Introduction
Osteoarthritis (OA) is a slow-developing degenerative joint disease with a high incidence, which is related to age, obesity, tension, strain, trauma, joint deformities, and other factors [
1,
2]. OA is characterized by structural change of subchondral bone, inflammation of synovitis, and destruction of cartilage matrix [
3,
4]. At present, methods such as anti-inflammatory analgesics and artificial joint replacement surgery are mainly used to reduce OA patient’s joint pain and control its progress; however, the side effects and high costs hinder their wide application [
5,
6]. Thus, identifying novel treatment strategies for OA is very necessary.
Circular RNAs (circRNAs) are a sort of non-coding RNAs (ncRNAs) with closed-loop structures, which can regulate gene expression by competitive targeting microRNAs (miRNAs) [
7,
8]. Emerging evidence has reported that circRNAs function as critical regulators in multiple human diseases, including OA. For example, circ_00050105 facilitated ECM degradation and inflammatory response in interleukin-1β (IL-1β)-treated chondrocytes by sponging miR-26a [
9]. Circ_0045714 accelerated chondrocyte proliferation and ECM synthesis and restrained apoptosis by modulating miR-193b and IGF1R [
10]. As for circ-BRWD1 (circ_0116061), the heatmap showed it was upregulated in OA cartilage tissues compared to normal cartilage tissues [
11]. However, whether the abnormal expression of circ-BRWD1 (circ_0116061) plays a role in OA is still undefined. Exosomes are tiny particles with a diameter of 30–150 nm which can be released by multiple cell types [
12]. It has been documented that circRNAs are abundant in exosomes and can be transferred into other cells to regulate biological functions [
13]. In this study, the functions of exosome-mediated circ-BRWD1 in OA were investigated.
As a class of small ncRNAs, miRNAs participate in regulating multiple biological processes via recognization of the 3′ untranslated region (3′UTR) of target mRNAs [
14]. In OA, miR-34a overexpression facilitated the apoptosis and suppressed the proliferation of chondrocytes in the pathophysiological process of OA via targeting SIRT1/p53 signaling pathway [
15]. MiR-101 inhibition reversed IL-1β-activation ECM degradation in chondrocytes through targeting Sox9 [
16]. Moreover, Wang et al. disclosed that miR-1277 alleviated ECM degradation in IL-1β-treated articular chondrocytes via interacting with MMP13 and ADAMTS5 [
17]. These findings indicated the vital role of miR-1277 in OA. TNF receptor-associated factor 6 (TRAF6) has been demonstrated to be targeted by miR-146a to regulate OA chondrocyte proliferation and apoptosis [
18]. By analyzing bioinformatics software circinteractome and Targetscan, miR-1277 was found to contain the binding sites of circ-BRWD1 and TRAF6, but the relationships among circ-BRWD1, miR-1277, and TRAF6 in OA development are still unclear.
The present study aimed to determine the expression profiles of exosomal circ-BRWD1, miR-1277, and TRAF6 in IL-1β-activated chondrocytes and further explore their functional roles in OA development.
Materials and methods
Tissues acquisition
The OA cartilage tissue specimens were harvested from 32 OA patients undergoing total knee arthroplasty and the normal cartilage tissue specimens were harvested from 32 traumatic amputees at the Second Hospital of Shanxi Medical University. The specimens were preserved at − 80 °C until further experiments. The work was approved by the Ethics Committee of Second Hospital of Shanxi Medical University. Written informed consents were provided by all patients.
Cell culture and IL-1β treatment
The chondrocyte cell line CHON-001 was acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) added with 10% fetal bovine serum (FBS; Sigma-Aldrich) and 1% penicillin-streptomycin (Sigma-Aldrich) in a humid incubator with 5% CO2 at 37 °C.
To stimulate OA chondrocyte model, CHON-001 cells were exposed to IL-1β (5 ng/mL, 10 ng/mL, and 20 ng/mL; Sigma-Aldrich) at 37 °C for 24 h. The untreated cells (0 ng/mL) were used as controls. 10 ng/mL IL-1β-treated cells were chosen for further experiments.
