RA patients (n=30; female, 22; male, 8; median age, 56.2 years; age range, 21–61 years) who were admitted to the Department of Rheumatology and Immunology, Tianjin Medical University General Hospital (Tianjin, China), between October 2015 and January 2017, were enrolled in the present study. To isolate synovial fibroblasts, synovial tissue specimens obtained immediately after opening the knee-joint capsule were minced and digested with dispase at 37°C for 60 min. After washing, the cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 100 IU/ml penicillin or streptomycin and 10% fetal calf serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA). RA-FLS cultures were maintained at 37°C under humid conditions with 5% CO₂. All FLS cultures were subjected to experimental procedures between passages 4 and 6.

miR-145-5p mimic (cat. no. miR10000437), miR-145-5p inhibitor (cat. no. miR20000437), mimic negative control (cat. no. miR01101), and inhibitor negative control (cat. no.miR02101) were provided by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Cells (2×10⁶) were transfected with miR-145-5p inhibitor (final concentration, 100 nM), miR-145-5p mimic (final concentration, 50 nM), and corresponding controls using RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.), following the manufacturer's instructions. Western blot analyses of factors in the NF-κB, p53/MDM2, JNK, and mitogen-activated protein kinase (MAPK) pathways were performed using proteins extracted from cells treated with miR-145-5p mimic and control mimic for 24 h. Chemical inhibitors were added to cultures 2 h prior to stimulation with miR-145-5p. Subsequently, the cells were used for functional assays or RNA/protein extraction.

TRIzol^® reagent (Takara Bio, Inc., Otsu, Japan) was used to isolate total RNA, including miRNA from FLS. A FastQuant RT Kit (Tiangen Biotech Co., Ltd., Beijing, China) was used to reverse-transcribe RNA into complementary DNA, following the manufacturer's instructions. SYBR-Green PCR Master Mix (Takara Bio, Ic.) was used to perform RT-qPCR, on a Light Cycler 480 (Roche Diagnostics, Indianapolis, IN, USA) with the following program: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec, 60°C for 30 sec, 95°C for 5 sec, and 60°C for 1 min. Specific primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). A forward primer was designed based on the mature miR-145-5p DNA sequence (5′-GUCCAGUUUUCCCAGGAAUCCCU-3′) from miRBase (http://www.mirbase.org), whereas a universal 3′ reverse primer (5′-AGTGCAGGGTCCGAGGTATT-3′; Guangzhou RiboBio Co., Ltd.) was used. Other primers were as follows: MMP-1 forward, 5′-TTCTACATGCGCACAAATCC-3′ and reverse, 5′-ACCGGACTTCATCTCTGTCG-3′; MMP-3 forward, 5′-GCGCCCTGGTCCTGGTGTCCAT-3′ and reverse, 5′-GAAACCACAATTCTGTCTTTCAC-3′; MMP-9 forward, 5′-CCTTCACTTTCCTGGGTAAG-3′ and reverse, 5′-CCATTCACGTCGTCCTTATG-3′; MMP-13 forward, 5′-TCCTGATGTGGGTGAATACAATG-3′ and reverse, 5′-GCCATCGTGAAGTCTGGTAAAAT-3′. GAPDH (forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′) and U6 (forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′ were used as internal controls for mRNA and miRNA, respectively. Relative quantification was performed based on differences in cross-threshold values (ΔCq) between the gene of interest and the endogenous control [i.e., Cq(gene of interest)-Cq (endogenous control)]. Relative expression was calculated using the comparative threshold cycle method (9).

The concentrations of MMP-1 (cat. no. 70-EK1M01-96), MMP-3 (cat. no. 70-EK1M03-96), MMP-9 (cat. no. 70-EK1M09-96) and MMP-13 (cat. no. 70-EK1M132) were assessed in cell supernatants by ELISA using the SensoLyte ELISA kit according to the instructions of the manufacturer (MultiSciences (Lianke) Biotech Co., Ltd., Hangzhou, China).

