Knockdown of LINC00963 targets miR-1252-5p to reduce inflammatory levels and ECM degradation in nucleus pulposus cells of IDD.

Background

Intervertebral disc degeneration (IDD) is a chronic, progressive disease that often affects middle-aged and older people, and is aleading cause of low back pain [ 1 , 2 ]. Approximately 80% of the world’s population experiences LBP at some point in their lives, and 10% of these cases can progress to long-term disability [ 3 – 5 ] making it a serious public health issue [ 6 ]. Current IDD treatments only relieve symptoms temporarily [ 7 – 10 ], and its pathogenic mechanism remains unclear.

Long non-coding intergenic RNAs (LncRNAs) are functional RNA molecules (> 200 nt, low coding potential) [ 11 ]. They are key regulators in biological processes like cell cycle control, apoptosis, and inflammation [ 12 ]. Several abnormally expressed lncRNAs have been identified in IDD, where they contribute to disease pathogenesis. For instance, lncRNA H19 promotes IDD by activating miR-139-3p/CXCR4/NF-κB signaling to induce cell apoptosis [ 13 ], while HOTAIR inhibits cell proliferation by targeting miR-130b [ 14 ]. These findings highlight lncRNAs as promising diagnostic and therapeutic targets [ 15 – 17 ]. Notably, LINC00963, located on human chromosome 9q34.11, has been linked to various malignancies [ 18 ]. Zhou et al. [ 19 ] reported that in bladder cancer, its overexpression drives tumor cell proliferation and invasion. Recent clinical studies have confirmed that LINC00963 exhibits abnormal expression in the tissues of IDD patients [ 20 ], but its specific mechanism of action in IDD requires further research.

Intervertebral discs, fibrous cartilage between vertebrae, include annulus fibrosus, nucleus pulposus (NP), and cartilage endplates [ 21 ]. Nucleus pulposus cells (NPCs) are susceptible to oxidative stress, aging, inflammation, and apoptosis. The extracellular matrix (ECM) secreted by NPCs helps uphold homeostasis [ 22 ]. When the equilibrium between synthesis and degradation is disrupted or if inflammatory mechanisms are dysregulated, this can lead to IDD [ 23 ]. Recently, the importance of microRNAs (miRNAs) in disease pathogenesis has become increasingly evident. Previous studies have confirmed that abnormal miRNA expression is not only closely associated with the occurrence and development of malignant tumors [ 24 ] but also serves as a major regulatory factor in skeletal system diseases [ 25 – 27 ]. For example, LINC00958 can accelerate NPCs proliferation and ECM degradation through the miR-203/SMAD3 pathway [ 28 ]; recent studies have shown that miR-1252-5p, located on chromosome 12, is downregulated in osteosarcoma cells and can significantly inhibit cancer cell proliferation, migration, invasion, and cell cycle progression [ 29 ].

Notably, both LINC00963 and miR-1252-5p have been confirmed to be involved in the regulation of bone-related diseases. Based on bioinformatics predictions, LINC00963 contains potential binding sequences for miR-1252-5p. Therefore, we hypothesize that LINC00963 influences IDD progression by regulating miR-1252-5p. This study aims to investigate the biological functions of LINC00963 and its mechanisms in IDD, offering new insights for prevention and treatment.

Methods

Clinical sample collection and ethical approval

This study included 102 patients with IDD and 88 patients with scoliosis who underwent lumbar discectomy at Zhucheng People’s Hospital between June 2021 and December 2022. The diagnosis of IDD was confirmed based on clinical symptoms and magnetic resonance imaging findings indicative of disc degeneration. Exclusion criteria: (1) History of spinal trauma, infection, or tumors; (2) Presence of systemic inflammatory diseases; (3) Previous spinal surgery at the same segment. The degree of disc degeneration was assessed using MRI and the modified Pfirrmann classification. All lumbar disc samples were collected in strict accordance with clinical guidelines. All procedures performed in studies involving human participants were in accordance with the 1964 Helsinki Declaration. Approval for this investigation was obtained from the Zhucheng People’s Hospital Ethics Committee. Before sample collection, each participant signed an informed consent form.

