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Research Article

Clinical Cohort Analysis of miR-215-5p as a Diagnostic Biomarker for Osteoarthritis and its Mechanism via EREG in IL-1β-Induced Chondrocyte Model

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DOI:

10.3791/72124

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July 31st, 2026

* These authors contributed equally

In This Article

Summary

This study aims to evaluate serum miR-215-5p as a diagnostic biomarker for osteoarthritis and to investigate its regulatory mechanism in IL-1β-induced chondrocytes.

Abstract

Osteoarthritis (OA) is a degenerative joint disease marked by degradation of joint cartilage and synovial inflammatory infiltration. This study aims to evaluate serum miR-215-5p as a diagnostic biomarker for OA and to investigate its regulatory mechanism in IL-1β-induced chondrocytes. 110 OA patients and 105 healthy controls were recruited. Serum miR-215-5p and EREG expression were determined by quantitative reverse transcription polymerase chain reaction. The diagnostic value of miR-215-5p for OA was assessed using a receiver operating characteristic curve. C28/I2 chondrocytes were stimulated with IL‑1β to construct an OA cell model. Cell viability, migration ability, and inflammatory cytokine levels were evaluated via Cell Counting Kit-8, Transwell assay, and an enzyme-linked immunosorbent assay (ELISA). The association between miR-215-5p and EREG was validated using a dual-luciferase reporter assay and an RNA immunoprecipitation (RIP) assay. In OA patients, serum miR-215-5p was upregulated, while EREG was downregulated. MiR-215-5p exhibited good diagnostic performance for OA (area under the curve, AUC = 0.883). In interleukin-1β (IL-1β)-treated C28/I2 cells, inhibition of miR-215-5p enhanced cell viability and migration, and attenuated IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) levels. EREG was confirmed to be a direct target of miR-215-5p. Knockdown of EREG partially reversed the promoting effect of miR-215-5p downregulation on the viability and migration capacity of IL-1β-treated C28/I2 cells and attenuated its inhibitory effect on inflammatory factors. miR-215-5p contributes to OA pathogenesis by regulating EREG and represents a promising diagnostic biomarker and potential therapeutic target for OA.

Introduction

Osteoarthritis (OA) is a degenerative joint disease, and its pathology is featured by cartilage degeneration with synovial inflammation1,2. Globally, the incidence of OA remains high, and relevant studies have reported a trend of increasing incidence year by year3,4. It has been established that factors such as obesity, aging, genetic susceptibility, and bone density are associated with the development of OA5. In terms of clinical symptoms, patients with OA often present with joint pain, swelling, limited mobility, and stiffness, which severely reduce the quality of life of patients6,7. It is noteworthy that different phenotypes of OA exist, and these phenotypes aptly reflect the diversity of the pathogenesis of the disease8. Currently, the treatment of OA is still centered on the improvement of symptoms, and commonly used clinical medications include analgesics and nonsteroidal anti-inflammatory drugs, which are only capable of relieving pain and cannot effectively reverse the progression of the disease9. Hence, exploring new OA biomarkers has become an urgent need for current research.

MicroRNAs (miRNAs) are a category of short-stranded RNA molecules that are not involved in protein coding10. They serve as important gene regulatory molecules in multicellular organisms and can influence the expression output of numerous protein-coding genes11. Numerous studies have confirmed that miRNAs exert a notable influence on the development of OA12. A previous study showed that miR-215-5p was overexpressed in preeclampsia and reduced the migration and invasive capacity of trophoblasts by regulating CDC6 expression13. Overexpression of miR-215-5p attenuated inflammatory injury in H9c2 cells in sepsis14. miR-215-5p could exert a protective effect in the pathogenesis of osteoporosis by inhibiting the expression of XIAP and promoting the differentiation of hBMSCs15. Notably, miR-215-5p was found to be upregulated when miRNA expression profiles of synovial tissues from OA patients were analyzed16. However, serum miR-215-5p expression and its potential as a diagnostic biomarker for OA have not yet been explored. The role of miR-215-5p in OA chondrocytes, particularly its effects on cell viability, migration, and the inflammatory response, remains unclear. No studies have reported whether miR-215-5p exerts its regulatory function in OA through specific downstream targets. Filling these gaps will provide novel insights into OA molecular mechanisms and identify new candidate therapeutic targets.

EREG is a member of the epidermal growth factor family and has been proven to be involved in tissue repair and inflammatory responses17,18. The study by Chen et al. reported that EREG expression was significantly reduced in cartilage and synovial tissue from OA patients compared with the control group19. However, whether EREG serves as a downstream target of miR-215-5p in OA chondrocytes and the role of this regulatory axis in the pathogenesis of OA remain unclear at present.

