Long noncoding RNA TUG1 regulates degradation of chondrocyte extracellular matrix via miR-320c/MMP-13 axis in osteoarthritis

2.1 Subjects

The OA cartilage samples (n = 40) were obtained from OA patients undergoing knee replacement surgery at The First People’s Hospital of Jingmen. These participants did not receive intraarticular steroid injections for nearly 3 months before surgery. Normal cartilages (n = 20) were gained from trauma patients without OA or rheumatoid arthritis history. The clinicopathological features of these patients are presented in Table 1.

1. Informed consent: Informed consent has been obtained from all individuals included in this study.

2. Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration and has been approved by the Ethics Committee of The First People’s Hospital of Jingmen.

2.2 Cell culture and treatment

Chondrocytes from OA or normal articular cartilage were separated using an in vitro two-step collagenase digestion method, then maintained in a sterile culture dish and washed thrice with phosphate-buffered saline (PBS; Gibco, Carlsbad, CA, USA). Additionally, complete medium was used to culture the sterile culture dish containing cartilage fragments of size 1 mm × 1 mm × 1 mm. Next the medium was transferred to a 50 mL centrifuge tube and centrifuged for 5 min at 104.67g under cold conditions. Then 5 mL of trypsin (Gibco) was added to digest the chondrocytes. According to the cell density, relative volume cell solution was added to the complete cell culture medium in a humidified CO₂ incubator (5% CO₂) for subculture. The isolated chondrocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Gibco), 100 IU/mL penicillin and 100 mg/mL streptomycin in an incubator at 37°C with 5% CO₂.

Human cartilage C28/I2 cells were obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). DMEM-containing Ham’s F12 nutrient medium (1:1, DMEM/F12; Thermo Fisher Scientific, Rockford, IL, USA) supplemented with 10% FBS (Gibco) and penicillin (100 IU/mL, Gibco)‒streptomycin (100 mg/mL, Gibco) was used to culture C28/I2 cells. Cells were incubated at 37°C in a humidified incubator with 5% CO₂. C28/I2 cells treated with 10 ng/mL IL-1β (Sigma, St. Louis, MO, USA) were used to establish OA cell model in vitro. Then the IL-1β-induced C28/I2 cells were cultured for 12 h for subsequent assay.

2.3 Quantitative reverse-transcriptase-polymerase chain reaction (qRT-PCR)

Total RNA was extracted from OA tissues, chondrocytes and C28/I2 cells using Trizol (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s introduction. To determine TUG1, miR-320c and fucosyltransferase 4 (FUT4) levels, reverse transcription (RT) and qRT-PCR kits were used. First, RT reactions were carried out utilizing TaqMan microRNA reverse transcription kit (Thermo Fisher Scientific), and cDNAs were collected in a final volume of 20 µL as the template for qPCR. Next TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) was employed to tenderly mix the reaction solutions according to the manuals. Subsequently, all reaction tubes were placed on a 7900 Real-time system (Applied Biosystems, Rockford, IL, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 snRNA was applied as the internal references. Relative expression of TUG1, miR-320c and FUT4 was calculated using the 2^−ΔΔCt method. The primers are listed as follows: TUG1 (forward, 5′-TCACAAGGCTGCACCAGATT-3′; reverse, 5′-GTCGGTCACAAAATGCATAGAGG-3'); miR-320c (forward, 5′-ACACTCCAGCTGGGAAAAGCTGGGTTGAGA-3′; reverse, 5′-ACACTCCAGCTGGGTCGCCCTC-3′); FUT4 (forward, 5′-CGGACGTCTTTGTGCCTTAT-3′; reverse, 5′-CGAGGAAAAGCAGGTACGAG-3′); GAPDH (forward, 5′-ATTCCATGGCACCGTCAAGGCTGA-3′; reverse, 5′-TTCTCCATGGTGGTGAAGACGCCA-3′); U6 (forward, 5′-ATTGGAACGATACAGAGAAGATT-3′; reverse, 5′-GGAACGCTTCACGAATTTG-3′). The above primers were synthesized by Genepharma (Shanghai, China).

