Degenerated IVD tissue was collected from 30 patients (14 male, 16 female; median age, 62.3 years; age range, 51–71 years) with IDD and normal NP tissue was obtained from 30 patients (17 male, 13 female; median age, 40.6 years; age range, 32–49 years) with spinal cord injury between June 2018 and December 2020 at the Shanghai Changzheng Hospital (Shanghai, China). The collected tissues were rapidly frozen in liquid nitrogen and stored at −80°C. The present study was approved by the Shanghai Changzheng Hospital (Shanghai, China), and followed the guidelines described in the Helsinki Declaration (31). Written informed consent was obtained for tissue sample collection from each participant.

Human NP cells were purchased from ScienCell Research Laboratories, Inc. (cat. no. 4800) and cultured in NP Cell Medium (both ScienCell Research Laboratories, Inc.) at 37°C with 5% CO₂ in a humidified incubator. NP cells had been initially isolated from NP of the human IVD; each vial contained >5×10⁵ cells in 1 ml and NP cells were allowed to expand for 15 passages according to the manufacturer's instructions. Human embryonic kidney 293T cells (American Type Culture Collection; cat. no. CRL-1573) were cultured in DMEM containing 10% FBS (both Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator with 5% CO₂. LINC00284 specific small interfering (si)RNA (si-LINC00284) was used to knockdown LINC00284 expression and scramble siRNA was used as negative control (si-NC) and human miR-205-3p and scrambled miR-205-3p mimic (mi-NC) were transfected into cells at the concentration of 20 µM using Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.) at 37°C for 24 h according to manufacturer's instructions. All siRNAs, miRNA mimics and NCs were obtained from Shanghai GenePharma Co., Ltd. The sequences of constructs were as follows: si-LINC00284 sense, 5′-GCAUGUUAAUUCACUAUUATT-3′ and antisense, 5′-UAAUAGUGAAUUAACAUGCTT-3′; si-NC sense, 5′-GUUGAAUUAACUUACACUUTT-3′ and antisense, 5′-AAGUGUAAGUUAAUUCAACTT-3′; miR-205-3p mimic sense, 5′-GAUUUCAGUGGAGUGAAGUU-3′ and antisense, 5′-AACUUCACUCCACUGAAAUC-3′ and mi-NC sense, 5′-GGUAGUGCGGAAAGUUUAUU-3′ and antisense, 5′-AAUAAACUUUCCGCACUACC-3′.

When cell confluence reached ~80%, transfected NP cells were plated into 6- or 96-well plates at 1×10⁵/ml for serum starvation overnight, then stimulated at 37°C using IL-1β (Prospec-Tany TechnoGene, Ltd.) at 5, 10 or 20 ng/ml for 24 h.

Total RNA was extracted from tissue or cells using TRIzol^® (Thermo Fisher Scientific, Inc.). LINC00284, MMP-3, aggrecan and collagen II mRNA levels were detected using SYBR-Green qPCR kit (Takara Bio, Inc.) according to the protocol. Briefly the one-step RT-qPCR reaction was: 10 µl of 2X One step TB Greem RT-PCR Buffer III, 0.4 µl of TaKaRa Ex Taq HS (5 U/µl), 0.4 µl of PrimeScript RT enzyme Mix II, 0.4 µl of forward primer (10 µM), 0.4 µl of reverse primer (10 µM), 2 µl of total RNA, and 6 µl of RNase Free dH₂O, then subjected to reverse transcription for 5 min at 42°C and initially denatured at 95°C for 10 sec, and then to 40 cycles of amplification with the condition of 95°C denature for 5 sec, and 60°C annealing for 30 sec. β-actin was used as the housekeeping gene. The mature miR-205-3p expression levels in tissue and cells were detected using Hairpin-it^™ miRNA One-step qPCR-PCR SYBR Green kit (Shanghai GenePharma Co., Ltd.) according to the protocol. Briefly the reaction system was setup as: 10 µl of One-Step SYBR Mix, 0.5 µl of Enzyme Mix, 0.5 µl of forward primer (2 µM), 0.4 µl of reverse primer (5 µM), 0.4 µl of ROX Reference Dye, 2 µl of RNA template, 6.2 µl of RNase Free dH₂O, then subjected to reverse transcription for 40 min at 42°C and initially denatured at 94°C for 3 min, and then to 40 cycles of amplification with the condition of: 94°C denature for 12 sec, 62°C annealing for 30 sec, and 72°C extension for 30 sec. Small U6 RNA was used as the endogenous control. RT-qPCR results were analyzed using the 2^−ΔΔCq method (32). The primers were synthesized by Biomics Biotechnologies Co. Ltd. and the sequences are listed in Table I.