The sample size for this study was determined based on a prior power analysis using GPower 3.1 software. A two-tailed independent samples t-test was set with an α level of 0.05 and an effect size of 0.3. To achieve 85% power, a minimum of 160 observations was required. The study ultimately enrolled 190 participants, exceeding the minimum sample size requirement and ensuring robust statistical power for the findings.

Harvesting and culturing NPCs from human tissue

IDD samples were washed with phosphate-buffered saline (PBS), then carefully cut into pieces of approximately 1 mm³ and digested sequentially with 0.25% trypsin (Sigma-Aldrich, USA) for 30 min and 0.2% type II collagen (Col-II, Invitrogen, USA) for 4–6 h at 37 °C with gentle agitation. The digested tissue was filtered through a 70-µm cell strainer. The isolated cells were cultured under standard conditions (37 °C, 5% CO₂, 95% air) in Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12) (HyClone, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The medium was replaced every three days. Upon reaching 80–90% confluence, cells were passaged at a 1:3 ratio using 0.25% trypsin-EDTA. All subsequent experiments employed third-generation NPCs to ensure phenotypic stability.

Designing a cell culture model to study disc degeneration

Before the experiment, all NPCs were pretreated overnight in fresh DMEM/F12 medium (containing 1% FBS). To construct an in vitro model of IDD in NPCs, NPCs were stimulated with IL-1β. Based on the literature [ 30 , 31 ] and pre-experimental validation, a treatment regimen of 5 nM IL-1β for 24 h was determined to establish the induction conditions. This protocol effectively induces NPCs to exhibit a degenerative phenotype without causing excessive cytotoxicity.

Cell transfection

When cell fusion reached approximately 70%, the LINC00963 cDNA fragment (pcDNA3.1-LINC) was cloned into the pcDNA3.1 expression vector. We then transiently transfected the cultured NPCs with miR-1252-5p mimic and its negative control. Following the standard procedure recommended by the manufacturer of the Lipofectamine 3000 transcription reagent (Invitrogen, USA). The relative expression levels of LINC00963 and miR-1252-5p were detected by quantitative real-time polymerase chain reaction (RT-qPCR) 24 h post-transfection to validate transfection efficiency.

RNA isolation followed by RT-qPCR

RNA extraction from tissue and cell samples was performed using Trizol reagent (Invitrogen, USA) as specified by the manufacturer. Following extraction, RNA was converted into complementary DNA (cDNA) using Prime Script RT premix. RT-qPCR analysis was performed by the SYBR Premix Ex Taq Reagent Kit (Takara, Dalian). GAPDH was used as the housekeeping gene to standardize the expression levels of LINC00963, Col-II, matrix metalloproteinases (MMP3, MMP13), along with aggrecan. Simultaneously, miR-1252-5p expression levels were corrected using U6 as a control group. To quantify relative gene expression, the 2 −ΔΔCt method was utilized.

Subcellular fractionation assay

1 × 10⁶ NPC cells were harvested, centrifuged, and washed with PBS. The PARIS™ Kit (Invitrogen, USA) was used to isolate RNA from the cytoplasm and nuclei of NPCs. Subsequently, RT-qPCR was employed to detect the expression levels of LINC00963 in both cytoplasmic and nuclear fractions, using GAPDH (cytoplasmic marker) and U6 (nuclear marker) as references, thereby determining the localization of LINC00963.

Western blot analysis (WB)

Total proteins were extracted from NPC using RIPA lysis buffer containing protease (GRF101, Epizyme, USA) and phosphatase (GRF102, Epizyme) inhibitors, and concentrations were determined by the BCA method. Equal amounts of protein were subjected to 10% SDS-PAGE electrophoresis and transferred to PVDF membranes (Millipore, USA). Primary antibodies were incubated overnight at 4 °C: Col-II (33340, SAB, USA), Aggrecan (45068, SAB, USA), MMP3 (32225, SAB, USA), MMP13 (32335, SAB, USA), and internal control GAPDH (AC001, abclonal, Wuhan). Subsequently, incubate with HRP-labeled goat anti-rabbit secondary antibody (AS014, abclonal, Wuhan) for two hours at room temperature. Visualize protein bands using the Millipore developing solution and quantify protein bands with ImageJ.

