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Abstract

Study Design

Retrospective Study.

Objective

Lumbar disc herniation (LDH) can cause low back pain and sciatica, and lead to nerve damage. This study aims to explore whether the severity of LDH and the risk of its onset are related to the dysregulation of miR-93-5p.

Methods

Firstly, the level of miR-93-5p in the nucleus pulposus (NP) of LDH patients and patients with traumatic lumbar fractures was detected by RT-qPCR. The compression degree of the lumbar disc on the nerve was evaluated by using the Pfirrmann classification. The VAS score and JOA score were used to evaluate the degree of pain and functional impairment. We also analyzed the relationship between the concentration of miR-93-5p and inflammatory factors (TNF-α, IL-6, IL-1β). Finally, the risk factors influencing LDH were explored through binary logistic regression.

Results

miR-93-5p was downregulated in LDH patients. The higher the Pfirrmann grade is, the lower the level of miR-93-5p is. Additionally, as the degree of pain increases, the level of miR-93-5p gradually begins to decrease. Contrary to this result, miR-93-5p was positively correlated with the JOA score. Inflammatory factors in the NP tissues of LDH patients increase and there is a negative correlation with miR-93-5p. More importantly, the decrease of miR-93-5p may be an independent risk factor leading to LDH.

Conclusion

The decrease of miR-93-5p is a risk factor for LDH and is negatively correlated with the clinical severity. The reduction of this molecule may lead to intensified pain and activation of inflammation.

Keywords

lumbar disc herniation pain miR-93-5p inflammation risk factors

Introduction

Lumbar Disc Herniation (LDH) is a common orthopedic disease, frequently occurring in people aged 20 to 50. When the fibrous ring ruptures and the internal nucleus pulposus protrudes and compresses the nerve roots, numerous pain symptoms will occur.¹ In recent years, the treatment methods for LDH have made significant progress in minimally invasive techniques, such as percutaneous endoscopic lumbar discectomy, radiofrequency ablation, and the combined treatment of these two minimally invasive approaches.^2,3 However, in recent years, with the widespread adoption of sedentary lifestyles, the incidence rate has shown a trend of becoming younger. L4-L5 and L5-S1 segments are the most commonly affected areas, with the lesion accounting for up to 95%.⁴ The most prominent core harm caused by LDH is mainly caused by nerve compression, resulting in lower back pain, radiating pain in the lower limbs, abnormal sensations, decreased muscle strength, and functional impairments such as limited movement, urinary and fecal dysfunction, etc.^5,6 Therefore, having LDH brings huge living and economic burdens to patients. As a type of non-coding RNA consisting of 18-25 nucleotides, miRNA regulates gene expression by binding to mRNA.⁷ It has been reported that more than 2000 types of miRNAs have been identified in the human genome, and miRNA can regulate approximately 60% of protein-coding genes.⁸ Due to this, miRNAs play a crucial role in a variety of cellular biological processes, including cell growth and differentiation.^9,10 Existing evidence confirms that abnormal dysregulation of miRNA is closely associated with neurological diseases,¹¹ cardiovascular diseases,¹² and some spinal-related diseases.¹³ miRNA has also been proven to affect LDH. For example, miR-223 has been confirmed to be released from the nucleus pulposus tissue and has the effect of reducing neuronal activity. Moreover, the dysregulation of miR-223 can indicate the risk of lumbar radicular pain caused by LDH.¹⁴ Furthermore, some recent studies have shown that in animal models, artificially injecting miRNA oligonucleotides can inhibit pain and inflammation caused by LDH, such as miR-185-5p and miR-146a.^15,16 In addition, some miRNAs, such as miR-4741, can also indicate the prognosis of LDH patients.¹⁷There are many studies that have confirmed that the uncontrolled and abnormal expression of miRNA is closely related to LDH. It is worth noting that the existing evidence indicates that miR-93-5p is abnormally expressed in spinal scoliosis and spinal stenosis patients.^18,19 And miR-93-5p has the effect of stimulating the synthesis of extracellular matrix in nucleus pulposus.²⁰ It is worth noting that the aging, necrosis, degeneration of nucleus pulposus, and the reduction of extracellular matrix synthesis are important aspects leading to the aggravation of LDH.

