MiR-10b-5p attenuates spinal cord injury and alleviates LPS-induced PC12 cells injury by inhibiting TGF-β1 decay and activating TGF-β1/Smad3 pathway through PTBP1

Abstract

Spinal cord injury (SCI) is a debilitating condition characterized by significant sensory, motor, and autonomic dysfunctions, leading to severe physical, psychological, and financial burdens. The current therapeutic approaches for SCI show limited effectiveness, highlighting the urgent need for innovative treatments. MicroRNAs (miRNAs) like miR-10b-5p are known to play pivotal roles in gene expression regulation and have been implicated in various neurodegenerative diseases, including SCI. Polypyrimidine tract binding protein 1 (PTBP1) has also been associated with neural injury responses and recovery. This study aims to explore the interaction between miR-10b-5p and PTBP1 in the context of SCI, hypothesizing that miR-10b-5p regulates PTBP1 to influence SCI pathogenesis and recovery using a rat model of SCI and lipopolysaccharide (LPS)-induced PC12 cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to measure miR-10b-5p levels, revealing its low expression in SCI rats. We then assessed neurological function, histopathological changes, and spinal cord water content. We found that administering the agomiR-10b-5p significantly improved neurological function and decreased the spinal cord water content and normal motor neuron loss in SCI rats. Additionally, we explored the functions of miR-10b-5p in LPS-treated PC12 cells. Our results showed that miR-10b-5p repressed LPS-stimulated apoptosis, inflammation, and oxidative stress in PC12 cells. PTBP1 was predicted as a potential target gene of miR-10b-5p using the TargetScan database. Pulldown and luciferase reporter assays further demonstrated that miR-10b-5p binds to the 3’ untranslated region (UTR) of PTBP1. RT-qPCR revealed that miR-10b-5p negatively modulated PTBP1 expression both in vivo and in vitro. Furthermore, rescue assays indicated that miR-10b-5p alleviated SCI in rats and LPS-triggered injury in PC12 cells by downregulating PTBP1. We also investigated the regulation of miR-10b-5p and PTBP1 on the transforming growth factor-beta 1 (TGF-β1)/small mother against decapentaplegic (Smad3) pathway. We found that miR-10b-5p targeted PTBP1 to repress TGF-β1 decay and facilitated TGF-β1/Smad3 pathway activation. In conclusion, our results demonstrate that miR-10b-5p alleviates SCI by repressing TGF-β1 decay and inducing TGF-β1/Smad3 pathway activation through PTBP1 downregulation. This study provides novel insights into potential targeted therapy plans for SCI.

Introduction

Spinal cord injury (SCI) is a common neurological disorder characterized by high mortality and morbidity [1], and it is complicated by the loss of sensory, motor, and autonomic functions [2]. SCI is a leading cause of paralysis and sensory impairment, resulting in serious physical and psychological trauma for patients, as well as significant financial burdens for their families [3]. It has been demonstrated that excessive oxidative stress, inflammatory responses in nerve cells, accumulation of neurotransmitters, loss of nerve axons, and apoptosis of nerve cells all contribute to the pathogenesis of SCI [4]. Current therapeutic methods for SCI include drug therapy, surgery, transplantation therapy, etc. [5]. However, these methods have limited therapeutic outcomes, highlighting the urgent need for new and effective therapeutic approaches.

MicroRNAs (miRNAs) are a class of highly conserved noncoding RNAs containing 18–22 nucleotides [6], which typically modulate gene expression at the post-transcriptional level [7]. MiRNAs can specifically bind to the 3' untranslated region (3'UTR) of target gene mRNA, thereby repressing or blocking its translation [8, 9]. Various miRNAs have been shown to play crucial roles in the proliferation, differentiation, apoptosis, and damage repair of central nervous system (CNS) stem cells [10]. Abnormally expressed miRNAs in SCI patients can serve as biomarkers for diagnosing the disease [11]. For instance, the depletion of miR-324-5p alleviates the loss of locomotion and degenerative progression after SCI through the elevation of Sirt1 [12]. Similarly, miR-378-3p mitigates contusion SCI by targeting ATG12 [13]. MiR-10b-5p, a multifunctional miRNA, is associated with breast cancer oncogenesis and is vastly expressed in the highly invasive MDA-MB-231 breast cancer cell line compared to the non-invasive MCF-7 breast cancer cell line [14, 15]. It also plays a significant role in the pathogenesis of neurodegenerative disorders such as Huntington’s disease [16]. Similarly, in Alzheimer’s disease (AD), miR-10b-5p is highly expressed, and inhibiting its expression decelerates the development of AD in rats [17]. Moreover, miR-10b-5p is reported to be enriched in the spinal cord, with its expression altered following SCI [18, 19]. A study by Yunta et al. demonstrated that miR-10b-5p is downregulated in rats at 3 and 7 days post-SCI, according to the analysis of the GSE19890 dataset [20]. Similarly, Tigchelaar et al. found that miR-10b is deregulated in the serum of SCI pigs, suggesting that it is a promising biomarker for distinguishing the severity of injury post-traumatic SCI [21]. A recent study also reported that miR-10b-5p is downregulated in SCI rat models, and its overexpression facilitates spinal cord tissue repair, promotes autophagy and inhibits neuron apoptosis following SCI by targeting UBR7 to inactivate the Wnt/β-catenin signaling [22]. However, the functional role and molecular mechanism of miR-10b-5p in SCI pathogenesis remain poorly understood.

