Patients and tissue samples

A total of 24 RA patients (12 females and 12 males, aged 34–58 years old) who underwent synovectomy and 24 healthy volunteers with trauma (all males, aged 34–48 years, mean age 39.9 years old) were enrolled at the Bengbu Medical College. RA was diagnosed on the basis of the 2010 criteria of the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR). All patients included in the study were in the active phase of the disease (Disease Activity Score 28 [DAS28] ≥ 2.6). Patients who had received antirheumatic medication and had no joint swelling and restricted in movement, or knee Larsen stage II surgery, were excluded. All RA patients underwent knee anteroposterior and lateral X-ray imaging prior to synovectomy, and the radiographs were evaluated by an experienced orthopedic surgeon. Synovial tissue samples were collected at the time of joint surgery. Table 1 presents comprehensive details regarding the characteristics of all participants. The study was conducted in accordance with the Declaration of Helsinki and its later amendments and approved by the ethics committee of the hospital (2021-089). All participants gave informed consent.

Table 1 Characteristics of patients with rheumatoid arthritisCell culture

RASFs were obtained from the RA patients during synovectomy. The synovial tissue specimens were washed five times with Hank’s buffer (pH 7.5), minced, and digested with 120 μl type II collagenase in 4 ml DMEM at 37 °C for 6 h. The reaction was terminated with 3 ml EDTA-free trypsin, and the cells were centrifuged. The primary RASFs from passages 4–10 were used for subsequent experiments. The human fibroblast-like synoviocyte cell line (HFLS) was purchased from Cell Applications, Inc. (San Diego, CA, USA). All cells were cultured in DMEM (Gibco, Invitrogen) medium supplemented with 10% fetal bovine serum (Gibco, Invitrogen), 1000 U/ml streptomycin, and 1000 U/ml penicillin (Gibco, Invitrogen) at 37 °C with 5% CO2.

Overexpress LEF1-AS1 in HFLS

The LEF1-AS1 pcDNA3.1 vector was constructed using the pcDNA3.1 expression vector (Invitrogen, USA). The pcDNA3.1-LEF1-AS1 was synthesized by GeneChem (Shanghai, China). HFLS cells were seeded into 6-well plates and transfected with vectors using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. After 48 h of incubation, the cells were collected for further experiments. The experiment was repeated three times.

Luciferase assay

pGL3-WT lncRNA LEF1-AS1 and pGL3-MUT lncRNA LEF1-AS1 were synthesized by Shenggong Biotechnology (Shanghai) Co. For the transfection experiments, miR-30-5p and pGL3-WT lncRNA LEF1-AS1 or pGL3-MUT lncRNA LEF1-AS1 were cotransfected using Lipofectamine 3000 transfection reagent. After 12 h, the culture medium was replaced with fresh culture medium and incubated for an additional 24 h. Cell lysates were prepared by adding cell lysis buffer, followed by centrifugation at 12,000 rpm for 5 min. The supernatant was collected, and luciferase activity was measured. All assays were performed in triplicate.

Real-time PCR

Total RNA was extracted from the cells using TRIzol reagent (Invitrogen Technology, USA), and the concentration and purity of the samples were determined. Reverse transcription was performed with 2–5 mg RNA using a kit (BEENbio, Shanghai, China) in 20-ml reaction mix. The conditions of the reverse transcription reaction were as follows: 25 °C, 5 min; 42° C, 60 min; and 70 °C, 15 min. The primers were designed and synthesized by Shanghai Biology Engineering Corporation and are shown in Table 2.