The above studies have shown that miRNA, as an important regulatory factor of gene expression, plays a crucial role in the occurrence and development of LDH. Among them, miR-93-5p may play an important role in spinal-related diseases. However, no studies have yet revealed the expression of miR-93-5p in patients with LDH, nor have they explored the correlation between this molecule and the severity of LDH. This study aims to reveal the relationship between the severity of LDH and the expression level of miR-93-5p. This study is expected to provide new ideas and methods for targeted treatment and prognosis assessment of LDH patients.

Materials and Methods

Participants

This study included 79 patients with LDH from February 2021 to February 2024. Inclusion criteria: Diagnosed with LDH by MRI; having a history of chronic low back pain accompanied by radiating pain; the straight leg lift test is positive; aged 18 years or above; able to communicate normally and cooperate with treatment. Exclusion criteria: Those with other spinal diseases that could cause similar symptoms, such as fractures, tumors, severe osteoporosis, etc.; those who have previously undergone lumbar spine surgery; those with serious life-threatening diseases such as severe heart, brain, liver, kidney and blood system disorders; those currently with infections, infectious diseases, skin damage, etc.; pregnant or lactating patients. We also recruited 27 patients with traumatic lumbar fractures as the control group for.

Data Collection and Clinical Assessment

Collect all the basic information of the subjects, including age, gender, BMI, as well as the status of smoking and drinking. Additionally, two professional orthopedic doctors used the modified Pfirrmann grading system to assess the degree of intervertebral disc lesions in patients with LDH.

Sample Collection

During the surgery, Lumbar disc tissues were collected, and the nucleus pulposus tissues were immediately separated. Subsequently, the nucleus pulposus tissues were rapidly frozen using liquid nitrogen and then stored in a −80°C refrigerator.

Additionally, the peripheral venous blood of all subjects was collected and placed in vacuum blood collection tubes without anticoagulants. The tubes were then left at room temperature to stand for half an hour. After centrifugation at 3000×g for 15 minutes, the blood sample was divided into three layers (the upper layer being the serum). Subsequently, the upper layer of serum was carefully aspirated and immediately aliquoted into sterile cryovials for cryopreservation.

cDNA Synthesis

The total RNA was extracted using the TRIzol reagent (Cat# 15596026CN, Thermofisher Scientific, USA). After obtaining the total RNA, the purity and concentration of the RNA samples were immediately determined using Nanodrop 2000 ultramicro spectrophotometer (Thermofisher Scientific, USA). For miRNA, we used the miRNA First-Strand cDNA Kit (Cat# KR211, TIANGEN BIOTECH, China) to amplify the cDNA containing miR-93-5p. For mRNA, we used the FastKing gDNA Dispelling RT Super Mix (Cat# KR118, TIANGEN BIOTECH, China) to amplify the cDNA. The amount of RNA added to each sample was 1 μg. The cDNA was temporarily stored at −80°C in the refrigerator.

Quantitative Real-Time Polymerase Chain Reaction (Q-PCR)

We used the miRcute Plus miRNA qPCR Kit (Cat# FP411, TIANGEN BIOTECH, China) and SuperReal PreMix Plus (Cat# FP205, TIANGEN BIOTECH, China) to amplify miRNAs and mRNAs. The amplification program for miRNAs was: 95°C for 15 minutes; 94°C for 20 seconds; 60°C for 30 seconds. The amplification program for mRNAs was: 95°C for 15 minutes; 95°C for 10 seconds; 60°C for 30 seconds. Each sample was set up with three replicate wells to minimize experimental errors caused by experimental operations. Correspondingly, U6 and GAPDH were used as internal controls for miRNA and mRNA. After amplification, we used the 2^-ΔΔCT method to calculate the levels of miR-93-5p, TNF-α, IL-1β and IL-6. The primer sequence information can be found in Supplemental Table 1.

Assessment of Clinical Severity

Evaluation of Pain Severity

In this study, the visual analogue scale (VAS) was used to assess the pain condition of patients with LDH. This scale employs a 10-cm ruler to quantify the degree of pain. The ends of the ruler are marked as “0” (no pain) and “10” (severe pain).

Evaluation of Lumbar Spine Function

The JOA score is a method developed by the Japanese Orthopedic Association for assessing lumbar spine function. Its content includes four aspects: subjective symptoms, clinical manifestations, daily life, and bladder function. The higher the score, the better the lumbar spine function.

Statistical Analysis

IBM SPSS Statistics 23 was used for statistical analysis of the data and for analyzing differences. Count data were analyzed using the chi-square test. Measurement data were analyzed using the t-test. The correlation between two variables was evaluated using Pearson correlation analysis. When P < 0.05, it was considered that the difference was significant.

