Abstract
Background
Previous studies show that about 80% of lumbar disc degeneration (LDD) patients experience back or leg pain, affecting their quality of life. However, the causes of LDD are not fully understood. This study focused on exploring the expression level of miR-340-5p and SRY-related high-mobility-group box 4 (SOX4) in LDD and attempts to clarify its potential mechanism in the pathological process of LDD.
Methods
A total of 88 patients with LDD and 80 non-LDD patients were continuously included in this study. The miR-340-5p levels in nucleus pulposus (NP) tissues were measured using Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR). The potential diagnostic significance of miR-340-5p for LDD was evaluated by generating a Receiver Operating Characteristic (ROC) curve. The impacts of miR-340-5p on Lipopolysaccharide (LPS)-stimulated NP cells were analyzed through the Cell Counting Kit-8 (CCK-8) method, and apoptosis levels were quantified using flow cytometry. Additionally, the concentrations of inflammatory cytokines and extracellular matrix (ECM) remodeling markers were assessed with Enzyme-Linked Immunosorbent Assay (ELISA) kits.
Results
miR-340-5p was abnormally down-regulated in LDD patients and the ROC curve showed that miR-340-5p has diagnostic value for LDD (AUC = 0.909, sensitivity = 81.8%, specificity = 85.0%, cutoff value = 0.815). Additionally, the enforced expression of miR-340-5p promoted proliferation and ECM remodeling and inhibited the level of apoptosis and pro-inflammatory factors in LPS-damaged NP cells. However, overexpression of SOX4 counteracted the effects of miR-340-5p overexpression on LPS-induced NP cells.
Conclusions
miR-340-5p alleviates the damage effect of LPS on NP cells by negatively regulating the SOX4, and miR-340-5p may be a potential therapeutic target for LDD.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13018-025-06069-4.
Keywords: miR-340-5p, SOX4, Lumbar disc degeneration, Inflammation, Extracellular matrix
Background
The lumbar region serves as the primary locus of axial movement and is susceptible to degenerative changes associated with advancing age, repetitive stress, and excessive mechanical load [1]. Lumbar disc degeneration (LDD) is commonly presented as a clinical syndrome characterized by significant low back pain [2]. Prior research indicates that approximately 80% of individuals with LDD experience varying levels of back or leg pain, significantly impacting their overall quality of life. Nevertheless, the etiology of LDD remains incompletely understood. Multiple factors are believed to contribute to the pathogenesis of LDD, including lifestyle choices, advancing age, and genetic susceptibility [3]. Currently, the management of LDD primarily involves conservative and surgical approaches, with no universally accepted treatment protocols established either domestically or internationally [4]. Thus, a thorough and comprehensive understanding of the underlying mechanisms of LDD is crucial for developing more effective clinical treatment strategies.
miRNAs exhibit abnormal expression in patients with LDD and are thus involved in the generation and development of the disease [5]. Studies have reported that miRNA has therapeutic potential in tendon injuries [6], and miRNA is involved in the regulation of genes and the control of homeostatic pathways in osteoarthritis [7]. Empirical evidence indicates that miR-340-5p is intricately involved in the pathogenic mechanisms underlying several diseases. For instance, miR-340-5p has been shown to mitigate myocardial oxidative stress in sepsis-induced cardiomyopathy [8], mediate corneal neovascularization [9], and alleviate brain injury resulting from subarachnoid hemorrhage-induced neuroinflammation [10]. Furthermore, miR-340-5p may serve as a potential regulator and could be involved in regulation of mouse skeletal muscle following aerobic exercise [11]. miR-340-5p also be involved in the differentiation process of osteoblasts [12]. Moreover, it is worth noting that miR-340-5p was downregulated by more than nine times in patients with intervertebral disc degeneration [13]. However, the level of miR-340-5p in LDD and its specific role in LDD have not yet been clarified.
The SRY-related high-mobility-group box 4 (SOX4) is closely related to bone-related diseases. For instance, studies have found that SOX4 expression is upregulated in rheumatoid arthritis, and SOX4 seems to be a diagnostic biomarker for arthritis [14]. Additionally, SOX4 is involved in regulating the function of osteoblasts during fracture healing [15]. Moreover, inhibiting the expression of SOX4 can alleviate intervertebral disc degeneration and low back pain [16]. SOX4 inhibition reduces IL-1β-induced nucleus pulposus (NP) cell degeneration [17]. Bioinformatics database reveals the presence of potential binding sites for miR-340-5p within the SOX4 sequence. Nevertheless, the regulatory effect of miR-340-5p on SOX4 expression and its potential role in LDD pathogenesis remain to be elucidated.
