The clinical application potential of miR-1287-5p in spinal cord injury resulting from spinal trauma and its involvement in modulating the inflammatory response

Abstract

Background

Spinal cord injury (SCI) is a severe nervous trauma and the underlying mechanism of miR-1287-5p in its pathogenesis remains incompletely elucidated.

Objective

This research seeks to elucidate the molecular mechanism of miR-1287-5p in regulating inflammatory responses following SCI.

Methods

The expression levels of miR-1287-5p and MAP3K9 were measured using qRT-PCR. Pearson correlation tests scrutinised their associations with clinical indices. ROC curve analysis was conducted to evaluate it diagnostic value for SCI. CCK-8 and ELISA assays were employed to evaluate cell viability and protein expression levels, respectively. The direct interaction between miR-1287-5p and MAP3K9 was confirmed through dual-luciferase reporter assays.

Results

miR-1287-5p was markedly down-regulated in SCI patients and correlated with pain scores, LEMS, and ASIA grade.ROC curve analysis indicated that miR-1287-5p has potential as a diagnostic biomarker for SCI. Overexpression of miR-1287-5p enhances cell viability, reduces the production of inflammatory cytokines, and specifically binds to MAP3K9, thereby inhibiting its expression.

Conclusion

miR-1287-5p serves as a potential diagnostic biomarker for SCI and modulates the inflammatory response in SCI through direct targeting of MAP3K9.

Introduction

Spinal cord injury (SCI) represents a critical injury affecting the central nervous system, marked by consistently elevated disability and fatality rates, primarily caused by external injuries [1, 2]. The pathophysiology of SCI unfolds in acute and chronic phases, encompassing ischaemia, oxidative stress, exuberant inflammation, apoptosis, and motor dysfunction [3]. These processes are closely interconnected and ultimately result in neurological impairments below the level of injury, including sensory and motor deficits, as well as various systemic complications [4]. Acute spinal cord injury promptly initiates a cascade of secondary injury processes, which may progress to chronic pathological changes, resulting in various complications and an increased mortality rate compared to age-matched healthy individuals [5].This not only significantly compromises patients’ quality of life but also imposes substantial physical, emotional, and financial burdens on individuals, families, and society [1]. In clinical practice, magnetic resonance imaging (MRI) and the American Spinal Injury Association (ASIA) Impairment Scale are currently regarded as the gold standard diagnostic tools for SCI [6]. However, their reliability can be affected by factors such as poor patient baseline conditions, concomitant injuries, and the influence of pharmacological and surgical interventions [7]. Therefore, further exploration of the mechanisms underlying SCI and the discovery of new predictive biomarkers are crucial for improving the accuracy and effectiveness of clinical diagnosis and management of SCI patients.

In recent years, the rapid advancement of molecular biology techniques has significantly accelerated research on non-coding RNAs, including circRNA, siRNA, and miRNA [8, 9]. Among these, miRNA has garnered particular attention in the scientific community due to its extensive regulatory roles in various diseases, with growing research interest observed in this field [10]. miRNA plays a crucial role in the development and progression of various orthopedic conditions, including tendon injury, arthritis. and spinal cord injury [11, 12]. In the context of SCI, accumulating evidence indicates that miRNA expression profiles are significantly altered, with certain miRNAs demonstrated to modulate key pathological processes such as inflammation, apoptosis, and neural regeneration following SCI [3]. For example, upregulation of miR-223 has been shown to regulate macrophage phenotypic transformation, thereby mitigating inflammatory damage [13]. miR-124 contributes to neural functional recovery by regulating apoptosis after SCI, while also influencing angiogenesis and neural repair [14]. miR-1287-5p has been demonstrated to play a key regulatory function in the development and progression of multiple pathological conditions. For instance, the decreased expression of miR-1287-5p has been associated with chronic sinusitis [15]. miR-1287-5p triggers ferroptotic cell death in osteosarcoma cells by suppressing GPX4 activity [16].Emerging studies suggest that miR-1287-5p expression is reduced after SCI, indicating its potential involvement in the pathophysiological mechanisms of the disease [17]. Moreover, miR-1287-5p serves as a key regulator in the control of osteogenic differentiation in mesenchymal stem cells [18]. Therefore, further investigation into the specific functions and underlying mechanisms of miR-1287-5p in SCI may uncover novel molecular targets and therapeutic strategies for the treatment of this condition.

