In the present study, 40 male SPF Sprague-Dawley (SD) rats (6-8 weeks old; 200±20 g) were purchased from the Anhui Experimental Animal Center. The rats were housed individually under a set temperature (18-22°C) and humidity (40-60%) with a 12-h light/dark cycle, free access to standard laboratory food and water, and environmental noise kept to a minimum range. All animal experiments were approved by the Animal Ethics Committee of Anhui University of Chinese Medicine (Anhui, China).

After one week of adaptive feeding, all rats were randomly divided into a control group (n=10) and a model group (n=30). Except for the control group, all rats were intracutaneously injected with 0.1 ml Freund's complete adjuvant (FCA) into the right hind metatarsal footpad to induce adjuvant-induced arthritis (AA) and 0.9% saline was administered to the control group rats as a placebo. The rats were anesthetized with 1.0% sodium pentobarbital by a 60 mg/kg intraperitoneal injection and were sacrificed after blood was collected from the abdominal aorta on the 20th day after immunization (22,23).

Then, the synovial tissue was obtained and cultured in complete DMEM medium with 20% fetal bovine serum (lot no. 1715752; cat. no. 10099141C; Gibco; Thermo Fisher Scientific, Inc.) at 37°C and 5% CO₂ by the tissue explant method (24). After a large number of cells were dissociated near the synovial tissue, the tissue mass was discarded and the cells continued to be cultured. When the cell density reached 80-90%, the cells were digested with 0.25% trypsin and passaged at 1:2 and FLS used in the experiment were 3-5 generations. The primary and sub-cultured FLS were observed under an inverted phase contrast microscope (Olympus Corporation), the sub-cultured synovial cells were identified by immunofluorescence staining for vimentin, and the specific operation was carried out according to the instructions of the kit as previously described (25,26). The cells were fixed 20 min with 4% paraformaldehyde (PFA), permeabilized 20 min using 0.1% Triton X-100 and then blocked in 5% BSA for 30 min; all operations aforementioned were performed at room temperature. The cells were then incubated with anti-vimentin (1:100; product code ab92547; Abcam) overnight at 4°C, followed by a further incubation at room temperature for 1 h with FITC-labeled Goat Anti-Rabbit IgG (1:500; cat. no. A0562; Beyotime Institute of Biotechnology). Nuclear DNA was labelled in blue with DAPI. The cell viability was detected by trypan blue staining as follows: Cell survival rate (percentage)=nonblue-stained cells/(blue-stained cells + nonblue-stained cells) ×100%.

To suppress lncRNAS56464.1, specific small interfering RNAs (siRNAs) targeting lncRNAS56464.1 were synthesized and purchased from Shanghai GenePharma Co., Ltd., and were as follows: siRNA1 forward sequence, 5′-GCU ACU UUG UGG UAU CAA UTT-3′ and reverse sequence, 5′-AUU GAU ACC ACA AAG UAG CUU-3′; siRNA2 forward sequence, 5′-GAG GAG AUA GGU AGA ACC UTT-3′ and reverse sequence, 5′-AGG UUC UAC CUA UCU CCU CUU-3′; siRNA negative control (NC) forward sequence, 5′-UUC UUC GAA CGU GUC ACG UTT-3′ and reverse sequence, 5′-ACG UGA CAC GUU CGG AGA ATT-3′. The cells were inoculated on a 6-well plate, and the cell density reached 60-80% confluence before the transfection of siRNA (20, 50, 100 and 200 nM). The mixture of transfection reagents of Lipofectamine 2000 (cat. no. 11668027; Invitrogen; Thermo Fisher Scientific, Inc.), Opti-MEM (cat. no. 31985070; Gibco; Thermo Fisher Scientific, Inc.) and siRNA was slowly dripped into the 6-well plate. After being cultured at 37°C and 5% CO₂ for 4-6 h, the mixed transfection reagents were replaced with complete DMEM (without antibiotics), and the follow-up experiment was carried out after 24 h of transfection as previously described (27).

The effect of lncRNAS56464.1 on FLS proliferation was detected by an MTT assay as previously described (28). The transfected FLS (1×10⁵/ml) were cultured in complete DMEM medium with 20% fetal bovine serum for 12, 24 and 48 h in 96-well plates. After culturing, 20 µl MTT (5 mg/ml) (cat. no. ST316; Beyotime Institute of Biotechnology,) was added into each well and cultured at 37°C and 5% CO₂ for 4 h. Then, 150 µl dimethyl sulfoxide (DMSO) was utilized to dissolve the formazan after discarding the liquid in each well as previously described (29,30). Determination of the absorbance at 490 nm was performed using a microplate reader (BioTek Instruments, Inc.). The cell proliferation inhibition rate was calculated as follows: Inhibition rate = [1−(OD siRNA group-OD blank control group)/(OD control group-OD blank control group)] ×100%. The blank control group consisted of MTT reagents without FLS and the control group refers to FLS treated with DEPC water, which is the solvent of siRNA.

