miR-369-3p Regulates Microglia Polarization and Neuroinflammation in Traumatic Spinal Cord Injury by Targeting PELI1

Abstract

Study Design

Conducted in vivo and in vitro modeling investigations.

Objective

The present research aims to explore the regulatory role of microRNA (miR)-369-3p in spinal cord injury inflammatory response and its targeting mechanism.

Methods

A female mouse model with T8-T10 spinal cord injury (SCI) was established. The motor function assessment (BMS) score was employed to evaluate motor function. BV2 microglial cells were treated with lipopolysaccharide (LPS) in vitro to construct an inflammatory cell model. Real-time fluorescence quantitative PCR was applied to assess miR-369-3p, M1 (CD86, iNOS), and M2 (Arg-1) polarization markers. Enzyme-linked immunosorbent assay (ELISA) determined the concentration of inflammatory factors (TNF-α, IL-6, and IL-1β). Additionally, RNA pull-down, RNA immunoprecipitation, and Dual-luciferase reporter experiments were performed to verify that miR-369-3p targets Pellino E3 ubiquitin protein ligase 1 (PELI1).

Results

miR-369-3p was noticeably down-regulated in SCI mice spinal cord tissues and LPS-induced BV2 cells, while PELI1 expression was upregulated. Raising miR-369-3p improved BMS scores (for moto function) and reduced inflammatory cytokines in spinal cord tissues. Mechanistically, miR-369-3p targeted PELI1. LPS treatment increased inflammatory factor mRNA levels and concentrations, which were significantly reversed by raising miR-369-3p and restored by PELI1. Also, raising miR-369-3p suppressed CD86 and iNOS and induced Arg-1 expression in LPS-activated microglia, while PELI1 reversed this effect.

Conclusion

miR-369-3p mitigates inflammation and suppresses microglia polarization by targeting PELI1, ultimately mitigating the progression of spinal cord injury. Our research suggests miR-369-3p as a potential therapeutic target for spinal cord injury.

Keywords

miR-369-3p PELI1 microglia polarization neuroinflammation BV2

Introduction

Traumatic-triggered spinal cord injury (SCI) severely damages the central nervous system, which can lead to permanent neurological dysfunction and bring a heavy burden to the family and society.^1,2 The pathophysiological process after SCI is extremely complex, among which neuroinflammation plays a key role in secondary injury.³ Neuroinflammation not only exacerbates tissue destruction initially at the location of the injury but also expands the extent of damage and hinders the recovery of nerve function.^4,5 In addition, microglia, as a key constituent of the innate immune system within the CNS, become activated following injury and play pivotal roles in the pathological progression and development of CNS trauma. Activated microglia are typically categorized into 2 phenotypes: pro-inflammatory (M1) and anti-inflammatory (M2).⁶ It is believed that reducing M1 polarization while promoting M2 polarization is crucial for optimal healing and functional restoration after SCI.⁷ Hence, a thorough investigation into the molecular mechanisms underlying neuroinflammation in SCI, along with microglial polarization, is imperative for the development of effective therapeutic strategies and the improvement of patient outcomes.

MicroRNAs (miRNAs), as a category of short ncRNAs, have attracted much attention for their regulatory roles in various physiological and pathological processes.⁸ Emerging evidence has identified that miRNAs exert regulatory functions in SCI and hold promise as potential molecular targets,⁹ including miR-223-5p,¹⁰ miR-138-5p,¹¹ and miR-494.¹² Among the many miRNAs, miR-369-3p has gradually entered the field of research.¹³ Previous studies have found that miR-369-3p exhibits unique functions in the regulation of inflammation in a variety of diseases.^14,15 In the aspect of cardiovascular diseases, it disrupts cardiomyocyte function and modulates inflammatory reactions.¹⁶ In the field of nervous system diseases, although few studies have targeted miR-369-3p in SCI, it can regulate the survival of nerve cells and inflammation-related signaling pathways in other nerve injury models.^17,18 Nevertheless, the specific underlying role of miR-369-3p in the inflammatory response of SCI remains to be further explored.