Cultured FLS were lysed in radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail (both from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and boiled. The protein concentration was measured using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Equal volumes (25 µg) of concentrated samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 8–10% gels, before transfer to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Non-fat dried milk powder in Tris buffered saline with 0.1% Tween-20 was used to block membranes at room temperature for 2 h, and membranes were then incubated with primary antibodies at 4°C overnight. Incubation with horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. 7074S; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature was used to assess the protein levels. Enhanced chemiluminescence (Amersham Life Sciences; GE Healthcare Life Sciences, Little Chalfont, UK) reagent was used to visualize signals, and individual protein band intensities were quantified using ImageJ software (version 1.6.0; National Institutes of Health, Bethesda, MD, USA). Primary antibodies used were as follows: MMP-1 (1:1,000; cat. no. ab137332), MMP-3 (1:1,000; cat. no. ab52915), MMP-9 (1:1,000; cat. no. ab38898), MMP-13 (1:3,000; cat. no. ab39012), IκBα (1:2,000; cat. no. ab32518), p65 (1:3,000; cat. no. ab32536), and p-p65 (1:700; Ser536; cat. no. ab28856) were all purchased from Abcam (Cambridge, MA, USA). β-actin antibody (1:2,000; cat. no. ab8227; Abcam) was applied as a loading control.

Male DBA/1 mice (15–20 g; 8 weeks old) were used in the present study. A total of 30 animals were housed at 24±1°C with a 12:12-h light/dark cycle and a relative humidity of 56±5%. Animals were provided access to food and water ad libitum, and were fed a commercial diet according to the guidelines of the Animal Ethical and Welfare Committee. All mice were housed individually. Lyophilized bovine type II collagen (Chondrex, Inc., Redmond, WA, USA) was dissolved overnight at 4°C in 0.05 M acetic acid under constant stirring. Next, the collagen was emulsified using Freund's complete adjuvant (Chondrex, Inc.) to provide a final concentration of 2 mg/ml. Mice were injected in the tail with 0.1 ml of emulsion on day 1 and booted intraperitoneally on day 21 with collagen. On day 30, an equal volume of mir-145-5p agomir or scrambled miRNA agomir (15 nM; Guangzhou RiboBio Co., Ltd.) was injected in the tail vein of mice at 1-week intervals. Four weeks after the first injection, the mice were sacrificed by injecting pentobarbital sodium at a dose of 100 mg/kg, then various indications such as respiration, heartbeat, pupil reflection, and nerve reflex were observed in the mice, and when all the aforementioned reactions disappeared, death was judged. Next, the right hind limb was dissected and synovial tissue was collected for subsequent experiments.

IHC analysis of MMPs was conducted using 3-µm-thick sections from 8-week-old male DBA/1 mice, obtained from Hua Fukang Co., Ltd. (Beijing, China). Sections were fixed in 4% methanol at 4°C for 24 h, washed in PBS and treated with retrieval solution (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) for 20 min at 90°C. Following incubation in 5% BSA (cat. no. SW3015; Beijing Solarbio Science & Technology Co., Ltd.) at 4°C for 30 min, the sections were treated with MMP-1 (1:1,000; cat. no. ab137332), MMP-3 (1:1,000; cat. no. ab52915), MMP-9 (1:1,000; cat. no. ab38898) and MMP-13 (1:3,000; cat. no. ab39012) primary antibodies overnight at 4°C. Sections incubated with Alexa Fluor^® 568-conjugated rabbit anti-goat immunoglobulin G (1:1,000; cat. no. A-11079; Molecular Probes; Thermo Fisher Scientific, Inc.) as a secondary antibody for 1 h at room temperature. Antibodies were diluted with 5% BSA. DAPI solution was applied for 5 min at room temperature for nuclear staining. Negative controls were prepared in the same manner, but without primary antibody. Images were captured using an Olympus IX2-UCB microscope (magnification, ×40; Olympus Corporation, Tokyo, Japan).

Specific inhibitors of various signaling pathways were purchased from Beijing Solarbio Science & Technology Co., Ltd. and used to analyze the effects of these pathways on levels of MMP-3, MMP-9, and MMP-13. Briefly, specific inhibitors of NF-κB (20 µM, BAY11-7082), p53/MDM2 (10 µM, Nutlin-3), JNK (10 µM, SP600125), and MAPK (10 µΜ, SB203580) pathways (all from Beyotime Institute of Biotechnology, Haimen, China) were added to FLS cultures 2 h prior to miR-145-5p stimulation, following the manufacturer's instructions. RT-qPCR and western blotting were used to assess the expression levels of MMP-3, MMP-9, and MMP-13 as aforementioned.

