MicroRNA-17-5p contributes to osteoarthritis progression by binding p62/SQSTM1.

Autophagy has been reported to be widely involved in the pathogenesis of osteoarthritis (OA). Increasing evidence suggested the important role of microRNAs (miRs) in the progression of OA. However, the functional role of miR-17-5p in OA development has remained to be fully elucidated. First, a mouse model of OA was established and the relative level of miR-17-5p was determined using PCR. Safranin O-fast green staining was applied to determine cartilage degeneration. TargetScan software and a dual luciferase reporter assay were applied to determine potential target genes of miR-17-5P. Autophagy measurement was performed using green fluorescent protein-microtubule-associated protein 1 light chain 3 (LC3) dot analysis. The results demonstrated that the relative expression of miR-17-5p was significantly decreased in OA model mice. In addition, the level of miR-17-5p was decreased in SW1353 human chondrosarcoma cells treated with interleukin-1β. Furthermore, autophagy was found to be suppressed in the knee joints of experimental OA model mice. The dual luciferase reporter assay confirmed that p62/sequestosome 1 was a target gene of miR-17-5p. Of note, miR-17-5p inhibitor-induced reduction of LC3 dots was markedly reversed by knockdown of p62 in SW1353 cells. In conclusion, decreased miR-17-5p expression in chondrocytes induced autophagy mainly through suppressing the expression of p62, thereby contributing to OA progression.

Introduction

As the most common aging-associated joint condition, osteoarthritis (OA) is well known as a degenerative joint disease, which is characterized by cartilage extracellular matrix (ECM) degradation (1,2). In the initiation and progression of OA, other joint tissues are also widely involved (3,4). For instance, chondrocytes have a key role in maintaining the structure of adult articular cartilage (4). Chondrocytes are the only cell population of adult articular cartilage that is able to generate the normal cartilage matrix architecture (5). However, the normal function of chondrocytes declines with aging and fails to respond to abnormal stimuli. Thus, the protection of chondrocytes may have a great potential for the treatment of OA.

Autophagy has a key role in cellular homeostasis through the removal of damaged macromolecules and organelles (6). Accumulating evidence indicated that autophagy is tightly correlated to the pathogenesis of OA (6,7). In articular cartilage, constitutive activation of autophagy has been widely observed, indicating an important protective role of autophagy in maintaining chondrocyte survival (6,8). Autophagy has been reported to be decreased in joint aging and OA in humans and mice, and to be accompanied by enhanced chondrocyte apoptosis (9). p62 [also known as sequestosome-1 (SQSTM-1) or A170] has emerged as a crucial regulator in the regulation of ubiquitination and autophagy (10) Reduced autophagy results in defects of protein and organelle quality control, which leads to the accumulation of p62 (11).

MicroRNAs (miRs) are small non-coding RNAs of ~22 nucleotides in length, which widely modulate gene expression through binding the 3′ untranslated region (3′UTR) of target mRNAs (12). Abnormal expression of miRs has been widely reported in the progression of OA (13–15). For instance, miR-139 was reported to be upregulated in OA by targeting eukaryotic translation initiation factor 4 gamma 2 and insulin-like growth factor 1 receptor (14). miR-365 prompts osteoarthritis mainly by binding histone deacetylase 4 (13). Aberrant expression of miR-17-5p has been extensively explored in various tumors types, including gastric cancer, thyroid cancer and follicular lymphoma (16–18). However, functional role of miR-17-5p in OA progression has remained to be fully elucidated. The present study identified that the expression of miR-17-5p was significantly decreased during the progression of OA in mouse models. More importantly, reduced expression of miR17-5p in chondrocytes was found to suppress autophagy mainly by suppressing p62, which then contributed to the defects of autophagy supervision.

Materials and methods

Experimental OA in mice

All mouse procedures were approved by the Animal Ethics Committee of Ruian Municipal People's Hospital. To establish the experimental OA animal model, 10 two-month-old male wild-type C57BL/6J mice (12.3±2.1 g) were purchased from the Peking University Health Science Center (Beijing, China). Mice were housed in a temperature (20–24°C) and humidity-controlled (45–55%) environment with free access to food and water. A 12 h light/dark cycle was maintained in the animal housing rooms. C57Bl/6J mice were transected by the medial meniscotibial ligament and the medial collateral ligament in the right knee (19). The left knee was not subjected to surgery and used as a control. Ten weeks after the knee surgery, the animals were euthanized and the tissues were collected.