Pellino E3 ubiquitin protein ligase 1 (PELI1), as a key E3 ubiquitin ligase, is involved primarily in mediating the activation of various intracellular pathways, especially in the process of inflammatory signal transduction.^19,20 Existing literature has identified that PELI1 can affect the activation of inflammation-related signaling pathways by ubiquitinating specific substrate proteins, thereby affecting the production and release of inflammatory factors.^20,21 In the pathological environment of SCI, the expression changes of PELI1 and its regulatory role in inflammatory response have gradually attracted attention.²² Bioinformatics database predicts that miR-369-3p has a binding site with PELI1. However, whether miR-369-3p is involved in the pathological progression of traumatic-triggered SCI by targeting PELI1 is unknown.

This study aims to systematically evaluate the regulatory role of miR-369-3p in the inflammatory response and microglia polarization of traumatic-triggered SCI and to explore the underlying molecular mechanism through which miR-369-3p may contribute to traumatic-triggered SCI pathogenesis, specifically by examining whether its effects are mediated via targeting PELI1. The findings of this research may offer novel theoretical insights and potential therapeutic targets for traumatic-triggered SCI management.

Materials and methods

Establishment of the SCI Mouse Model

Female C57BL/6 mice aged 6-8 weeks were procured from Shanghai Lingchang Biotechnology and maintained in a sterile facility with controlled environment conditions: temperature at (22 ± 2)°C, humidity at (50 ± 5) %, and a 12-hour light. The experimental plan was assessed and endorsed by the Research Ethics Committee, ensuring adherence to the animal welfare standards set by the National Institutes of Health in China.

The mice were randomly divided into Sham and SCI model groups. Anesthesia was induced via intraperitoneal 3% pentobarbital injection. A midline skin incision was made, followed by muscle dissection. A T8-T10 laminectomy was performed, and moderate mid-thoracic (T8-10) spinal cord injury was induced in model mice using a modified Allen weight-drop device (6.0 g weight at a vertical height of 45 mm, 5g × 50 m), consistent with established protocols.²³ Successful model construction was confirmed by the presence of spastic tail flicking and bilateral hindlimb paralysis. In contrast, sham mice underwent identical surgical preparation without spinal cord compression.

To investigate the impact of miR-369-3p on SCI-induced inflammation, the second batch of SCI mice were randomized into the SCI, SCI + NC agomir, and SCI + miR-369-3p agomir groups. Three days before modeling, the latter 2 groups were intrathecally injected with NC agomir (Cat#miR4N0000001-4-5, RiboBio, Guangzhou, China) and miR-369-3p agomir (Cat#miR40003186-4-5, RiboBio, Guangzhou, China), respectively. After 14 days of modeling, mice were anesthetized with 3% sodium pentobarbital (Cat#P5178, Sigma-Aldrich, USA), sacrificed by cervical dislocation, and spinal cord tissues were harvested, partly for RNA extraction and storage at −80°C. Mice were excluded from the analysis if they died before the experimental endpoint. In this study, no animals were excluded, and all 60 mice completed the protocol.

Motor Function Assessment (BMS) Score

Based on prior research, locomotor function in SCI mice was assessed using the BMS score.²⁴ On days 1, 3, 7, and 14 after SCI, 2 professionally trained and independent observers were arranged to place the mice in a quiet open field for 4 minutes of free movement. During this period, observers scored the movement of the hind limbs of the mice according to the BMS scoring standard. BMS scores range from 0 to 9, with a score of 0 indicating that the mouse has no movement ability in its hind limbs, while a score of 9 means that the mouse has completely normal motor function and can perform normal walking, running, and other sports. After each observer scored independently, the average value was taken as the BMS score of the mice at that time point.

Cell Model Construction and Simulation

BV2 microglia cells (Cat# CRL-2469, ATCC, USA) were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Cat#26140, Invitrogen, USA) and 1% penicillin-streptomycin (Cat#15140, Invitrogen, USA) at 37°C in a 5% CO₂ and humidity over 95%. The state of the cells was observed daily, and when the confluence reached 80%-90%, the cells were passaged, rinsed with PBS buffer, digested with 0.25% trypsin-EDTA, and inoculated at 1:3-1:5. Subsequently, logarithmic-phase cells were stimulated with lipopolysaccharide (LPS, Cat# L2654, Sigma-Aldrich, USA) at concentrations of 1, 10, 100, and 1000 ng/mL for 24h, after which the expression levels of miR-369-3p and PELI1 were analyzed. Furthermore, based on prior research,²² BV2 cells were induced with 100 ng/mL LPS to establish an in vitro cellular model for subsequent experimental analysis.