Results

Comparison of the Basic Characteristics of the Subjects

The basic clinical data of the two groups of subjects are presented in Table 1. The relevant results show that there are no significant differences between the subjects in the control group and the LDH group in terms of gender ratio, age, BMI, and the number of people who smoke and drink (P > 0.05). Additionally, the Pfirrmann grades in the LDH patients included in this study were between Grade3 and Grade7 respectively. The VAS score and JOA score of patients with LDH were 6.01 ± 1.46 and 12.63 ± 3.18, respectively. This related result indicates that the LDH patients included in this study were accompanied by moderate pain and had varying degrees of functional impairment.

Table 1.

Basic Characteristics of LDH Patients and Controls

Parameters	Controls	LDH patients	P Value
Gender (n, %)
Male	16 (59.26%)	47 (59.49%)	1.000
Female	11 (40.74%)	32 (40.51%)	1.000
Age (years)	43.00 ± 11.46	46.77 ± 5.15	0.109
BMI (kg/m2)	22.84 ± 1.83	22.67 ± 1.82	0.681
Smoking (n, %)	9 (33.33%)	20 (25.32%)	0.458
Drinking (n, %)	9 (33.33%)	16 (20.25%)	0.193
Pfirrmann grades (n, %)
Grade3	/	26 (32.91%)	/
Grade4	/	17 (21.52%)	/
Grade5	/	27 (34.18%)	/
Grade6	/	6 (7.59%)
Grade7	/	3 (3.80%)
VAS scores	/	6.01 ± 1.46	/
JOA scores	/	12.63 ± 3.18	/

Notes. LDH, lumbar disc herniation; BMI, body mass index; VAS scores, Visual Analogue Scale; JOA scores, Japanese Orthopedic Association score.

miR-93-5p Abnormally Decreased in the Nucleus Pulposus of Patients with LDH

Through RT-qPCR, we compared the levels of miR-93-5p in patients with LDH and those without LDH. The relevant results showed that miR-93-5p abnormally decreased in the nucleus pulposus of patients with LDH (P < 0.001, Figure 1A). However, no significant change of miR-93-5p was found in the serum (P > 0.05, Figure 1B). Subsequently, based on the average level of miR-93-5p in LDH patients, we further divided LDH patients into two subgroups: the miR-93-5p high-expression group and the miR-93-5p low-expression group, and further compared the differences in Pfirrmann grades between the two subgroups. The results showed that the Pfirrmann grades in the high-expression group were significantly lower (P < 0.001, Figure 2A), indicating that the degree of intervertebral disc degeneration in the high-expression group was lower. Further analysis results indicated that there was a relatively significant negative correlation between the local expression level of miR-93-5p and Pfirrmann grades (r = −0.535, P < 0.001, Figure 2B). Binary logistic regression indicated that the decreased level of miR-93-5p was an independent risk factor for LDH (P < 0.05, Table 2).

Figure 1.

miR-93-5p Levels in Patients With Lumbar Disc Herniation (LDH). (A) miR-93-5p Levels in Nucleus Pulposus (NP) Tissues of Both LDH Patients and Non-LDH Patients. (B) The Expression Pattern of miR-93-5p In the Serum of Subjects. ***P < 0.001

Figure 2.

The Relationship Between miR-93-5p Levels in Nucleus Pulposus (NP) Tissues and Pfirrmann Grades. (A) The Differences of Pfirrmann Grades Between the miR-93-5p High-Expression Group and the Low-Expression Group. (B) The Correlation Between the Level of miR-93-5p And Pfirrmann Grades was Investigated Through Pearson Correlation Analysis. **P < 0.01

Table 2.

Logistic Regression Analysis of Factors for LDH Risk

Parameters	Univariate logistic regression			Multivariate logistic regression
Parameters	OR	95%CI	P	OR	95%CI	P
Age	0.444	0.182-1.082	0.074	0.440	0.171-1.127	0.087
Gender	0.990	0.407-2.410	0.983	0.755	0.264-2.161	0.600
BMI	1.159	0.481-2.788	0.743	0.949	0.348-2.591	0.919
Smoking	0.678	0.263-1.748	0.421	0.840	0.185-3.819	0.821
Drinking	0.508	0.193-1.340	0.171	0.537	0.131-2.201	0.388
MiR-93-5p	3.488	1.362-8.933	0.009	3.442	1.306-9.067	0.012

Note. BMI, body mass index.

