Exosomal miR‐95‐5p regulates chondrogenesis and cartilage degradation via histone deacetylase 2/8

Abstract MicroRNAs play critical roles in the pathogenesis of osteoarthritis, the most common chronic degenerative joint disease. Exosomes derived from miR‐95‐5p‐overexpressing primary chondrocytes (AC‐miR‐95‐5p) may be effective in treating osteoarthritis. Increased expression of HDAC2/8 occurs in the tissues and chondrocyte‐secreted exosomes of patients with osteoarthritis and mediates cartilage‐specific gene expression in chondrocytes. We have been suggested that exosomes derived from AC‐miR‐95‐5p (AC‐miR‐95‐5p‐Exos) would enhance chondrogenesis and prevent the development of osteoarthritis by directly targeting HDAC2/8. Our in vitro experiments showed that miR‐95‐5p expression was significantly lower in osteoarthritic chondrocyte‐secreted exosomes than in normal cartilage. Treatment with AC‐miR‐95‐5p‐Exos promoted cartilage development and cartilage matrix expression in mesenchymal stem cells induced to undergo chondrogenesis and chondrocytes, respectively. In contrast, co‐culture with exosomes derived from chondrocytes transfected with an antisense inhibitor of miR‐95‐5p (AC‐anti‐miR‐95‐5p‐Exos) prevented chondrogenic differentiation and reduced cartilage matrix synthesis by enhancing the expression of HDAC2/8. MiR‐95‐5p suppressed the activity of reporter constructs containing the 3ʹ‐untranslated region of HDAC2/8, inhibited HDAC2/8 expression and promoted cartilage matrix expression. Our results suggest that AC‐miR‐95‐5p‐Exos regulate cartilage development and homoeostasis by directly targeting HDAC2/8. Thus, AC‐miR‐95‐5p‐Exos may act as an HDAC2/8 inhibitor and exhibit potential as a disease‐modifying osteoarthritis drug.

Recent studies have indicated significant regulatory roles of miRNAs and HDACs in chondrogenesis and cartilage degeneration. For example, miR-92a-3p regulates the expression of cartilage-specific genes by directly targeting HDAC2 in chondrogenesis and cartilage degradation, 17 and miR-455-3p modulates cartilage development and degeneration through HDAC2/8. 18 Furthermore, miRNA-222 modulates MMP-13 expression by inhibiting HDAC4 during the initiation and progression of OA, 25 and miR-140 and miR-381 target HDAC4 to regulate cartilage and bone formation. 26,27 HDAC inhibitors (HDACi) up-regulate the level of miRNA-146a and repress IL-1βinduced signalling and cytokine secretion in OA fibroblast-like synoviocytes. 28 However, changes in the exosomal miRNA content associated with cartilage development and degeneration have not yet been described. The primary goal of this study was to illustrate differences in exosomal miRNAs in normal and OA cartilage-secreted exosomes and to explore their biological functions.
In this study, we used a miRNA microarray to analyse the miRNA profiles of normal and OA cartilage-secreted exosomes and observed the down-regulation of miR-95-5p in OA. We found that miR-95-5p has the potential to regulate HDAC2/8 expression with miRNA target prediction software. However, no previous studies on the function of miR-95-5p have been conducted. Given the role of exosomal miRNAs in regulating cartilage homoeostasis and chondrogenesis, we hypothesized that exosomal miR-95-5p may play a role in both cartilage development and OA pathogenesis. In this study, we aimed to determine whether exosomal miR-95-5p regulates cartilage-specific gene expression by targeting HDAC2/8 in chondrogenesis and cartilage degradation.

| Ethics
All procedures were approved by the ethical committee of the First Affiliated Hospital of SunYat-Sen University (IRB: 2014C-028) and the Declaration of Helsinki (2000). All volunteers provided written informed consent.