Cell Transfection

When BV2 cells reached a density of 50-70%, transfection was carried out. miR-369-3p mimic and mimic NC (100 nM) were mixed with Lipofectamine™ 3000 reagent in Opti-MEM medium. The mixture was supplemented sequentially for 5 min and 20 min to generate complexes, which were directly added to the cell culture system in a dropwise manner. After 6-8 hours of transfection, the medium was replaced. BV2 was collected according to the experimental design for subsequent experimental detection.

When BV2 cells reached a density of 50-70%, transfection was carried out. To regulate miR-369-3p levels, a miR-369-3p mimic (5′-AAUAAUACAUGGUUGAUCUUU-3′, Cat#miR1003186-1-5, RiboBio, Guangzhou, China) and mimic NC (5′-CUGAACUGCUAGGACGCGUA-3′, Cat#miR1N0000001-1-5, RiboBio, Guangzhou, China) were mixed with Lipofectamine™ 3000 reagent (Cat#L300015, Invitrogen, Carlsbad, CA, USA) in Opti-MEM medium. The mixture was supplemented sequentially for 5 min and 20 min to generate complexes, which were directly added to the cell culture system in a dropwise manner. For PELI1 overexpression experiments, a PELI1 overexpression vector (oe-PELI1) was constructed by inserting the PELI1 sequence into pcDNA3.1. An empty pcDNA3.1 vector served as the negative control (oe-NC), which was obtained from GenePharm (Shanghai, China). These plasmids (2.0 μg) were similarly mixed with Lipofectamine™ 3000 reagent in Opti-MEM medium, added sequentially for 5 and 20 min to form complexes, and then directly added dropwise to the cell culture system. After 6-8 hours of transfection, the medium was replaced. BV2 was collected according to the experimental design time for subsequent experimental detection.

Real-Time Fluorescence Quantitative PCR (RT-qPCR)

Total RNA isolation was carried out on spinal cord tissues or cells employing TRIzol™ Reagent (Cat#AG21102, Accurate Biology, China). For quality assessment, the RNA concentration and purity were measured using Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and RNA samples with absorbance of >1.8 at 260 and 280 nm were deemed suitable for further use. The concentration of tRNA was determined after purification by chloroform extraction and isopropanol precipitation. For miRNA, cDNA was created by the Mir-XTM miRNA first-strand Synthesis Kit (Cat#638313, Takara, Tokyo, Japan). For mRNA, cDNA was generated using the Exo M-MLV RT kit (Cat#AG11706, Accurate Biology, China) and gDNA Clean for qPCR II. The SYBR Green Premix Pro Taq HS qPCR Kit (Cat#AG11707, Accurate Biology, China) and Light Cycler 480II system (Roche, Switzerland) were used to amplify the target genes. U6 and GAPDH acted as background genes. miR-369-3p, Cluster of differentiation 86 (CD86), Inducible nitric oxide synthase (iNOS), Arginase-1 (Arg-1), Tumor necrosis factor-α (TNF-α), Interleukin (IL)-1β, and IL-6 levels were calculated by the 2 ^-ΔΔCT method. All primer sequences used in RT-qPCR are presented in the Supplemental Table within Supplemental Material 1.

Western Blotting

The cells were rinsed three times with phosphate-buffered saline (PBS). Subsequently, cold radioimmunoprecipitation assay (RIPA) lysis buffer (Cat#ab156034, Abcam, Cambridge, MA, USA), supplemented with 1% protease and phosphatase inhibitors (Cat#1861284, Invitrogen, Carlsbad, CA, USA), was added to the cells or spinal cord tissues. The mixture was then incubated on ice for about 20 min to facilitate total protein extraction. The extracted protein was quantified using a BCA kit (Cat#23227, Thermo Scientific, Waltham, MA, USA) and separated via 10% SDS-PAGE. After separation, the proteins were transferred onto a PVDF membrane (Cat#IPFL00010, Millipore, Shanghai, China). The membrane was blocked with 5% bovine serum albumin (BSA, Cat#ST025-5 g, Beyotime Biotechnology, Shanghai, China) for 1 h. Next, the membrane was incubated overnight at 4°C with primary antibodies: anti-PELI1 (Cat#12053-1-AP, 1:1000, Proteintech, Wuhan, China), anti-iNOS (Cat#ab178945, 1:1000, Abcam, USA), anti-CD86 (Cat#ab239075, 1:1000, Abcam, USA), anti-Arg-1 (Cat#ab281603, 1:1000, Abcam, USA), and anti-β-actin (Cat#ab8226, 1:3000, Abcam, USA). Afterward, the membrane was incubated with a secondary antibody (HRP-labeled; Cat#ab288151, 1:5000, Abcam, USA) for 1 h. Finally, protein bands were visualized using a can ECL detection kit (Cat#180-5001, Tanon, Beijing, China). Quantification of the western blot band intensity was performed using Image J 1.45 software (NIH) according to the manufacturer’s instructions. All uncropped original Western blot figures are presented in Supplemental Figures S1-S5 within Supplemental Material 1.