Quantitative real-time polymerase chain reaction (qRT-PCR)
The RNA extraction was conducted through the usage of TRIzol reagent (Beyotime, Shanghai, China). RNA treatment was performed on total RNA with 3 U/μg RNase R (Epicenter Biotechnologies, Madison, WI, USA) for 15 min at 37 °C. Then the M-MLV Reverse Transcriptase Kit (Promega, Madison, WI, USA) or TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was adopted for the reverse transcription of RNA samples. QRT-PCR analysis was manipulated with BeyoFast™ SYBR Green qPCR Mix (Beyotime) and indicated primers (GeneCopoeia, Guangzhou, China). The primers were circ-BRWD1: (F: 5′-AGCACGGATTTGGAGATTTG-3′ and R: 5′-CGTAGCAAAGACTGCCTTCC-3′); BRWD1: (F: 5′-CCAGCGCATCGGTCCTATG-3′ and R: 5′-CTTCCTGCACCAAGTAAAGAAGT-3′); miR-1277: (F: 5′-ACACTCCAGCTGGGAAATATATATATATATGT-3′ and R: 5′-TGGTGTCGTGGAGTCG-3′); TRAF6: (F: 5′-TGTTGCAGCAGCTATTTTGC-3′ and R: 5′-CTTCTCGAGGGCACTAGCAC-3′); GAPDH: (F: 5′-GGAAGGTGAAGGTCGGAGTC-3′ and R: 5′-CGTTCTCAGCCTTGACGGT-3′); U6: (F: 5′-GCTCGCTTCGGCAGCACATA-3′ and R: 5′-ACGCTTCACGAATTTGCGT-3′). The expression was estimated via the 2-ΔΔCt strategy with GAPDH or U6 as a negative control.
Subcellular fraction assay
The isolation of nuclear and cytoplasmic fractions was conducted with the PARIS Kit (Life Technologies, Grand Island, NY, USA) in line with the protocols of the manufacturers. The RNAs isolated from the fractions of CHON-001 cells were subjected to the aforementioned qRT-PCR analysis for circ-BRWD1, U6 (a control for nuclear fraction) and GAPDH (a control for cytoplasmic fraction) levels.
Isolation of exosomes
The ExoQuick precipitation kit (System Biosciences, Mountain View, CA, USA) was used to isolate exosomes from the culture media of CHON-001 cells. Briefly, the media were collected and centrifuged at 1000×g for 10 min to sediment the cells. Then, the media were centrifuged at 10,000×g for 30 min to remove the dead cells and cellular debris. After that, the ExoQuick solution was supplemented into the supernatant for 30 min at 4 °C and then centrifuged for 40 min at 2000×g. Subsequently, the supernatant was removed and the exosome-containing pellet was washed with PBS (Beyotime) and then resuspended in PBS (Beyotime).
Transmission electron microscopy (TEM)
The exosomal morphology was analyzed by TEM (JEOL Ltd., Tokyo, Japan) using negative staining according to the previous report [
19]. The images were observed using the FEI TecnaiG2 spirit transmission electron microscope (Thermo-Fischer, Waltham, MA, USA) operated at 80 kV.
Western blot assay
The extraction of total protein was done utilizing RIPA buffer (Beyotime) and quantified utilizing the BCA protein assay kit (Tiangen, Beijing, China). An equal amount of protein was resolved with sodium dodecyl sulfonate-polyacrylamide gel (Solarbio, Beijing, China) and then transferred onto polyvinylidene difluoride membranes (Sigma-Aldrich). After blocking in 5% defatted milk for 1 h at indoor temperature, the membranes were incubated with primary antibodies against CD9 (ab223052; Abcam, Cambridge, MA, USA), CD63 (ab68418; Abcam), CyclinD1 (ab226977; Abcam), Bax (ab104156; Abcam), matrix metalloprotein 13 (MMP13; ab39012; Abcam), aggrecan (ab36861; Abcam), TRAF6 (ab137452; Abcam) or GAPDH (ab37168; Abcam) overnight at 4 °C and indicated secondary antibody (ab6789; Abcam) for 1.5 h at indoor temperature. The ECL kit (Beyotime) was employed for chemiluminescence imaging.