| Culture of human mesenchymal stem cells and chondrogenesis in micromass culture
Bone marrow samples were obtained by iliac crest aspiration from 6 normal human donors (mean age: 35 years; range: 31-39 years; male: 3, female: 3). The isolation of human mesenchymal stem cells (hMSCs) was carried out using a density gradient centrifugation method. 17  Cells were cultured at 37°C under a 5% CO 2 atmosphere, and the medium was changed every 3 days. The hMSCs were induced to differentiate into chondrocytes in micromass culture within three passages as described previously. 17

| Isolation and identification of exosomes
Exosome isolation was carried out using ultracentrifugation. 29 In brief, chondrocytes culture supernatants were subjected to successive centrifugations at 3000 g (30 minutes) and 10 000 g (30 minutes). Exosomes were then pelleted at 64 000 g for 110 minutes using an SW28 rotor (Beckman Coulter, California, USA). Exosome pellets were resuspended in 0.32 mol/L sucrose and centrifuged at 100 000 g for 1 hour (SW60Ti rotor; Beckman Coulter). The exosome pellet was then resuspended in phosphate-buffered saline (PBS). Nanosight 2000 analysis and transmission electron microscopy (TEM) were used to identify exosomes. RNA and proteins were extracted from exosomes using a Total Exosome RNA & Protein Isolation Kit (Invitrogen, Carlsbad, CA, USA) for further analysis. The BCA protein assay kit was used to quantify the exosomes.

| Primary chondrocyte collection, isolation and cell culture
Degraded joint cartilage samples were obtained from patients (n = 6; mean ± standard deviation [SD] age: 58.2 ± 1.24 years; male: 3, female: 3) with OA knee joints during total knee replacement operations. Normal cartilage samples were taken from patients (n = 6; mean ± SD age: 53.6 ± 1.48 years; male: 3, female: 3) with no previous history of OA or rheumatoid arthritis, who underwent total hip replacement surgery because of fractures of the femoral neck. The cartilages were dissected away from the subchondral bone and then digested by 4 mg/mL protease and 0.25 mg/mL collagenase P as described previously. 17

| Chondrocyte proliferation assay
The effect of AC-Exos and AC-miR-95-5p-Exos on the proliferation of human chondrocytes was evaluated using the Cell Counting Kit-8 (CCK-8; Dojindo, Kyushu Island, Japan) as described previously. 30 Chondrocytes were seeded into 96-well plates at 2 × 10 3 cells/well. After 12 hours, different doses of AC-Exos or AC-miR-95-5p-Exos were added to the wells. Cell proliferation curves were constructed by measuring the amount of formazan dye generated by cellular dehydrogenase activity with a microplate reader at a wavelength of 450 nm.

| RNA extraction, reverse transcription and quantitative real-time polymerase chain reaction (qRT-PCR)
RNA extraction and reverse transcription were performed as described previously. 17 CDNA was generated using the PrimeScript miRNA cDNA Synthesis Kit (Takara Biotechnology, Shiga, Japan) following the manufacturer's instruction. Transcript levels were normalized to that of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH; for mRNA) or the small U6 RNA (for miRNA). The specific primers used for these analyses are listed in Table 1. Gene expression was calculated using the 2 −ΔΔCt method, and each experiment was performed in triplicate.

| Microarray analysis
The miRNA expression profiling assays from six samples exosomes

| Western blot analysis and immunohistochemical analysis
Western blot analysis was carried out as described previously. 17 Briefly, total proteins were isolated from normal or OA chondrocytes Immunohistochemical analysis was performed as described previously. 17 Cartilage tissue sections were blocked in PBS plus 0.025% Tween 20 with 10% foetal bovine serum, followed by incubation with rabbit anti-human HDAC2/8 antibody (1:200; Abcam).
T A B L E 1 Primers for quantitative real-time polymerase chain reaction (qRT-PCR)

| Statistical analysis
Data are presented as means ± standard deviations (SD) of the results of at least three independent experiments. Student's t tests and Mann-Whitney U tests were used to identify differences between groups as appropriate. One-way analysis of variance (ANOVA) and Kruskal-Wallis tests were carried out for multiple group comparisons. A P-value <0.05 was considered statistically significant. 31 All analyses were performed using SPSS Version 20 (IBM Corporation, Armonk, NY, USA).

| MiRNA expression profiles in OA and normal chondrocyte-secreted exosomes
The expression profiles of miRNAs in OA and normal chondrocyte-secreted exosomes were detected using a miRNA microarray.   Figure 6A). This reduction was correlated with a decrease in cartilage HDAC2/8 protein expression, as well as increases in the expression of COL2A1, aggrecan and SOX9, as shown in Figure 6B.
These data indicate that the down-regulation of HDAC2/8 promotes increases in the expression of cartilage-specific proteins.  and increases the expression of cartilage matrix genes.

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In conclusion, the results presented here demonstrate that the overexpression of exosomal miR-95-5p is essential during chondro-