Based on the above background, this study focused on exploring the expression level of miR-340-5p on SOX4 in LDD and attempts to clarify its potential mechanism in the pathological process of LDD, thereby laying a foundation for future research and application.
Methods
Participants
The statistical analysis of sample size was conducted using G*Power software. When the effect size d = 0.5, α = 0.05, power value = 0.8, and the ratio of sample sizes between groups was set to 1, the total calculated sample size was 128, with at least 64 subjects in each group. From January 2022 to January 2025, a total of 88 patients with LDD underwent surgical treatment at Xingtai People’s Hospital and were continuously included in this study. The inclusion criteria for patients include patients aged between 30 and 70 years old and were diagnosed with LDD based on their medical history, clinical examination and imaging studies, and historical examination verified that the herniated nucleus pulposus (HNP) was caused by degenerative changes. This study excluded patients with spinal trauma, infection, deformity or a history of spinal surgery, as well as those with autoimmune diseases, malignant tumors, severe osteoporosis or liver and kidney dysfunction. In addition, 80 non-LDD patients who underwent lumbar intervertebral disc surgery due to trauma were included as the control group. Basic information of all participants was collected, and NP tissues were obtained and stored in liquid nitrogen during the operation. This study has been approved by the ethics committee of Xingtai People’s Hospital, and all patients participating in the study have signed the informed consent form.
Cell culture and treatment
Primary human NP cells were cultured in DMEM (Gibco, MA, USA) supplemented with 10% FBS (HyClone, UT, USA) and were incubated at 37 °C in 5% CO2. NP cells were transfected with miR-340-3p mimic, a miR-340-3p inhibitor, plasmids overexpressing SOX4 (oe-SOX4) or corresponding negative control (NC) (GenScript, Piscataway, NJ, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). According to previous studies [18, 19], to establish an in vitro model of LDD, cells were treated with 10 ng/mL lipopolysaccharide (LPS, Sigma-Aldrich, Saint Louis, MO, USA) for a duration of 24 h while being incubated at 37 °C with 5% CO2. The supernatants were harvested after 24 h of treatment.
RT-qPCR
Total RNA was isolated using TRIzol reagent (Takara, Japan). cDNA was synthesized utilizing the miScript Reverse Transcription kit (Qiagen GmbH, Hilden, Germany) as the provided guidelines. Quantitative PCR (qPCR) was subsequently performed using the Maxima SYBR-Green/ROX qPCR Master Mix (Thermo Fisher Scientific). U6 and GAPDH served as internal controls for miRNA and mRNA expressions, respectively. Relative expression levels were calculated using the 2−ΔΔCt method. The primer sequences and corresponding annealing temperatures and organized into Supplementary Table 1.
Dual-luciferase reporter assay
Starbase database was employed to predict potential targets of miR-340-5p. The vectors containing either the wild-type (wt-SOX4) or mutated (mut-SOX4) 3’UTR of SOX4 were constructed. NP cells were co-transfected with either wt-SOX4 or mut-SOX4, along with miR-340-5p mimic, miR-340-5p inhibitor or its negative control by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Forty-eight hours post-transfection, luciferase activity was measured with the Dual-Luciferase Reporter Assay kit (Beyotime, Jiangsu, China).
CCK-8 assay
NP cells were plated into 96-well plates and incubated for 24 h following transfection. Cell viability was assessed every 24 h over a period of three days by adding 10µL CCK-8 (Beyotime, Jiangsu, China) reagent. After a 4-hours incubation at 37˚C, spectrophotometric detection was employed to quantify absorbance at 450 nm.
Flow cytometry assay
Cell apoptosis was assessed using the Annexin V/propidium iodide (PI) method. At 24 h post-transfection, cells were dual-stained with Annexin V-FITC and PI to determine the rate of apoptosis. Flow cytometry (BD Biosciences, NJ, USA) was employed for cell analysis.
ELISA assay
NP cells were collected to measure the levels of TNF-α, IL-1β, IL-6, MMP13 (Abcam, USA), Collagen II and Aggrecan (Beyotime, Jiangsu, China). The concentrations proteins across different groups were determined using ELISA kits, following the manufacturer’s protocols.
Statistical analysis
All data were analyzed using SPSS 26.0 and Graphpad Prism 9.0. The diagnostic role of miR-340-5p for LDD was analyzed by the ROC curve. For continuous variables, the Shapiro-Wilk test was used to assess normality of the data, and the T-test was employed to analyze the differences between two groups, while one-way or two-way ANOVA was utilized to examine the statistical differences among multiple groups. For categorical variables, the chi-square test was applied. The relationship between miR-340-5p and SOX4 was evaluated by calculating the Pearson correlation coefficient. In cell experiments, each group was repeated three times. All P values less than 0.05 indicated significant differences.