In summary, SCI is a complex disorder of the central nervous system that involves multiple pathological and physiological processes. The purpose of this research is to explore the expression profile and clinical diagnostic potential of miR-1287-5p in SCI, as well as its potential molecular mechanism in regulating inflammation. These findings may provide novel therapeutic targets and a theoretical foundation for the treatment of SCI.

Materials and methods

Clinical sample collection

A total of 109 patients with spinal trauma causing in SCI, diagnosed in the Zhucheng People’s Hospital from March 2022 to December 2024, were enrolled in the SCI group. During the corresponding timeframe, 95 patients with spinal fractures but without SCI were selected to serve as the control group (SF group).The inclusion criteria included the following: meeting the American Spinal Cord Injury Association (ASIA) [19] injury classification criteria, injury duration ≤ 48 h, age between 18 and 65 years, and absence of other severe systemic diseases. Baseline clinical data, including age, gender, body temperature, injury site, cause of injury, ASIA classification, Lower Extremity Motor Score (LEMS), and pain score, were collected for both groups.

Five milliliters of venous blood were collected from each participant immediately upon admission (within 48 h of injury); serum was promptly separated and stored at − 80 °C until analysis.2.2 Extraction of total RNA and quantitative PCR.

Isolation of miRNA and quantitative PCR: miRNA was isolated from samples using the MolPure^® Serum/Plasma miRNA Kit (19332ES50, Yeasen). The extracted miRNA was reverse-transcribed into cDNA using the miRNA 1st Strand cDNA Synthesis Kit (by tailing A) (MR201-02, Vazyme). The relative expression level of miR-1287-5p was quantified using ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme), with U6 serving as the internal reference.

RNA Extraction and qPCR: Total RNA was extracted from the samples using the FastPure Complex Tissue/Cell Total RNA Isolation Kit (RC113-01, Vazyme). The extracted RNA was reverse transcribed into complementary DNA (cDNA) using the HiScript III RT SuperMix for qPCR (+ gDNA wiper) (R323-01, Vazyme). The relative expression level of LONP2, MAP3K9, VIM, LCOR, H2AFX, GAS7, RFX7, GNG12, and MYO1C, was quantified using ChamQ Universal SYBR qPCR Master Mix(Q711-02, Vazyme), with GAPDH serving as the internal reference.

Cell culture and transfection

PC12 cells were obtained from the Cell Biology Institute at the Chinese Academy of Sciences in Shanghai and were maintained in DMEM medium enriched with 10% fetal bovine serum (FBS). For experimental induction, an inflammatory model was established by treating PC12 cells with 5 µg/ml lipopolysaccharide (LPS). The cells were then divided into five groups: the normal control group (Control), LPS model group (LPS), miR-1287-5p mimic group (LPS + miR-1287-5p mimic), miR-1287-5p inhibitor group (LPS + miR-1287-5p inhibitor), and the negative control group (LPS + NC mimic/inhibitor). The aforementioned vectors (miRNA mimic, inhibitor, and negative control) were synthesized by Reebok Bio and transfected into cells using Lipofectamine 3000 (L3000015, Thermo Fisher).

CCK-8 assay for cell viability detection

PC12 cells were treated with CCK-8 reagent (40203ES76, YEASEN) at 0, 24, 48, and 72 h post-incubation. After each treatment, the cells were further incubated for 60 min. Subsequently, the absorbance at 450 nm was measured for each well using a microplate reader to evaluate the proliferative capacity of the cells.

ELISA detection of inflammatory factors

After cell treatment and transfection, the levels of inflammatory factors were determined using enzyme-linked immunosorbent assay (ELISA). Specifically, culture supernatants from each group were collected, and the levels of inflammatory cytokines, specifically TNF-α, IL-1β, and IL-6, were measured in accordance with the instructions provided by the manufacturer of the ELISA kit (Thermo Fisher).