Based on the above background, we hypothesize that serum miR-215-5p expression is upregulated in OA patients and drives chondrocyte inflammatory dysfunction by directly targeting EREG, while miR‑215‑5p inhibition relieves interleukin-1β (IL-1β)-induced chondrocyte injury. This study adopted unified measurable endpoints and judgment criteria for clinical and cellular validation: clinically, 110 OA patients and 105 healthy controls were enrolled; serum miR‑215‑5p and EREG levels were quantified via qRT‑PCR, and ROC curves were applied to assess diagnostic performance (valid if AUC ≥ 0.7, P < 0.05). Correlations were analzed via Chi‑square tests between miR‑215‑5p and OA clinical features, and multivariate logistic regression identified independent OA risk factors (adjusted P < 0.05). In vitro, C28/I2 chondrocytes were treated with 10 ng/mL IL‑1β for 24 h to establish an OA model, followed by transfection with miR‑215‑5p inhibitor/mimic and si‑EREG. CCK‑8, Transwell, and ELISA assays were used to evaluate cell viability, migration, and inflammatory cytokine levels. Rescue experiment criteria: miR‑215‑5p inhibitor restored chondrocyte viability and migration (P < 0.05), and EREG silencing partially abrogated this protective effect (P < 0.05). Bioinformatic TargetScan prediction, dual‑luciferase reporter assays, and RIP assays further validated their direct binding: significant reduction in WT‑EREG luciferase activity after miR‑215‑5p mimic transfection and markedly elevated miR‑215‑5p/EREG enrichment in Ago2 precipitates versus IgG control (both P < 0.05) were defined as criteria for target interaction. Collectively, this work systematically clarified the diagnostic value and molecular mechanism of miR‑215‑5p in OA, thereby identifying novel therapeutic candidates.

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Protocol

The study was performed in line with the principles of the Declaration of Helsinki. The Ethics Committee of Xianyou County General Hospital approved the study before it began (Approval No. 202514, on 18 June 2025).

A summary of the protocol is provided in Supplementary Figure 1.

Specimen

A total of 110 OA patients treated at this hospital and 105 healthy controls were recruited for this research. The inclusion criteria for the OA group were as follows: diagnosis of OA confirmed according to the classification criteria established by the American College of Rheumatology20. All patients underwent X-ray imaging and Kellgren-Lawrence (K-L) imaging grading; no antibiotic treatment within 3 months before admission; complete clinical data; and written informed consent was obtained from all patients. The exclusion criteria for the OA group included patients who had received relevant treatment or were in the terminal stages of the disease; those suffering from other types of joint diseases such as gout, rheumatoid arthritis, etc.; those with a combination of tumors, severe hepatic and renal insufficiency, autoimmune diseases, infectious diseases, neurological diseases, or severe cardiorespiratory diseases; and those with conditions of physical disability that prevented them from taking care of themselves. At the time of enrollment, baseline data of all participants were recorded, including age, sex, body mass index (BMI), smoking, drinking, family history, course of disease, K-L grade, Visual Analog Scale (VAS) score, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score, and lesional site. The relevant data were analyzed and summarized in Table 1. All subjects had 8 mL of venous blood drawn in a fasting state, and left to stand for 3 h at 4 °C, then centrifuged at 3000 × g for 15 min at 4 °C to isolate serum. The serum was gently harvested and kept at −80 °C until further testing.

Human blood/serum (Category B infectious biological material) contains human body fluids that may carry unknown infectious pathogens and may cause percutaneous or mucosal infections through splashing, needle pricks, or contact with damaged skin. Must wear disposable nitrile gloves, disposable medical isolation gowns, goggles or face masks, and surgical masks. After being subjected to high-pressure sterilization, it is centrally collected and incinerated by certified medical waste disposal institutions.

Cell culture and OA cell model construction

Human C28/I2 chondrocytes were maintained in DMEM/F12 medium containing 10% fetal bovine serum (FBS) at 37 °C within a humidified incubator filled with 5% CO₂. All experiments were performed using cells at passages 3–6 to maintain a stable cellular phenotype. Subsequent experimental manipulations were conducted once cell confluence reached 80%. Cells in the experimental group were treated with 10 ng/mL of IL-1β at 37 °C for 24 h to establish an in vitro OA model21, while cells in the control group were left untreated.