The full-length sequences of lncRNAS56464.1 and mature miRNAs such as miR-152-3p, miR-20a-3p, miR-17-2-3p as well as others which were differentially expressed key miRNAs in the experimental arthritis in our previous study (16), were obtained from NCBI Refseq and miRbase (31), respectively. We predicted the miRNA target of lncRNAS56464.1 using OG-local tools based on the RNAhybrid algorithm (32) and revealed that miR-152-3p to lncRNAS56464.1 had the highest binding degree and lowest free energy. Then, the full fragment of lncRNAS56464.1 with the binding site of miR-152-3p was amplified through PCR, including the wild-type or mutant lncRNAS56464.1 sequence binding site. Then, the constructed plasmid and miR-152-3p mimic with the Lipofectamine 2000 reagent were co-transfected into 293T functional cells (cat. no. FH0244; Shanghai FuHeng Biology). The cells were collected at 48 h post-transfection, and the luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega Corporation) and normalized to the Renilla luciferase activity as previously described (33).

Total RNA was extracted from FLS using TRIzol (lot no. 90802; cat. no. 15596018; Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNAs using the Reverse Transcription kit (K1621; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. RT-qPCR was performed to amplify the cDNA templates by 2X Universal SYBR-Green Fast qPCR Mix (cat. no. RK21203; ABclonal) and using the PIKOREAL 96 fluorescence quantitative PCR instrument (Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Pre-denaturation at 95°C for 3 min, 40 cycles at 95°C for 5 sec and 60°C for 30 sec. The relative expression of each target gene was quantified and normalized by calculating (cycle of quantification) the values and the level of each index as previously described (34,35). The primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and are presented in Table I.

FLS was extracted using RIPA lysis buffer (product no. P0013B; Beyotime Institute of Biotechnology) containing PMSF (100 mM) (product no. ST506; Beyotime Institute of Biotechnology). FLS were washed three times with cold PBS and digested with 100 µl protein lysate. The protein concentration was determined using the BCA method. The lysates (20 µg of protein loaded per lane) were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride membrane (PVDF membrane). The PVDF membrane was washed with 1X TBST for 2 min, sealed with 5% skim milk at room temperature for 2 h, and incubated with anti-Wnt1 (1:5,000; product no. ab63934; Abcam), anti-β-catenin (1:500; ab68183; Abcam), anti-c-Myc (1:500; BM4042; Boster Biological Technology), anti-cyclin D1 (1:500; ab40754), anti-GSK3β (1:2,000; product code ab93926), anti-GSK3β (phospho S9) (1:10,000; product code ab75814), anti-SFRP4 (1:200; ab154167), and anti-β-actin (1:1,000; ab8226; all from Abcam) antibodies overnight at 4°C. Then, the membrane was washed three times with TBST and incubated with a second anti-mouse or anti-rabbit antibody HRP (1:10,000; cat. nos. AS003 and AS014, respectively; ABclonal) for 2 h at room temperature. The protein was detected with an enhanced chemiluminescence kit (EMD Millipore). Each protein band was a representative picture of three replicates. Western blotting data were quantified with ImageJ software (version 1.52, National Institutes of Health) (30).

The cells were fixed in 4% paraformaldehyde for 30 min, then 50-100 µl 0.25% Triton X-100 was added and incubated for 10 min; all steps aforementioned were performed at room temperature. The sections were washed with PBS for three times, and immunostained with primary antibodies, and incubated overnight in a wet chamber at 4°C. The dilutions of the antibodies (as aforementioned) were as follows: Wnt1, 1:100; β-catenin, 1:200; c-Myc, 1:200; cyclin D1, 1:500; p-GSK-3β (Ser9), 1:200; SFRP4, 1:200; and β-actin, 1:200. Then, the sections were stained with the corresponding secondary antibody (as aforementioned) (1:10,000), and incubated at room temperature for 50 min. The nucleus was stained for 5 min with DAPI at room temperature. Six fluorescence images for semi-quantitative analysis and the average optical density were used to reflect the fluorescence intensity of immune response of different indexes as previously described (36).