2.4 Transient transfection

Small interfering RNA against TUG1 (si-TUG1) and its corresponding negative control (si-NC), miR-320c mimic (miR-320) and its inhibitor (anti-miR-320), as well as the relative control (miR-NC or anti-miR-NC), overexpression sequences of TUG1 and FUT4 (pcDNA-FUT4), empty pcDNA3.1 vector (pcDNA) synthesized by Genepharma were transfected into C28/I2 cells using Lipofectamine™ 3000 (Invitrogen).

2.5 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay

For cell proliferation assay, 5,000 C28/I2 cells were seeded onto the 96-well plate. After 12 h incubation, the MTT solution was added into each well and then the cells were cultured for another 4 h. Then the supernatants were replaced with 150 µL of dimethyl sulfoxide (Sigma). Finally, the optical density of the solution was identified via a microplate reader (Thermo Fisher Scientific).

2.6 Flow cytometry

Cell apoptosis was measured by using fluorescein isothiocyanate-labeled Annexin V/propidium iodide (Annexin V-FITC/PI) detection kit (Sangon Biotech, Shanghai, China). At 12 h post-transfection, cells were collected and washed with ice-cold PBS. Subsequently, 1× binding buffer was used to resuspend the cells and 5 µL of annexin V and PI was utilized to stain the apoptotic cells in the dark, according to the manufacturer’s protocol. Finally, the proportion of apoptotic cells was counted via a flow cytometry machine (BD Biosciences, San Jose, CA, USA).

2.7 Western blot assay

The total proteins from tissue samples, chondrocytes and C28/I2 cells were harvested. The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (12%) and transferred onto a nitrocellulose membrane (Thermo Fisher Scientific). Then the membranes were blocked by 5% skim milk powder (Sangon Biotech) for 2 h at room temperature. Subsequently, the membranes were incubated with specific primary antibody primary antibodies overnight at 4°C. The primary antibodies included proliferation-relative protein of cyclin D1 (ab16663, 1:150; Abcam, Cambridge, MA, USA), apoptosis protein of cleaved-caspase 3 (cleaved-casp-3; ab2302, 1:500; Abcam), and ECM degradation-relative proteins of MMP-13 (ab39012, 1:4,500; Abcam), type II collagen (ab34712, 1:3,000 collagen II; Abcam), Aggrecan (ab3778, 1:100; Abcam) and FUT4 (ab181461, 1:1,000; Abcam), and GAPDH (ab8245, 1:5,000; Abcam) were the endogenous references. The next day, the uncombined antibodies were washed trice with tris-buffered saline Tween-20 and then the membranes were covered with matched secondary antibody at room temperature for 1 h. After washing, the blots were visualized using enhanced chemiluminescence (ECL) substrates (Millipore, Bedford, MA, USA) and then photographed.

2.8 Dual-luciferase reporter assay

The biological prediction software of starBase was employed to identify the binding sites between miR-320c and TUG1 or FUT4. In accordance with obtained results, the common fragments of miR-320c, wild-type (WT-TUG1) or mutant (MUT-TUG1) TUG1, and 3′-UTRs common sequence of wild-type (WT-FUT4) and mutant (MUT-FUT4) of FUT4 were amplified and cloned into cloning sites downstream of pmirGLO (Promega Corporation, Madison, WI, USA). In this assay, WT-TUG1, MUT-TUG1, WT-FUT4 or MUT-FUT4 was co-transfected with miR-con or miR-320c mimic into cells using Lipofectamine™ 3000 (Invitrogen). Relative dual-luciferase activity assay was analyzed based on the introduction provided by Promega dual-luciferase assay kit. pRL-TK vector (Promega) was used to adjust the cell number and transfection efficiency.

2.9 RNA immunoprecipitation (RIP) assay

RIP assay was implemented utilizing Magna RIP RNA-binding protein immunoprecipitation kit (Millipore) and AGO2 antibody in accordance with the producer’s descriptions, and immunoglobulin G antibody was used as the control. Cells were transfected with TUG1, miR-320c mimic or FUT4 and cultured for 24 h, and the abundance was evaluated via qRT-PCR.