Cell culture

SW1353 human chondrosarcoma cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Leibovitz's L-15 medium (GE Healthcare Life Sciences, Little Chalfont, UK) supplemented with 10% (v/v) fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 80 U/ml penicillin and 80 µg/ml streptomycin, at 37°C in a humidified atmosphere with 5% CO2.

Transient transfection

Firstly, 6×105 cells were equally seeded in 6-well plates with 2 ml Leibovitz's L-15 medium (GE Healthcare Life Sciences) containing 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin and streptomycin. At the same time, a specific siRNA targeting p62 (GGC UGA AGG AAG CUG CCU U) or a non-target control siRNA (GenePharma, Shanghai, China) were mixed with HiperFect transfection reagent (Qiagen, Hilden, Germany) and incubated at room temperature for 10 min. Then, the complex was respectively transfected into SW1353 cells for 48 h.

Histological analysis of mouse knee joints

The knee joints were fixed in 10% zinc-buffered formalin (Solarbio, Beijing, China) for 24 h, followed by paraffin embedding. The paraffin-embedded joints were cut into serial sections (4 µm) and stained with Safranin O-fast green (OriGene Technologies, Inc., Rockville, MD, USA). The histopathological changes were analyzed using a semiquantitative scoring system as described as previously (1).

Immunofluorescence

The slides were fixed with 4% paraformaldehyde at room temperature for 30 min, followed by incubation in 0.5% Triton X-100 for 30 min. After blocking with 5% bovine serum albumin (Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 30 min, 50 µl primary rabbit anti-microtubule-associated protein 1 light chain 3 (LC3) polyclonal antibody (1:50 dilution, cat. no. L8918; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added, followed by incubation at 4°C overnight. Subsequently, samples were stained with fluorescently labeled goat anti-rabbit secondary antibody (1:5,000, cat. no. ZB2306; OriGene Technologies, Inc.) at 37°C for 30 min, and the nuclei were stained with Hoechst 33258 (1:1,000, Invitrogen; Thermo Fisher Scientific, Inc.) for 5 min. All of the steps were followed by three washes with PBS for 5 min each. The fluorescence intensity was examined under a fluorescence microscope.

Cellular OA model induced by interleukin (IL)-1β

To establish a cellular OA model, the cells were incubated with IL-1β (Sigma-Aldrich; Merck KGaA) at a final concentration of 10 ng/ml. Cells without any treatment were used as controls.

Cells were seeded in 6-well plates at 106 cells/well. miR-17-5p mimics, inhibitor or the respective negative control (Genepharma) or siRNA targeting p62 (CCA GCC GGU UGA GGU AGA AGU AGU A) or non-target control siRNA (GenePharma) were mixed with HiperFect transfection reagent (Qiagen, Hilden, Germany) and incubated at room temperature for 10 min. The complex was then added to the culture medium, followed by incubation for 48 h.

RNA extraction and reverse-transcription quantitative polymerase chain reaction (RT-qPCR)

The total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions and complementary DNA was transcribed using a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). A quantitative real-time PCR assay was performed using SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in a Bio-Rad iCycler iQ Real-Time PCR Detection system (Bio-Rad Laboratories, Inc.) as previously described (20,21). Nucleotide primers used for reverse transcription were as follows (5′-3′): miR-17-5p, GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACT GGA AGA C; U6, GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACA AAT ATG. The primers used for qPCR were as follows (5′-3′): miR-17-5p forward, GCG CTA AAG TGC TTA TGG AAG AC; U6 forward, GCG CGT CGT GAA GCG TTC; universal reverse primer, GTG CAG GGT CCG AGG T. A total of 1 mg cDNA was used for qPCR using SYBR Green Master mix (Roche Diagnostics, Basel, Switzerland) on a Roche Lightcycler 480 (Roche Diagnostics) at 95°C for 10 min followed by 50 cycles of 95°C for 10 sec, 55°C for 10 sec, 72°C for 5 sec, 99°C for 1 sec, 59°C for 15 sec, 95°C for 1 sec; then cooling to 40°C. Relative miRNA expression of miR-132-3p was normalized against the endogenous control, U6, using the 2−ΔΔCq method (21).