Polypyrimidine tract binding protein 1 (PTBP1), first discovered in HeLa cell nuclear extracts in 1989 [23], belongs to the heterogeneous nuclear ribonucleoproteins (hnRNPs) family and can freely shuttle between nucleus and cytoplasm, with an evident nuclear localization function [24]. PTBP1 has been shown to be involved in neuronal development and induces neuronal apoptosis by upregulating BAK protein expression [25], and play a key role in the pathogenesis of various neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease [26,27,28,29]. In SCI, PTBP1 has been shown to regulate injury responses and sensory pathways in adult peripheral neurons [30]. The knockdown of PTBP1 has been suggested as a therapeutic strategy to enhance motor function recovery after SCI [31]. Furthermore, PTBP1 interacts with the TNF-α/NF-κB axis, which is crucial for repair mechanisms in SCI models [32].

Given the regulatory roles of both miR-10b-5p and PTBP1 in gene expression and cellular responses to injury, it is plausible that miR-10b-5p may target PTBP1 in the context of SCI. Zhao et al. [33] demonstrated that miR-124 induces the formation of neurogenic cells from bone marrow mesenchymal stem cells via the downregulation of PTBP1. This suggests that miRNAs can directly regulate PTBP1 expression, impacting neural repair processes. In SCI, the downregulation of miR-10b-5p observed post-injury, according to the analysis of the GSE19890 dataset [20], could lead to the upregulation of PTBP1, thereby influencing the injury response and recovery mechanisms. However, the precise molecular interactions between miR-10b-5p and PTBP1 in SCI remain elusive and need further investigation.

In the present study, our objective was to investigate the functional role and molecular mechanism of miR-10b-5p and PTBP1 underlying inflammation, oxidative stress, and apoptosis in SCI, which may provide novel insight for seeking targeted therapy plans for treating SCI.

Materials and methods

Animals

The Animal Ethics Committee of Zhongda Hospital approved this research (approval number: 20210228037). A total of 56 adult male Sprague–Dawley SD rats weighing 250–300 g at 6–8 weeks were purchased from SLAC Laboratory Animal Co., Ltd (Shanghai, China). The rats were kept in a 12-h light/dark cycle at 25 °C in a humid atmosphere of 40–60%, with free access to food and water. All surgeries were performed under anesthesia, and every effort was made to reduce pain. 56 rats were classified into two groups, with 8 rats in the sham group and 48 rats in the SCI rat model group. All rats were fasted but had free access to water for 12 h before surgery.

Establishment of the SCI rat model

Rats were placed on their ventral surface in a U-shaped stabilizer and anesthetized with 0.3% pentobarbital sodium (30 mg/kg). The hair on the back at position T9–T10 was shaved using an electric shaver and then received a T10 laminectomy. SCI was induced following the modified Allen method [34, 35]. A customized IH-0400 Spinal Cord Impactor (PSI, Lexington, KY, USA) was used to introduce the contusion at the spinal cord T10 spinous process. The rod weighed 20 g and had a 2.5 mm diameter. The striking force was 20 × 2.5 g•cm and the contact time was 1 s. In order to determine whether or not the model was successful, we looked for the following symptoms of spinal cord injury: body tremors after the free weight fall, with rapid retraction and bouncing of the posterior limbs; tail erecting and then falling; red petechiae on the spinal cord surface; paralysis of the lower limbs. Rats in the sham group received laminectomy only without the contusive injury [36, 37]. After surgery, all rats were placed on a heating pad until they fully recovered from anesthesia. Animals received intraperitoneal injections of penicillin (100 U/day, #ST2555, Beyotime, Shanghai, China) for 3 days to avoid infection.

Injection of agomir constructs and grouping

To explore the effects and underlying mechanism of miR-10b-5p on SCI rats in vivo, SCI rats were classified into four groups in a random manner: Control group (n = 8), agomiR-NC group (n = 16), agomiR-10b-5p group (n = 16), and agomiR-10b-5p + oe-PTBP1 group (n = 8). After SCI modeling for 3 days, SCI rats in the agomiR-10b-5p group received an intrathecal injection of mixed miR-10b-5p agomir (20 µM, 2 µL; GenePharma, Shanghai, China) at a speed of 0.2 μL/min with a glass micropipette (tip diameter: 20–40 µm). For rats in the agomiR-10b-5p + oe-PTBP1 group, agomiR-10b-5p (2 µL) and lentivirus carrying oe-PTBP1 (2 µL) were diluted in PBS and intrathecally injected with a glass micropipette. Rats in the agomiR-NC group were injected with an empty vector as a negative control, and rats in the control group were administered an equal volume of PBS. The injections were administrated once daily for 3 consecutive days. The PE-10 polyethylene catheter at the length of 15 cm was inserted into the subarachnoid space [38]. The catheter was passed through a slit into the atlanto-occipital membrane and pushed ahead 11 cm to ensure that the tip was caudal to the conus medullaris. The needle was left in place for another 5 min before being withdrawn slowly. During injection, blood vessels and nerves were avoided.

Measurement of neurological function

Neuronal function recovery was, respectively, measured at 1, 3, 7, and 14 days post-SCI by two researchers who were blind to all operations. Hindlimb motor function was evaluated in accordance with Basso, Beattie, and Bresnahan (BBB) scoring system. BBB scores ranged from 0 (complete paralysis) to 21 (normal exercise) points. Each rat was placed in an open field and observed for hind limb movements for more than 3 min. Hind limb joint movement, weight support, plantar stepping, coordination, paw position, and trunk and tail control were evaluated. All rats were confirmed to have no baseline defects before modeling.

Assessment of spinal cord edema

Water content in spinal cord tissue was used to determine the degree of edema. A 1.5-cm spinal cord tissue band centered around the injury site was weighed at 3 and 7 days post-SCI. Then spinal cord tissue dry weight was acquired at 100 °C for 24 h. Wet-to-dry weight ratio was calculated as follows; wet-to-dry weight ratio = [(wet weight−dry weight)/wet weight] × 100%.

