Mechanical response microRNA-145a-5p alleviates osteoarthritis by inhibiting inflammation and promoting chondrogenesis

Animal experiments

After inducing anesthesia via intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight), followed by a stabilization period of ≥10 min to ensure surgical anesthesia, destabilization of the medial meniscus (DMM) or sham surgery was performed on forty 8-week-old male C57BL/6J mice (obtained from the department of laboratory animal science of Peking University Health Science Center) using standardized surgical protocols, as previously described (Glasson, Blanchet & Morris, 2007). The protocol was complied with the Guide for the Care and Use of Laboratory Animals published by the National Academy Press (National Institutes of Health Publication No. 85–23, revised 1996), and the procedures involving animal experiments were reviewed and approved by the Peking University Biomedical Ethics Committee (Grant number: PUIRB-LA2022629). The mice were placed separately (five mice per cage) and kept at ambient temperature (23 ± 3 °C) with 12-h light/dark cycle and free access to a standard pellet diet and water. For agomir delivery, eighteen mice in the DMM group were randomly assigned to experimental groups (DMM+NC-agomir, DMM+agomir-145a-5p, OA) using a computer-generated random number sequence, and intra-articular injection of 10 µg of agomir-NC (miR-NC, GenePharma, Suzhou, China), or agomir-145a-5p (B06023; miR-145a-5p, GenePharma, Suzhou, China) at 1-, 3-, 5-, and 7-weeks post-operation, the flowchart was shown in Fig. S1. Nine weeks after DMM surgery, the mice were anesthetized with pentobarbital sodium (50 mg/kg body weight) for at least 10 min, followed by humane euthanasia via cervical dislocation to minimize suffering, and then their knee joints were collected. No surviving animals remained at study conclusion.

Histological, immunofluorescence, and RNA FISH analyses

Mouse knee joints were collected and fixed immediately with 4% paraformaldehyde for 48 h, decalcified with 10% EDTA for 10 days, dehydrated for paraffin embedding, and continuous sagittal cross-sections sequentially from medial to lateral orientations were collected. These sections were cut to a thickness of 5 microns, dewaxed, and stained with hematoxylin and eosin (HE), toluidine blue, safranin O and fast green. HE and safranin O and fast green stained tissue sections were used for OARSI scoring. The OARSI scoring system 50 was performed by two observers blinded to the experimental groups to evaluate cartilage destruction in the medial joints (Arden et al., 2021). Immunofluorescence (IF) staining was performed on paraffin-embedded joint sections or cultured cells. After antigen retrieval (for tissues) or permeabilization (for cells), samples were blocked with 5% BSA and 10% normal goat serum, followed by incubation with primary antibodies against YAP (1:50, sc-101199), MMP13 (1:50, 18165-1-AP), and COL2A1 (1:100, ARG20787) overnight at 4 °C. Species-specific Alexa Fluor-conjugated secondary antibodies (1:500, 4408S/8889; Cell Signaling Technology, Danvers, MA, USA) were applied for 1 h at room temperature, and nuclei were counterstained with DAPI (C0065; Solarbio, Beijing, China). The RNA FISH assay was performed via an RNA FISH kit (GenePharma, Suzhou, China) according to the manufacturer’s instructions. Images were acquired on a ZEISS LSM 880 with Airyscan (Carl Zeiss Microscopy GmbH, Jena, Germany). Fluorescence intensity was measured in 3–5 microscopic fields per joint using ImageJ software, with all analyses conducted by an operator blinded to the group identity. The median value for each mouse was then calculated and reported (Zhang et al., 2022).

Cell culture and treatment

ATDC5 cells (American Type Culture Collection, ATCC, Manassas, VA, USA) were maintained at 37 °C in a humidified incubator with 5% CO₂ and were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, SV30208.02; HyClone, Logan, UT, USA) before differentiation. To induce chondrogenic differeclenium (NO. 41400045; Thermo Fisher, Waltham, MA, USA), was added to the growth medium of ATDC5 cells, and alcian blue staining and q-PCR were performed after 7 days. For the treatment of IL-1β, 10 ng/ml IL-1β (MCE, HY-P7073A) was added to the chondrogenic differentiation induction medium of ADTC5. The medium was changed every 2 days (Shen et al., 2017).

Primary human articular chondrocytes (HACs) were isolated from macroscopically non-lesional cartilage fragments obtained during arthroscopic procedures, following written informed consent under protocols approved by the Peking University Third Hospital Medicine Science Research Ethics Committee (grant number: M2023779). Tissues were processed within 4 h. After thorough washing and dissection to isolate the chondral layer, fragments were minced and digested with 0.2% collagenase type II (17101015; Gibco, Waltham, MA, USA) in DMEM overnight (37 °C, 5% CO₂). The resulting suspension was filtered (100 µm), washed extensively in PBS, and pelleted cells were resuspended in DMEM supplemented with 15% fetal bovine serum (SV30208.02; FBS, HyClone, Logan, Utah, USA) and 1% penicillin-streptomycin (C0222; Beyotime, Shanghai, China). Cells were cultured at 37 °C, 5% CO₂ and used at passage 1 (P1) or P2 for experiments to minimize dedifferentiation (Gan et al., 2024).