Protein extraction and western blot analysis

Protein samples were extracted in radioimmunoprecipitation assay buffer (1% TritonX-100, 15 mmol/l NaCl, 5 mmol/l EDTA and 10 mmol/l Tris-HCl, pH 7.0; Solarbio, Beijing, China) supplemented with a protease and phosphatase inhibitor cocktail (Sigma-Aldrich; Merck KGaA) and then separated by 10% SDS-PAGE, followed by electrophoretical transfer to a polyvinylidene difluoride membrane. After incubation with 8% milk in PBS containing Tween-20 (pH 7.5) for 2 h at room temperature, the membranes were incubated with the following primary antibodies: For 24 h overnight: Anti-p62 (cat. no. 13121, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-beclin 1, anti-LC3 (1:1,000, cat. no. L8918; Sigma-Aldrich; Merck KGaA) and anti-GAPDH (cat. no. 2118, 1:5,000; Cell Signaling Technology, Inc.). Following several washes with Tris-buffered saline with 1% Tween-20, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (all 1:5,000, SP-9000D; OriGene Technologies, Inc.) for 2 h at room temperature and then washed. Immunodetection was performed using an enhanced chemiluminescence detection system (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. The housekeeping gene GAPDH was used as an internal control ImageJ v64 software (National Institutes of Health, Bethesda, MD, USA) was used for density analysis.

Luciferase reporter assay

DNA was extracted from mouse bone tissue using GenElute Mammalian Genomic DNA Miniprep kits (Sigma-Aldrich; Merck KGaA). The 3′UTR of p62 containing the predicted binding site was cloned into the pmirGLO luciferase reporter vector (Promega, Madison, WI, USA). The PCR procedures were as follows: A hot start step at 95°C for 10 min, 40 cycles of 95°C for 15 sec and 55°C for 45 sec, and a final step of 72°C for 30 sec. PCR products were maintained at 4°C. To construct the mutant vector, the Fast Mutagenesis System (TransGen Biotech, Beijing, China) was applied.

For the luciferase reporter assay, cells were seeded in 24-well plates at 5×104 cells/well in a volume of 500 µl and incubated for 18 h. The modified firefly luciferase vector (500 ng/µl) was mixed with Vigofect transfection reagent (Vigorous Biotechnology, Beijing, China) according to the manufacturer's instructions. After transfection for 48 h, the dual-luciferase reporter assay system (Promega) was used to determine the changes of relative luciferase units in the wild-type luciferase vector + miRNA mimics or NC. Renilla activity was used as the internal control.

Autophagy measurement using green fluorescent protein (GFP)-LC3

SW1353 cells were transfected with GFP-LC3 expression plasmid (Invitrogen; Thermo Fisher Scientific, Inc.). After 24 h, cells were treated with miR-17-5p mimics or NC, and the fluorescence of GFP-LC3 was observed under a fluorescence microscope. The LC3 punctate spots were counted 2 days later. The percentage of cells undergoing autophagy was calculated from the ratio of autophagic cells to normal cells bearing GFP-LC3 fluorescence.

Statistical analysis

Values are expressed as the mean ± standard deviation from 3 independent experiments or 5 mice. Statistical analysis was performed with Student's t-test using GraphPad Prism 5 (GraphPad, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Decreased miR-17-5p in OA mice and cell models

First, safranin O-fast green staining was applied to determine whether the mouse models of OA were successfully established. As shown in Fig. 1A, the knee joints of the mice demonstrated significant cartilage degeneration, with depletion of proteoglycan, surface lamina and fibrillations in OA models. Compared with the control group, the histological scores of the OA mice were markedly enhanced, indicating severe of cartilage injury (Fig. 1B). Furthermore, the relative expression of miR-17-5p was found to be significantly decreased in the knee joints tissues of the OA model mice (Fig. 1C). In addition, the level of miR-17-5p was also decreased in human chondrosarcoma SW1353 cells treated with IL-1β (Fig. 1D).