Enzyme-linked immunosorbent assay (ELISA)

Centrifuge the NPCs’ culture to obtain the supernatant. Follow the manufacturer’s testing protocol to quantitatively analyze the concentrations of target cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) using the ELISA method. Finally, using an ELISA reader, record the optical density of all wells at 450 nm, and calculate the concentrations of each inflammatory marker based on the standard curve.

Determination of dual luciferase activity

A predictive analysis using the ENCORI database identifies that LINC00963 binds to miR-1252-5p and simultaneously predicts potential binding sites between miR-1252-5p and the 3’ untranslated region of MAPK1. Based on these predictive results, luciferase reporter gene vectors for the wild-type (WT) and mutant-type (MUT) of LINC00963 and MAPK1 were constructed separately. These vectors were co-transcribed in NPCs with the miR-1252-5p mimic, mimic negative control (mimic NC), inhibitor negative control (inhibitor NC), or miR-1252-5p inhibitor. Measurements of luciferase activity in the reporter gene vector from all cell groups were conducted with the Dual-Luciferase Reporter (DLR) Assay System (Promega, USA) paired with a microplate reader.

RNA immunoprecipitation (RIP) assay

Under the manufacturer’s recommended procedure, we performed a RIP experiment using the Magna RIP kit (Millipore, USA) to investigate the potential binding relationship between LINC00963 and miR-1252-5p. First, after transcription, NPCs were lysed using RIP lysis buffer. The lysate was then separately treated with magnetic beads that had been coated with anti-IgG antibodies in one case and anti-Ago2 antibodies in the other. Following immunoprecipitation, the complex was digested with protease K. Finally, the abundance of LINC00963 and miR-1252-5p in the immunoprecipitate was detected using RT-qPCR.

Statistical analysis

All data were obtained through at least three independent replicate experiments. Statistical analyses were performed using SPSS 22.0 and GraphPad 9.0 software. Normality of continuous data was assessed using the Kolmogorov-Smirnov test, and comparisons between two groups were performed using Student’s t-test. For analysis involving two independent variables and more than two groups, two-way analysis of variance (ANOVA) was employed. Following identification of significant interactions, simple effects analyses with Bonferroni correction were performed to address multiple comparisons. One-way ANOVA was used for comparisons among multiple groups, followed by Tukey’s HSD post hoc tests. Normally distributed data are presented as mean ± standard deviation (SD), while categorical data are expressed as frequency and percentage. Group comparisons were assessed using chi-square tests. The diagnostic efficacy of LINC00963 in patients with IDD was evaluated using receiver operating characteristic (ROC) curve analysis. Multivariate logistic regression analysis was employed to identify independent predictors of IDD. Pearson correlation analysis was conducted to investigate the relationship between LINC00963 and miR-1252-5p in IDD patients. The level of statistical significance was set at 0.05.

Results

Expression levels of LINC00963 in cases of IDD and its diagnostic potential

A comparison of baseline data for patients with scoliosis and patients with IDD showed that the two groups were comparable in age, body mass index, gender composition, tobacco use, and alcohol intake, with no significant statistical differences ( P > 0.05, Table 1 ). In particular, the expression level of LINC00963 was significantly higher in the IDD group than in the scoliosis group (control group) ( P < 0.0001, Fig. 1 A). In addition, IDD patients were categorized into three severity groups (mild, moderate, and severe) based on the Pfirrmann grading system, as shown in Fig. 1 B. LINC00963 expression increased significantly as degeneration progressed ( P < 0.0001). LINC00963 also showed high diagnostic efficacy with an area under the curve (AUC) of 0.8692, specificity of 73.86%, and sensitivity of 86.27% (Fig. 1 C), demonstrating its practical utility in distinguishing patients with scoliosis from those with IDD. To evaluate whether LINC00963 expression could serve as an independent predictor for IDD after adjusting for potential confounders, multivariate logistic regression analysis was performed. The results showed that LINC00963 ( P = 0.003, OR = 3.955, 95% CI: 1.603–9.758) was a significant independent predictive factor for the occurrence of IDD (Table 2 ).

