The role of miR-320a and IL-1β in human chondrocyte degradation

Specimen selection and chondrocyte isolation

OA articular cartilage samples were harvested from 15 OA patients, aged 56 to 74 years, who underwent joint replacement or joint surgery. Normal articular cartilage was collected from 15 individuals, aged 33 to 55 years, who underwent amputation resulting from trauma, with no history of any form of secondary OA or inflammatory joint diseases. OA patients were diagnosed according to the American College of Rheumatology (ACR) criteria.¹⁵ This study was approved by our local ethics committee, and written informed consent was obtained from all subjects for the use of their articular cartilage samples for research.

Cartilage specimens were cut into 1 mm to 2 mm³ pieces. For detection of miR-320a level, cartilage pieces were milled in liquid nitrogen, and total RNA were extracted using the Rneasy Fibrous Tissue Mini Kit (Qiagen, Valencia, California) according to the manufacturer’s instructions. For chondrocyte culture, cartilage pieces were predigested with 1 mg/mL trypsin (Sigma-Aldrich, St Louis, Missouri) for 30 minutes and followed by digestion with 1 mg/mL collagenase (Sigma) for 16 hours at 37°C with gentle shaking. Afterwards, the suspension was filtered through a 100 mm nylon cell strainer (BD Falcon, BD Biosciences, San Jose, California) and cells were collected by centrifugation.

Cell culture

Chondrocytes were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 medium (Gibco, Invitrogen Life Technologies, Carlsbad, California) supplemented with 10% foetal bovine serum (FBS; Gibco), 100 units/mL penicillin, and 100 μg/mL streptomycin (Gibco).¹⁵ The immortalised human juvenile chondrocyte cell line C28/I2 was obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. C28/I2 cells were grown in DMEM (Gibco) supplemented with 10% FBS (Gibco), 100 units/mL penicillin, and 100 μg/mL streptomycin (Gibco).¹⁶ All cells were incubated in a humidified incubator with 5% CO₂ at 37°C.

Cell transfection and treatment. Cells were plated onto 60 mm dishes and after cells were grown to 80% confluence, miR-320a, antisense oligonucleotides (ASO) of miR-320a or their negative controls (Shanghai Genechem Co., Shanghai, China) were transfected into the cells.The transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, California) according to the manufacturer’s instructions. After 48 hours of transfection, cells were serum starved for 12 hours and then treated with 5 ng/mL of IL-1β (PeproTech, Rocky Hill, New Jersey) or 5 μM MG132 (Sigma-Aldrich), an antagonist of nuclear factor kappa B (NF-κB) for 24 hours. Cells were then collected for the forthcoming analyses.

Luciferase reporter assays. The 3’-UTR of Pre-B-Cell Leukemia Homeobox 3 (PBX3) was amplified by PCR and placed in the pMIR-Report vector (Ambion, Austin, Texas). These vectors were co-transfected with miR-320a, ASO-miR-320a or their negative controls into cells using Lipofectamine 2000. After 48 hours of transfection, luciferase assays were carried out using the Dual-Luciferase Reporter Assay System (Promega, Madison, Wisconsin).

Real-time reverse transcription polymerase chain reaction (RT-PCR)

Total RNA of chondrocytes were isolated by using TRIzol reagent (Invitrogen), and total RNA of cartilage tissue were extracted as described above. Synthetic of cDNA was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). RT-PCR was performed in the ABI PRISM 7500 Real-time PCR System (Applied Biosystems, Foster City, California) and by using FastStart Universal SYBR Green Master (ROX) (Roche), according to the manufacturer’s instructions. Data were normalised to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or U6 snRNA expression, and were analysed using the 2-∆∆Ct method.

Western blotting

Cellular protein was extracted with a lysis buffer (Beyotime, Shanghai, China), and equal amounts of protein samples were mixed with a sample buffer (Beyotime) and boiled for five minutes. Protein samples were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bed-ford, Massachusetts). After blocking within 5% skim milk for one hour, the membranes were incubated with primary antibodies for PBX3 or GAPDH (dilution: 1:1,000; Santa Cruz Biotechnology, Santa Cruz, California) overnight at 4°C. Afterwards, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for one hour at room temperature. The blots were visualised by Super Signal Femto (Pierce, Rockford, Illinois) according to the manufacturer’s instructions.