Cell transfection

Negative control inhibitor (inhibitor-NC), miR-215-5p inhibitor, mimic-NC, miR-215-5p mimic, negative control small interfering RNA (si-NC), and si-EREG were commercially synthesized. Transfection assays were performed in 6-well plates using a transfection reagent during the logarithmic growth phase of the cells. Specifically, 50 nM of the inhibitor/mimic or negative controls was diluted in 125 µL of Opti-MEM medium per well, combined with 5 µL of transfection reagent dissolved in 125 µL of Opti-MEM, and kept at room temperature for 15 min to form a complex. The mixture was then added to the cells. Cells were harvested 48 h after transfection for subsequent experiments.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR assay)

Total RNA was isolated using TRIzol reagent following the manufacturer’s protocols, and samples included serum and cells. For reverse transcription, 1 µg of total RNA was used in each reaction, in accordance with the manufacturer’s protocol of PrimeScript Reverse Transcriptase. After completion of reverse transcription, amplification reactions were carried out on a real-time PCR instrument using SYBR-Green Real Master mix as the reaction reagent. The thermal cycling parameters were as follows: an initial denaturation at 95 °C for 30 s, then 40 cycles of 95 °C for 5 s and 60 °C for 30 s. qRT‑PCR was performed in technical triplicate for all samples. For quantitative detection of miRNA and mRNA levels, cycle threshold (Ct) values were normalized using U6 and β-actin as endogenous references, and the final data analysis was quantified with the 2-ΔΔCT method. The primer sequences utilized for qRT-PCR are presented in Supplementary Table 1.

TRIzol reagent is a hazardous chemical. It is highly corrosive; direct contact can cause severe burns to the skin and eyes, and its volatile fumes can irritate the respiratory tract. It is necessary to wear chemical-resistant nitrile gloves, laboratory coats, and chemical splash-proof goggles; it can only be used in a fume hood. The waste liquid is stored in sealed plastic chemical waste containers with labels and is properly disposed of in accordance with local regulations.

Cell viability assay

Cell viability was evaluated via the Cell Counting Kit-8 (CCK-8). Cells were plated into 96-well plates at 5 × 103 cells/well. At 0, 24, 48, and 72 h of incubation, 10 µL of CCK-8 solution was added to each well. After incubating the cells at 37 °C for 1 h, the optical density of each well was measured at 450 nm.

Transwell assay

Cell migration capacity was evaluated using a Transwell chamber with 8 µm pore-size membranes. Briefly, 5 × 104 cells/well were introduced into the upper inserts containing serum-free medium, while the lower inserts were then supplemented with complete medium. The chambers were incubated at 37 °C for 24 h. Cells that did not migrate, remaining on the upper membrane surface, were gently wiped off with a cotton swab. Cells that migrated to the lower surface were fixed with 4% formaldehyde for 10 min and stained with 0.1% crystal violet for 20 min. Finally, the cells that penetrated the membrane were observed and counted using an optical microscope (×200). For each model, five non-overlapping fields of view were randomly selected for counting. The average number of migrating cells in each experimental group was calculated and recorded.

Formaldehyde (carcinogen, skin/respiratory irritant, volatile toxic organic compound). Its volatile gas can cause mucosal irritation, dermatitis, and allergic reactions. All operations must be carried out in a well-ventilated fume hood. Wear chemical-resistant gloves, a laboratory coat, anti-fog goggles, and an activated carbon respirator. Waste containing PFA should be treated in accordance with hazardous waste regulations. Waste should be disposed of in accordance with local regulations.

Crystal violet is a biological stain. It is toxic when absorbed through the skin; it has long-term harmful effects on the environment. Avoid direct contact with skin and random discharge. Wear disposable nitrile gloves and a laboratory coat.

Enzyme-linked immunosorbent assay (ELISA)

IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) levels were measured via human ELISA kits following the operational guidelines specified in the product manual.

Dual luciferase reporter gene assay

The TargetScan database was utilized to predict candidate target genes of miR-215-5p. The interaction of miR-215-5p with the target gene EREG was analyzed via the dual luciferase assay kit: the wild-type and mutant sequences of EREG were amplified and cloned into the pGL3 vector. Subsequently, constructed recombinant plasmids were co-transfected into C28/I2 cells with mimic-NC or miR-215-5p mimic, respectively. Luciferase activity was detected after 48 h of incubation.

RNA immunoprecipitation (RIP)

The interaction between miR-215-5p and EREG was validated via EZ-Magna RIP Kit. The steps were as follows: cells were first lysed with RIP lysate. Subsequently, cell lysates were co-incubated with magnetic beads conjugated to anti-argonaute 2 (Ago2) or anti-immunoglobulin G (IgG) antibodies (human) in RIP buffer. Then, the RNA was treated with proteinase K and purified. Finally, miR-215-5p and EREG levels were measured using qRT-PCR.