Hematoxylin and eosin (HE)

All rats received anesthetization 3 and 7 days post-SCI to explore the histological changes over time. The entire spinal cord received dissection, with a mark at snout/caudal position. A 3-cm piece of tissue was removed from the spinal cord at the injured center. The spinal cord tissue was then immersed in 0.1 mol/L PBS containing 4% paraformaldehyde and stored at 4 °C for 7 days. Paraffin-embedded tissue was cut into 5-μm sections. For assessing histopathological changes, sections were stained with hematoxylin and eosin (HE) using HE Staining Kit (#C0105S, Beyotime) following the manufacturer’s instructions and then observed under the optical microscope.

Cell culture and treatment

Rat adrenal pheochromocytoma cell line (PC12) (#CRL-1721, ATCC, Manassas, VA, USA) was cultured in RPMI-1640 medium (#11875093, Gibco, Grand Island, NY, USA) supplemented with 10% FBS (#26010074, Gibco) in a humidified environment of 5% CO₂ at 37 °C. To induce the differentiation of PC12 cells into sympathetic neuron-like cells, after cell passage for 5 generations, the medium was changed to differentiation medium, RPMI-1640 supplemented with 50 ng/mL nerve growth factor (#N0513, Sigma-Aldrich. Saint Louis, MO, USA), 1% horse serum (#16050130, ThermoFisher Scientific, Rochester, NY, USA), and 1% penicillin–streptomycin (#ST488S, Beyotime) for a week [39]. The culture medium was changed every 2–3 days. All subsequent experiments were performed using neuronal differentiated PC12 cells. For inducing inflammatory injury, cells were stimulated with 5 μg/mL.

Cell transfection

PC12 cells were divided into the Ctrl, agomiR-NC, agomiR-10b-5p, agomiR-10b-5p + oe-PTBP1, oe-NC, and oe-PTBP1 groups and were seeded into 6-well plates (3 × 10⁵ cells/well) before transfection. When the cells reached approximately 80% confluency, PC12 cells in the agomiR-NC, agomiR-10b-5p, and agomiR-10b-5p + oe-PTBP1 groups were transfected with 20 nM of agomiR-NC, 20 nM agomiR-10b-5p, or 20 nM agomiR-10b-5p combined with 2 μg of pcDNA-PTBP1 (oe-PTBP1) at 37 °C using Lipofectamine^® 2000 (#11668019, Invitrogen, Carlsbad, CA, USA) for 48 h, followed by stimulation with lipopolysaccharide (LPS) (#L2630, Sigma-Aldrich) for 12 h [39, 40]. Subsequently, PC12 cells in the oe-NC and oe-PTBP1 groups were only transfected with 2 μg of empty pcDNA vector or pcDNA-PTBP1 vector for 48 h using Lipofectamine^® 2000 without LPS treatment. All plasmids were provided by GenePharma (Shanghai, China). The PC12 cells without transfection and LPS treatment were used as the Ctrl groups.

RNA extraction and reverse transcription real-time polymerase chain reaction (RT-qPCR)

Total RNA was extracted from spinal cord tissue and PC12 cells using the TRIzol reagent (#15596026, Invitrogen, USA) following the manufacturer's protocol. For complementary DNA (cDNA) synthesis, the reverse transcription reaction was performed using the PrimeScript RT reagent kit (#RR037A, Takara, Kyoto, Japan). Quantitative PCR (qPCR) assays were prepared using the SYBR^® Premix Ex TaqTM II kit (#RR390A, Takara) and were conducted on an ABI 7500 real-time PCR system (#4359286, Applied Biosystems, USA).

For miR-10b-5p analysis, miRNA reverse transcription was carried out using the TaqMan MicroRNA Reverse Transcription Kit (Cat# 4366596, Thermo Fisher Scientific, USA). For qRT-PCR, the reaction mixture consisted of 5 μL of cDNA (diluted 1:3 with nuclease-free water), 4.5 μL of TaqMan Fast Advanced Master Mix, and 0.73 μL of each specific miRNA primer. This mixture was processed in a ViiA7 PCR system (Applied Biosystems, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The thermal cycling conditions were set as follows: predenaturation at 95 °C for 10 min; denaturation at 95 °C for 15 s; annealing at 60 °C for 35 s; and extension at 72 °C for 10 s for a total of 40 cycles. GAPDH served as a loading control for mRNAs, and U6 was used as a loading control for miRNA. The relative gene expression levels were obtained using the 2^−ΔΔCt method. The primer sequences used were as follows:

MiR-10b-5p forward: 5’-GATTTAGGTATTTTATTTTGGGTGG-3’;

MiR-10b-5p reverse: 5’-CTCCATATCGCACTTTAATCTCTAACT-3’;

U6 forward: 5’-CTCGCTTCGGCAGCACATATACT-3’;

U6 reverse: 5’-ACGCTTCACGAATTTGCGTGTC-3’;

PTBP1 forward: 5’-AGTTCTTCCAGAAGGACCG-3’;

PTBP1 reverse: 5’-GCAGCTCAATTAGTGCCTG-3’;

TGF-β1 forward: 5’-TTACCTTGGTAACCGGCTG-3’;

TGF-β1 reverse: 5’-CTGTATTCCGTCTCCTTGGT-3’;

SMAD3 forward: 5’-ACACAATAACTTGGACCTACAG-3’;

SMAD3 reverse: 5’-TGGTTCAGCTCGTAGTAGG-3’;

GAPDH forward: 5’-AACTCCCATTCTTCCACCT-3’;

GAPDH reverse: 5’-TTGTCATACCAGGAAATGAGC-3’;

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay

After the indicated treatment, PC12 cells were fixed with 4% formaldehyde (#P0099, Beyotime) for 15 min, and then cells were stained with a TUNEL Bright-Red Apoptosis Detection kit (#A113, Vazyme, USA) following manufacturer instructions. TUNEL-positive cells were observed through fluorescence microscopy (Leica, Germany) to measure the apoptosis rate.