Figure 1.

Decreased miR-17-5p in OA mice and cell models. (A) Safranin O-fast green staining demonstrated marked cartilage degeneration with depletion of proteoglycan, surface lamina and fibrillations of knee joints of OA model mice. Magnification, ×20. (B) Compared with the control group, the scores of OA mice were markedly enhanced. (C) The relative expression of miR-17-5p was significantly decreased in OA model mice. (D) The level of miR-17-5p was also decreased in SW1353 human chondrosarcoma cells treated with IL-1β. Values are expressed as the mean ± standard error (n=6 in each group). **P<0.01 vs. Con. OA, osteoarthritis; miR, microRNA; IL, interleukin; Con, control.

Suppressed autophagy in knee joints of experimental OA model mice

The present study then explored the expression of LC3 in OA tissues by immunofluorescence. A decrease in LC3 expression was identified in mouse knee joints of OA models as shown by reduced LC3 puncta, indicating suppression of autophagy in articular cartilage (Fig. 2A). Furthermore, in consistency with decreased autophagy in the knee joints of murine OA models, western blot analysis demonstrated that the protein level of Beclin1 was markedly decreased, while the expression of p62 was significantly increased (Fig. 2B).

Figure 2.

Suppressed autophagy in mouse knee joints of experimental OA models. (A) Reduced light chain 3 puncta were observed in mouse knee joints of experimental OA models compared with controls (magnification, ×10 or ×100). (B) Western blot analysis demonstrated that the protein levels of Beclin 1 were markedly decreased in knee joints of experimental OA model mice compared with controls. Values are expressed as the mean ± standard error (n=6 in each group). *P<0.05, **P<0.01. OA, osteoarthritis; Con, control.

p62 is a direct target of miR-17-5p

Next, the present study sought to identify possible target genes of miR-17-5p associated with autophagy. According to TargetScan, a conserved binding site of miR-17-5p was identified in the 3′UTR of p62/SQSTM1 (Fig. 3A). In order to verify this, the 3′UTR of p62/SQSTM1 was cloned into the pmirGLO plasmid. As shown in Fig. 3B, transfection of miR-17-5p mimics significantly decreased the luciferase activity of pmirGLO-p62-3′UTR. In comparison, no luciferase activity changes of the plasmid containing the mutated sequence, pmirGLO-p62-mut-3′UTR, were detected in the presence of miR-17-5p. In line with the result of the luciferase reporter assay, overexpression of miR-17-5p significantly suppressed the protein expression of p62 in SW1353 cells (Fig. 3C). Furthermore, inhibition of miR-17-5p markedly increased the relative expression of p62 in SW1353 cells (Fig. 3D). These results indicated that p62 was a target gene of miR-17-5p.

Figure 3.

p62 was the target gene of miR-17-5p. (A) According to TargetScan, a conserved binding site of miR-17-5p was identified in the 3′UTR of p62/SQSTM1. (B) Transfection of miR-17-5p mimics significantly decreased the luciferase activity of pmirGLO-p62-3′UTR. (C) Overexpression of miR-17-5p significantly suppressed the protein expression of p62 in SW1353 cells. (D) Inhibition of miR-17-5p markedly increased the relative level of p62 in SW1353 cells. Values are expressed as the mean ± standard error (n=6 in each group). *P<0.05, **P<0.01 and ***P<0.001. UTR, untranslated region; SQSTM, sequestosome; NC, negative control; miR, microRNA; Mut, mutant; RLU, relative luciferase unit.

miR-17-5p induces autophagy through suppressing the expression of p62

It was further determined whether miR-17-5p induces autophagy in SW1353 human chondrosarcoma cells. As shown in Fig. 4A, overexpression of miR-17-5p significantly increased LC3 dots in SW1353 cells. Furthermore, a specific small interfering siRNA targeting p62 or NC was applied. In SW1353 cells, inhibition of miR-17-5p markedly increased the protein level of p62 (Fig. 4B). However, transfection of miR-17-5p inhibitors failed to increase p62 expression when a specific siRNA targeting p62 was applied in SW1353 cells. In another experiment, SW1353 cells were cultured in serum-free medium for 24 h. As shown in Fig. 4C, starvation obviously increased LC3 dots in SW1353 cells. However, this effect was significantly abolished when miR-17-5p inhibitor was transfected into SW1353 cells. More importantly, miR-17-5p inhibitor-induced reduction of LC3 dots was markedly reversed by knockdown of p62 in SW1353 cells (Fig. 4C). These results indicated that miR-17-5p induced autophagy through suppressing the expression of p62.