Statistics

Data analysis and image plotting were performed using SPSS and GraphPad Prism. The Shapiro-Wilk test was applied to assess the normality of distribution before statistical analysis. Measurements following a normal distribution were expressed as mean ± standard deviation (SD). Student's t-test was used for two-group comparisons, while analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used for multiple-group analyses. Non-normally distributed continuous variables were reported as medians and interquartile ranges (IQR) and compared using the Mann-Whitney U test. Categorical variables were expressed as percentages (n (%)), and intergroup differences were examined via the chi-square test. The diagnostic efficacy of miR-215-5p for OA was assessed using receiver operating characteristic (ROC) curves. The association between miR-215-5p and clinical characteristics of OA patients was evaluated via the chi-square test. Risk factors for the development of OA were explored using multivariable logistic regression. The association between miR-215-5p and EREG was assessed through Spearman correlation analysis. P < 0.05 was considered statistically significant.

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Results

Baseline characteristics

105 patients were included in the control group, including 58 (55.24%) males and 47 (44.76%) females, with a mean age of 52.81 ± 12.57 years and a BMI of 24.25 ± 2.71 kg/m2. The OA group contained 110 patients, 69 (62.73%) males and 41 (37.27%) females, with a mean age of 54.42 ± 12.33 years and a BMI of 24.88 ± 3.51 kg/m2. Additional baseline information for the OA group was presented in Table 1.

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Discussion

OA, a prevalent joint disorder in clinical settings, has become the third most important cause of organic disability after diabetes and dementia22,23. However, the pathogenesis of OA is complex, and its diagnosis often requires a combination of clinical symptoms, imaging examinations, and other means, and the lengthy diagnostic cycle seriously delays the timely treatment of patients24. Thus, it is clinically important to explore biomarkers...

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Disclosures

The authors have no financial or proprietary interests in any material discussed in this article.

Acknowledgements

The authors received no specific funding for this work.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
96-well platesCorning/USA3903
Anti-Ago2Abcam/USAAB_867543
RRID: ab186733
Anti-IgGAbcam/USAAB_10673832
RRID: ab133470
C28/I2 chondrocytesMerck Millipore/USASCC043
CCK-8 KitBeyotime/ChinaC0038Cell viability assay kit
Crystal violetSigma-Aldrich/USAV5265
DMEM/F12 mediumGibco/USA11320033
Dual-Luciferase Reporter Assay KitPromega/USAE1910
EZ-Magna RIP KitMerck Millipore/USA17701
FBSThermo Fisher Scientific/USA26010074
FormaldehydeSigma-Aldrich/USAF8775
Graphpad Prism 9.0GraphPad Software Inc. /USAhttps://www.graphpad.com/features
IL-1βSigma-Aldrich/USASRP3083
IL-1β  ELISA kitNanjing Jiancheng/ChinaH002-1-2
IL-6  ELISA kitNanjing Jiancheng/ChinaH007-1-2
IncubatorThermo Fisher Scientific/USABB150-2TCS-L
Inhibitor-NCGenePharma (custom synthesis) /Chinahttps://www.genepharma.com/
Light Cycler 480Roche/Switzerlandhttps://lifescience.roche.com/global/en/article-listing/article/lightcycler-480-system-technology.htmlReal-time PCR instrument
Lipofectamine 3000Invitrogen/USAL3000015The transfection reagent used in cell transfection
Microplate readerThermo Fisher Scientific/USAVA000010C
MicroscopyNikon/Japanhttps://www.nikon.com.cn/sc_CN/
Mimic-NCGenePharma (custom synthesis) /Chinahttps://www.genepharma.com/
MiR-215-5p inhibitorGenePharma (custom synthesis) /Chinahttps://www.genepharma.com/
MiR-215-5p mimicGenePharma (custom synthesis) /Chinahttps://www.genepharma.com/
Opti-MEMGibco/USA31985070
pGL3 vectorPromega/USAE1751
PrimeScript Reverse TranscriptaseTakara/Japan2680Reverse transcription kit
Si-EREGGenePharma (custom synthesis) /Chinahttps://www.genepharma.com/
Si-NCGenePharma (custom synthesis) /Chinahttps://www.genepharma.com/
SPSS 24.0IBM Corp/USAhttps://spssau.com/index.html
SYBR Green SupermixBio-Rad/USA1725121
TNF-α  ELISA kitNanjing Jiancheng/ChinaH052-1-2
Transwell chamberCorning/USA3422
TRIzol reagentThermo Fisher Scientific/USA15596026RNA isolation reagent

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Osteoarthritis BiomarkerEREG ExpressionIL-1β Chondrocyte ModelSerum miRNADiagnostic PerformanceCell Viability AssayInflammatory CytokinesDual-Luciferase AssayRNA Immunoprecipitation