Synovia were obtained from 5 patients with RA and 5 patients with osteoarthritides (OA) that fulfilled the American College of Rheumatology Criteria for RA or OA (22), as well as one patient undergoing reconstruction of the anterior cruciate ligament (as a control). Synovial tissues were collected from 3 patients with RA as they underwent total knee arthroplasty (TKA) and 2 patients with RA as they underwent synovectomy. All synovial tissue samples taken from patients with OA were collected during TKA. The mean age of patients with RA and OA were 62.9±10.9 (39–78) and 71.6±4.9 (72–81) years, respectively. As the mean age of patients in the two groups differed, a multivariate logistic regression model with adjustment for age was used to evaluate miR-143-3p expression in the synovium of patients with RA as well as those with OA. Among the patients with RA, there were 2 males and 3 females; however, all patients with OA were female. A further patient (male, 45 years old) undergoing leg amputation but with a normal knee joint was included as the control. The synovial tissues were harvested from the control patient during the amputation surgery. The present study was approved by the Ethics Committee of Shandong Academy of Medical Science (Jinan, China) and written informed consent was obtained from all patients that participated in the study.

The human RA synovial cell line MH7A was procured from the American Type Culture Collection (Manassas, VI, USA). MH7A cells were stained positively for intercellular adhesion molecule-1 (ICAM-1), interleukin (IL)-1R, cluster of differentiation (CD)16, CD40, CD80 and CD95 (23); and have been previously used to investigate the molecular mechanisms underlying RA (23). Cells were maintained in RPMI 1640 medium (Hyclone; GE Healthcare, Logan, UT, USA), supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin/streptomycin (1:100, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), at 37°C in a relative humidity of 95% and in 5% CO₂ (24).

To assess the effects of miR-143-3p abnormal expression on MH7A cells, the cells were transfected with miR-143-3p mimic, miR-143-3p inhibitor or their controls (mimic NC or inhibitor NC). Briefly, MH7A cells (1×10⁵-2×10⁵) were transfected with 90 pmol hsa-miR-143-3p mimic (Takara, Okinawa, Japan) and the mimic control (mimic NC; 90 pmol) (Takara) three times over 7 days. Cells were also transfected with mirVana^™ hsa-miR-143-3p inhibitor (Thermo Fisher Scientific, Inc.) (150 pmol), inhibitor NC (150 pmol), small interfering (si)RNA against IGF1R (si-IGFR; 100 pmol) or IGFBP5 (si-IGFBP5; 100 pmol) or the si-NC control (100 pmol) (Thermo Fisher Scientific, Inc.) for 72 h. All transfections were performed using Lipofectamine^® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. The cells in the control group received no additional treatment. The sequences of the miRs and siRNA used were as follows: miR-143-3p mimic sense 5′-GGUGCAGUGCUGCAUCUCUGGU-3′ and anti-sense, 5′-CAGAGAUGCAGCACUGCACCUU-3′; mimic NC sense 5′-UCACAACCUCCUAGAAAGAGUAGA-3′; mimic NC antisense 5′-UCUACUCUUUCUAGGAGGUUGUGA-3′; miR-143-3p inhibitor 5′-ACCAGAGAUGCAGCACUGCACC-3′; inhibitor NC, 5′-UCUACUCUUUCUAGGAGGUUGUG-3′. si-IGFR 5′-GCGGAGAGAUGUCAUGCAAGU-3′; si-IGFBP5 5′-GGAUCUGUCUCCUCCUCUAGC-3′. si-NC: 5′-UUCUUCGAAGGUGUCACGUTT-3′.

MH7A cells were seeded in 96-well plates at 5,000 cells/well for 72 h transfected with miR-143-3p inhibitor, si-IGF1R, si-IGFBP5 or corresponding controls (inhibitor NC or si-NC) for 24 h. Subsequently, cells were treated with tumor necrosis factor (TNF)-α (10 ng/ml; Sigma-Aldrich; Merck KGaA) at room temperature for 24 h to construct a model of RA. The cells in the control group were treated with normal RPMI 1640 medium. Subsequently, CCK-8 solution (Invitrogen; Thermo Fisher Scientific, Inc.) was added to each well and plates were incubated at 37°C for 2 h. The absorbance of each well was measured at 450 nm using a microplate reader (25).

Cells or corresponding controls were harvested following transfection with miR-143-3p inhibitor, si-IGF1R or si-IGFBP5 for 24 h. Cells were treated with TNF-α, washed twice with pre-chilled PBS and resuspended in 100 µl binding buffer at a concentration of 1×10⁶ cells/ml. Annexin V and propidium iodide (PI) double-staining was performed using an Annexin V-fluorescein isothiocyanate Apoptosis Detection kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's protocol. Apoptosis analysis was performed using the BD LSRII Flow Cytometer System with FACSDiva Software version 6.0 (BD Biosciences) within 1 h (25).