The local expression level of miR-93-5p is associated with the degree of pain and dysfunction in patients with LDH.

Based on the relevant results of the VAS score, all LDH patients were divided into the following two subgroups according to their pain manifestations: moderate pain group and severe pain group. The results of RT-qPCR indicated that the level of miR-93-5p in the severe pain group was significantly lower than that in the moderate pain group (P < 0.01, Figure 3A). The level of miR-93-5p was confirmed to have a certain negative correlation with the VAS score (r = −0.467, P < 0.001, Figure 3B). Additionally, we used the JOA score to assess the degree of functional impairment in LDH patients. The results showed that miR-93-5p was positively correlated with the JOA score (r = 0.374, P < 0.001, Figure 4), meaning that the higher the level of miR-93-5p, the less severe the functional impairment caused by LDH.

Figure 3.

The Relationship Between the Level of miR-93-5p In the Nucleus Pulposus (NP) Tissues of Patients With Lumbar Disc Herniation (LDH) and the Degree of Pain. (A) The Levels of miR-93-5p In LDH Patients With Different Degrees of Pain. (B) The Correlation Between the Level of miR-93-5p And the VAS Score was Investigated Through Pearson Correlation Analysis. *P < 0.05, **P < 0.01

Figure 4.

The Correlation Between the miR-93-5p Level in Nucleus Pulposus (NP) Tissues and the JOA Score of Patients With Lumbar Disc Herniation (LDH)

The level of miR-93-5p in nucleus pulposus is negatively correlated with nucleus pulposus inflammation.

The mRNA levels of pro-inflammatory factors in the nucleus pulposus tissues of LDH patients all increased to varying degrees (P < 0.001, Figure 5A). Through Pearson correlation analysis, we further investigated the relationship between miR-93-5p and nucleus pulposus inflammation. The results showed that the relative expression level of miR-93-5p was significantly negatively correlated with TNF-α (r = −0.686, P < 0.001, Figure 5B), IL-6 (r = −0.346, P < 0.01, Figure 5C) and IL-1β (r = −0.328, P < 0.01, Figure 5D).

Figure 5.

The Level of miR-93-5p In Patients With Lumbar Disc Herniation (LDH) is Negatively Correlated With Nucleus Pulposus (NP) Inflammation. A. The mRNA Levels of Pro-inflammatory Factors in NP. B-D. The Correlation Between the Level of miR-93-5p In NP and the mRNA Levels of TNF-α, IL-6 and IL-1β. ***P < 0.001

Discussion

miRNA, as a non-coding RNA molecule with regulatory functions on gene expression, was discovered as an important milestone in modern molecular biology. miRNA precisely regulates approximately one-third of human genes by degrading target mRNAs or inhibiting translation.²¹ Abnormal expression of miRNA is not only closely related to the onset and progression of diseases, but has also become a hot area in the development of disease diagnosis and treatment targets.²² LDH is a disease caused by the rupture of the annulus fibrosus. Its characteristic is that the inner nucleus pulposus tissue protrudes outward and subsequently compresses the spinal cord or nerves. The deterioration of LDH is also related to miRNA. For example, miR-146a alleviates pain hypersensitivity caused by LDH in the rat model by inhibiting the release of inflammatory factors.¹⁶ While miR-17 aggravates the nerve root pain caused by LDH through high expression in the nucleus pulposus.²³ Some miRNAs have also been confirmed to be related to the aging of nucleus pulposus cells, such as miR-4741¹⁷ and miR-495.²⁴ Thus, it can be seen that miRNA in LDH may be closely related to the aggravation of pain, the aging of nucleus pulposus cells, and the activation of inflammation.