2.10 Statistical analysis

Data from at least three independent assays were processed by SPSS 19.0 software and expressed as mean ± standard deviation. Student’s t-test or one-way analysis of variance was performed to analyze the differences in significant group, and P < 0.05 was considered statistically remarkable.

3.1 Increase in TUG1 in OA tissues and cell lines

In order to investigate the biological role of TUG1 in OA development, this study first measured TUG1 level in OA patients, and the result showed that TUG1 expression was efficiently augmented in OA tissues compared with the normal control (Figure 1a). Meanwhile, chondrocytes were isolated from OA and normal tissues. qRT-PCR suggested an increase in TUG1 in isolated OA chondrocytes (Figure 1b). Subsequently, C28/I2 cells and IL-1β were used to establish OA cell model, and the expression of TUG1 was significantly elevated in IL-1β-stimulated C28/I2 cells (Figure 1c). Overall, TUG1 was highly expressed in OA, which may play a critical role in the progress of human OA.

Figure 1

High expression of TUG1 in OA tissues and cell lines. (a) The level of TUG1 was determined via qRT-PCR in OA tissues. (b) qRT-PCR was employed to detect TUG1 level in isolated chondrocytes. (c) Expression of TUG1 in IL-1β-induced C28/I2 cells was identified utilizing qRT-PCR. *P < 0.05.

3.2 TUG1 deletion promoted cell proliferation and inhibited apoptosis and ECM degradation in IL-1β-induced C28/I2 cells

Due to the aberrant expression of TUG1 in OA tissues, the functions of TUG1 were researched in vitro. The knockdown efficiency of si-TUG1 was remarkably evident (Figure 2a). The results showed that IL-1β could impede cell proliferation, while this inhibitory effect of IL-1β on cell proliferation was restored by TUG1 knockdown (Figure 2b). Apoptotic cells were also examined by flow cytometry, and the results uncovered that the promotion effect of IL-1β treatment on cell apoptosis was regained via TUG1 silencing (Figure 2c and d). Furthermore, proliferation-related protein of cyclin D1, apoptosis of cleaved-casp-3 and ECM degradation-related proteins of MMP-13, collage II and Aggrecan were measured and quantified in IL-1β-treated C28/I2 cells. The proliferation and apoptosis tendency was in line with the results of MTT and flow cytometry assays, and Western blot results also determined that the acceleration impact of IL-1β on ECM degradation was abrogated by downregulation of TUG1 in vitro (Figure 2e and f). In brief, TUG1 knockdown facilitated cell proliferation and relieved apoptosis and ECM degradation in IL-1β-induced C28/I2 cells.

Figure 2

TUG1 deletion promoted cell proliferation and inhibited apoptosis and ECM degradation in IL-1β-induced C28/I2 cells. (a) The knockdown efficiency of si-TUG1 was confirmed via qRT-PCR. (b) MTT assay measured cell proliferation in IL-1β-induced C28/I2 cells. (c and d) Flow cytometry analyzed IL-1β-stimulated C28/I2 cell apoptosis in vitro. (e and f) Expression of cyclin D1, cleaved-casp-3, MMP-13, collage II and Aggrecan was measured using Western blot assay. *P < 0.05.

3.3 miR-320c was a target gene of TUG1

StarBase software predicted that miR-320c harbored the binding sites for TUG1 (Figure 3a). Dual-luciferase reporter assay indicated that the luciferase activity decreased in the WT-TUG1 group, but not in the MUT-TUG1 group, providing evidence that miR-320c was a target gene of TUG1 (Figure 3b). Furthermore, RIP assay results also concluded that TUG1 directly targeted miR-320c (Figure 3c). Moreover, our data exhibited that TUG1 silencing triggered miR-320c expression, while miR-320c expression was suppressed by the overexpression of TUG1 (Figure 3d). Furthermore, the results demonstrated that the miR-320c level was evidently inhibited in OA cartilage tissues and separated OA chondrocytes (Figure 3e and f). The level of miR-320c was also decreased in IL-1β-treated C28/I2 cells (Figure 3g). Besides, the expression of miR-320c was negatively correlated with the TUG1 level (Figure 3h). All the above data indicated that TUG1 exerted its role in IL-1β-treated C28/I2 cells via downregulating miR-320c.