The protein levels of MMP-3, aggrecan, collagen II and Wnt1, β-catenin and β-actin were detected using western blotting. Total proteins from IL-1β-treated cells were lysed in RIPA buffer with 1% PMSF and InStab^™ Protease Cocktail (Shanghai Yeasen Biotechnology Co., Ltd.) and protein concentration was quantified using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Then, 20 µg/lane protein was resolved using 10% SDS-PAGE and transferred onto a PVDF membrane (MilliporeSigma), followed by blocking with EZ-Buffers N 1X BLOCK BSA in TBS (Sangon Biotech Co., Ltd.) for 2 h at room temperature. The blots were probed with the following antibodies (all 1:1,000; all from Abcam): Anti-MMP-3 (cat. no. ab52915), anti-collagen II (cat. no. ab188570), anti-aggrecan (cat. no. ab3778), anti-Wnt1 (cat. no. ab15251), anti-β-catenin (cat. no. ab223075) or anti-β-actin (cat. no. ab8226) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit (cat. no. ab7090) for MMP-3, collagen II, Wnt1 and β-catenin detection or anti-mouse IgG (cat. no. ab47827) for aggrecan and β-actin for 1.5 h at room temperature. ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.) was used to visualize the signals. The mean gray values of blots were measured using ImageJ version 1.8.0 software (National Institutes of Health). β-actin was used for normalization.

Cell proliferation was detected using a Cell Counting Kit (CCK)-8 assay. After seeding into 96-well plates (5×10³ cells/well) for 24 h, cells were transfected at 37°C with si-LINC00284, miR-205-3p mimics or NC for 24, 48 and 72 h. At 48 h post-transfection, 10 µl CCK-8 reagent (Abcam) was added per well and cells were incubated for 4 h at 37°C. The absorbance at 450 nm was measured. Untreated NP cells were used as the control.

Annexin V-FITC/Propidium Iodide (PI) staining and flow cytometry analysis were used to detect NP cell apoptosis. At 48 h post-transfection, 1×10⁶ cells/well were harvested, washed in PBS and plated in a 6-well plate. Annexin V-FITC Apoptosis Detection kit (MilliporeSigma) was used to measure cell apoptosis according to the manufacturer's instructions. Briefly, after washing with PBS twice, cells were resuspended in Annexin-V binding buffer, stained with the Annexin V-FITC/PI for 15 min at room temperature in the dark and analyzed using FACSCalibur and BD CellQuest^™ Pro software version 6.0 (both BD Biosciences), and the apoptotic rate was calculated as the numbers of early + late apoptotic cells/total numbers of cell.

LINC00284 sponging of miR-205-3p was predicted using DIANA-LncBase V2 (carolina.imis.athena-innovation.gr) (33). The binding of LINC00284 with miR-205-3p was validated using dual-luciferase reporter assay. Wild-type (wt) or mutated (mut) LINC00284 sequence was constructed and inserted into a pGL3 vector (Promega Corporation) as luciferase reporter gene vectors. Briefly, 293T cells were cultured at 37°C overnight to 70–80% confluence, then ~2×10⁵ cells were seeded into 24-well plates. After culturing at 37°C for 24 h, cells were co-transfected with wt-LINC00284 or mut-LINC00284 reporter vector and miR-205-3p mimics or mi-NC using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.) according to manufacturer's instructions. At 48 h post-transfection, luciferase reporter assay was performed using a Dual-Luciferase Reporter Assay System (Promega Corporation), the results were normalized with Renilla luciferase activity).

To investigate the effect of LINC00284 in IDD in vivo, si-LINC00284 and si-NC were inserted into short hairpin (sh)RNA lentiviral vectors and packaged as sh-LINC00284 and sh-NC lentiviruses by Shanghai GenePharma Co., Ltd. A total of 24 male Sprague-Dawley rats (weight, 200–250 g; age, 3 months) were obtained from Nantong University (Nantong, China) for in vivo experiments and were housed under the condition of 18–26°C and 40–70% humidity, with a 12 h light/dark cycle and free access to food and water. The rats were randomly divided into three groups (n=8/group): sham, treated with sh-NC lentivirus (sham + sh-NC); IDD model, treated with sh-LINC00284 lentivirus (IDD + sh-LINC00284) and IDD NC, treated with sh-NC lentivirus treatment (IDD + sh-NC). All surgical procedures were performed as previously described (34). Briefly, to induce IDD, rats were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg), then a midline longitudinal incision was made on the back. The left facet joint between the fourth and fifth lumbar (L4/5) vertebrae was removed to uncover the L4/5 IVD. Then, a 21-gauge needle was inserted into the IVD parallel to the endplate to a depth of 3 mm and held in place for 30 sec. The animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (35) and were approved by the Animal Care and Use Committee of Nantong University (Nantong, China).