Results
Comparison of basic information
Data on age, gender, BMI, smoking, hypertension and diabetes for two groups were collected. The control group consisted of 43 males and 37 females, with a mean age of 50.91 ± 12.77. The LDD group included 49 males and 39 females, with an average age of 52.41 ± 11.94. There were no significant differences in age, gender, BMI, smoking status, and the prevalence of hypertension and diabetes between the two groups (Table 1).
Table 1.
Comparison of the basic information of the two groups
| Controls (n = 80) | LDD (n = 88) | P value | |
|---|---|---|---|
| Age (years) | 50.91 ± 12.77 | 52.41 ± 11.94 | 0.433 |
| Gender (male/female) | 43/37 | 49/39 | 0.802 |
| BMI (kg/m2) | 23.41 ± 2.95 | 23.21 ± 3.17 | 0.671 |
| Smoking (yes/no) | 28/52 | 34/54 | 0.626 |
| Hypertension (yes/no) | 26/54 | 31/57 | 0.709 |
| Diabetes (yes/no) | 19/61 | 26/62 | 0.397 |
LDD, Lumbar disc degeneration; BMI, Body Mass Index. The data are presented as mean ± standard deviation or n and were analyzed by t-test or chi-square test. A P value < 0.05 indicates a significant difference
Expression level and diagnostic role of miR-340-5p in LDD
The level of miR-340-5p was downregulated in the NP tissues of patients with LDD (Fig. 1A). The abnormally down-regulated level of miR-340-5p had an outstanding diagnostic effect on LDD, with an AUC of 0.909 on the ROC curve, indicating its high diagnostic accuracy. In addition, the sensitivity was determined to be 81.8%, its specificity was calculated at 85.0%, and the cutoff value was 0.815 (Fig. 1B).
Fig. 1.
Expression level and diagnostic role of miR-340-5p in LDD. ***P<0.001. The expression level of miR-340-5p in Control group (n = 80) and LDD patients (n = 88) (A). The ROC curve of miR-340-5p for diagnosing LDD (B)
miR-340-5p negatively regulated the expression of SOX4
In LDD patients, the expression level of SOX4 was markedly upregulated (Fig. 2A), and exhibited a moderate negative correlation with miR-340-5p (Fig. 2B, r = -0.688). By consulting the StarBase database, potential binding sites were predicted between miR-340-5p and SOX4 (Fig. 2C). Overexpression of miR-340-5p inhibited the luciferase activity of wt-SOX4, while inhibition of miR-340-5p enhanced its luciferase activity (Fig. 2D). In addition, following transfection with miR-340-5p mimic, the expression level of miR-340-5p was significantly upregulated, whereas the expression of SOX4 was markedly downregulated. However, overexpression of SOX4 restored the SOX4 expression but did not significantly affect the expression level of miR-340-5p (Fig. 2E and F).
Fig. 2.
miR-340-5p negatively regulated the expression of SOX4. ***P<0.001, **P<0.01, ns: no significant difference. The expression level of SOX4 in Control group (n = 80) and LDD patients (n = 88) (A). The correlation between the expression levels of miR-340-5p and SOX4 in patients with LDD (B). The binding site of miR-340-5p and SOX4 (C). The binding relationship between miR-340-5p and SOX4 was verified by the dual-luciferase assay (D). The regulatory effect of miR-340-5p on the expression level of SOX4 (E-F)
Effects of miR-340-5p and SOX4 on the proliferation and apoptosis of LPS-induced NP cells
The findings demonstrated that the expression of miR-340-5p in NP cells decreased in a concentration-dependent manner with the increase of LPS concentration (Supplementary Fig. 1). We chosed a concentration of 10 ng/mL for subsequent experiments. Transfection with a miR-340-5p mimic led to an upregulation of miR-340-5p levels (Fig. 3A). Furthermore, SOX4 expression was observed to be elevated in LPS-treated cells, SOX4 expression was inhibited by the overexpression of miR-340-5p, an effect that was counteracted by the transfection of an oe-SOX4 construct (Fig. 3B). In addition, after LPS treatment of cells, the proliferation ability of NP cells weakened, and the apoptosis rate increased. Overexpression of miR-340-5p enhanced the cell proliferation ability and reduced cell apoptosis, while overexpression of SOX4 counteracted the effects of miR-340-5p on cell proliferation and apoptosis (Fig. 3C and D).
Fig. 3.