Enzyme-Linked Immunosorbent Assay (ELISA)

An ELISA kit (IL-1β Cat#E-EL-M0037; IL-6 Cat#E-EL-M0044; TNF-α Cat#E-EL-M3063, Elabscience, Wuhan, China) was utilized to quantify the concentration of IL-1β, IL-6, and TNF-α in mouse spinal cord tissue lysates and BV2 cell supernatants. Samples and calibration standards were diluted and dispensed into enzyme-linked immunosorbent assay plates, followed by a 1-hour incubation at 37°C. Biotinylated detection antibodies were added, and a second incubation step was performed. The reaction was carried out in the dark for 15 minutes, and the termination solution was supplemented. The OD value was recorded by the SpectraMax i3x microplate reader (Supplemental materials 2).

RNA Pull-Down

MiRNAs were labeled with biotin according to an RNA pull-down kit (Cat#KT103-01, Guangzhou, Saicheng Biotechnology, China) instructions, mixed with cell lysates, and incubated overnight at 4°C. Add Streptavidin-agarose beads and shake for 3 h at 4°C to enrich the complex. The agarose beads were washed with precooling washing buffer to remove impurities. RT-qPCR was employed to quantify PELI1 to determine the targeting relationship. Negative controls were set to exclude non-specific binding interference.

Dual-Luciferase Reporter (DLR) Assay

TargetScan predicted an inter-targeting relationship between miR-369-3p and the 3’UTR of PELI1. Subsequently, dual-luciferase reporter plasmids with wild-type (WT) and mutant (MT) sequences of PELI1 were constructed using the pGL3 dual luciferase miRNA target expression vector. The target fragment was amplified by PCR, both the target fragment and the vector were digested with appropriate restriction enzymes, and finally they were ligated. When BV2 cell density reached 50-70% confluence, WT-PELI1 or MT-PELI1 was transfected with miR-369-3p mimic or mimic NC, respectively. Luciferase activity was measured 48 h post-transfection.

RNA Immunoprecipitation (RIP) Assay

RIP assay was performed based on the Magna RIP Kit (Cat#MAGNARIP03, Millipore, MA, USA). BV2 microglia lysates were collected and lysed and then incubated with magnetic beads containing anti-Ago2 antibody or rabbit anti-IgG antibody (negative control) overnight at 4°C to form immune complexes. The magnetic beads were washed to remove impurities, and miR-369-3p and PELI1 levels were quantified by RT-qPCR.

Statistical Analysis

After data collection was completed in all the above experiments, SPSS 23.0 was used for statistical analysis. Statistical comparisons were performed using a T-test for two-group analyses and ANOVA with post hoc Tukey’s test for multiple group comparisons. Data are presented as mean ± SD from at least three independent experiments. Statistical significance was defined as P < 0.05.

Results

Abnormal Down-Regulation of miR-369-3p in SCI Animals and LPS-Induced Cells

Compared with the sham, miR-369-3p expression in the spinal cord tissue of the SCI mice was significantly diminished (P < 0.001, Figure 1A). In BV2 microglia cells exposed to LPS at concentrations of 1, 10, 100, and 1000 ng/mL, the levels of miR-369-3p were gradually depressed as the LPS concentration increased (P < 0.001, Figure 1B).

Figure 1.

The Expression of miR-369-3p was Down-Regulated in the SCI Mice Model. (A) The Expression of miR-369-3p in the Spinal Cord Tissue of SCI Mice was Detected by RT-qPCR. (B) The Expression of miR-369-3p in BV2 Microglia Induced by Different Concentrations of LPS was Detected by RT-qPCR. n = 6 Per Group; **P < 0.01, ***P < 0.001

miR-369-3p Affects Motor Function and Inflammatory Factors in Mice With Spinal Cord Injury

The levels of miR-369-3p in the SCI group were persistently lower than that in the Sham group, but miR-369-3p agomir was effective in restoring its expression (P < 0.01, Figure 2A). In terms of BMS score, SCI mice had a lower BMS score than the Sham, and the increased miR-369-3p could significantly restore the BMS score on day 14 after injury (P < 0.05, Figure 2B). Furthermore, the spinal cord tissues levels of inflammatory factors in SCI mice were noticeably elevated, but this evaluation was greatly alleviated by rising miR-369-3p expression (P < 0.01, Figure 2C–E).