Enzyme-linked immunosorbent assay (ELISA)

The culture supernatant was harvested after PC12 cells received the indicated treatment. The contents of proinflammatory cytokines including IL-1β, IL-6, and TNF-α were assessed using corresponding human ELISA detection kits (#PI305, #PI330, and #PT518, Beyotime) following manufacturer instructions.

Western blot

Total protein was extracted from spinal cord tissue and PC12 cells using RIPA buffer containing protease and phosphatase inhibitors (#P0013C, Beyotime). Proteins were loaded and separated on SDS-PAGE (#P0012A, Beyotime) and then transferred onto 0.2 µm PVDF membranes (#1620177, BIO-RAD). Primary antibodies used in this study included anti-Bax (ab32503, 1:1000, Abcam), anti-Bcl-2 (ab196495, 1:1000, Abcam), anti-cleaved-caspase-3 (#9661, 1:1000, Cell Signaling Technology), anti-cleaved-PARP (ab32064, 1:1000, Abcam), anti-TGF-β1 (ab215715, 1:1000, Abcam), anti-p-SMAD3 (ab254407, 1:1000, Abcam), anti-SMAD3 (ab40854, 1:1000, Abcam), anti-PTBP1 (1:1000, ab244207, Abcam) and anti-β-actin as a loading control. Next morning, after incubating the membranes with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody anti-IgG (ab209010, 1:5000, Abcam) for 60 min, the ECL Plus Reagent (ab65623, Abcam) was applied for the visualization of protein bands and the ImageJ software was used for quantification.

Measurement of intracellular reactive oxygen species (ROS) levels

Intracellular ROS level was assessed using a Reactive Oxygen Species Assay Kit (#S0033M, Beyotime). This kit uses probe DCFH-DA, which can freely pass through the cell membrane. After entering the cell, DCFH-DA is hydrolyzed by the esterase into DCFH. DCFH cannot permeate the cell membrane, making it easy for the probe to be loaded into the cell. Intracellular ROS can oxidize non-fluorescent DCFH to produce fluorescent DCF. After indicated transfection, PC12 cells were incubated with DCFH-DA under the manufacturer’s guidance. The fluorescence signal was detected and observed using microscopy (Olympus BX53, Japan) and images were acquired. The Indica Labs—Area Quantification FL v2.1.2 module in Halo v3.0.311.314 analysis software was used to quantify the target area of each section separately, and the mean intensity was calculated.

RNA/protein pull-down

The mixture of protein from PC12 cells and 50 pmol of biotinylated miR-10b-5p (Bio-miR-10b-5p) or biotinylated arbitrary single nucleotide (Bio-NC) received incubation for 1 h with 50 µL of Dynabeads™ M-280 streptavidin agarose beads (#11206D, Life Technologies) at 4 °C. The associated RNA–protein complexes were eluted with biotin elution buffer and boiled in SDS for 10 min. The elute was then examined with RT-qPCR. Protein pull-down was conducted using a Pierce Magnetic RNA–Protein Pull-Down Kit (#20164, Thermo Fisher). PTBP1 was captured with streptavidin magnetic beads and received incubation with PC12 cell lysates at 4 °C for 6 h. Then, the mixture received washing and elution. The eluate was measured using a western blot.

Dual luciferase gene reporter assay

The sequence of PTBP1 3'UTR was subcloned to pmirGLO dual-luciferase miRNA target expression vector (#E1330, Promega, USA). The luciferase vector was co-transfected with agmiR-NC or agomiR-10b-5p into PC12 cells. After 48 of transfection, luciferase activity was assessed using the Dual-Luciferase^®Reporter (DLR^™) Assay System (#E1910, Promega) under the manufacturer's guidance. Luciferase activities of Photinus pyralis were detected followed by that of Renilla reniformis. Relative luciferase activity was presented as luciferase activity of Photinus pyralis/Renilla reniformis.

Radioimmunoprecipitation (RIP) assay

A Magna RIP^™ RNA-Binding Protein Immunoprecipitation Kit (17–700, Sigma-Aldrich) was used for RIP assay. After the indicated treatment, PC12 cell lysates received extraction on ice for 2 h, centrifugation, and incubation with antibody-conjugated beads at 4 °C overnight. Antibody-bead complexes were washed 5 or 6 times with cold lysis buffer. qPCR was subsequently performed to detect enrichment of TGF-β1 in IgG- or anti-PTBP1-immunoprecipitated complexes.

Fluorescence in situ hybridization (FISH) assay

PC12 cells were seeded in a glass-bottom dish. Next, cells were incubated with CY3-labeled miR-10b-5p probe and FAM-labeled PTBP1 probe (20 μM, Ribobio, Guangzhou, China) in a hybridization buffer overnight. The slides were then washed, dehydrated and stained with DAPI (Beyotime, China). The images were captured using a scanning confocal microscope (Leica, Wetzlar, Germany).

Statistical analysis

Data were expressed as the mean ± standard deviation of three independent assays. Statistical analysis was conducted using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA). Comparisons between two groups were assessed with Student's t-test and comparisons among multiple groups were assessed with one-way ANOVA followed by Tukey's post hoc test. Each experiment was repeated at least three times. A statistical significance was presented upon P < 0.05.