Sulfated glycosaminoglycans (sGAG) analysis

In order to quantitatively assess sGAG content released due to breakdown of the cartilage extracellular matrix. C28/I2 cells were digested with 300 μg/mL papain solution (Sigma) at 65°C for four hours. Afterwards, the lysates were removed by centrifugation and the sGAG content was measured by using the Blyscan sGAG Assay Kit (Biocolor, Newton Abbey, UK) according to the manufacturer’s instructions. Chondroitin 4-sulfate was used as a standard.¹⁷

Enzyme-linked immunosorbent assay (ELISA). The level of matrix metallopeptidase 13 (MMP-13) secreted by C28/I2 cells in the culture supernatants was quantified using an MMP-13 ELISA kit (CUSABIO, Wuhan, China) according to the manufacturer’s instructions. The MMP-13 concentration was normalized to the concentrations of the standard controls provided by the manufacturer.

Statistical analysis

All data are expressed as means and standard deviations (sd) from at least three independent analyses. Statistical comparisons were made between two groups by using the two-tailed paired t-test and GraphPad Prism 5 software (GraphPad, San Diego, California). A value of p < 0.05 was considered significant.

miR-320a was elevated during OA

To explore the role of miR-320a in the pathogenesis of OA, the expression of miR-320a in OA cartilage tissues and OA chondrocytes, as well as in IL-1β-stimulated C28/I2 cells, was assessed by RT-PCR. As results show in Figures 1a to 1c, the expression of miR-320a was significantly elevated in OA chondrocytes, and in IL-1β-stimulated C28/I2 cells, when compared with that in normal chondrocytes and C28/I2 cells without IL-1β stimulation, respectively (p < 0.05 or p < 0.01). These results indicated that miR-320a might play a pivotal role in the pathogenesis of OA, or that miR-320a might serve as a biomarker during OA.

Fig.

miR-320a was elevated in OA. (a) Expression of miR-320a in normal (n = 15) and OA cartilage cells (n = 15) was measured by RT-PCR. (b) The level of miR-320a in normal and OA chondrocytes was measured by RT-PCR. (c) C28/I2 cells were stimulated with IL-1β for 24 hours, and then the level of miR-320a was determined by RT-PCR. *, p < 0.05; **, p < 0.01. Statistical analysis was performed using two-tailed paired t-test.

miR-320a overexpression enhanced IL-1β-induced matrix degradation factors

To explore the effects of miR-320a on IL-1β-induced matrix degradation factors, C28/I2 cells were transfected either with miR-320a or ASO-miR-320a and then stimulated with IL-1β. RT-PCR results showed that IL-1β significantly downregulated the expression of Col2α1 and ACAN (p < 0.01) (Figs 2a and 2b). miR-320a overexpression significantly enhanced the downregulative effects of IL-1β on Col2α1 and ACAN expression (p < 0.05), while miR-320a suppression displayed the opposite results (p < 0.01). Moreover, sGAG content and MMP-13 expression were determined. The results in Figures 2c and 2d showed that IL-1β led to lower concentrations of sGAG (p < 0.01) and higher concentrations of MMP-13 (p < 0.01) when compared with the control group. miR-320a overexpression enhanced the concentration of sGAG and the increase of MMP-13 induced by IL-1β (p < 0.01). As expected, miR-320a suppression displayed contrary results (p < 0.01). Thus, we speculated that miR-320a overexpression might aggravate IL-1β-induced cartilage matrix degradation through controlling the levels of Col2α1 and ACAN and the concentrations of sGAG and MMP-13.

Fig.

miR-320a overexpression enhanced IL-1β-induced matrix degradation factors. C28/I2 cells were transfected either with miR-320a or ASO-miR-320a and then stimulated with IL-1β. mRNA level expressions of (a) Col2α1 and (b) ACAN were determined by RT-PCR. The concentrations of (c) sGAG and (d) MMP-13 were measured using the Blyscan sGAG Assay Kit and MMP-13 ELISA kit. *, p < 0.05; **, p < 0.01. Statistical analysis was performed using two-tailed paired t-test.