The data were inputted into SPSS17.0 software (SPSS, Inc.) for statistical processing, and the results were expressed as the mean ± SD (standard deviation) ( x ¯ ± s ). The data were analyzed using one-way ANOVA (one-way analysis of variance) followed by Tukey's multiple comparison test to detect the differences between groups. The result was considered statistically significant when P<0.05.

After more than two passages, the characteristics of the FLS were stable, the species were mainly fibroblasts, the shape was mainly fusiform, and the polar processes were slender, as revealed in Fig. 1A. Under a fluorescence microscope, the passage of FLS was positive for vimentin antibody labeling, the cells were fusiform, and the nucleus was oval and located in the center of the cells, as revealed in Fig. 1B. In addition, the results of trypan blue staining revealed that the survival rate of the FLS was more than 90% (data not shown).

As revealed in Fig. 2A, the highest efficiency of lncRNAS56464.1 siRNA1 interference on FLS was ~30%; the highest efficiency of lncRNAS56464.1 siRNA2 interference on FLS was >60%, and the interference efficiency of 100 nM was more than 50%. According to the interference efficiency, lncRNAS56464.1 siRNA2 (100 nM) was selected for the following experimental study. As revealed in Fig. 2B, with the increasing interference time of the lncRNAS56464.1, the inhibition rate of FLS increased continuously. The inhibition rate significantly increased between 12 and 24 h as well as 12 and 48 h. However, there was no significant increase between 24 and 48 h. Thus, 24 h was selected as the stimulation time for the follow-up experiment.

lncRNAs are considered to bind competitively with miRNAs, resulting in a series of biological effects (37,38). In Fig. 3A, the present results revealed using RNAhybrid, that miR-152-3p was identified as having a potential binding site of lncRNAS56464.1. Then, in the subsequent luciferase experiment, the luciferase constructor containing the wild-type or mutant lncRNAS56464.1 was co-transfected with miR-152-3p or control miRNAs to verify whether lncRNAS56464.1 could target miR-152-3p. As revealed in Fig. 3B, upon overexpressing miR-152-3p in FLS, the luciferase activity of wild-type lncRNAS56464.1 was lower than that of mutant lncRNAS56464.1, which also indicated that lncRNAS56464.1 could directly target miR-152-3p and combine with it.

As revealed in Fig. 4, compared with the control group, the expression of lncRNAS56464.1 was significantly increased (Fig. 4A) and that of miR-152-3p was significantly decreased (Fig. 4B) in the model group. Compared with the model group, the expression of lncRNAS56464.1 was significantly decreased (Fig. 4A) and that of miR-152-3p significantly increased (Fig. 4B) in the siRNA group.

β-catenin is a key gene in the classical Wnt signaling pathway, which plays a central role in signal transduction (40). As revealed in Fig. 6, the mRNA and protein expression of β-catenin in the model group and siRNA (NC) group was significantly higher than that in the control group. The expression levels of β-catenin at both the protein and mRNA level were significantly lower in the siRNA group than that in the model group.

c-Myc is a proto-oncogene that is a downstream target gene of the Wnt signaling pathway (41). As revealed in Fig. 7, compared with the control group, the mRNA and protein expression of c-Myc in the model group and siRNA (NC) group was significantly increased. After lncRNAS56464.1 interference, compared with the model group, the mRNA and protein expression of c-Myc was significantly decreased.

The main function of cyclin D1 is to promote cell proliferation, and its overexpression can lead to cell loss of control (42). To detect the level of cyclin D1, RT-qPCR and western blotting were employed. As revealed in Fig. 8, the mRNA and protein expression of cyclin D1 in the model group and siRNA (NC) group was significantly higher than that in control group. The expression of cyclin D1 protein and mRNA in the siRNA group was significantly lower than that in the model group.

GSK-3β is a serine/threonine kinase that can regulate cell proliferation and differentiation (43). As revealed in Fig. 9, compared with the control group, the mRNA and protein expression of GSK-3β in the model group and siRNA (NC) group had no significant change, but the protein level of p-GSK-3β (Ser9)/GSK-3β was significantly increased. Compared with the model group, the mRNA and protein expression of GSK-3β in the siRNA group had no significant change, but the protein level of p-GSK-3β (Ser9)/GSK-3β was significantly decreased.

SFRP4 is an antagonist of the Wnt signaling pathway, which is closely related to cell proliferation and differentiation (44). As revealed in Fig. 10, the mRNA and protein expression of SFRP4 in the model group and the siRNA (NC) group was significantly lower than that in the control group. After lncRNAS56464.1 interference, the expression of SFRP4 at both protein and mRNA levels was significantly higher than that in the model group.