Target gene prediction

A computational bioinformatics approach was used to identify potential target genes of miR-1287-5p. TargetScan 7.2(https://www.targetscan.org/vert_72/), miRTarBase(https://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTarBase_2025/php/index.php), and TarBase(https://dianalab.e-ce.uth.gr/tarbasev9/interactions) were utilized to analyze the binding sites and sequence complementarity between miR-1287-5p and mRNAs through algorithmic methods. Subsequently, venn analysis was performed to identify overlapping prediction results from the three databases.

Dual-luciferase reporter assay

A dual-Luciferase reporter assay was performed to verify the binding interaction between miR-1287-5p and the 3’UTR region of MAP3K9. For this assay, plasmids containing either the wild-type (MAP3K9-WT) or mutant (MAP3K9-MUT) 3’UTR sequences of MAP3K9 were constructed. These plasmids were then co-transfected into cells with the miR-1287-5p mimic, miR-1287-5p inhibitor, or negative control, respectively. At 48 h post-transfection, intracellular luciferase activity was measured according to the protocol provided with the Dual-Luciferase Reporter Assay Kit (11402ES60, Yeasen). The direct binding of miR-1287-5p to the MAP3K9 3’UTR was assessed by comparing luciferase activity across the different transfection groups.

Statistical analysis

Data were analyzed and visualized using SPSS and GraphPad software. Quantitative data were presented as the mean ± standard deviation (mean ± SD). The t-test was used to assess differences between two groups, while one-way or two-way ANOVA was used to analyze variations across multiple groups. Categorical data analyzed using with the chi-square (χ²) test. Pearson’s correlation analysis was performed to investigate the relationship between miR-1287-5p expression levels and clinical characteristics. Receiver operating characteristic (ROC) curves were generated to evaluate the diagnostic accuracy of miR-1287-5p in SCI. A P-value < 0.05 was considered statistically significant.

Results

Comparison of clinical characteristics

The baseline characteristics and clinical indicators of the two groups were statistically analyzed (Table 1). The results showed no statistically significant differences between the groups in terms of age, sex, body temperature, injury location, or cause of the injury (all P > 0.05), indicating that the groups were comparable. For key clinical indicators, the pain score, LEMS score, and ASIA classification in the SCI group were significantly different from those in the SF group (all P < 0.001).

Table 1.

Comparison of clinical data between the SF group and the SCI group

Variables	SF group(n = 95)	SCI group(n = 109)	Statistic	P-value
Age	40.62 ± 13.91	41.08 ± 14.18	t = 0.234	0.82
Gender(Male/Female)	46/49	50/59	χ²=0.132	0.72
Body temperature	37.95 ± 1.14	38.04 ± 1.23	t = 0.520	0.60
Cause of injury(Else/Accident)	31/64	38/71	χ²=0.113	0.74
Injured part(Thoracolumbar/Cervical)	54/41	47/62	χ²=3.824	0.05
Pain score(0–10)	3.16 ± 1.51	6.94 ± 1.77	t = 14.410	<0.001
LEMS(0–50)	35.53 ± 8.76	18.95 ± 6.13	t = 15.810	<0.001
ASIA			χ²=214	<0.001
A	0	34
B	0	24
C	0	37
D	0	24
E	95	0

LEMS, Lower extremity motor score; ASIA, American Spinal Cord Injury Association

miR-1287-5p is downregulated in SCI group and exhibits diagnostic potential

The expression level of miR-1287-5p in clinical samples was measured by qRT-PCR. The results showed a significant decrease in serum miR-1287-5p levels in SCI patients compared with the SF group (t = 6.501, P < 0.001, Fig. 1A).

Fig. 1.

Expression analysis of miR-1287-5p in individuals with SCI and SF, along with its prospective role as a diagnostic indicator. (A) miR-1287-5p expression is markedly reduced in SCI patients, ***P<0.001; (B) miR-1287-5p demonstrates significant diagnostic value for identifying SCI, AUC = 0.734 (95% CI: 0.665–0.802), sensitivity = 62.4%, specificity = 73.7%

To evaluate its diagnostic potential, ROC curve analysis was performed. The area under the curve (AUC) of miR-1287-5p for diagnosing SCI was 0.734, with a sensitivity of 62.4% and a specificity of 73.7% (Fig. 1B).