Figure 2.

Therapeutic Effects of miR-369-3p Overexpression in SCI Mice. (A) Relative Expression of miR-369-3p in Different Groups of mice. (B) Assessment of Motor Ability Recovery in SCI Mice by BMS Score on the 14th Day After Injury. (C–E) The Concentration of Inflammatory Factors TNF-α, IL-6, and IL-1β in Spinal Cord Tissues was Examined by the ELISA. n = 6 or 12 Per Group; ***P < 0.001 vs Sham; P < 0.001 vs SCI, ##P < 0.01 vs SCI + NC Agomir

Effect of miR-369-3p on the Levels of M1 Polarization Markers and Inflammatory Factors in Microglial Cells Stimulated by LPS

In LPS-stimulated microglia, LPS suppressed miR-369-3p expression, which was effectively abolished by miR-369-3p mimic (P < 0.001, Figure 3A). What’s more, LPS significantly promoted mRNA expression of the M1 polarization markers CD86 and iNOS while suppressing that of the M2-polarisation marker Arg-1, which were additionally suppressed by miR-369-3p (P < 0.001, Figure 3B). Consistent results were also observed in the protein levels. LPS increased the protein levels of CD86 and iNOS but decreased Arg-1 protein expression. Meanwhile, miR-369-3p mitigated the regulatory impact of LPS on the protein levels of both M1 and M2 polarization markers (P < 0.001, Figure 3C–D, and Supplemental Figure S1). Finally, LPS promoted the mRNA and protein concentrations of TNF-α, IL-1β, and IL-6 in BV2, but this promotion was partially attenuated by miR-369-3p (P < 0.05, Figure 3E–F).

Figure 3.

miR-369-3p Inhibits LPS-Induced M1 Polarization of Microglia and the Expression of Inflammatory Factors. A. Effect of miR-369-3p Mimic Transfection on the Expression of miR-369-3p in LPS-Induced BV2 Cells. B-D. The Expression of M1 Polarization Markers CD86 and iNOS, and M2 Markers Arg-1 mRNA and Protein Levels in Response to the Combined Effects of LPS and miR-369-3p Mimic. E-F. The mRNA and Concentration of the Inflammation Factor TNF-α, IL-6, and IL-1β in the LPS-Induced BV2 Cells. n = 6 Per Group; ***P < 0.001 vs Control; ###P < 0.001 vs LPS + NC Mimic

PELI1 is the Direct Target of miR-369-3p

Through the miRDB website, we predicted that miR-369-3p could target PELI1. In the RIP assay, the mRNA levels of miR-369-3p and PELI1 in the anti-Ago2 antibody group were significantly higher (P < 0.001, Figure 4A). Furthermore, in RNA pull-down assay, PELI1 levels in the Bio-miR-369-3p group was significantly higher (P < 0.001, Figure 4B). Figure 4C shows the predicted binding sites between miR-369-3p and the 3’UTR of PELI1 mRNA. The dual-luciferase reporter presenting miR-369-3p mimic distinctly inhibited the luciferase activity of PELI1-WT (P < 0.001, Figure 4D). The mRNA and protein expression levels of PELI1 in the spinal cord tissue of SCI mice were broadly expressed (P < 0.01, Figure 4E–G, and Supplemental Figure S2). Moreover, both mRNA and protein expression levels of PELI1 exhibited a dose-dependent increase following LPS stimulation (P < 0.001, Figure 4H–J, and Supplemental Figure S3).

Figure 4.

Validation of the Targeting Relationship Between miR-369-3p and PELI1. (A) mRNA Expression of miR-369-3p and PELI1 in RIP Assay. (B) Expression of PELI1 in RNA Pull-Down Assay. (C) Binding Sites of miR-369-3p and PELI1. (D) A Dual Luciferase Assay was Employed to Detect the Targeting Relationship Between miR-369-3p and PELI1. (E–G) MRNA and Protein Expression of PELI1 in the Spinal Cord Tissue of SCI Mice was Explored. (H–J) MRNA and Protein Expression of PELI1 in Microglia Induced by Different Concentrations of LPS. n = 6 per group; ***P < 0.001 vs Anti-IgG, ***P < 0.001 vs Bio-NC, **P < 0.01 vs NC Mimic, ***P < 0.001, **P < 0.01 vs control.