The potential target genes of miR-143-3p were analyzed by TargetScan Human 7.0 (targetscan.org/vert_71/). A cut-off value of 0.7 was applied to predict the target genes. To develop a luciferase reporter construct, the 3′-UTR fragments of IGF1R or IGFBP5, which contained putative binding sites for miR-143-3p, were inserted downstream of firefly luciferase in the pGL3 vector (Promega Corporation). Mutant 3′-UTR, which carried the mutated sequence in the complementary site for miR-143-3p, was generated using the polymerase chain reaction (PCR) method (26) and inserted downstream of firefly luciferase in a separate pGL3 vector. 293A cells (1×10⁵; American Type Culture Collection) were cultured in a 48-well plate were co-transfected with miR-143-3p with a luciferase reporter comprising wild-type or mutant 3′-UTR of the target gene. The luciferase assay was performed as previously reported (26). 293A cells were co-transfected with miRs and wild-type 3′-UTR or mutant 3′-UTR luciferase reporter, using pRL-TK (Promega Corporation) as a control vector. Following 48 h transfection, luciferase activity was measured using the Dual Luciferase Assay kit (Promega Corporation) with a β-counter luminometer. Relative luciferase activity was calculated as the ratio of the raw firefly luciferase activity and Renilla luciferase activity (27).

Total RNA was isolated from MH7A cells using the commercial total RNA miniprep kit (Axygen; Corning Inc., Corning, NY, USA) according to the manufacturer's protocol. Each sample was reverse transcribed using a cDNA synthesis kit (Takara). qPCR was performed using SYBR Green PCR Premix Ex Taq II reagents (Takara) on a Light Cycler 480 II real-time system (Roche Diagnostics, Indianapolis, IN, USA). The house-keeping gene GAPDH was used for normalization. The mRNA expression was quantified by the comparative 2^{−(∆∆Cq)} method (28). The primers used for target amplification were as follows: MiR-143 forward, 5′-TGAGATGAAGCACTGTAGCTC-3′ and reverse, 5′-TGGTGTCGTGGAGTCG-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; B-cell lymphoma (Bcl)-2 forward, 5′-GATGCAGTGCCGGCCTAAG-3′ and reverse, 5′-TTCTCTTGTACGCACGAGCT-3′; Bax forward, 5′-CTGAGCTGACCTTGGAGC-3′ and reverse, 5′-GACTCCAGCCACAAAGATG-3′; Caspase-3 forward, 5′-ACCGATGTCGATGCAGCTAA-3′ and reverse, 5′-AGGTCCGTTCGTTCCAAAAA3′. GAPDH forward, 5′-CCACCCATGGCAAATTCCATGGCA-3′ and reverse, 5′-TCTAGACGGCAGGTCAGGTCCACC-3′; IGF1R forward, 5′-GCGAGCTCTCTGGGATAGAAATGTTTAGGTGTA-3′ and reverse, 5′-GCAAGCTTCAGGTGCTGAGAAAGGTGAGATGT-3′; IGFBP5 forward, 5′-ACGCGTCGACATGGGCTCCTTCGTGCAC3′ and reverse, 5′-CGCGGATCCATCACTCAACGTTGCTGCTG-3′. The thermocycling conditions were as follows: 95°C for 1 min followed by 35 cycles of 95°C for 10 sec and 58°C for 40 sec, with a final extension step of 10 min at 68°C.

(29). The culture medium was collected and the concentration of the cytokines was determined using commercial ELISA kits for IL-1β (ab100704), IL-6 (ab46042), IL-8 (ab46032), MMP-1 (ab100603) and MMP-13 (ab100605) (all Abcam, Cambridge, UK) following the manufacturer's protocol. Values were calculated on the basis of a standard curve constructed for each assay.

Cells were solubilized with radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.) and protein concentration was measured using a BCA kit (Thermo Fisher Scientific, Inc.). Approximately 50 µg of protein from each sample was separated by 12% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Burlington, MA, USA). Following blocking with 5% bovine serum albumin (1:100; Sigma-Aldrich; Merck KGaA) in Tris-buffered saline with Tween for 2 h at room temperature, membranes were incubated with Bcl-2 (ab178529), Bax (ab53154), pro-caspase-3 (ab32150) and active-caspase-3 (ab181418), phosphorylated (p)-p38 (ab4822), p38 (ab31828) and Ras (ab52939) primary antibodies at a dilution of 1:1,000 overnight at 4°C (all Abcam, Cambridge, UK), followed by incubation with goat anti-rabbit horseradish peroxidase conjugated secondary antibodies (at a dilution of 1:2,000; ab191866; Abcam) for 1 h at room temperature. GAPDH (ab8245; dilution 1:2,000; Abcam) was used as an internal control. Protein blots were visualized and analyzed using a chemiluminescence system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and autoradiography films (Kodak Image Station 440; Kodak, Rochester, NY, USA).