The expression level of miR-1287-5p was notably correlated with pain score, LEMS, and ASIA injury classification

Pearson correlation analysis revealed that miR-1287-5p expression was significantly correlated with multiple clinical parameters (Table 2). Specifically, miR-1287-5p expression showed a significant negative correlation with pain score (r = -0.787, P < 0.001), a significant positive correlation with LEMS score (r = 0.809, P < 0.001), and a significant inverse correlation with ASIA classification (r = -0.801, P < 0.001).

Table 2.

Pearson correlation analysis was performed to investigate the correlations between miR-1287-5p and various clinical indicators in patients

Indicators	Pearson correlation coefficient	P-value
Pain score(0–10)	-0.787	< 0.001***
LEMS(0–50)	0.809	< 0.001***
ASIA	-0.801	< 0.001***

LEMS, Lower extremity motor score; ASIA, American Spinal Cord Injury Association

Elevated expression of miR-1287-5p promotes cell viability

To further explore the functional role of miR-1287-5p in cellular mechanisms, we established an LPS-induced PC12 cell model. The level of miR-1287-5p in PC12 cells was significantly reduced following LPS treatment. However, miR-1287-5p overexpression led to a notable increase in its expression (P < 0.001, Fig. 2A).

Fig. 2.

The influence of miR-1287-5p on cell proliferation and inflammatory cytokine regulation. (A) Enhanced expression of miR-1287-5p significantly increased its concentration in LPS-stimulated cells; (B) Upregulation of miR-1287-5p improved the proliferative ability of cells exposed to LPS; (C) Elevated miR-1287-5p levels led to a decrease in TNF-α production in LPS-treated cell models; (D) Overexpression of miR-1287-5p resulted in reduced IL-6 levels in LPS-exposed cells; (E) Increased miR-1287-5p expression also lowered IL-1β levels in LPS-treated cell systems. ***P＜0.001, LPS compared to control; ###P＜0.001, LPS + miR-1287-5p mimic compared to LPS + mimic NC. n = 6, the experiment was independently repeated three times

The effect of miR-1287-5p on cell viability was evaluated using the CCK-8 assay. The results showed that LPS administration markedly suppressed cell proliferation after 72 h, while miR-1287-5p overexpression significantly restored this proliferative capacity (P < 0.001, Fig. 2B).

Overexpression of miR-1287-5p reduces the levels of inflammatory cytokines in PC12 cells

The effect of miR-1287-5p on inflammatory factor expression was evaluated using ELISA. Following LPS treatment, the concentrations of inflammatory cytokines, including TNF-α, IL-1β, and IL-6, were markedly increased in the cells. In contrast, the concentrations of these key inflammatory cytokines, were markedly reduced in the culture supernatants of cells with overexpression of miR-1287-5p (P < 0.05, Fig. 2C-E).

MAP3K9 is a direct target of miR-1287-5p

To explore the molecular targets of miR-1287-5p associated with the regulation of SCI, this study predicted its potential target genes using TargetScan, miRTarBase, and TarBase. Following Venn diagram intersection analysis, nine candidate target genes were identified (Fig. 3A). qPCR was performed to measure the expression levels of the above-mentioned genes, and MAP3K9 was found to be significantly upregulated in the cells (P < 0.001, Fig. 3B). Among these, MAP3K9, which has a relatively high prediction score and is known to participate in inflammatory signaling pathways, was chosen for subsequent experimental verification (Fig. 3C).

Fig. 3.

MAP3K9 is directly regulated by miR-1287-5p. (A) Nine potential target genes of miR-1287-5p were identified through prediction across three databases; (B) qPCR analysis enables the prediction of target gene expression levels.;(C) Identification of the specific binding site between miR-1287-5p and the 3’UTR of MAP3K9; (D) Dual-luciferase reporter assay validated the direct interaction between miR-1287-5p and MAP3K9; (E) miR-1287-5p negatively controls the mRNA expression of MAP3K9, ***P<0.001

Results from the dual-luciferase reporter assay indicated that overexpression of miR-1287-5p markedly decreased luciferase activity in cells transfected with the MAP3K9-WT construct (P < 0.001, Fig. 3C). In contrast, inhibition of miR-1287-5p resulted in a marked enhancement of luciferase activity (P < 0.001, Fig. 3D). No significant change in luciferase activity was observed in the MAP3K9-MUT group (Fig. 3E), confirming that miR-1287-5p directly binds to the 3’UTR region of MAP3K9.