Results

MiR-10b-5p alleviates neurological impairment in SCI rats

Through the GSE19890 dataset, (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE19890), miR-10b-5p was discovered to present a marked downregulation at 3- and 7-days post-SCI. Therefore, we hypothesize that miR-10b-5p may be involved in SCI progression. To elucidate the potential function of miR-10b-5p underlying SCI pathogenesis, we constructed an SCI rat model to mimic SCI characteristics. First, we measured the neurological function and histopathological changes of rats using BBB scoring and HE staining. Results demonstrated that BBB scores of rats presented reduced motor rehabilitation 3 days and 7 days after SCI compared to the sham group (Fig. 1A), suggesting that SCI resulted in neurological damage of rats. Additionally, a remarkable reduction of spared tissues in the rostral/caudal direction from the injury site was discovered in SCI rats relative to controls (Fig. 1B). Moreover, water contents in spinal cord tissues presented elevation along with time in SCI rats relative to the control group (Fig. 1C). These results highlight the successful construction of the SCI rat model. Then, we examined miR-10b-5p level in rat spinal cord tissues (1 cm around injury epicenter) through RT-qPCR. We found that miR-10b-5p gene expression was low in SCI rats relative to the control sham group (Fig. 1D). We then determined the role of miR-10b-5p in the pathogenesis of SCI. For this purpose, SCI rats received injection with overexpression constructs agomiR-10b-5p and negative control agomiR-NC. RT-qPCR confirmed the successful elevation of miR-10b-5p in spinal cord tissues of agomiR-10b-5p-injected SCI rats relative to agomiR-NC-injected ones (Fig. 1E). Furthermore, we discovered that miR-10b-5p upregulation elevated BBB scores and reduced water contents in the spinal cord tissues of SCI rats (Fig. 1F, G). Additionally, HE staining revealed that miR-10b-5p overexpression alleviated the damage at the lesion site (Fig. 1H). Meanwhile, we also evaluated the normal motor neuron index in all motor neurons (normal + abnormal neurons) using HE staining. Indeed, the normal motor neuron index in SCI rats presented elevation under miR-10b-5p overexpression (Fig. 1I). Moreover, we detected the expression of apoptosis-related proteins in rat spinal cord tissues using a western blot. The results showed that miR-10b-5p overexpression induced the upregulation of Bcl-2 while downregulating Bax, cleaved caspase-3, and cleaved-PARP protein expression (Fig. 1J), suggesting that miR-10b-5p overexpression promoted cell survival post-SCI. Collectively, this data indicates that miR-10b-5p alleviates neurological impairment and spinal cord damage in rats after SCI.

Fig. 1

MiR-10b-5p alleviates neurological impairment and spinal cord damage in SCI rats. A BBB scoring was used to measure the neurological function of rats in the sham (n = 8) or SCI groups (n = 8). B HE staining was performed to evaluate the histopathological changes of rats in each group (n = 8). Scale bar: 1 mm (upper), 50 μm (left lower), 100 μm (right lower). C Spinal cord water contents of rats in each group (n = 8). D RT-qPCR was performed to measure the miR-10b-5p level in the spinal cord tissue of rats in each group (n = 8). E RT-qPCR measured miR-10b-5p level in the spinal cord tissue of SCI rats in agomiR-NC or agomiR-10b-5p groups (n = 8). F BBB scoring for the neurological function of SCI rats in each group (n = 8). G Spinal cord water contents of SCI rats in each group (n = 8). H HE staining was used to assess the histopathological changes of rats in each group (n = 8). I Normal motor neuron index of SCI rats in each group (n = 8). J Western blot was used to detect the protein levels of apoptosis-related proteins (Bcl-2, Bax, cleaved-caspase-3, cleaved-PARP) in rat spinal cord tissues in each group. **p < 0.01, ***p < 0.001

MiR-10b-5p suppresses LPS-triggered PC12 cell injury

To further clarify the potential function of miR-10b-5p underlying SCI, we constructed the cellular model to mimic SCI characteristics. To this end, rat adrenal pheochromocytoma cells (PC12) were first stimulated with 5 μg/mL LPS for 12 h and then transfected with agomiR-10b-5p or agomiR-NC, while the untreated cells were taken as controls. Subsequently, we examined miR-10b-5p level in PC12 cells under indicated treatment through RT-qPCR. MiR-10b-5p presented downregulation under LPS stimulation, however, this effect was countervailed by agomiR-10b-5p transfection (Fig. 2A). Moreover, TUNEL staining demonstrated that LPS treatment resulted in elevation of TUNEL-positive PC12 cells, whereas miR-10b-5p reduced the increased TUNEL-positive PC12 cells under LPS treatment (Fig. 2B). Similarly, ELISA demonstrated that the contents of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in PC12 cells were high upon LPS stimulation, which were reduced by miR-10b-5p overexpression (Fig. 2C). Next, we measured the abundance of pro-apoptotic proteins (Bax, cleaved-caspase-3, and cleaved-PARP) and anti-apoptotic protein (Bcl-2) in PC12 cells. We noticed that LPS stimulation resulted in the elevation of Bax/Bcl-2 ratio, cleaved-caspase-3, and cleaved-PARP levels, whereas miR-10b-5p counteracted this effect in LPS-treated PC12 cells (Fig. 2D, E). Furthermore, DCFH-DA staining depicted that LPS treatment facilitated ROS production in PC12 cells, and this effect was neutralized by miR-10b-5p upregulation (Fig. 2F). Taken together, these results suggest that miR-10b-5p represses LPS-stimulated PC12 cell apoptosis, inflammation, and oxidative stress.

Fig. 2

MiR-10b-5p suppresses LPS-triggered PC12 cell injury. A RT-qPCR was used to measure miR-10b-5p level in PC12 cells in Ctrl, LPS + agomiR-NC or LPS + agomiR-10b-5p groups. B TUNEL staining detected PC12 cell apoptosis rate in indicated groups. Scale bar: 50 μm. C ELISA was performed to detect inflammatory cytokine levels in PC12 cells under indicated transfection. D Western blotting detected apoptosis-related protein abundances in PC12 cells under indicated transfection. E The ratio of Bax/Bcl-2 from western blot results. F DCFH-DA staining was used to detect ROS levels in PC12 cells under indicated transfection. Scale bar: 50 μm. The data are representative of at least three independent experiments. **p < 0.01, ***p < 0.001, compared with the Ctrl group; ##p < 0.01, ###p < 0.001, compared with the LPS + agomiR-NC group