Exosomes or GW4869 treatment
CHON-001 cells were plated into 6-well plates (1 × 105 cells/well) and then the exosomes (20 μg) derived from 10 ng/mL IL-1β-triggered CHON-001 cells were added into the culture media for 48 h. The control media were treated with PBS (Beyotime).
To block exosome secretion, IL-1β-treated CHON-001 cells were treated with 20 μM GW4869 (Sigma-Aldrich) for 2 h prior to IL-1β (Sigma-Aldrich) exposure. After 48 h, the supernatants were harvested for circ-BRWD1 expression via qRT-PCR analysis.
Cell transfection
Circ-BRWD1 short interfering RNA (si-circ-BRWD1) and scramble control (si-NC), the overexpression vector of circ-BRWD1 (circ-BRWD1) and its control (pCD5-ciR), miR-1277 mimics (miR-1277) and control mimics (miR-NC), miR-1277 inhibitors (anti-miR-1277) and anti-miR-NC, the overexpression vector of TRAF6 (TRAF6) and pcDNA were purchased from GeneCopoeia. CHON-001 cells (1 × 104 cells/well) were seeded into 24-well plates and the oligonucleotides (50 nM) or vectors (2 μg) were transfected into CHON-001 cells utilizing Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturers’ instructions.
Cell Counting Kit-8 (CCK-8) assay
Following relevant treatment, CHON-001 cell viability was tested by CCK-8 assay. In brief, CHON-001 cells were plated into 96-well plates (1 × 104 cells/well) and then cultivated for 72 h. 20 μL CCK-8 (Sigma-Aldrich) was treated into each well for further 3 h. After that, the OD value was measured at 450 nm with a microplate reader (BioTek, Winooski, VT, USA).
Flow cytometry analysis
CHON-001 cell apoptosis was assessed with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit (Beyotime) according to the manufacturers’ guidelines. Briefly, CHON-001 cells were collected and rinsed with PBS (Beyotime) after relevant treatment and then resuspended in binding buffer. After that, 5 μL AnnexinV-FITC and 5 μL PI were added and maintained for 15 min in the dark. At last, cell apoptosis was examined with a FACScan® flow cytometry (BD Biosciences, San Jose, CA, USA).
5-Ethynyl-2′-deoxyuridine (EDU) assay
EDU assay was conducted to evaluate cell viability through the usage of EDU assay kit (Solarbio, Beijing, China). In short, the treated CHON-001 cells were seeded into 12-well plates and incubated with EDU buffer for 2 h at 37 °C. Then the cells were fixed with 4% formaldehyde for 30 min, permeabilizated for 20 min using 0.1% Triton X-100. Thereafter, the cells were exposed to EDU solution for 30 min and then stained cell nuclei using 5 μg/mL Hoechst 33342 for 20 min. The images were captured with a fluorescence microscope (Olympus, Tokyo, Japan) and the EDU-positive cells were counted.
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of interleukin-6 (IL-6) and interleukin-8 (IL-8) were conducted utilizing Human IL-6 ELISA Kit (ab178013; Abcam) and Human IL-8 ELISA Kit (ab214030; Abcam) according to the manufacturers’ instructions.
Dual-luciferase reporter assay
The regions of circ-BRWD1 (or TRAF6 3′UTR) including or lacking the predicted miR-1277 binding sequences were amplified and inserted into pmirGLO vector (Promega), generating WT-circ-BRWD1, MUT-circ-BRWD1, TRAF6 3′UTR-WT, and TRAF6 3′UTR-MUT. The generation of WT-circ-BRWD1, MUT-circ-BRWD1, TRAF6 3′UTR-WT, and TRAF6 3′UTR-MUT was accomplished by GeneCopoeia. Then the generated vectors were transfected into CHON-001 cells in combination with miR-1277 or miR-NC. After the post-transfection for 48 h, dual-luciferase assay kit (Promega) was adopted for renilla and firefly luciferase activities.
RNA immunoprecipitation (RIP) assay
The Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) was adopted for RIP assay. CHON-001 cells were lysed in RIP buffer and then incubated overnight with beads coated with antibody immunoglobulin G (IgG) or argonaute-2 (anti-Ago2) at 4 °C. Then the RNAs on the beads were retrieved for circ-BRWD1, miR-1277, and TRAF6 levels via qRT-PCR assay.