Further qRT-PCR analysis revealed that miR-1287-5p overexpression significantly downregulated the mRNA expression level of MAP3K9, whereas miR-1287-5p inhibition significantly upregulated MAP3K9 mRNA expression (P < 0.001, Fig. 3D).

Discussion

SCI frequently leads to permanent neurological impairments, significantly compromising patients’ quality of life [20, 21]. In clinical practice, MRI and ASIA are currently regarded as the gold standards for diagnosing SCI [7]. However, the reliability of these tools may be compromised by patients’ poor baseline conditions, the presence of concomitant injuries, and the limited efficacy of existing pharmacological and surgical interventions [22–24]. Therefore, identifying an objective, sensitive, and easily measurable biomarker has substantial potential to improve early diagnosis and prognostic assessment of SCI.

miRNA has attracted significant attention as a promising biomarker candidate due to its high stability and tissue-specific expression in biological fluids, exhibiting broad potential in the assessment and prediction of outcomes for various medical conditions [25]. An increasing body of research suggests that microRNAs are significantly involved in the pathophysiological processes following SCI. For example, miRNA-486 has been identified as a novel therapeutic target for human SCI [26]. Approximately 50% of miRNAs detected in the cerebrospinal fluid and blood of patients with acute SCI exhibit significant alterations in expression levels, further underscoring the potential utility of miRNAs in SCI research [27]. Previous studies have demonstrated that miR-1287-5p is significantly involved in the progression of non-small cell lung cancer and could potentially function as a diagnostic biomarker [28]. miR-1287-5p is commonly found to be downregulated in anaplastic astrocytomas and glioblastomas, suggesting its potential as a diagnostic biomarker [29], this finding is consistent with the role of miR-1287-5p observed in this study.The study revealed that miR-1287-5p was significantly downregulated in the serum of SCI patients, and its expression level was significantly correlated with pain scores, LEMS, and ASIA classification. These findings align with previous reports showing downregulation of miR-1287-5p following SCI [17], suggesting its potential involvement in the pathophysiological mechanisms of SCI and its value as a diagnostic and prognostic biomarker. Furthermore, ROC curve analysis demonstrated that miR-1287-5p exhibits strong diagnostic performance for SCI. Given its stable expression in clinical samples, miR-1287-5p represents a promising non-invasive biomarker with substantial clinical application potential for the early diagnosis and monitoring of SCI.

The inflammatory response following SCI constitutes a central component of secondary injury [30]. Excessive activation of this response can exacerbate neuronal damage and functional deficits by releasing pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), as well as by promoting the infiltration of inflammatory cells [31]. In recent years, a growing body of evidence has indicated that miRNAs play critical roles in regulating the post-SCI inflammatory response [32, 33]. For example, miR-21-5p reduces cell apoptosis and inflammation in SCI-induced rats by activating the PI3K/AKT signaling pathway [34]. MiR-16-5p enhances the suppression of neuronal apoptosis and inflammation following SCI by inactivating the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway [35]. Moreover, miR-23b, transported via exosomes derived from bone marrow mesenchymal stem cells (BMSC-EVs), suppresses the activation of the nuclear factor-kappa B (NF-κB) pathway by targeting TLR4, thereby alleviating inflammation and improving neurological outcomes in SCI rats [36]. Previous studies have reported, Increased expression of miR-1287-5p downregulates HMGB1 and SNAI1, leading to decreased secretion of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α, while also suppressing the epithelial-mesenchymal transition (EMT) process [15]. Additionally, in research on microscopic polyangiitis (MPA), upregulation of miR-1287-5p significantly enhances the expression of inflammatory factors [37].Our in vitro experiments confirmed that elevated levels of miR-1287-5p notably decrease the expression of inflammatory cytokines, including TNF-α, IL-1β, and IL-6, while enhancing cell viability. These findings align with recent trends in miRNA-based regulation of inflammation in SCI research [38, 39], It has been demonstrated that miR-1287-5p may exert regulatory effects on spinal cord injury through the modulation of inflammatory responses. Therefore, miR-1287-5p, as a newly identified regulator of inflammation, holds promise for further investigation as a potential therapeutic target in SCI treatment.