MiR-10b-5p targets PTBP1

We then attempted to elucidate the molecular mechanism by which miR-10b-5p modulated LPS-triggered PC12 cell injury. As well known, miRNAs can specifically bind to target gene mRNA 3'UTR, thereby repressing or blocking target gene mRNA translation [8]. Based on this, we hypothesized that miR-10b-5p may exert a role in LPS-treated PC12 cells in such a manner. Thus, we screened the putative target molecule of miR-10b-5p using TargetScan website (https://www.targetscan.org/vert_72/) and discovered the binding sequence of miR-10b-5p on PTBP1 3’UTR (Fig. 3A). As revealed by the FISH assay, miR-10b-5p was colocalized with PTBP1 in the cytoplasm of PC12 cells (Fig. 3B). RT-qPCR depicted that LPS stimulation upregulated PTBP1 in PC12 cells compared to the control group whereas miR-10b-5p downregulated PTBP1 levels in LPS-treated PC12 cells (Fig. 3C). RNA pull-down illustrated that PTBP1 3'UTR presented enrichment in pulled-down products by biotinylated miR-10b-5p in LPS-treated PC12 cells in contrast to the control group (Fig. 3D), suggesting the binding of PTBP1 3'UTR to miR-10b-5p in LPS-treated PC12 cells. Subsequently, we measured the interaction between miR-10b-5p and PTBP1 in LPS-treated PC12 cells using a luciferase reporter assay. We found that miR-10b-5p upregulation resulted in decreased luciferase intensity of LPS-treated PC12 cells harboring pmirGLO-PTBP1 3'UTR-wt constructs relative to controls (Fig. 3E). Collectively, these findings highlight that miR-10b-5p negatively modulates PTBP1 via targeting PTBP1 3'UTR in LPS-treated PC12 cells.

Fig. 3

MiR-10b-5p targets PTBP1. A TargetScan predicted binding fragment of miR-10b-5p on PTBP1 3'UTR. B FISH assay was used to determine the colocalization of miR-10b-5p and PTBP1. C RT-qPCR detected PTBP1 level in PC12 cells under indicated transfection. D RNA pull-down assay detected PTBP1 3'UTR enrichment pulled down by Bio-NC/Bio-miR-10b-5p in PC12 cellular model. E Dual luciferase reporter assay detected the interaction between miR-10b-5p and PTBP1 3'UTR in PC12 cellular model. The data are representative of at least three independent experiments. ***p < 0.001 vs control; ###p < 0.001, compared with the LPS + agomiR-NC group

MiR-10b-5p alleviates LPS-induced PC12 cell damage by repressing PTBP1

In the next step, we attempted to clarify whether miR-10b-5p mediates PC12 cellular processes by regulating PTBP1. To this end, LPS-stimulated PC12 cells were transfected with oe-NC or oe-PTBP1 vector. RT-qPCR and western blot confirmed the successful elevation of PTBP1 in the oe-PTBP1-transfected PC12 cellular model (Fig. 4A). Then, we conducted a series of rescue experiments under the indicated treatment. We discovered that the inhibitory impact of miR-10b-5p overexpression on LPS-induced PC12 cell apoptosis (Fig. 4B) and inflammation (Fig. 4C) were countervailed through PTBP1 upregulation. Moreover, the inhibitory influence of miR-10b-5p overexpression on LPS-induced elevation in Bax/Bcl-2, cleaved-caspase-3, and cleaved-PARP upregulation were neutralized through PTBP1 upregulation (Fig. 4D, E). Furthermore, through PTBP1 upregulation, the inhibitory effect of miR-10b-5p overexpression on LPS-induced oxidative stress was mitigated (Fig. 4F). Together, this data indicates that miR-10b-5p alleviates LPS-triggered PC12 cell injury through PTBP1 downregulation.

Fig. 4

PTBP1 mediates LPS-induced PC12 cell damage repressed by miR-10b-5p. A RT-qPCR and western blot detected the overexpression efficacy of oe-NC or oe-PTBP1 in the PC12 cellular model. B TUNEL staining was conducted to detect apoptosis rate in LPS-treated PC12 cells in agomiR-NC, agomiR-10b-5p or agomiR-10b-5p + oe-PTBP1 groups. Scale bar: 50 μm. C ELISA detected inflammatory cytokine levels in LPS-treated PC12 cells under indicated transfection. D Western blotting detected apoptosis-related protein abundances in LPS-treated PC12 cells under indicated transfection. E The ratio of Bax/Bcl-2 from western blot results. F DCFH-DA staining detected ROS levels in LPS-treated PC12 cells under indicated transfection. Scale bar: 50 μm. The data are representative of at least three independent experiments. **p < 0.01, ***p < 0.001, compared with the oe-NC or agomiR-NC group; #p < 0.05, ###p < 0.001, compared with the agomiR-10b-5p group

miR-10b-5p alleviated spinal cord injury in rats by targeting PTBP1

To explore whether miR-10b-5p exerts protective effects against SCI by modulating PTBP1, SCI rats were injected with agomiR-10b-5p alone or in combination with oe-PTBP1. RT-qPCR results revealed that miR-10b-5p expression was elevated in the spinal cord tissues of the agomiR-10b-5p group, with no significant alteration observed after PTBP1 overexpression (Fig. 5A). However, PTBP1 levels were downregulated in the agomiR-10b-5p group and significantly elevated in the agomiR-10b-5p + oe-PTBP1 group compared to the agomiR-10b-5p group (Fig. 5B). The BBB score, elevated by agomiR-10b-5p in SCI rats, was restored to baseline levels following PTBP1 overexpression (Fig. 5C). Furthermore, the reduction in spinal cord water content induced by miR-10b-5p overexpression was reversed after PTBP1 upregulation (Fig. 5D). HE staining further indicated that PTBP1 overexpression diminished the protective effects of miR-10b-5p on spinal cord lesions in SCI rats, and the increase in the normal motor neuron index caused by miR-10b-5p was reversed by overexpressing PTBP1 (Fig. 5E, F). Additionally, the upregulation of Bcl-2 and the downregulation of Bax, cleaved caspase-3, and cleaved PARP induced by agomiR-10b-5p showed opposite changes after PTBP1 overexpression in SCI rats (Fig. 5G). Overall, these results indicate that miR-10b-5p modulates PTBP1 to alleviate spinal cord injury in vivo.