Figure 3

miR-320c was a target gene of TUG1. (a) The interrelation between miR-320c and TUG1 was predicted using starBase software. (b and c) Dual-luciferase reporter and RIP assays verified the interaction between miR-320c and TUG1. (d) Level of miR-320c was examined by qRT-PCR in C28/I2 cells transfected with si-NC, si-TUG1, pcDNA or TUG1, respectively. (e and f) qRT-PCR was utilized to determine the level of miR-320c in OA tissues and isolated chondrocytes. (g) Expression of miR-320c in IL-1β-treated C28/I2 cells was measured by qRT-PCR. (h) The correlation between miR-320c and TUG1 was analyzed by Spearman’s correlation analysis. *P < 0.05.

3.4 FUT4 was directly targeted by miR-320c

StarBase showed that FUT4 was a downstream of miR-320c (Figure 4a). Dual-luciferase reporter assay was used to verify the interaction between miR-320c and FUT4, and the luciferase activity in the WT-FUT4 group was inhibited by miR-320c, while no change was observed in the MUT-FUT4 group (Figure 4b). RIP assay results also demonstrated that FUT4 was targeted by miR-320c (Figure 4c). Furthermore, miR-320c inhibitor apparently boosted FUT4 mRNA level and protein expression, while the effects of miR-320c mimic on FUT4 mRNA level and protein expression were opposite to that of miR-320c inhibitor (Figure 4d and e). Moreover, FUT4 mRNA level specially increased in OA cartilage tissues (Figure 4f). The mRNA level and protein expression of FUT4 were augmented in separated chondrocytes (Figure 4g and h). In addition, the mRNA and protein levels of FUT4 also upregulated in IL-1β-stimulated C28/I2 cells (Figure 4i and j). Our data indicated that FUT4 expression was negatively related to the miR-320c level (Figure 4k). In short, these findings revealed that miR-320c directly targeted FUT4.

Figure 4

FUT4 was directly targeted by miR-320c. (a) The target relationship between miR-320c and FUT4 was predicted using starBase software. (b and c) The relationship between miR-320c and FUT4 was confirmed via dual-luciferase reporter and RIP assays. (d and e) Expression of FUT4 in C28/I2 cells transfected with miR-320 mimic or its inhibitor was assessed via qRT-PCR and Western blot assays. (f) TUG1 level in OA was analyzed by qRT-PCR. (g–j) The mRNA and protein expression of FUT4 in separated chondrocytes and IL-1β-stimulated C28/I2 cells were examined by qRT-PCR and Western blot assays, respectively. (k) Spearman’s correlation analysis was used to analyze the interrelation between miR-320c and TUG1. *P < 0.05.

3.5 FUT4 overexpression reversed the effects of miR-320c on cell proliferation, apoptosis and ECM degradation in IL-1β-treated C28/I2 cells

To further investigate the regulatory mechanism of miR-320c and FUT4 in the progression of OA, miR-NC, miR-320c, miR-320c + pcDNA or miR-320c + FUT4 was transfected into IL-1β-stimulated C28/I2 cells. The results showed that the inhibiting impacts of miR-320c mimic on the FUT4 mRNA level and protein expression were both rescued by overexpression of FUT4 (Figure 5a and b). Moreover, upregulation of FUT4 relieved the promotion effect of miR-320c mimic on cell proliferation in IL-1β-treated C28/I2 cells (Figure 5c). Cell apoptosis hindered by miR-320c mimic was abrogated by overexpression of FUT4 (Figure 5d). The expression of cyclin D1 and cleaved-casp-3 confirmed the above conclusion of cell proliferation and apoptosis in MTT and flow cytometry assays. In addition, ECM-relative proteins of MMP-13, collage II and Aggrecan were examined by Western blot, and the results indicated that FUT4 upregulation restored the inhibiting effect of miR-320c mimic on degradation of ECM in IL-1β-stimulated C28/I2 cells (Figure 5e and f). These data demonstrated that miR-320c regulated cell proliferation, apoptosis and ECM degradation in IL-1β-treated C28/I2 cells by targeting FUT4.

