis and cartilage degradation by directly regulating the TGF-β1/CTGF/p38MAPK signaling cascade [39].

MiR-409-5p is a miRNA that is upregulated in multiple diseases, including diabetes and breast cancer [40, 41]. Notably, prior studies have indicated that miR-409-5p may promote the development and bone metastasis of human prostate cancer [42]. Importantly, miRNA sequencing experiments revealed the up-regulation of miR-409-5p in cartilage tissue from OA patients [30]. However, its specific functional role and molecular mechanisms in OA remain unclear. Therefore, this study first explores the role of miR-409-5p in OA and its molecular mechanisms. Currently, several miRNAs have been identified as potential diagnostic biomarkers for OA. For instance, miR-151a-3p (AUC = 0.921) and miR-320c (AUC = 0.942) exhibit high diagnostic accuracy [27, 43]. In this study, we found miR-409-5p also possesses significant diagnostic potential (AUC = 0.844). Previous studies have demonstrated that miR-409-5p overexpression may inhibit osteoblast differentiation by targeting Lrp-8, suggesting its role as an osteogenesis inhibitor [29]. Therefore, we ventured to hypothesise that miR-409-5p may have an important role in the pathogenesis of OA.

Studies have revealed that inflammatory mediators such as IL-1β could induce chondrocyte dysfunction, leading to accelerated degradation of key ECM components and exacerbating OA [44, 45]. Aggrecan serves as an essential factor for promoting chondrocyte differentiation, reflecting the degradation and formation of joint tissue [46]. MMP13 possesses a specific ability to cleave type II collagen, making it one of the primary factors contributing to ECM degradation [47]. Alterations in the expression levels of these two genes indirectly reflect the severity of OA. Disruption of ECM homeostasis represents a core pathological feature of OA, and multiple miRNAs have been shown to participate in this process by targeting specific mRNAs [48]. For instance, overexpression of miR-8485 may mitigate IL-1β-induced chondrocyte damage by suppressing CRLF1 expression [49]. In contrast to miR-8485, this study suggested that inhibiting miR-409-5p may promote chondrocyte viability while alleviating inflammatory responses and ECM degradation processes.

Furthermore, our study provides evidence of a potential binding relationship between miR-409-5p and DLST. DLST participates in the tricarboxylic acid cycle and plays a crucial role in cellular energy metabolism [50]. It is noteworthy that cuproptosis has been found to play a significant role in the development and progression of OA [51]. As one of the cuproptosis-related genes, DLST is highly likely to be involved in OA progression [52]. Furthermore, previous studies have revealed that DLST expression is down-regulated in both RA and OA [53, 54]. The findings of this study supported the hypothesis that knockdown of DLST reversed the beneficial effects resulting from miR-409-5p inhibition, including improvements in cellular viability, cartilage matrix metabolism, and inflammatory responses. Therefore, we hypothesize that miR-409-5p contributes to OA pathogenesis, at least in part, through regulating DLST.

Despite these insights, the present study exhibits certain limitations. Firstly, the analysis focused solely on peripheral blood samples from OA patients, lacking validation at the tissue level (such as synovial fluid or cartilage tissues), which may restrict the generalisability of conclusions. Moreover, the absence of WB analysis on patient samples could undermine the integrity of findings. Secondly, the research was confined to the cellular level without validation through animal models. Finally, the study stopped at establishing correlations between the miR-409-5p/DLST axis and phenotypic alterations, without delving into DLST downstream pathways. Moreover, the cellular function experiments only assessed cell viability, lacking direct proliferation assays for validation. Future research should complement these findings by measuring miR-409-5p and DLST expression levels in patient synovial fluid or cartilage tissues. Furthermore, establishing an osteoarthritis animal model is essential to validate the miR-409-5p/DLST axis’s mechanism of action under in vivo conditions. Additionally, the precise downstream mechanisms through which DLST influences chondrocyte function and inflammatory responses warrant further investigation.

In summary, this study suggested that the up-regulation of miR-409-5p may serve as a potential diagnostic biomarker for OA. Furthermore, this research proposed for the first time the hypothesis that the miR-409-5p/DLST axis may play a role in osteoarthritis.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

JP Z, QY W, Y Z and H Z designed this study. JP Z, QY W, Q Y, BH Z and H Z conducted the experiment and analyzed the data. JP Z and QY W wrote the manuscript. H Z revised the manuscript. All authors reviewed and approved for publication.

Funding

The authors did not receive support from any organization for the submitted work.

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 authors state that they have obtained West China Hospital, Sichuan University review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Consent for publication

All patients provided written informed consent.

Competing interests

The authors declare no competing interests.