Statistical analysis
The experiments were carried out in triple times. The collected data were analyzed by GraphPad Prism 7 software and presented as mean ± standard deviation. The linear correlation between the levels of miR-1277 and circ-BRWD1 or TRAF6 was estimated by Pearson’s correlation coefficient analysis. The differences between two groups were estimated by Student’s t test, while those among three groups were estimated by one-way analysis of variance (ANOVA). The differences were defined as significant when P < 0.05.
Discussion
Up to date, numerous circRNAs have been identified to be associated with the progression of human diseases. Nevertheless, the biological roles of circRNAs in OA are not well known. In the research, we focused on the effects of exosomal circ-BRWD1 in OA pathogenesis. IL-1β was utilized to treat CHON-001 cells to construct an OA model in vitro, as previously mentioned [
20,
21]. We found that IL-1β treatment repressed CHON-001 cell viability and induced apoptosis, inflammation and ECM degradation. Moreover, exosomal circ-BRWD1 level was elevated in IL-1β-activated CHON-001 cells. Circ-BRWD1 knockdown protected CHON-001 cells from IL-1β-activated injury by miR-1277/TRAF6 axis.
The vital roles of exosome-mediated circRNAs have been demonstrated in human illnesses. For instance, exosomal circ_MMP2 accelerated the malignancy of hepatocellular carcinoma by modulating miR-136-5p/MMP2 axis [
22]. Exosome-mediated circ_0044516 contributed to prostate cancer carcinogenesis [
23]. Moreover, several circRNAs, such as circ_0045714 [
10], circ_0136474 [
11] and circGCN1L1 [
24], have been reported to play vital roles in OA. Herein, we elucidated the role of exosome-mediated circ-BRWD1 in OA progression. Circ-BRWD1 level was raised in OA cartilage tissues and IL-1β-induced CHON-001 cells. Circ-BRWD1 level was also increased in the exosomes derived from IL-1β-stimulated CHON-001 cells. Moreover, exosome treatment elevated circ-BRWD1 level in IL-1β-activated CHON-001 cells, while GW4869 treatment reduced circ-BRWD1 level, indicating exosomes mediated the transmission of circ-BRWD1. Functionally, deficiency of circ-BRWD1 promoted cell viability and impeded cell apoptosis and inflammatory factors release mediated by IL-1β in CHON-001 cells. In addition, we measured the levels of MMP13 and aggrecan, which play crucial roles in cartilage ECM production during OA development [
25,
26]. Our results exhibited that circ-BRWD1 silencing decreased MMP13 level and increased aggrecan level in IL-1β-stimulated CHON-001 cells, indicating ECM degradation was inhibited. Taken together, exosomal circ-BRWD1 knockdown blocked OA development.
Subsequently, the potential mechanisms of circ-BRWD1 in regulating OA progression were explored. The data indicated that circ-BRWD1 served as the sponge for miR-1277 to positively alter TRAF6 expression. MiR-1277 was found to be decreased in OA tissues and IL-1β-stimulated chondrocytes, and its overexpression restrained the degradation of ECM [
17]. Consistently, we observed that miR-1277 was weakly expressed in OA cartilage tissues and IL-1β-activated CHON-001 cells. Moreover, miR-1277 overexpression promoted cell viability and hampered apoptosis, inflammatory response and ECM degradation in IL-1β-activated CHON-001 cells. MiR-1277 suppression alleviated the impacts of circ-BRWD1 deficiency on cell viability, apoptosis, inflammation and ECM degradation in IL-1β-activated CHON-001 cells, suggesting that circ-BRWD1 deficiency blocked OA progression by sponging miR-1277. Additionally, TRAF6 was identified to be the target gene of miR-1277 for the first time, though it could be targeted by multiple miRNAs [
27‐
29]. Of note, we demonstrated that TRAF6 upregulation abrogated the impact of miR-1277 on the progression of IL-1β-activated CHON-001 cells, indicating that miR-1277 regulated OA development by targeting TRAF6.
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