As a critical upstream kinase in the MAPK signaling pathway, MAP3K9 regulates the expression of multiple inflammatory factors by activating downstream pathways such as NF-κB and p38 MAPK, thereby playing a central role in modulating inflammatory responses [40]. Previous studies have shown that in SCI models, abnormal activation of MAP3K9 is strongly associated with the progression of neuroinflammation and increased neuronal apoptosis, establishing it as a crucial molecular mechanism involved in secondary SCI damage [41]. siRNA can be utilized to identify molecular targets and is extensively applied in the study and treatment of arthritis and osteoporosis [42, 43]. In this study, through a combination of bioinformatics prediction and experimental validation, we clearly demonstrated that miR-1287-5p specifically interacts with and binds to the 3’UTR of MAP3K9, suppressing its expression via post-transcriptional regulation. qRT-PCR results further confirmed that miR-1287-5p-mediated inhibition of MAP3K9 effectively blocks the activation of downstream inflammatory pathways, thereby exerting anti-inflammatory effects. This regulatory mechanism is highly consistent with the miRNA-MAP3K9 interaction networks observed in other pathological conditions. For example, miR-125b-5p suppresses the progression of laryngeal squamous cell carcinoma and glucose metabolism disorders by targeting MAP3K9 [44], while dysregulated miR-361-5p exacerbates inflammation in shoulder arthritis through the same pathway [45]. Based on these findings, it is proposed that during the pathophysiological process of SCI, miR-1287-5p may inhibit inflammatory pathway activation via the “miR-1287-5p/MAP3K9” axis, thereby reducing neuroinflammation and cellular damage and exerting neuroprotective effects. The elucidation of this mechanism not only expands the theoretical framework of SCI molecular pathology but also clarifies the molecular basis for miR-1287-5p as a potential therapeutic target, offering novel insights and experimental support for the development of miRNA-based precision therapies for SCI.

However, this study has several limitations. First, the relatively small sample size may introduce potential biases. Second, the LPS-induced PC12 cell model reflects key inflammatory and viability changes, but it does not fully explain the complex mechanisms of secondary injury after spinal cord injury. Future studies will use animal models to validate the in vivo role of miR-1287-5p. Third, it remains to be determined whether miR-1287-5p participates in the pathophysiological process of SCI through additional targets beyond MAP3K9. These limitations should be addressed in future research to further substantiate the findings.

In summary, miR-1287-5p exhibits significant clinical relevance and biological functionality in SCI. By targeting MAP3K9 and modulating the MAPK/NF-κB inflammatory signaling pathway, it exerts neuroprotective effects through the suppression of inflammatory factor release. Given current advancements in SCI research, miR-1287-5p has dual functionality, serving as both a potential diagnostic biomarker and a promising therapeutic target for SCI.

Abbreviations

SCI

spinal cord injury

MRI

magnetic resonance imaging

ASIA

American spinal injury association

MiRNAs

microRNAs

LEMS

lower extremity motor score

TNF-α

tumor necrosis factor-alpha

IL-1β

interleukin-1 beta

IL-6

interleukin-6

EMT

epithelial-mesenchymal transition

MPA

microscopic polyangiitis

Author contributions

RZ D and YN L made substantial contributions to the conception and design, acquisition of data or analysis and interpretation of data. GG W and WCL contributed with interpretation of the results and critically revised the manuscript. All the authors approved the final version of the manuscript.

Funding

This work was supported by Yunnan Province Clinical Research Center for Orthopaedic and Athletic Rehabilitation(202102AA310068) and protective mechanism of DHEA against Brucella induced intervertebral disc destruction through the TLR2-MAPK/NF κB pathway (2024D01D25).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Zhucheng People’s Hospital before the study began. The participants’ right to be informed about the study was ensured and agreed to participate in the study.

Consent for publication

All patients provided written informed consent.

Competing interests

The authors declare no competing interests.