Elevated expression of LINC00963 enhances inflammatory responses in NPCs of IDD and promotes ECM degradation

To study the mechanism of action of LINC00963 in NPCs, IDD NPCs were transfected with the empty vector pcDNA3.1 or the recombinant vector pcDNA3.1-LINC00963. RT-qPCR results demonstrated that transfection with pcDNA3.1-LINC00963 significantly increased LINC00963 expression, confirming transfection efficacy ( P < 0.0001, Fig. 2 A). Quantitative detection of key inflammatory mediators (IL-6, TNF-α, and IL-1β) using the ELISA technique showed that overexpression of LINC00963 significantly promoted the secretion of these inflammatory factors ( P < 0.001, Fig. 2 B). Furthermore, RT-qPCR and WB analysis of molecules related to ECM synthesis (Col-II and Aggrecan) showed that overexpression of LINC00963 significantly inhibited matrix synthesis ( P < 0.001, Fig. 2 C). Further analysis of markers related to ECM degradation (MMP3 and MMP13) showed that both mRNA and protein levels of these two matrix metalloproteinases (MMPs) were significantly activated with increased expression of LINC00963 ( P < 0.0001, Fig. 2 D).

miR-1252-5p is a target of LINC00963

Subcellular localization analysis revealed that LINC00963 is primarily distributed in the cytoplasm (Fig. 3 A). Fig. 3 B shows the potential binding region between LINC00963 and miR-1252-5p. To investigate whether they directly interact, we used a DLR assay. Experimental data showed that when both the miR-1252-5p mimic and LINC00963-WT plasmid were co-transfected, the luciferase activity was significantly reduced; however, when the miR-1252-5p inhibitor was introduced, the luciferase activity was significantly increased. However, neither the miR-1252-5p mimic nor the inhibitor had a significant effect on fluorescence activity when co-transfected with the mutant LINC00963 plasmid ( P < 0.0001, Fig. 3 C). To further clarify the target relationship, RIP experiments revealed that the enrichment levels of LINC00963 and miR-1252-5p in the Ago2 antibody group were significantly higher than those in the IgG control group, confirming their direct binding ( P < 0.0001, Fig. 3 D). Additionally, the expression level of miR-1252-5p was markedly downregulated in IDD patients compared to the controls ( P < 0.0001, Fig. 3 E). Pearson correlation analysis further revealed that the expression of LINC00963 and miR-1252-5p in IDD patients was highly negatively correlated ( r = -0.7486, 95%CI: -0.8232to -0.6485, P < 0.0001, Fig. 3 F).

LINC00963 influences inflammation and ECM degradation in NPCs of IDD through interaction with miR-1252-5p

To investigate whether LINC00963 regulates NPCs function via miR-1252-5p, we first detected miR-1252-5p expression levels by RT-qPCR after transfecting cells with mimic NC and miR mimic. Results showed that transfection with the miR-1252-5p mimic significantly upregulated miR-1252-5p expression ( P < 0.001, Fig. 4 A), indicating transfection efficiency. Furthermore, transfection of pcDNA3.1-LINC00963 into NPCs significantly increased LINC00963 expression. This upregulation was not altered by co-transfection with the miR-1252-5p mimic ( P < 0.001, Fig. 4 B), further validating transfection efficacy. Further analysis of the effects of LINC00963 overexpression and miR-1252-5p overexpression on miR-1252-5p expression levels revealed that overexpression of LINC00963 significantly reduced the expression level of miR-1252-5p and that the addition of the miR-1252-5p mimic effectively reversed this negative regulatory effect ( P < 0.0001, Fig. 4 C). Furthermore, increased LINC00963 expression significantly elevated the production of inflammatory mediators (IL-6, TNF-α, IL-1β), but the introduction of a miR-1252-5p mimic significantly blocked this inflammatory effect ( P < 0.001, Fig. 4 D). Regarding ECM metabolism, elevated LINC00963 levels reduced mRNA and protein expression of anabolic proteins (Col-II and Aggrecan), while increasing mRNA and protein expression of catabolic proteins (MMP3 and MMP13). However, these metabolic abnormalities were significantly improved after an increase in miR-1252-5p levels ( P < 0.001, Fig. 4 E-F).