Data are presented as the mean ± standard deviation from at least three independent experiments. All statistical analyses were performed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). Student's t test was performed to assess whether differences between two groups was significant, whereas one-way analysis of variance followed by a post-hoc Tukey's test was performed to evaluate whether differences among three or more groups were significant. P<0.05 was considered to indicate a statistically significant difference.

The expression of miR-143-3p in RA was significantly higher than in patients with OA and the control (P<0.01; Fig. 1A). MH7A cells were treated with 10 ng/ml TNF-α to construct a model of RA and the results demonstrated that the expression of miR-143-3p was significantly increased in TNF-α-induced RA cells compared with the untreated control cells (P<0.05, Fig. 1B).

Transfection of the miR-143-3p inhibitor in MH7A cells significantly decreased miR-143-3p expression (P<0.01), indicating that transfection was successful (Fig. 2A). Treatment with TNF-α significantly increased the proliferation and inhibited the apoptosis of MH7A cells (P<0.05; Fig. 2B and C). The effect of TNF-α on cell proliferation and apoptosis was significantly reversed following inhibition of miR-143-3p (P<0.05; Fig. 2B-D). In addition, the results demonstrated that transfection with the miR-143-3p inhibitor significantly promoted the expression of Bax and inhibited the expression of Bcl-2 (P<0.01; Fig. 2E and F). The expression of cell apoptosis-associated proteins, including Bcl-2 and pro-caspase-3 was significantly increased in the TNF-α group (P<0.01) compared with the control group, whereas the expression of Bax and active-caspase-3 were significantly decreased (P<0.05; Fig. 2E and F). Furthermore, following transfection with miR-143-3p inhibitor, the expression of pro-caspase-3 was significantly inhibited and the expression of active-caspase-3 was significantly promoted.

As presented in Fig. 3, TNF-α treatment significantly increased levels of inflammatory factors, including interleukin (IL)-1β, IL-6, IL-8, matrix metalloproteinase (MMP)-1 and MMP-13 (P<0.05). However, cells transfected with miR-143-3p inhibitor exhibited significant decreases in levels of these inflammatory factors, compared with control cells (P<0.05; Fig. 3).

The TargetScan Human 7.0 (targetscan.org/vert_71/) was used to analyze the potential target gene of miR-143-3p, and the sequence analysis data indicated that IGF1R and IGFBP5 were targets of miR-143-3p (Fig. 4A and B). The results of the luciferase reporter assay confirmed that the 3′-UTR regions of IGF1R and IGFBP5 possess hsa-miR-143-3p binding sites (P<0.05; Fig. 4C and D). Moreover, the expression levels of IGF1R and IGFBP5 were significantly inhibited following transfection of miR-143-3p mimics (P<0.05), whereas the transfection of miR-143-3p inhibitors significantly increased the levels of IGF1R and IGFBP5 (P<0.01; Fig. 4E and F). Reduced levels of IGF1R and IGFBP5 mRNA and protein expression were observed in RA models following treatment with TNF-α (P<0.05; Fig. 4G and H).

As presented in Fig. 5A-D, levels of IGF1R and IGFBP5 expression were significantly suppressed following transfection with siRNAs against IGF1R and IGFBP5, respectively (P<0.01), indicating efficient transfection. Furthermore, suppression of IGF1R or IGFBP5 expression significantly reversed the effect of miR-143-3p inhibition on MH7A cell proliferation and apoptosis (P<0.05; Fig. 5E-H). These results indicate that miR-143-3p regulates MH7A cell proliferation and apoptosis by targeting IGF1R or IGFBP5.

The effect of TNF-α treatment and the miR-143-3p inhibitor on the Ras/p38 signaling pathway was investigated. The results demonstrated that TNF-α treatment significantly increased the expression of Ras (P<0.01) and p-p38/p38 (P<0.001; Fig. 6). These increases were significantly reversed by the miR-143-3p inhibitor, indicating that the miR-143-3p inhibitor inhibits the Ras/p38 MAPK signaling pathway in a TNF-α-induced RA model.