Fig. 5

MiR-10b-5p targets PTBP1 to alleviate neurological impairment and spinal cord damage in SCI rats. RT-qPCR detected the expression of A miR-10b-5p and B PTBP1 in the spinal cord tissue of rats in each group. C BBB scoring was used to measure the neurological function of rats in each group. D Spinal cord water contents of rats in each group. E HE staining was performed to evaluate the histopathological changes of rats in each group (scale bar: 50 μm). F Normal motor neuron index of SCI rats in each group. G Western blot was used to detect the protein expression of apoptosis-related proteins (Bcl-2, Bax, cleaved-caspase-3, cleaved-PARP) in rat spinal cord tissues in each group. **p < 0.01, ***p < 0.001, compared with the agomiR-NC group; ##p < 0.01, ###p < 0.001, compared with the agomiR-10b-5p group

MiR-10b-5p suppresses TGF-β1 decay and activates TGF-β1/Smad3 pathway

Previously, it has been shown that TGF-β1/Smad3 pathway contributes to SCI repair [41], and one report has revealed that miR-10b-5p facilitates the TGF-β1/Smad3 pathway activation [42]. Thus, we further elucidated whether miR-10b-5p exerted its function via regulating this pathway in SCI. First, we detected the expression levels of TGF-β1/Smad3 pathway-related genes (TGF-β1 and p-Smad3) in rat spinal cord tissues. We found that TGF-β1 and p-Smad3 protein levels were reduced in the SCI group, which was significantly reversed after miR-10b-5p overexpression (Fig. 6A). Then we determined TGF-β1/Smad3 pathway-related gene (TGF-β1 and p-Smad3) expression levels in LPS-treated PC12 cells under indicated transfection using western blotting and RT-qPCR. Results showed that TGF-β1 and p-Smad3 protein levels were high in LPS-treated PC12 cells under miR-10b-5p overexpression, however, PTBP1 upregulation reversed this effect (Fig. 6B). RT-qPCR results displayed a similar trend as western blotting results (Fig. 6C), suggesting that miR-10b-5p facilitates TGF-β1 expression and TGF-β1/Smad3 pathway activation via PTBP1 in SCI cellular model. We further clarified the mechanism by which PTBP1 downregulates TGF-β1. Since PTBP1 can mediate mRNA degradation as an RBP [43], it was speculated that PTBP1 may suppress TGF-β1 mRNA stability. For this purpose, we examined mRNA half-life of TGF-β1 or β-actin (internal control) using RT-qPCR. We observed that PTBP1 overexpression facilitated TGF-β1 mRNA degradation in LPS-treated PC12 cells (Fig. 6D, E), suggesting the inhibitory impact of PTBP1 on TGF-β1 mRNA stability in LPS-treated PC12 cells. Additionally, RNA pull-down depicted that PTBP1 protein presented enrichment in pulled-down products by TGF-β1 sense rather than TGF-β1 anti-sense in LPS-treated PC12 cells (Fig. 6F). Similarly, RIP demonstrated that TGF-β1 presented enrichment in complexes immunoprecipitated with anti-PTBP1 rather than anti-IgG in LPS-treated PC12 cells (Fig. 6G), indicating that PTBP1 bound to TGF-β1 to destabilize TGF-β1 in LPS-treated PC12 cells. Collectively, these results suggest that miR-10b-5p suppresses TGF-β1 decay and activates TGF-β1/Smad3 pathway in SCI.

Fig. 6

MiR-10b-5p suppresses TGF-β1 decay and activates TGF-β1/Smad3 pathway. A Western blot detected protein levels of TGF-β1/Smad3 pathway-related proteins in rat spinal cord tissues in each group. B Western blot detected TGF-β1/Smad3 pathway-related protein levels in LPS-treated PC12 cells in agomiR-NC, agomiR-10b-5p or agomiR-10b-5p + oe-PTBP1 groups. C RT-qPCR detected TGF-β1/Smad3 pathway-related gene mRNA level in LPS-treated PC12 cells under indicated transfection. D, E RT-qPCR detected TGF-β1 or β-actin mRNA half-life in LPS-treated PC12 cells in oe-NC or oe-PTBP1 groups. F RNA pull-down assay was conducted to detect PTBP1 enrichment pulled down by TGF-β1 sense/anti-sense in LPS-treated PC12 cells. G RIP detected TGF-β1 enrichment in complexes immunoprecipitated with anti-PTBP1 or anti-IgG in LPS-treated PC12 cells. The data are representative of at least three independent experiments. **p < 0.01, ***p < 0.001 compared with the control group; ###p < 0.001, compared with the agomiR-10b-5p group

Discussion

SCI is the most common complication of spine injury [44, 45]. SCI includes primary injury and secondary injury, among which the secondary injury is majorly nerve cell injury associated with inflammation and oxidative stress; prevention and mitigation of nerve injury is of great significance for improving the prognosis of SCI patients [46, 47]. Herein, BBB scores of rats presented an evident depletion 3 days and 7 days after SCI. There was a remarkable reduction of spared tissue in the rostral/caudal direction from the injury site in SCI rats. The increased water content in spinal cord tissue is a common factor for edema often observed in SCI [48], and we found water contents in spinal cord tissue presented elevation with time in SCI rats. PC12 cells, a cell line cloned from rat adrenal medulla pheochromocytoma, can differentiate into nerve-like cells under induction of cell growth factors, with good nerve cell characteristics, which are often used as a cellular model to elucidate neurological diseases [49]. LPS, the main component of Gram-negative bacteria cell wall, can induce excessive inflammatory response in normal animal immune system, and thus is commonly used to induce inflammatory response in cells [50]. Increasing studies have used LPS-treated PC12 cells to study the underlying mechanism involved in neuron inflammation and apoptosis in vitro [51,52,53]. In the present study, LPS resulted in the elevation of PC12 cell apoptosis, inflammation, and oxidative stress.