Effects of miR-340-5p and SOX4 on the proliferation and apoptosis of LPS-induced NP cells. ***P<0.001, **P<0.01, *P<0.05, ns: no significant difference. The expression of miR-340-5p in LPS-induced NP cells (A). The effect of overexpressing miR-340-5p on SOX4 in LPS-induced NP cells (B) The effects of miR-340-5p and SOX4 on LPS-induced cell proliferation (C). The effects of miR-340-5p and SOX4 on LPS-induced cell apoptosis (D)
Effects of miR-340-5p and SOX4 on inflammation and ECM remodeling in LPS-induced NP cells
LPS induced inflammatory responses in NP cells and enhanced the levels of pro-inflammatory factors (TNF-α, IL-1β, and IL-6). Overexpression of miR-340-5p suppressed the concentrations of inflammatory factors, while SOX4 overexpression counteracted the effect of miR-340-5p mimic on these factors (Fig. 4A, B and C). In addition, LPS treatment increased the levels of MMP13 and decreased the levels of Aggrecan and Collagen II. miR-340-5p mimic alleviated the influence of LPS on the levels of remodeling molecules in NP cells, while oe-SOX4 counteracted the levels of MMP13, Aggrecan and Collagen (Fig. 4D, E and F).
Fig. 4.
Effects of miR-340-5p and SOX4 on inflammation and ECM remodeling in LPS-induced NP cells. ***P<0.001, **P<0.01, *P<0.05. The effects of miR-340-5p and SOX4 on LPS-induced TNF-α, IL-1β, and IL-6 levels (A-C). The effects of miR-340-5p and SOX4 on the levels of MMP13 (D), Aggrecan (E) and Collagen II (F)
Discussion
The lumbar vertebrae are essential for providing structural support to the body’s trunk and play a fundamental role in enabling typical human physical activities [20]. Research has indicated that early intervention, guided by effective predictive indicators, can enhance patient outcomes [21]. In this study, miR-340-5p was abnormally down-regulated expressed in LDD patients and showed an excellent diagnostic effect for LDD. Additionally, miR-340-5p overexpression promoted cell proliferation and extracellular matrix (ECM) remodeling, and inhibited the apoptotic level and inflammatory response in LPS-induced NP cells by negatively regulating the level of SOX4, and thus might be involved in the development of LDD.
Recent studies have shown that multiple miRNAs are dysregulated in LDD patients and may act as potential biomarkers for LDD and participate in the occurrence of LDD. For instance, miR-589-3p has been found to be upregulated and acts as a promoter in the context of LDD [18]. Conversely, miR-155-5p is downregulated in LDD and has been identified as a novel biomarker for LDD [22]. Additionally, the expression of miR-4458 is elevated in LDD patients, contributing to the progression of the disease [23]. In this study, miR-340-5p expression was decreased in the NP tissue of LDD patients, and it could be used to diagnose the occurrence of LDD. According to the classic literature [24], an AUC of 0.5 suggests no discrimination, 0.7 to 0.8 is considered acceptable, 0.8 to 0.9 is considered excellent, and more than 0.9 is considered outstanding. In this study, the AUC of the ROC curve was 0.909, indicating that miR-340-5p demonstrated outstanding diagnostic ability in diagnosing LDD. The data from our investigation aligns with prior findings indicating a reduction in miR-340-5p expression within the context of intervertebral disc degeneration [13]. This consistency reinforces the potential involvement of miR-340-5p in the pathogenesis of LDD and suggests its utility as a significant biomarker for the condition. However, due to the insufficient sample size in this study, this conclusion may still require a larger sample size to be verified.
miR-340-5p regulates the expression of SOX4 and regulates the functions of gallbladder cancer cells [25]. Additionally, miR-340-5p regulates the level of SOX4 and mediates the metastasis of breast cancer cells as well [26]. Additionally, according to a guide to appropriate use of Correlation coefficient in medical research [27, 28], the strength of correlation coefficients is defined as: 0–0.3 (Negligible correlation), 0.3–0.5 (Low correlation), 0.5–0.7 (Moderate correlation), 0.7–0.9 (High correlation), 0.9–1.0 (Very high correlation). This study verified the moderate negative correlation of miR-340-5p on SOX4 through various experiments, thereby further clarifying overexpression of SOX4 counteracted the effects of miR-340-5p overexpression on LPS-induced NP cells. Moreover, this interaction may play a significant role in the disease progression of LDD.