PBX3 was a directed target of miR-320a

In order to elucidate how miR-320a impacted IL-1β-induced cartilage matrix degradation, the microRNA.org database was used¹⁸ to predict the target genes of miR-320a. As the analysis results showed in Figure 3a, PBX3 is a candidate target gene of miR-320a. To confirm this prediction and explore whether PBX3 was a directed target of miR-320a, a double-luciferase assay was performed (Figs 3b and 3c). We found that the PBX3 3’-UTR luciferase expression was significantly decreased by miR-320a overexpression (p < 0.01), while it was increased by miR-320a suppression (p < 0.01). Furthermore, in order to investigate how miR-320a targeted PBX3, cells were transfected either with miR-320a or ASO-miR-320a, and then the mRNA and protein levels of PBX3 were monitored (Figs 3d and 3e). We found that both the mRNA and protein levels of PBX3 were downregulated by miR-320a overexpression (p < 0.05), but upregulated by miR-320a suppression (p < 0.01). Taken together, PBX3 might be a direct target of miR-320a, and was negatively regulated by miR-320a.

Fig.

PBX3 was a directed target of miR-320a. (a) microRNA.org database was used to predict the target genes of miR-320a. (b) miR-320a or (c) ASO-miR-320a was co-transfected with the pMIR-Report vector containing full length PBX3 3’-UTR or a mutated seed site within pMIR-PBX3 into C28/I2 cells. Subsequently, a dual-luciferase reporter assay was performed. miR-320a or ASO-miR-320a was transfected into C28/I2 cells, and the (d) mRNA and (e) protein level expression of PBX3 were detected by RT-PCR and Western blot analysis, respectively. NS, no significance; *, p < 0.05; **, p < 0.01. Statistical analysis was performed using two-tailed paired t-test.

miR-320a overexpression enhanced IL-1β-induced matrix degradation by targeting PBX3

In order to further explore the correlation between miR-320a and PBX3 in IL-1β-induced matrix degradation factors, C28/I2 cells were transfected with miR-320a and/or PBX3 vector and then stimulated with IL-1β. The changes in Col2α1 and ACAN expressions, as well as in sGAG and MMP-13 production, were evaluated. We found that PBX3 significantly attenuated the regulatory effects of miR-320a overexpression on the expression of Col2α1 and ACAN and the production of sGAG and MMP-13 (p < 0.01) (Figs 4a to 4d). Therefore, we deduced that PBX3 might be involved in the regulation of miR-320a overexpression on IL-1β-induced matrix degradation factors.

Fig.

miR-320a overexpression enhanced IL-1β-induced matrix degradation by targeting PBX3. C28/I2 cells were transfected with miR-320a and/or PBX3 vector and then stimulated with IL-1β. mRNA level expressions of (a) Col2α1 and (b) ACAN were determined by RT-PCR. The concentrations of (c) sGAG and (d) MMP-13 were measured by Blyscan sGAG Assay Kit and MMP-13 ELISA kit, respectively. *, p < 0.05; **, p < 0.01. Statistical analysis was performed using two-tailed paired t-test.

miR-320a overexpression enhanced IL-1β-induced matrix degradation by regulating nuclear factor kappa B (NF-κB)

To explore the underlying mechanism of miR-320a on IL-1β-induced cartilage matrix degradation further, C28/I2 cells were treated with MG132, a NF-κB pathway inhibitor, and/or transfected with miR-320a, and then the expression level of Col2α1 and the concentration of MMP-13 were measured. As results showed in Figure 5a, the level of miR-320a was negatively regulated by MG132 (p < 0.01). In addition, MG132 significantly attenuated the regulatory effects of miR-320a on the expression of Col2α1 and the concentration of MMP-13 (Figs 5b and 5c; p < 0.01). These findings suggested that miR-320a overexpression impacted IL-1β-induced matrix degradation factors might be through NF-κB signaling pathway.

Fig.

miR-320a overexpression enhanced IL-1β-induced matrix degradation by regulating NF-κB. (a) C28/I2 cells were treated with MG132 for 24 hours, and the expression of miR-320a was monitored by RT-PCR. C28/I2 cells were transfected with miR-320a and/or treated with MG132, and the expression of (b) Col2α1 and the concentration of MMP-13 (c) were measured by RT-PCR and MMP-13 ELISA kit, respectively. *, p < 0.05; **, p < 0.01. Statistical analysis was performed using two-tailed paired t-test.