MiRNAs participate in the regulation of neuronal apoptosis, inflammation, and oxidative stress and have a close relation to the occurrence and development of neurological injury diseases [54, 55]. As a multifunctional miRNA, miR-10b-5p has been revealed to be involved in the development of multiple diseases. For the regulation of biological processes, miR-10b-5p is revealed to antagonize cardiomyocyte apoptosis under hypoxia through PTEN downregulation [56]. Multiple studies have also shown that miR-10b-5p represses cell apoptosis and promotes cell growth in several cancers such as glioma, liver cancer, and colorectal cancer [57,58,59]. MiR-10b-5p is also indicated as a biomarker associated with inflammation and oxidative stress [60, 61]. Inhibition of miR-10b-5p is revealed to hinder the development of Alzheimer's disease progression via mitigating neuronal apoptosis, inflammatory response, and oxidative stress [17]. In line with the results of these studies, herein, we also observed that miR-10b-5p facilitated neurological function repair and attenuated spinal cord damage in rats after SCI. Additionally, miR-10b-5p repressed apoptosis by downregulating Bax, cleaved caspase-3, and elevating Bcl-2 levels. MiR-10b-5p also attenuated the LPS-induced inflammation by reducing the production of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α. Additionally, we found that miR-10b-5p alleviated the LPS-induced oxidative stress by reducing ROS production. These findings suggest that miR-10b-5p exerts a protective role against SCI in rats, highlighting the therapeutic potential of miR-10b-5p in repair following SCI and may also be used as a diagnostic biomarker for SCI.

PTBP1 is a member of the hnRNPs family, and its main binding sequence consists of 15–25 pyrimidine bases, mainly U and C-rich sequences on pre-mRNA, thus termed a polypyrimidine region binding protein [62]. In our study, PTBP1 was identified as a target of miR-10b-5p and harboring binding site at PTBP1 3'UTR. Mechanistically, PTBP1 was found to be upregulated in SCI rats and in LPS-stimulated PC12 cells, whereas overexpressing miR-10b-5p downregulated PTBP1 expression both in vivo and in vitro. We also found that miR-10b-5p is bound to PTBP1 in LPS-treated PC12 cells. These findings imply that miR-10b-5p negatively modulates PTBP1 through targeting PTBP1 3'UTR. Previously, PTBP1 has been shown to be involved in various biological processes in cells such as cell proliferation, migration, invasion, neuronal development, and so on [63,64,65]. It was reported that PTBP1 downregulation was sufficient to convert cultured mouse fibroblasts and N2a cells into functional neurons [66]. PTBP1 depletion resulted in the restoration of visual function following excitotoxic NMDA damage [67]. Recent studies reported that downregulation of the RNA binding protein, PTBP1, converts astrocytes into neurons in situ in multiple mouse brain regions, consequently improving pathological phenotypes associated with Parkinson’s disease [68]. In our study, rescue assays demonstrated that miR-10b-5p exerted protective effects against spinal cord injury in vivo by downregulating PTBP1, as evidenced by improved BBB scores, reduced spinal cord water content, decreased spinal cord lesions, and altered expression of apoptosis-related proteins, which were reversed upon PTBP1 overexpression. Additionally, in vitro assays revealed that the inhibitory effects of miR-10b-5p overexpression on LPS-triggered PC12 cell apoptosis, inflammation, and oxidative stress were counteracted by PTBP1 upregulation, suggesting that miR-10b-5p alleviated LPS-triggered PC12 cell injury through PTBP1 downregulation. These results are consistent with the aforementioned studies, suggesting the role of PTBP1 as a potential therapeutic target for motor function recovery after SCI.

TGF-β is a multifunctional protein activating the downstream Smad pathway [69]. TGF-β1/Smad3 pathway has a close relation to the onset of inflammation, oxidative stress, and apoptosis [70]. The TGF-β1/Smad3 pathway contributes to SCI repair [71]. This indicates that SCI may be mitigated through modulating the TGF-β1/Smad3 pathway. Herein, miR-10b-5p upregulated TGF-β1 mRNA and protein levels to facilitate TGF-β1/Smad3 pathway activation via PTBP1 depletion in the SCI cellular model. PTBP1 has been shown to play a key role in post-transcriptional regulation, uncapped translational regulation, RNA localization, and RNA stability in the cytoplasm [72]. Herein, PTBP1 bound to TGF-β1 to attenuate TGF-β1 mRNA stability in LPS-treated PC12 cells. These outcomes imply that miR-10b-5p targeted PTBP1 to repress TGF-β1 degradation and facilitate TGF-β1/Smad3 pathway activation in SCI (Fig. 7).

Fig. 7

The scientific concept map for this study. MiR-10b-5p targets PTBP1 to repress TGF-β1 degradation and facilitate TGF-β1/Smad3 pathway activation in spinal cord injury (SCI)

In conclusion, the findings of this study demonstrate that miR-10b-5p relieves SCI through repressing TGF-β1 decay and inducing TGF-β1/Smad3 pathway activation through PTBP1 downregulation, providing a novel insight for seeking targeted therapy plans of SCI. However, the limitation of this study is that it does not further explore whether there are regulatory factors upstream of miR-10b-5p, which will be investigated in the future.