miR-93-5p has been proven to play an important role in various human diseases such as cancer,²⁵ arthritis,²⁶ and myocardial infarction.²⁷ However, the relationship between the level of this molecule and the clinical severity of LDH patients has not yet been studied. The results of this study indicate that the level of miR-93-5p in the nucleus pulposus tissues of LDH patients is decreased. However, the level of miR-93-5p in the serum did not show significant changes. Therefore, we believe that the local miR-93-5p may be closely related to LDH. The results of binary logistic regression analysis further confirmed that the decrease in miR-93-5p is a risk factor for triggering LDH. The typical feature of LDH is that the nucleus pulposus tissue protrudes outward and compresses the spinal cord or nerves, causing neuropathic pain. Clinically, Pfirrmann grades are used to assess the degree of intervertebral disc degeneration. While VAS scores are used for pain assessment. The results of this study show a significant negative correlation between the level of miR-93-5p and Pfirrmann grade or VAS score. That is, the decrease in miR-93-5p level may be an inducing factor for the increase in the degree of intervertebral disc degeneration and the aggravation of pain. Studies have confirmed that overexpression of miR-93-5p can alleviate neuropathic pain and reduce pain sensitivity in rat models with chronic sciatic nerve compression injury.²⁸ This study indirectly supports our findings that an increase in miR-93-5p levels can alleviate the pain caused by LDH.

Studies have shown that the radicular pain after lumbar intervertebral disc protrusion may be related to the local inflammatory response caused by nucleus pulposus cells.²⁹ And miR-93-5p seems to act as an anti-inflammatory factor by acting on multiple signaling pathways and exerting anti-inflammatory effects in the disease. For example, overexpression of miR-93-5p significantly inhibits the activation of microglia and reduces the release of pro-inflammatory cytokines in the optic nerve injury model.³⁰ Moreover, miR-93-5p has also been confirmed to inhibit the production of inflammatory cytokines in macrophages.³¹ Additionally, miR-93-5p can exert neuroprotective effects in the spinal cord injury by inhibiting neuroinflammation.³² Based on these research backgrounds, we detected the levels of inflammatory factors. The results showed that the mRNA levels of TNF-α, IL-6, and IL-1β were negatively correlated with miR-93-5p. Therefore, the results of this study indicate that the low expression of miR-93-5p in nucleus pulposus tissue can help promote inflammation in the nucleus pulposus tissue. Furthermore, studies have confirmed that miR-93-5p can inhibit the apoptosis of nucleus pulposus cells in lumbar intervertebral disc lesions.³³ It is worth noting that the apoptosis of nucleus pulposus cells is not only a driving factor for intervertebral disc degeneration but also a key link in the pathological chain of LDH. Therefore, the inhibitory effect of miR-93-5p on inflammation and nucleus pulposus cell apoptosis may well explain why a decrease in miR-93-5p levels is the reason for the aggravation of pain and functional impairment in LDH patients. In conclusion, this study confirmed that miR-93-5p was abnormally downregulated in the nucleus pulposus tissue of LDH patients but not in the serum. The abnormal downregulation of miR-93-5p in the nucleus pulposus is a risk factor for LDH. The level of this molecule is negatively correlated with the severity of LDH and may be closely related to the aggravation of pain and activation of inflammation. This study provides a new molecular target for the prognosis of LDH and the development of targeted therapeutic drugs for LDH. However, the small sample size is a major limitation of this study. In the future, it is still necessary to explore the efficacy of this molecule in indicating the severity of LDH in a larger population. Additionally, although significant progress has been made in targeted therapeutic drugs for miRNA in recent years, there are still many challenges, such as off-target effects, safety, and how to efficiently and precisely deliver miRNA. In the future, a more comprehensive assessment of the role of miR-93-5p in lumbar disc herniation needs to be conducted, and functional verification should be carried out in animal models.

Supplemental Material

Supplemental Material - The Expression Level of miR-93-5p in Patients With Lumbar Disc Herniation is Related to the Severity of the Disease

Supplemental Material for The Expression Level of miR-93-5p in Patients With Lumbar Disc Herniation is Related to the Severity of the Disease by Zhen Wang, Jichen Liu, Haishan Xu, Wei He, Xijing He in Global Spine Journal.

Footnotes

ORCID iD

Xijing He

Ethics Approval

The study was given the go-ahead by the Ethics Committee of People’s Hospital of Ningxia Hui Autonomous Region, Ningxia Medical University (No.2020-NZR-177).

Informed Consent

All participants gave their informed consent.

Author contributions

Xijing He developed the original idea and the protocol, abstracted and analyzed data, wrote the manuscript, and is a guarantor. Zhen Wang, Jichen Liu, Haishan Xu and Wei He contributed to the development of the protocol, abstracted data, and prepared the manuscript.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Key R&D Projects in Ningxia Hui Autonomous Region, China(2022BEG03086) and Major Scientific and Technological Achievement Transformation Project of the Autonomous Region(2022CJE09010).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

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

Supplemental Material

Supplemental Material for this article is available online.

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