Table 2 Primers used in the articleWestern blot

Equal amounts of protein (20 µg) per sample were separated by SDS-PAGE at 80 V and 120 V and transferred to PVDF membranes (Millipore, Billerica, MA, USA) for 120 min at 100 V. After blocking with 5% skim milk and washing thrice with TBST, the membranes were incubated overnight with primary antibodies at 4 °C, followed by incubation with HRP-labeled secondary antibodies for 1 h at room temperature. The following primary antibodies were used: rabbit anti-PIK3R2 (PA5-84807, Thermo Fisher, USA), anti-PI3K (3811S, CST, USA), anti-p-PI3K (4228S, CST, USA), anti-AKT (4691S, CST, USA), anti-p-AKT (4060S, CST, USA), anti-GAPDH (ab8245, Abcam, USA), and anti-LAIR1 (ab189412, Abcam, USA).

Enzyme-linked immunosorbent assay (ELISA)

The blood samples were centrifuged to obtain the serum, and 200 µl aliquots were dispensed into each well of a 96-well plate. The levels of IL-6 (ab46027), IL-1β (ab255730), TNF-α (ab181421), and IL-10 (ab214566) were detected using specific ELISA kits according to the manufacturer’s instructions. The absorbance of the wells was measured at 532 nm using a microplate reader set (Z742711-1EA, Sigma-Aldrich, Merck KGaA).

Apoptosis assay

The cells were seeded in 6 cm dishes at the density of 6 × 104 cells/dish and incubated overnight. Following treatment with 100 µg/ml lncRNA LEF1-AS1, (Zn-Adenine)@Ab, or (Zn-Adenine)@Ab@lncRNA LEF1-AS1 for 48 h, the cells were washed thrice with PBS and incubated with Annexin V-FITC and propidium iodide (PI) in the dark for 10 min. The stained cells were analyzed by flow cytometry, and 10,000 cells were acquired per sample.

CCK-8 assay

RASFs were seeded into a 6-well plate and cultured until reaching approximately 75% confluence. The cells were then subjected to the following treatments: (1) control group: no treatment was administered; (2) lncRNA LEF1-AS1 group: pcDNA3.1 lncRNA LEF1-AS1 at a final concentration of 100 pg/ml; and (3) (Zn-Adenine)@Ab group and (Zn-Adenine)@Ab@lncRNA LEF1-AS1 group: NPs at a final concentration of 100 µg/ml. After 24 and 48 h, the viability of the cells was assessed using the CCK-8 assay. Additionally, at different time points (0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h), cell counts were performed, and cell proliferation curves were generated. Finally, hematoxylin and eosin (H&E) staining was conducted for cell staining and subsequent photomicroscopic observation.

Colony formation assay

Cells in the logarithmic growth phase were incubated with 100 µg/ml of the different nano-carriers for 48 h, harvested using 0.25% trypsin, and resuspended in fresh medium at the density of 1 × 106 cells/L. The cell suspensions were serially diluted, and 200 cells were seeded in each culture dish. After culturing for 2–3 weeks, the ensuing colonies were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, and then stained with crystal violet for 10 min. The excess dye solution was rinsed off with running water, and the plates were air-dried. The colonies were observed under a microscope and counted.

Preparation of (Zn-Adenine)@Ab@lncRNA LEF1-AS1

Solutions of the lncRNA (2 µg/ml), HEPES buffer (50 mM, pH 7.4), Zn(NO3)2,·6H2O (50 mM), adenine (10 mM), and antibody (1 mg/ml) were prepared. Thereafter, 4 ml aqueous adenine solution, 4 ml lncRNA solution, and 4 ml aqueous Zn(NO3)2·6H2O solution were sequentially added to 20 ml HEPES buffer. The reaction mixture was stirred vigorously in the dark for 2 h at 800 rpm/min. The precipitate was washed thrice with deionized water, and the (Zn-Adenine)@lncRNA LEF1-AS1 NPs were collected. The Zn-Adenine NPs were prepared by the same method. To link the antibody, (Zn-Adenine)@lncRNA LEF1-AS1 NPs were dispersed in 5 ml deionized water and poured into a small beaker containing 20 ml antibody solution. The mixture was stirred for 4 h away from light. After removing the unlinked antibodies by centrifuging for 30 min, the bottom residue was washed thrice with deionized water to obtain the (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs.