PELI1 Significantly Attenuates the Functions of miR-369-3p on LPS-Triggered M1 Polarization of Microglia and Neuroinflammation

The elevation of miR-369-3p significantly depressed the LPS-triggered elevation of PELI1 protein levels, but this depression was effectively counteracted by overexpression of PELI1 (P < 0.001, Figure 5A, and Supplemental Figure S4). Elevation of miR-369-3p remarkably suppressed the mRNA and protein expression of LPS-activated CD86 and iNOS while upregulating Arg-1. This miR-369-3p effect was substantially reversed by PELI1 overexpression (P < 0.001, Figure 5E–I, and Supplemental Figure S5). Elevation of PELI1 partially counteracted the inhibitory impact of elevated miR-369-3p on the mRNA and protein levels of inflammatory factors (P < 0.01, Figure 6A–B).

Figure 5.

miR-369-3p Inhibits M1 Polarization of Microglia and Inflammation by Targeting PELI1. (A-B) Relative Protein Expression of PELI1 in Different Treatment Groups. (C-E) Relative Expression of CD86, iNOS, and Arg-1 mRNA in Different Treatment Groups Detected by RT-qPCR. (F-I) Relative Expression of CD86, iNOS, and Arg-1 Protein Expression in Different Treatment Groups was Detected by Western blot Analysis. n = 6 Per Groups; **P < 0.01 vs LPS + NC Mimic; P < 0.001 vs LPS + miR-369-3p Mimic + oe-NC; **P < 0.01 vs LPS + NC Mimic; ###P < 0.001 vs LPS + miR-369-3p Mimic + oe-NC

Figure 6.

miR-369-3p Inhibits Inflammation by Targeting PELI1. (A) mRNA Expression of TNF-α, IL-1β, and IL-6 in Different Treatment Groups Detected by RT-qPCR. (B) Concentrations of TNF-α, IL-1β, and IL-6 in Culture Medium Supernatant of Different Treatment Groups Detected by ELISA. n = 6 Per Group; **P < 0.01 vs LPS + NC Mimic; ###P < 0.001 vs LPS + miR-369-3p Mimic + oe-NC; **P < 0.01 vs LPS + NC Mimic; ###P < 0.001 vs LPS + miR-369-3p Mimic + oe-NC

Discussion

Traumatic spinal cord injury is an extremely destructive condition. Around the world, numerous patients endure permanent neurological dysfunction because of it. This not only gravely undermines their self-care capabilities but also substantially heightens the economic strain on families and society.²⁵ Being a representative of the miRNA family, miR-369-3p has been implicated in several physiological and pathological regulatory processes in previous studies.²⁶ In the field of cardiovascular disease, its regulatory effect on vascular endothelial cell function and inflammatory response has been recognized.^13,27 In the nervous system, although studies on SCI are limited, it has also been shown to regulate nerve cell survival and inflammatory signaling pathways in other nerve injury models.^17,18 Here, we report that miR-369-3p was abnormally expressed in the SCI model. This study offers the inaugural proof that miR-369-3p targets and suppresses PELI1, dampening neuroinflammation and curbing M1 microglial polarization, which in turn hinders SCI recovery. In SCI mice (in vivo), it reduced spinal inflammation and aided moto recovery. In inflammatory cell models (in vitro), it curbed M1 microglial polarization and neuroinflammatory secretion via PELI1 (Figure 7). This indicates that miR-369-3p takes a vital action in controlling the inflammatory response and promoting the recovery of neurological function in SCI, which is consistent with the general role of miRNAs in disease-related inflammation regulation as reported in previous literature.²⁸

Figure 7.

miR-369-3p Mitigates Traumatic Spinal Cord Injury via PELI1 Targeting, Suppressing Neuroinflammation and Microglial M1 Polarization. In Vivo, miR-369-3p Reduces Spinal Cord Inflammation and Facilitates Motor Function Restoration in SCI Mice. In Vitro, in Inflammatory Cell Models, it Targets PELI1 to Inhibit M1 Polarization and Inflammatory Reactions in Microglia