Figure 5

FUT4 overexpression reversed the effects of miR-320c on cell proliferation, apoptosis and degradation in IL-1β-treated C28/I2 cells. (a–f) C28/I2 cells underwent IL-1β stimulation were transfected with miR-NC, miR-320c, miR-320c + pcNDA or miR-320c + FUT4, respectively. (a and b) The mRNA and protein levels of FUT4 were evaluated by qRT-PCR and Western blot assays, respectively. (c) Cell proliferation was measured using MTT assay. (d) Apoptotic cells were calculated via flow cytometry. (e and f) Western blot assay was carried out to detect the protein expression of cyclin D1, cleaved-casp-3, MMP-13, collage II and Aggrecan. *P < 0.05.

3.6 Effects of TUG1 silencing on cell proliferation, apoptosis and ECM degradation were abrogated by miR-320c inhibitor or FUT4 overexpression in IL-1β-stimulated C28/I2 cells

First, the correlation between FUT4 and TUG1 was analyzed and the results showed that FUT4 expression was positively related to the TUG1 level (Figure 6a). Then the molecular mechanism between TUG1 and miR-320c or FUT4 was explored, and si-NC, si-TUG1, si-TUG1 + anti-miR-NC, si-TUG1 + anti-miR-320c, si-TUG1 + pcDNA or si-TUG1 + pcDNA-FUT4 was transfected into IL-1β-treated C28/I2 cells. The FUT4 mRNA and protein expression were restrained by TUG1 deletion, which was recovered by miR-320c inhibitor or FUT4 overexpression (Figure 6b and c). Moreover, the acceleration effect of TUG1 silencing on cell proliferation in IL-1β-treated C28/I2 cells was blocked by miR-320c inhibitor or FUT4 upregulation (Figure 6d). The inhibitory effect of si-TUG1 on cell apoptosis was abrogated by miR-320c inhibitor or overexpression of FUT4 in C28/I2 cells under IL-1β-treated condition (Figure 6e). Furthermore, the expression of cyclin D1 and cleaved-casp-3 also verified the previously obtained results regarding cell proliferation and apoptosis. Besides, ECM-relative proteins of MMP-13, collage II and Aggrecan were also measured by Western blot assay, and the results displayed that the repression impact of TUG1 deletion on degradation of ECM was overturned by miR-320c inhibitor or FUT4 upregulation in C28/I2 cells with IL-1β stimulation (Figure 6f and g). In short, the influence of TUG1 knockdown on cell proliferation, apoptosis and degradation of ECM was abolished by miR-320c inhibitor or FUT4 overexpression in IL-1β-induced C28/I2 cells.

Figure 6

Effects of TUG1 silencing on cell proliferation, apoptosis and ECM degradation were abrogated by miR-320c inhibitor or FUT4 overexpression in IL-1β-stimulated C28/I2 cells. (a) The association between TUG1 and FUT4 was analyzed via Spearman’s correlation analysis. (b–g) si-NC, si-TUG1, si-TUG1 + anti-miR-NC, si-TUG1 + anti-miR-320c, si-TUG1 + pcDNA or si-TUG1 + pcDNA-FUT4 was introduced into IL-1β-induced C28/I2 cells. (b and c) The mRNA level and protein expression were measured by qRT-PCR and Western blot assays, respectively. (d) MTT assay was performed to estimate cell viability in vitro. (e) The apoptotic rate was measured by flow cytometry. (f and g) The relative proteins of cyclin D1, cleaved-casp-3, MMP-13, collage II and Aggrecan were determined using Western blot. *P < 0.05.