miR-1252-5p targets MAPK1

Figure 5 A shows the WT/MUT-MAPK1 (mitogen-activated protein kinase 1) reporter vector constructed based on the miR-1252-5p binding site predicted by the ENCORI website. To validate their interaction, we performed a DLR assay. Results showed that miR-1252-5p inhibitor significantly enhanced WT-MAPK1 fluorescence activity, while miR-1252-5p mimic markedly suppressed it; however, neither treatment significantly affected MUT-MAPK1 fluorescence activity ( P < 0.0001, Fig. 5 B). RIP results showed that miR-1252-5p was significantly enriched with MAPK1 in the Ago2 compared to the IgG control, further confirming the interaction between MAPK1 and miR-1252-5p ( P < 0.0001, Fig. 5 C). Furthermore, we predicted potential downstream target genes of miR-1252-5p using four databases (miRDB, miRWalk, CTD, and ENCORI). Among the top 10 overlapping target genes ranked by detection scores, MAPK1 exhibited the highest expression level in IDD patients ( P < 0.0001, Fig. 5 D). Further experiments demonstrated that upregulating LINC00963 expression significantly enhanced both mRNA and protein expression of MAPK1, while transfecting a miR-1252-5p mimic markedly reversed the increase in MAPK1 expression induced by LINC00963 upregulation ( P < 0.0001, Fig. 5 E-F).

Discussion

IDD poses a serious threat to human health and significantly impairs patients’ quality of life, but clinical treatment options remain limited. The pathological mechanism underlying this disease has not yet been established, but existing studies suggest that an irregular ECM metabolism and the inflammatory response may be important pathological factors [ 23 , 32 ]. Based on these findings, modulating inflammatory pathways and ECM metabolic balance presents a new therapeutic strategy for IDD. This study first reports that LINC00963 acts as a ceRNA in IDD, synergistically exacerbating NPCs inflammation and ECM degradation by binding miR-1252-5p and relieving its inhibition on MAPK1. This discovery not only provides a potential molecular biomarker for the early diagnosis of IDD but also opens up new avenues for the development of targeted therapeutic strategies.