Inflammation is known to be a pivotal element in the pathophysiology of SCI.²⁹ Previous studies have reported that inflammation can exacerbate secondary injury after SCI, leading to further nerve cell death and functional impairment.^5,30 In the current research, for the first time, we demonstrated that miR-369-3p could participate in the regulation of SCI by modulating the inflammatory response. The abnormal down-regulation of miR-369-3p in SCI animals and LPS-induced cells was associated with increased levels of inflammatory factors, suggesting that miR-369-3p may be a potential regulator of the inflammatory process in SCI. Microglial polarization contributes to the microenvironment of spinal cord injury.³¹ M1-polarized microglia secrete pro-inflammatory cytokines, which contribute to the progression of inflammation and tissue damage, while M2-polarized microglia are associated with anti-inflammatory and tissue-repair functions.^32-34 Here, we investigated the ability of miR-369-3p to inhibit LPS-induced M1 polarization of microglia.

PELI1, a crucial E3 ubiquitin ligase, plays a significant role in multiple intracellular signaling pathways, with a particular emphasis on inflammatory signaling.³⁵ In the context of other related diseases such as cancer and autoimmune diseases, previous reports have demonstrated that PELI1 regulates the activation of inflammation-related signaling cascades. It does so by ubiquitinating specific substrate proteins, thereby influencing the production and release of inflammatory factors.³⁶ In SCI, our study revealed that PELI1 expression was notably up-regulated in the spinal cord tissues of affected mice and LPS-induced microglia. For the first time, we identified a targeting relationship between miR-369-3p and PELI1. This interaction is involved in modulating the inflammatory response and microglial polarization in SCI. Specifically, miR-369-3p inhibits the M1 polarization of microglia and the associated pro-inflammatory response by targeting PELI1, indicating that the miR-369-3p-PELI1 axis could be a key regulatory mechanism in the pathogenesis of SCI. Several limitations in this study warrant attention. Firstly, the researchers conducted RT-qPCR and Western blot analyses yet failed to employ flow cytometry or immunohistochemistry to further evaluate polarization markers. This oversight may restrict the generalizability of the findings. Secondly, inhibiting a single miRNA (miR-369-3p) could potentially result in compensatory upregulation of other functionally similar miRNAs, which might obscure their true roles. Thirdly, using only one time point (14 dpi) may oversimplify the dynamic nature of spinal cord injury pathology, representing another limitation of this study. Furthermore, the failure to conduct longitudinal investigations into underlying mechanisms, such as signaling pathways, represents a further shortcoming. In terms of future research, follow-up studies should extend the analysis to earlier times (eg, 3-7 dpi) or later time points (eg, 28 dpi) to enhance the study’s rigor. This is particularly important since 7 dpi marks the peak of neuroinflammation after SCI, enabling a more in-depth analysis of M1 and M2 polarization. Additionally, incorporating research methods such as immunohistochemistry and immunofluorescence can be used to accurately assess polarization markers and analyze changes in multiple miRNAs and alterations in signaling pathways.

Collectively, the current investigation is the first to reveal that miR-369-3p is markedly decreased in SCI. The decreased miR-369-3p may drive SCI progression by regulating PELI1, thereby promoting microglia polarization and neuroinflammatory reactions. Regulating miR-369-3p may interfere with the pathological mechanism of SCI and may offer a novel strategy for the treatment of traumatic-triggered SCI.

Supplemental Material

Supplemental Material - miR-369-3p Regulates Microglia Polarization and Neuroinflammation in Traumatic Spinal Cord Injury by Targeting PELI1

Supplemental Material for miR-369-3p Regulates Microglia Polarization and Neuroinflammation in Traumatic Spinal Cord Injury by Targeting PELI1 by Yalun Li, Guiping Sun, Rubing Lin, Yingxuan Huang in Global Spine Journal.

Supplemental Material

Supplemental Material - miR-369-3p Regulates Microglia Polarization and Neuroinflammation in Traumatic Spinal Cord Injury by Targeting PELI1

Footnotes

ORCID iD

Yingxuan Huang

Ethical Approval

The experimental plan was assessed and endorsed by The Affiliated Hospital of Youjiang Medical University for Nationalities Research Ethics Committee (approval number: 2023E18), ensuring adherence to the animal welfare standards set by the National Institutes of Health in China.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Declaration of Conflicting Interests

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

Supplemental Material

Supplemental material for this article is available online.

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