Characterization of the NPs

The particle size and dispersion coefficient of the (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs were evaluated using a laser nano-particle size analyzer. The NPs were morphologically characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Briefly, the suitably diluted NP solution was spread over a carbon-coated copper grid and incubated for 1 min. After absorbing the residual liquid, 2% (W/V) phosphotungstic acid-negative staining solution was added for 1 min, and the residual liquid was removed. The copper grid was air-dried and observed by TEM and SEM. To determine the entrapment efficiency (EE%) and drug loading (LE%) of lncRNA, the (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs were centrifuged at 10,000 rpm for 35 min, and the supernatants were analyzed by fluorescence spectrophotometry and UV-visible spectroscopy. The in vitro drug release from (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs was analyzed by dialysis. Briefly, 1 ml of the solution was placed in a dialysis bag and dialyzed in 100 ml release medium (pH 7.4 PBS, 0.1%W/W SDS) at 100 rpm. At the time points of 1, 2, 4, 6, 8, 12, 24, 48, and 72 h, 1 ml aliquots were taken and replaced with the same volume of the buffer. The samples were serially diluted, and the concentration of the drug was determined by UV spectrophotometry. The cumulative release rate of the drug was calculated at 72 h.

Establishment of collagen-induced arthritis (CIA) model

The Institutional Animal Care and Use Committee of Bengbu Medical College (2021-192) approved all animal experiments. Female Sprague-Dawley rats (8 weeks old, 180–200 g) were provided by the Model Animal Research Center of Nanjing University. The animals were housed in sterile chambers under a 12 h light/dark cycle and provided standard laboratory food and water ad libitum. To establish the CIA model, the rats were subcutaneously injected into the tail root with 100 µl of a 1:1 mixture of 1 mg/ml bovine collagen II (Chondrex, USA) dissolved in 0.05 mol/L acetic acid and complete Freund’s adjuvant (Chondrex, USA). One week later, 100-µl bovine type 2 collagen was injected to enhance immunity. The animals in the control group were injected with the same volume of PBS (pH 7.5). Paw and ankle swelling were observed weekly, and the symptoms were classified as none, weak, mild, moderate, and severe. Each paw was evaluated and scored individually on a scale of 0–4, and the maximum possible cumulative score of each rat was 16 points. The animals with stable joint symptoms were selected for the experiment. Fourteen days after the initial immunization, the rats were randomly divided into the control, (Zn-Adenine)@Ab, RA, MTX + RA, (Zn-Adenine)@Ab + RA, and (Zn-Adenine)@Ab@lncRNA LEF1-AS1 + RA groups (n = 6 each). The respective drugs were given 100 µl once every 2 days for 14 days, and the control and RA model groups were injected with normal saline. Fur around knee joint was shaved and skin sterilized with iodophor. The knee joint of rats was flexed to approximately 70~80°, which resulted in the widening of the joint space. The puncture points were determined as the lower margin of the patella and the medial part of the patellar ligament. A syringe was held, and the needle was inserted perpendicularly through the skin. The injection was stopped upon experiencing a noticeable resistance [14]. The specific administration method and dosage are shown in Table 3.

Table 3 Group administration method and dose for RA ratsHistological examination

The suitably treated rats were sacrificed under anesthesia (2–4% isoflurane), and both ankles were removed. The specimens were fixed in 10% formalin solution for 24 h, decalcified in 12% EDTA for 3 days, neutralized in 5% sodium thiosulphate for 5 h, and rinsed with water for 12 h. The tissues were dehydrated, embedded in paraffin wax, and then sliced into 6 μm sections. The samples were stained with H&E and safranin O/fast green and observed under a light microscope.

Statistical analyses

The quantitative data were presented as mean ± SD (standard deviation) of at least 3–6 independent samples. All statistical analyses were performed using SPSS version 17.0, and the data were compared using ANOVA or Student’s T-test. P < 0.05 was considered statistically significant.