The lentivirus was injected into the anterior midline area of IVD 7 days after puncture using a microliter syringe with a 26-gauge needle (10 µl, Shanghai Gaoge Industry and Trade Co., Ltd.) via the anterior transperitoneal midline, as previously described (36,37).

At 7, 14 and 28 days after initial IVD puncture, six animals were randomly selected for X-ray and MRI examination. The animals were kept in a prone position with tails straightened on a molybdenum target radiographic image unit (GE Healthcare). IVD height was measured according to the disc height index (DHI) as previously described (38). DHI change (%) was calculated relative to sham + sh-NC group. Transverse relaxation time (T2) midsagittal MRI images were obtained via Siemens 3.0T Magneto Trio MRI System (Siemens Healthineers). T2 MRI images were analyzed by two independent radiologists blinded to the experimental conditions according to Pfirrmann MRI-grade system as previously described (39).

IVD tissue was obtained following model rat sacrifice by intraperitoneal injection of sodium pentobarbital (100 mg/kg) 28 days after initial disc puncture. Tissue was fixed in 10% formalin for 48 h at room temperature, decalcified using 10% EDTA solution for 30 days at room temperature and embedded in paraffin wax. The paraffin blocks of IVD tissue were sectioned into 5 µm coronal sections containing NP, annulus fibrosus (AF) and cartilaginous endplate. The sections were heated at 60°C for 30 min, and then dewaxed with xylene twice for 20 min each, and rehydration in descending alcohol series stained using a Hematoxylin-Eosin (H&E) Staining kit (Beijing Solarbio Science & Technology Co., Ltd.) or Modified Safranine O-Fast Green FCF Cartilage Stain kit (Beijing Solarbio Science & Technology Co., Ltd.) according to manufacturer's protocols. For histological grading, staining was categorized by two independent pathologists blinded to the experimental conditions on a scale of 4 (normal) to 12 points (severe degeneration) as described by Masuda et al (40) under a light microscope (Olympus CX43; Olympus Corporation; magnification, ×100) and Olympus cellSens software version V3.2 (Olympus; Olympus Corporation) was used for analysis.

All data are presented as the mean ± standard deviation of three independent experiments and were analyzed using SPSS version 19.0 (IBM Corp.). Differences between two groups were evaluated using unpaired Student's t-test; differences between ≥3 groups were compared using one-way ANOVA followed by post hoc Dunnett's or Tukey's. Spearman's rank correlation analysis was used to determine the correlation between LINC00284 and miR-205-3p expression. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of LINC00284 in IDD and normal tissue was evaluated using RT-qPCR. LINC00284 expression was significantly upregulated in 30 IDD samples compared with that in normal NP tissues (30 samples obtained from patients with spinal cord injury; Fig. 1). The results indicated that LINC00284 was overexpressed in NP tissues of IDD patients.

Human NP cells were treated with 5, 10 or 20-ng/ml IL-1β and LINC00284 expression was detected using RT-qPCR. LINC00284 expression was low in normal NP cells (control) but significantly increased following IL-1β treatment, with 5 ng/ml exerting the most significant increase (Fig. 2A). Thus, 5 ng/ml IL-1β was used in subsequent experiments.

To investigate whether IL-1β promotes IDD by stimulating NP cells and determine the role of LINC00284 in development of IDD, LINC00284 expression was knocked down using siRNA (Fig. 2B) and expression levels of MMP-3 and other ECM markers were measured using RT-qPCR and western blotting. IL-1β inhibited expression of aggrecan and collagen II but increased MMP-3 expression at both the mRNA and protein level. Additionally, in IL-1β-induced NP cells, LINC00284 knockdown decreased MMP-3 expression but increased aggrecan and collagen II expression levels compared with si-NC treated cells (Fig. 2C-I). The results indicated that LINC00240 may promote the development of IDD.

To determine the role of LINC00284 in IDD, proliferation of IL-1β-induced NP cells following LINC00284 knockdown was detected using CCK-8 assay and Annexin V-FITC/PI staining with flow cytometry analysis. Compared with the control group, proliferation was inhibited in cells treated with 5 ng/ml IL-1β for 24, 48 and 72 h; however, in IL-1β-induced NP cells, si-LINC00284 promoted cell proliferation (Fig. 3A) but significantly decreased apoptosis (Fig. 3B and C), compared with si-NC-treated cells. The results indicated that LINC00240 may inhibit the proliferation, but promote the apoptosis, of NP cells in IDD patients.