FLS cells were fixed in 4% methanol at 4°C for 15 min, washed three times (5 min each) with PBS, permeabilized with 0.1% Triton X-100 for 15 min, then, incubated overnight at 4°C with anti-NF-κB p65 primary antibody (1:100; cat. no. ab32536; Abcam), followed by incubation with FITC-labeled secondary antibody (cat. no. ab7086; Abcam) at room temperature for 1 h. Then, the cells were counterstained with DAPI for 5 min. Images were captured using a Leica TCS SP5 confocal fluorescence microscope (magnification, ×80). The negative control was prepared in the same manner without the primary antibody.

TargetScan (version 7.2; http://www.targetscan.org/vert_72/docs/help.html) and mirDB (version 6.0; http://www.mirdb.org/download.html) were employed to search for putative target sequences of miR-145-5p in the NF-κB pathway.

Data were expressed as the mean ± standard error of the mean (SEM) from at least three independent experiments. The difference among groups was determined by analysis of variance (ANOVA) followed by Bonferroni's test, for qPCR experiments and western blotting, and differences between the two groups were analyzed using a two-tailed Student's t-test. Differences were considered statistically significant for P-values <0.05 (P<0.05, P<0.01, P<0.001).

FLS samples isolated from synovial tissue derived from patients with RA were used to investigate the effect of miR-145-5p on synoviocytes in vitro. FLS were incubated with miR-145-5p mimic, miR-145-5p inhibitor, and their corresponding negative controls for 48 h. MMP-3, MMP-9, and MMP-13 RNA levels were significantly increased by miR-145-5p mimic, while they were downregulated by miR-145-5p inhibitor; however, no change in MMP-1 was detected (Fig. 1A and B). Consistent with the changes in secreted protein levels in cell culture supernatants (Fig. 1C-F), levels of intracellular MMP-3, MMP-9, and MMP-13 proteins were markedly enhanced by treatment with miR-145-5p mimic (Fig. 2A and B), but decreased after treatment with miR-145-5p inhibitor (Fig. 2C and D). These results indicated that miR-145-5p regulates various MMPs in FLS.

miR-145-5p agomir or scrambled miRNA agomir was injected into CIA mice in the tail vein. It was observed that miR-145-5p agomir significantly upregulated MMP-3, MMP-9, and MMP-13 compared with the mice treated with the scrambled miRNA agomir, however, the level of MMP-1 underwent no apparent alteration (Fig. 3).

IHC analysis (Fig. 4) revealed increased MMP-3, MMP-9, and MMP-13 levels in the hind ankles of mice treated with miR-145-5p agomir, compared with those in the right hind ankles of control mice.

To explore whether there is a direct functional connection among miR-145-5p, cell signaling pathways, and MMP expression, the effects of NF-κB, p53/MDM2, MAPK and JNK signaling pathways were assessed using specific inhibitors targeting NF-κB (BAY11-7082, 20 µM), p53/MDM2 (Nutlin-3, 10 µM), MAPK (SB203580, 10 µM), and JNK (SP600125, 10 µM). In RA-FLS stimulated by miR-145-5p, the NF-κB inhibitor, BAY11-7082, significantly inhibited MMP-9 levels compared to those in the controls (Fig. 5A-C), while inhibitors of the other three cell signaling pathways had no marked effect on the levels of MMP-3, MMP-9, or MMP-13 (Fig. 5D-F).

To explore the mechanism underlying the involvement of miR-145-5p in MMP activation, the effects of the key NF-κB pathway factors were assessed, including p65, p-p65, and IkB-a, in response to miR-145-5p control of MMP-9 synthesis. Levels of p-p65 were significantly increased, while those of IkB-a were markedly decreased in response to miR-145-5p (Fig. 6A and B). Furthermore, reduction of miR-145-5p led to marked downregulation of p-p65 levels, while it increased those of IkB-a (Fig. 6C-D). In conclusion, miR-145-5p can contribute to arthritis progression by activating NF-κB signaling, which controls MMP-9 expression.

An immunofluorescence assay was conducted to investigate p65 nuclear translocation in FLS. miR-145-5p induced NF-κB p65 nuclear translocation in FLS; however, this change was reversed by treatment with the NF-κB pathway inhibitor, BAY11-7082 (Fig. 7). These findings demonstrated that BAY11-7082 can attenuate miR-145-5p-induced NF-κB activation.