Cyclic stretch stress

ATDC5 cells in three groups (Ctrl, 10% Stretch and 20% Stretch) were cultured in BioFlex 6-well culture plates precoated with type I collagen for 24 h. When the ATDC5 cells reached a density of 50–60%, they were stretched with a Flexcell-4000T vacuum stretching device at an amplitude of 10% (normal mechanical stimulation, 10% stretch) or 20% (Huang et al., 2025) (excessive mechanical stimulation, 20% stretch) elongation and a frequency of 0.5 Hz for 4 h per day for 4 days (Na et al., 2024), ctrl group keep static in the same incubator.

Transfection of miR-145a-5p mimics, inhibitors, and negative controls

When ATDC5 cells in 12-well plates reached 30–40% confluence, 50 nM mmu-miR-145a-5p mimics (miR-mimic), inhibitors (miR-inhibitor), or negative controls (miR-NC) (all purchased from GenePharma) were transfected into ATDC5 cells via Lipofectamine 3000 (L3000008; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, after which the medium was changed to chondrogenic differentiation induction medium after 6 h.

RNA extraction and quantitative real-time PCR

Total RNA was extracted via TRIzol reagent (15596026CN; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The RNA concentration and purity were assessed via a Nanodrop spectrophotometer (ND-1000; Thermo Scientific, Waltham, MA, USA). The values of A260/A280 ratio between 1.8 and 2.0 indicating high purity. Reverse transcription reactions were performed using two distinct protocols for mRNA and miRNA synthesis. For mRNA cDNA synthesis, total RNA samples (2 μg) were reverse transcribed with GoScript™ Reverse Transcriptase (A2800; Promega, Madison, WI, USA) following the manufacturer’s protocol. The reaction system (20 μL final volume) contained 2 μg of purified total RNA samples, 4 μL GoScript™ Reaction Buffer (Random Primer), 2 μL GoScript™ Enzyme Mix, and nuclease-free water. Thermal cycling conditions were programmed as follows: primer annealing at 25 °C for 5 min, cDNA synthesis at 42 °C for 60 min, and enzyme inactivation at 70 °C for 15 min. For miRNA-specific cDNA synthesis, the miRNA First Strand cDNA Synthesis Kit (B532451; Sangon Biotech, Shanghai, China) was employed. A 20 μL reaction mixture containing 2 μg total RNA, 10 μL 2× miRNA P-RT Solution Mix, and 2 μL miRNA P-RT Enzyme Mix was subjected to the following thermal profile: 37 °C for 60 min followed by enzyme denaturation at 85 °C for 5 min. All cDNA products were stored at −20 °C for subsequent analysis. Quantitative real-time PCR was performed using an ABI 7500 system (Applied Biosystems, Foster City, CA, USA) with NovoStart® SYBR Green SuperMix Plus (E166-01B; Novoprotein, Suzhou, China). Each 20 μL reaction contained 2 μL cDNA template, 10 μL SYBR Green Master Mix, 1 µL (200 nM) forward/reverse primers (Sangon Biotech, Shanghai, China sequences in Table S1), and 7 µL nuclease-free water. The thermal protocol consisted of an initial denaturation at 95 °C for 1 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The expression values were normalized to those of Gapdh or U6 via the 2^−ΔΔCt method (Kan et al., 2022).

Dual-luciferase assay

ATDC5 cells were cultured at a density of 1 × 10⁵ cells/well in 12-well culture plates and transfected with 2 μg of dual-luciferase reporter pmirGLO-IL6-WT or pmirGLO-IL6-Mut (all purchased from D-Nano Therapeutics), and co-transfected with 500 nM mmu-miR-145a-5p mimics or miR-NC using Lipofectamine 3000 (L3000008; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Six hours post-transfection, the transfection medium was removed and replenished with growth medium. Forty-eight hours post-transfection, luciferase activity was measured using the Dual Luciferase Reporter Assay Kit (DL101-01; Vazyme, Nanjing, China). Firefly and renilla luciferase activity were detected by a microplate reader (Varioskan Flash, Thermo, Waltham, MA, USA) Firefly luciferase activity was normalized to renilla luciferase activity (Jiang et al., 2022).

Statistical analysis

All data were obtained from three independent biological replicates, each with technical triplicates. The data are presented as the means ± standard deviations (SDs). One-way ANOVA with Tukey’s post-hoc test was applied for multi-group comparisons when data met normality (Shapiro-Wilk test) and homogeneity of variance (Levene’s test) assumptions. Student’s t test was used to analyze the differences between two groups. Statistical analyses were performed via GraphPad Prism 9 software. p < 0.05 was considered statistically significant.