LINC00963 has been implicated in bone metabolic disorders across multiple studies. For instance, it alleviates osteoporosis by promoting osteogenic differentiation through regulating the miR-760/ETS1 axis or inhibiting miR-506-3p [ 33 , 34 ]. Furthermore, this lncRNA upregulates MMP15 expression by sponging miR-608, accelerating cell growth and epithelial-mesenchymal transition, and promoting the progression of acute myeloid leukemia [ 35 ]. It also promotes proliferation and invasion of osteosarcoma cells by regulating the miR-204-3p/FN1 axis [ 36 ]. Wu et al. [ 20 ] used high-throughput sequencing combined with RT-qPCR technology to find that this lncRNA was significantly overexpressed in IDD patient tissues, which is highly consistent with the experimental results of this study. Our study demonstrates that LINC00963 (AUC = 0.8692) effectively distinguishes IDD patients from the control group. Compared to other commonly used markers in histological studies (whose standalone diagnostic values typically range from 0.75to0.85 [ 37 , 38 ]), LINC00963 exhibits superior diagnostic performance. Further multivariate logistic regression analysis confirmed that LINC00963 is an independent predictor of IDD (OR = 3.955), suggesting its potential clinical utility for diagnostic evaluation of IDD. Typical pathological changes in IDD disease include abnormal ECM metabolism, the formation of a chronic inflammatory microenvironment, and programmed cell death of NPCs [ 39 , 40 ]. Notably, Col-II and Aggrecan are core components maintaining ECM structural integrity, while MMPs act as primary proteolytic enzymes capable of disrupting the dynamic balance of these ECM components [ 41 ]. The results of this experiment indicate that overexpression of LINC00963 significantly inhibits Col-II and Aggrecan expression at both transcriptional and translational levels in NPCs, while substantially upregulating MMP3 and MMP13 expression consistently at these two levels. Combining previous reports on the degradative effects of MMPs and the ADAMTS protein family on NP tissue [ 42 , 43 ], we speculate that LINC00963 may promote ECM degradation by enhancing IL-1β-induced inflammatory responses. Ahmad et al. [ 44 ] also support this view, as they found that the abnormal expression of LINC00963 in rheumatoid arthritis patients was significantly correlated with inflammatory markers and immune cell infiltration. In this study, overexpression of LINC00963 led to a significant increase in pro-inflammatory factors such as IL-6, TNF-α, and IL-1β. Existing evidence suggests that the sustained accumulation of these inflammatory mediators in degenerative intervertebral disc tissue forms a vicious cycle, further inducing cell apoptosis and matrix destruction [ 45 ]. Abnormal elevation of inflammatory factors within NPCs has been recognized as a key factor driving the progression of IDD. This study confirmed that LINC00963 significantly promotes the secretion of inflammatory factors, ultimately exacerbating IL-1β-mediated ECM degradation. H19 primarily exacerbates IDD by promoting apoptosis and autophagy in NPCs [ 13 ].

The overproduction of inflammatory factors accelerates the degradation of the ECM and promotes the pathological development of IDD. To study the biological function of LINC00963 in NPCs in more detail, we examined its mechanism. Subcellular localization experiments indicate that LINC00963 is primarily distributed in the cytoplasm. Since cytoplasmic lncRNAs generally have a “sponge” function that binds miRNA [ 46 ], we used bioinformatics methods to predict the miRNA to which LINC00963 can bind. Database analysis indicated that miR-1252-5p may be its main target. Several miRNAs have been identified as being involved in the development of IDD [ 47 ], particularly miR-1252-5p whose overexpression is associated with various pathological processes. Research data indicate that miR-1252-5p expression is significantly downregulated in an ovarianectomy animal model [ 48 ]; its expression level decreases by over 80% in multiple myeloma U266 cells [ 49 ]. In osteoporosis, lncRNA PAX8-AS1 has been demonstrated to sponge miR-1252-5p to regulate osteoblast function [ 48 ]. However, the functional role of miR-1252-5p in IDD remains unexplored. Using expression profiling, we demonstrated for the first time a significant negative correlation between LINC00963 and miR-1252-5p. The expression of miR-1252-5p was significantly inhibited in IL-1β-stimulated NPCs. Transfection with miR-1252-5p mimics effectively obstructed the inflammatory response and ECM degradation induced by LINC00963 overexpression, suggesting that miR-1252-5p has an important protective effect on NPCs. In summary, our findings confirm that dysregulation of the LINC00963/miR-1252-5p axis simultaneously exacerbates inflammation and ECM degradation in NPCs. This mechanism parallels that of lncRNA OIP5-AS1 in promoting IDD [ 50 ]. Furthermore, this study confirms that MAPK1 is a downstream effector molecule of miR-1252-5p. Previous studies have demonstrated that MAPK1 plays a pro-cancerous role in various tumors, such as osteosarcoma [ 51 ], gastric cancer [ 52 ], and cervical cancer [ 53 ]. Recent studies further reveal abnormal regulation of MAPK1 in bone metabolism-related disorders. For instance, MAPK1 participates in spinal cord injury [ 54 , 55 ]and osteoarthritis [ 56 ], and may influence spinal cord remodeling by participating in ECM metabolism-related signaling pathway [ 57 ]. Furthermore, MAPK1 has been identified as a key hub gene in IDD [ 58 – 60 ]. Its overexpression may exacerbate inflammation or induce cellular senescence by activating downstream p38/NF-κB pathways, thereby impairing NPCs function and accelerating IDD progression [ 61 , 62 ]. These pathways play a pivotal role in the inflammatory response and ECM degradation associated with IDD. Further investigation into MAPK1-mediated regulation of these cascades holds promise for providing deeper mechanistic insights and identifying novel therapeutic targets. This study revealed that MAPK1 expression is upregulated in IDD patient tissues. Upregulating miR-1252-5p significantly reversed the elevated MAPK1 expression induced by LINC00963 overexpression. These findings suggest that LINC00963 may participate in the pathogenesis and progression of IDD by regulating the miR-1252-5p/MAPK1 axis.