Research has found that lumbar disc herniation is highly correlated with inflammation of low back pain [29]. The imbalance of ECM and the increase in inflammation have been identified as the occurrence and progression of intervertebral disc degeneration [30]. miR-589-3p enhances the production of pro-inflammatory factors in LPS-induced NP cells and inhibits aggrecan and collagen II expression [18]. miR-154 regulates the expression of collagen II, MMP13 and aggrecan thus promoting ECM degradation in intervertebral disc degeneration [31]. To investigate the regulatory roles of miR-340-5p and SOX4 in LDD, we conducted further experiments. Our study findings revealed that miR-340-5p overexpression significantly promotes the proliferation of NP cells by targeting and inhibiting SOX4 levels. miR-340-5p is also intricately linked to the process of the ECM, suggesting that it may enhance the balance of synthesis and degradation of ECM components such as Aggrecan and collagen II, thereby improving the function of damaged NP cells. Meanwhile, our experimental data further indicated that miR-340-5p effectively inhibits the apoptosis of NP cells and protects them from the toxic effects induced by LPS. LPS stimulation leads to the release of various pro-inflammatory cytokines by NP cells. The enforced expression of miR-340-5p weakened the inflammation by inhibiting SOX4, providing a potential therapeutic strategy for alleviating LPS-induced inflammation in NP cells. Studies have found that small interfering RNA has demonstrated significant potential in disease treatment [32–34]. Therefore, the use of siRNA technology to inhibit the expression of SOX4 may play a protective role in the treatment of LDD. These results provide a novel viewpoint for gaining a more profound comprehension of the pathogenesis of LDD and could potentially establish the groundwork for the creation of miRNA-based therapeutic strategies. Nevertheless, the pathway by which miR-340-5p modulates SOX4 to affect inflammation and ECM remodeling are still not fully understood and require further investigation.
While this study has achieved certain results, there are still some limitations that need further exploration and improvement. In this study, we calculated the minimum sample size required for statistical power using G*Power software before including patients, and the result was at least 64 people in each group Even if the sample size in this study has reached the minimum requirement calculated in advance through power analysis, from the perspective of scientific rigor, such a sample size is still far from sufficient to draw conclusions that are universally applicable and highly reliable. In statistics, although meeting the minimum sample size can ensure that the study has sufficient statistical power to detect the expected effect, a smaller sample size may be affected by random errors, individual differences, or other confounding factors, thereby limiting the external validity and generalizability of the research results. Therefore, in future studies, we plan to include a larger sample size to provide more supporting data for the conclusions of this study. Secondly, although our study indicates that miR-340-5p plays a significant role in the function of NP cells by regulating the SOX4 gene, miR-340-5p may have other yet undiscovered target genes. These potential target genes may also be involved in key biological processes such as inflammation, apoptosis, or ECM remodeling. Future studies need to design more experiments to thoroughly investigate the specific function of miR-340-5p in related signaling pathways. Moreover, we only investigated the effect of a single concentration of NP cells, which might limit our understanding of the complex changes during the disease development process. Different concentrations of LPS may exert varying degrees of influence on cell viability, inflammatory factor release, and matrix degradation, thereby simulating different stages of LDD. Therefore, we will systematically evaluate the impact of LPS gradient concentrations on the cell model to more comprehensively reflect the pathological process of LDD in future study.
Conclusions
miR-340-5p demonstrated a markedly decreased expression in LDD patients and demonstrated a robust diagnostic efficacy for LDD. Furthermore, miR-340-5p was observed to promote cell proliferation and ECM remodeling, while concurrently suppressing apoptosis and the inflammatory response in LPS-stimulated NP cells by inhibiting SOX4 levels, suggesting a potential involvement in the pathogenesis of LDD.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary Material 1: The expression of miR-340-5p in NP cells after treatment with LPS of different concentrations. ***P<0.001, **P<0.01, *P<0.05.
Acknowledgements
Not applicable.
Author contributions
YHL, LNH and ZTG was responsible for project development, data analysis, manuscript writing and editing. YNH, FLK and SBH was responsible for data acquisition and data analysis. ZL involved in interpretation of data and revising it critically for important intellectual content. All authors have read and approved the manuscript.
Funding
No funding was received to assist with the preparation of this manuscript.
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
This study has been approved by the ethics committee of Xingtai People’s Hospital, and all patients participating in the study have signed the informed consent form.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Yuhang Liu and Lina Huang share first authorship.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Contributor Information
Zhengtian Gao, Email: Gaozhengtianzt@163.com.
Zheng Li, Email: lizheng_dr@163.com.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplementary Material 1: The expression of miR-340-5p in NP cells after treatment with LPS of different concentrations. ***P<0.001, **P<0.01, *P<0.05.
Data Availability Statement
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.