To evaluate the therapeutic effect of LINC00284 knockdown in vivo, intradiscal injection of sh-LINC00284 lentivirus was performed weekly in a needle puncture rat IDD model. Compared with 7 days after initial puncture, X-ray examination showed that IDD rats treated with sh-NC exhibited more notable narrowing of disc height compared with sham rats treated with sh-NC on days 14 and 28. Following treatment of IDD rats with sh-LINC00284, the decline in disc height began to slow from days 7–28 (Fig. 4A) and the percentage DHI in the sh-LINC00284 group was 1.56, 1.28 and 1.32-fold higher than that in sh-NC IDD rats on days 7, 14 and 28, respectively (Fig. 4B). According to MRI examination, 14 and 28 days after initial puncture, intradiscal injection with sh-NC in IDD rats exhibited weaker MRI signal than 7 days. Compared with sh-NC-treated IDD rats, sh-LINC00284 treatment notably alleviated weak MRI signal intensity of the disc puncture, whereas MRI signal decreased significantly (Fig. 4C and D).

The histological structure of IVD was observed using H&E and safranin-O staining. On day 28, the sh-NC group showed a notable decrease in number of NP cells and destruction of AF lamella compared with sham (Fig. 4E and F). Compared with IDD rats treated with sh-NC, sh-LINC00284 treatment preserved the complete structure of the NP and AF; histological grade of the sh-LINC00284 group on days 7, 14 and 28 increased by 13.1, 15.5 and 17.5% respectively (Fig. 4G). The results indicated that LINC00240 may be a therapeutic target of IDD.

To investigate whether LINC00284 affected ECM degradation in IL-1β-induced NP cells, the target ncRNA of miR-205-3p was predicted using DIANA-LncBase V2 (Fig. 5A) and wt and mut binding sites between LINC00284 and miR-205-3p were established. The luciferase activity of wt-LINC00284 and miR-205-3p mimic co-transfected cells was significantly decreased compared with cells co-transfected with mi-NC transfected cells and mut-LINC00284 and miR-205-3p mimic co-transfected cells (Fig. 5B). These results indicated that LINC00284 was the direct target of miR-205-3p.

To validate the interaction between LINC00284 and miR-205-3p, the effect of transfection of miR-205-3p mimics on expression of LINC00284 was evaluated in NP cells. IL-1β induced upregulation of LINC00284 compared with control but miR-205-3p mimic transfection significantly inhibited LINC00284 expression levels compared with mi-NC-treated cells (Fig. 5C). RT-qPCR showed that miR-205-3p levels were significantly downregulated in 30 IDD compared with 30 spinal cord injury tissue samples (Fig. 5D). Moreover, there was a significant negative correlation between LINC00284 and miR-205-3p expression in 30 IDD tissue samples (Fig. 5E). The results indicated that miR-205-3p was low-expressed and was negative correlated with LINC00240 expression in NP tissues of IDD patients.

To confirm the effect of miR-205-3p-mediated regulation of LINC00284 on proliferation, apoptosis and ECM synthesis, miR-205-3p mimics were transfected into IL-1β-induced NP cells. Compared with mi-NC-treated cells, proliferation was increased by miR-205-3p overexpression (Fig. 6A). Furthermore, the cell apoptosis were promoted in IL-1β treated NP cells and mi-NC treated IL-1β-induced NP cells, but a suppressive effect on cell apoptosis was observed following miR-205-3p upregulation (Fig. 6B and C). Western blot analysis showed that, compared with control (untreated NP cells), MMP-3 protein levels were upregulated but aggrecan and collagen II protein levels were downregulated in IL-1β treated NP cells and mi-NC treated IL-1β-induced NP cells. Compared with mi-NC treated IL-1β-induced NP cells, MMP-3 protein levels were downregulated by miR-205-3p, whereas aggrecan and collagen II protein levels were upregulated in IL-1β-induced NP cells (Fig. 6D-G). The results indicated that LINC00240 can be inhibited by miR-205-3p in IDD through the promotion of NP cell proliferation and ECM synthesis.

Wnt/β-catenin signaling has been demonstrated to regulate inflammation in the musculoskeletal system and β-catenin is controlled by non-genomic mechanisms (26,27). To investigate if this signaling pathway participated in LINC00284-mediated rescue of ECM degradation via competitively binding with miR-205-3p, si-LINC00284 and/or miR-205-3p mimics were co-transfected into IL-1β-induced NP cells. Compared with control, the protein levels of MMP-3, Wnt1 and β-catenin were increased but aggrecan and collagen II were decreased in IL-1β treated NP cells and si-NC treated IL-1β-induced NP cells. Compared with si-NC treated IL-1β-induced cells, si-LINC00284 resulted in decreased MMP-3, Wnt1 and β-catenin but an increase in aggrecan and collagen II protein levels. Co-transfection of si-LINC00284 with miR-205-3p reversed this effect (Fig. 7). These results indicated that the effect of LINC00284 on promoting ECM degradation may partially be mediated via the Wnt/β-catenin signaling pathway.