Figure 4.

miR-17-5p induces autophagy through suppressing the expression of p62. (A) Overexpression of miR-17-5p significantly increased LC3 dots in SW1353 cells. (B) Transfection of miR-17-5p inhibitors failed to increased p62 expression when a specific si-p62 was applied on SW1353 cells. (C) miR-17-5p inhibitor-induced reduction of LC3 dots was markedly abolished by knockdown of p62 in SW1353 cells. Scale bars, 10 µm. Values are expressed as the mean ± standard error (n=6 in each group). **P<0.01 and ***P<0.001. LC3, microtubule-associated protein 1 light chain 3; miR, microRNA; si-p62, siRNA targeting p62; NC, negative control.

Discussion

OA has become one of the most common types of chronic joint disease worldwide (22). In previous years, substantial progress has been made toward the elucidation of the underlying mechanism and the development of effective treatments for the pathological progression in established OA (22). Studies have demonstrated that diseases associated with aging may result from failure of cellular homeostasis mechanisms, including autophagy, which lead to changes in gene expression patterns and extracellular matrix destruction (23,24). Thus, the present study explored whether autophagy is involved in the pathogenesis of OA and demonstrated markedly reduced autophagy in the knee joints of murine OA models.

The potential mechanism by which the level of autophagy is regulated in knee joints of murine OA models was also investigated. Abnormal expression of miRs has been widely explored in the progression of age-associated OA (25,26). For instance, downregulation of miR-411 was found to prompt OA progression mainly by binding matrix metalloproteinase-13 in chondrocytes (25). Furthermore, upregulation of miR-30a contributes to the pathogenesis of OA mainly via suppressing delta-like 4 expression in mesenchymal stem cells (26). The present study identified a novel miRNA, miR-17-5p, that was significantly suppressed in the knee joints of murine OA models and OA cell models. miR-17-5p belongs to the miR-17-92 family, which has been widely explored in cancer (27,28). A previous study reported that miR-17-5p modulates osteoblastic differentiation and cell proliferation via suppressing SMAD7 in non-traumatic osteonecrosis (29). However, there is little literature regarding the role of miR-17-5p in OA.

The present study found that overexpression of miR-17-5p significantly induced autophagy in SW1353 human chondrocyte cells, while inhibition of miR-17-5p in SW1353 cells suppressed autophagy after serum starvation. A previous study reported that when autophagy was present, the expression of p62 was reduced due to the ubiquitination-induced signaling pathway (30). The present study found that the expression of p62 was significantly upregulated in the knee joints of murine OA models, indicating a suppression of autophagy. Autophagy has a key role in maintaining cellular homeostasis (2,31). More importantly, autophagy has been widely reported to be correlated with various diseases, including Meckel's cartilage degradation and OA (32). By contrast, certain studies on the association between OA and autophagy have indicated that autophagy has a protective role (1,2). The present study showed that autophagy was suppressed in knee joints of murine OA models and that severe cartilage injury was present, indicating that autophagy exerts a protective role in the pathogenesis of OA progression.

The present study further explored the association between reduced miR-17-5p expression and decreased autophagy in chondrocytes. Bioinformatics predictions and a luciferase reporter assay confirmed that p62/SQSTM1 was a target gene of miR-17-5p. Analysis of GFP-LC3 dots indicated that inhibition of miR-17-5p contributed to autophagy suppression, which was likely to be at least in part via binding the 3′UTR of p62. Taken together, the present study reported that miR-17-5p was significantly upregulated in the knee joints of murine OA models. More importantly, miR-17-5p suppressed autophagy of chondrocytes mainly by targeting p62.