MiR-145a-5p is decreased in osteoarthritis

To investigate the function of miR-145a-5p, we design a series of experiments to confirm its role in OA progression. The flow diagram is shown in Fig. 1A. Toluidine blue staining reveals thinner and rougher articular cartilage in the OA group (Fig. 1B), confirming the successful establishment of the osteoarthritis mouse model. Then, qRT-PCR is performed to detect the expression level of miR-145a-5p in vivo; Fig. 1C shows that OA cartilage has a lower miR-145a-5p level. As depicted in Fig. 1D, the fluorescence intensity of miR-145a-5p in the cartilage of OA mice is faint, which is consistent with the statistical results presented in Fig. 1E.

MiR-145a-5p promotes chondrogenic differentiation and regulates the expression of Nrf2 and Il-6

MiR-145a-5p expression levels were detected at different time points of chondrogenesis (Fig. 2A). With increasing differentiation time, the miR-145a-5p expression levels gradually increased. Subsequently, miR-145a-5p mimic- and inhibitor-transfected cells were used to detect the effect of miR-145a-5p on chondrogenesis. As shown in Fig. 2C, after transfection with the miR-145a-5p mimic, the miR-145a-5p expression level significantly increased, whereas the miR-145a-5p expression level decreased in the inhibitor group. qRT‒PCR was performed 3days after cell chondrogenic induction, as shown in Figs. 2D–2F. miR-145a-5p mimic promotes the expression of Sox9, Acan, and Col2. Moreover, alcian blue staining reveals greater chondrogenic differentiation ability in the miR-145a-5p-mimic group than in the control group (Fig. 2B). The downstream genes of miR-145a-5p were predicted via miRWalk (http://mirwalk.umm.uni-heidelberg.de), and the predicted binding sequences were shown in Fig. 2G. As shown in Figs. 2H, 2I, the expression levels of Nrf2 and Il-6 were increased in the miR-145a-5p-inhibitor group but decreased in the mimic group, indicating that miR-145a-5p might alleviate OA through Nrf2 and Il-6. To validate the predicted miR-145a-5p binding site in the IL-6 3’UTR, we conducted dual-luciferase reporter assays. Co-transfection of miR-145a-5p mimic with the wild-type IL-6 3’UTR reporter significantly suppressed luciferase activity, whereas the mutant reporter showed no response, demonstrating direct interaction between miR-145a-5p and the IL-6 3’UTR (Fig. S2).

MiR-145a-5p can partially reverse the chondrogenic inhibition caused by IL-1 $\mathrm{\beta }$

IL-1β was added to the culture medium of ATDC5 cells. Results showed that IL-1β suppresses the expression of miR-145a-5p (Figs. 3A, 3B). Concurrently, the expression levels of Sox9, Col2, and Acan also indicate the inhibitory effect of IL-1β on chondrogenesis (Fig. 3C). Consistent with these findings, IL-1β similarly suppressed miR-145a-5p, Sox9, Col2, and Acan expression in HACs (Fig. S3). As shown in Fig. 3D, IL-1β reduced the expression of Sox9 and Col2, whereas miR-145a-5p increased the expression of Sox9, Col2, and Acan. Moreover, miR-145a-5p alleviated the inhibitory effect of IL-1β on chondrogenesis. Subsequently, alcian blue staining was performed on the cells. As shown in Fig. 3E, miR-145a-5p partially reversed the inhibitory effect of IL-1β on cartilage differentiation.

Cyclic stretch stress regulates chondrogenesis and the expression of miR-145a-5p

After stimulation with tensile mechanics, the intracellular distribution of YAP was detected by immunofluorescence to confirm the mechanical response of ATDC5 cells (Dupont et al., 2011). As shown in Fig. 4A, with the increase of tensile mechanics, the distribution of YAP in the nucleus was enhanced, and the 20% stretching significantly promoted the nucleolar residence of YAP, which gives us a clue that ATDC5 can receive the mechanical stimulation. Subsequently, alcian blue staining revealed that the cells in the 10% stretch group exhibited greater chondrogenic ability (Fig. 4B), while the 20% stretch inhibits chondrogenesis. As shown in Figs. 4C–4F, the application of physiological tensile mechanics (10% stretch) promoted the expression of miR-145a-5p, Sox9, Acan, and Col2, whereas overload (20% stretch) decreased their expression.

MiR-145a-5p rescues OA progression in vivo

To verify the role of miR-145a-5p in OA progression, ago-miR-145a-5p or ago-miR-NC was intra-articularly injected into the knees of mice after DMM surgery. As shown in Figs. 5A, 5B, the expression level of miR-145a-5p in the cartilage was increased in the miR-145a-5p group, and the administration of ago-miR-145a-5p indeed alleviated cartilage destruction and decreased OARSI scores at 9 weeks post DMM surgery (Figs. 5C, 5D). Moreover, miR-145a-5p partially improved the expression of COL2 and suppressed the expression of MMP13 (Figs. 5E–5G).