Although this study yielded some meaningful findings, several limitations remain that require refinement in future research. First, the cross-sectional design of the clinical sample analysis revealed correlations between LINC00963 expression levels and IDD severity/diagnostic value but it cannot establish causality with disease progression. Cross-sectional studies capture only a single time point, making it impossible to determine whether elevated LINC00963 precedes or follows degenerative processes. Prospective cohort studies are needed to validate its predictive value as a dynamic biomarker. Second, the in vitro cellular experimental model failed to fully replicate the complex mechanical microenvironment of the in vivo intervertebral disc. In daily life, the disc endures complex compressive, shear, and torsional stresses—key factors influencing non-cellular biological behavior and IDD progression. The IL-1β stimulation model employed in this study failed to incorporate the effects of mechanical loading. Future validation of the LINC00963/miR-1252-5p/MAPK1 signaling axis should be conducted in animal models integrating dynamic stress. Finally, this study did not thoroughly explore the role of MAPK1, a target gene of miR-1252-5p, in NPCs inflammation and ECM metabolism. Subsequent work will integrate single-cell sequencing with in vivo animal experiments to refine the functional regulation network of LINC00963/miR-1252-5p and explore the therapeutic potential of combining LINC00963 inhibitors with existing MAPK1 inhibitors, laying the foundation for developing targeted therapies that interfere with this axis.

This study demonstrated that LINC00963 expression is significantly elevated in patients with IDD, demonstrating excellent diagnostic value. It promotes IDD progression by targeting and inhibiting miR-1252-5p, thereby enhancing inflammatory responses and accelerating extracellular ECM degradation. These findings not only contribute to a better understanding of the pathological mechanism of IDD but also provide a theoretical basis for the development of new diagnostic and therapeutic strategies.

Conclusions

In summary, this study demonstrated, through clinical trials and in vitro experiments, that LINC00963 expression is significantly elevated in IDD, giving it excellent diagnostic value. Furthermore, it was discovered in NPCs that LINC00963 targets miR-1252-5p and regulates the level of inflammation and ECM degradation, providing new insights into the molecular mechanisms of IDD.

Supplementary Information

Supplementary Material 1

Supplementary Material 2

Acknowledgements

Not applicable.

Authors’ contributions

GGW designed the study, performed the experiment, and was a major contributor in writing the manuscript. CW and JCL re-evaluated the clinical data, revised the manuscript and performed the statistical analysis and revised the manuscript. QFZ involved in interpretation of data, drafting the manuscript and revising it critically for important intellectual content. All authors read and approved the final manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All procedures performed in studies involving human participants were in accordance with the 1964 Helsinki Declaration. Approval for this investigation was obtained from the Zhucheng People’s Hospital Ethics Committee. Before sample collection, each participant signed an informed consent form.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Chao Wei and Jiacheng Li are joint first author.

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Associated Data

Supplementary Materials

Supplementary Material 1

Supplementary Material 2

Data Availability Statement

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.