miR-99b-5p Targets AKT1 to Modulate Microglial Polarization: A Prognostic Biomarker for Spinal Cord Injury

miR-99b-5p Targets AKT1 to Modulate Microglial Polarization: A Prognostic Biomarker for Spinal Cord Injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article miR-99b-5p Targets AKT1 to Modulate Microglial Polarization: A Prognostic Biomarker for Spinal Cord Injury Ruye Li, Deming Chen, Shibo Feng, Chenge Xian This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8525596/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Study design: Cross-sectional study. Objectives This study aimed to investigate the mechanism by which miR-99b-5p regulates microglial polarization via targeting AKT1, and to evaluate its potential as a prognostic biomarker and therapeutic target for spinal cord injury (SCI). Setting: The study was conducted at Wuhan Hankou Hospital and Nedong District People's Hospital, China. Methods A total of 90 individuals with SCI and 70 healthy controls were enrolled. Serum levels of miR-99b-5p, inflammatory cytokines, and microglial polarization markers were quantified. Functional outcomes were assessed using the AIS, FIM, SCIM-III, and WISCI-II scales. In vitro investigations included cell transfection, LPS-induced polarization, co-culture, and dual-luciferase reporter assays. Results Serum miR-99b-5p was significantly elevated in SCI individuals (AUC = 0.872) and positively correlated with AIS grade and functional outcomes (FIM, SCIM-III, WISCI-II). The miR-99b-5p mimic selectively inhibited M1 polarization in microglia, downregulating markers such as iNOS and TNF-α, while exhibiting no significant effect on macrophages. AKT1 was identified as a direct target of miR-99b-5p, and its overexpression reversed the inhibitory effect of miR-99b-5p on microglial polarization. Furthermore, miR-99b-5p alleviated neuronal inflammation and apoptosis, as evidenced by decreased Bax and increased Bcl-2 expression, through its regulatory action on microglia. Conclusion miR-99b-5p targets AKT1 to suppress microglial M1 polarization, thereby alleviating neuroinflammation and neuronal damage in SCI. Its serum level serves as a promising prognostic biomarker and potential therapeutic target. Health sciences/Medical research/Biomarkers/Prognostic markers Biological sciences/Neuroscience/Spine regulation and structure/Spine motility Spinal cord injury miR-99b-5p microglial polarization AKT1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Following spinal cord injury (SCI), the complex pathological cascade is central to the development of long-term neurological deficits, with immune and inflammatory responses driving secondary injury being particularly critical [ 1 , 2 ]. In this process, the dynamic polarization of microglia and infiltrating macrophages profoundly influences the homeostasis of the local microenvironment and the outcomes of neural repair [ 3 , 4 ]. Therefore, elucidating the key molecular mechanisms that regulate the fate of these immune cells is an important direction for exploring novel therapeutic strategies for SCI. In recent years, microRNAs (miRNAs), as key post-transcriptional regulators of gene expression, have garnered increasing attention for their roles in the pathological progression of SCI [ 5 ]. miR-99b-5p has emerged as a molecule of particular interest due to its diverse regulatory roles across multiple disease contexts. Current research highlights its core function in modulating immune-inflammatory pathways and cell survival, with distinct roles reported in different pathological settings. In cancer, for example, miR-99b-5p acts as a tumor suppressor by targeting the mTOR/AR axis, thereby inhibiting the proliferation of prostate cancer cells and inducing autophagy [ 6 ]. miR-99b-5p can alleviate apoptosis and pyroptosis of neurons after paclitaxel chemotherapy by inhibiting the activation of NLRP3 and improving oxidative stress [ 7 ]. Beyond these, it participates in immune regulation in osteoarthritis and schizophrenia-like models, where it modulates macrophage polarization or microglial inflammatory responses via pathway-specific mechanisms [ 8 , 9 ]. Collectively, these findings establish miR-99b-5p as a key regulator of cell fate and immune homeostasis, with potential as a biomarker and therapeutic target across various diseases. Studies have shown that miR-99b-5p expression is upregulated in mice after SCI and that it can target the mTOR signaling pathway [ 10 ]. Inhibition of miR-99b-5p can reverse the injury-induced downregulation of mTOR, thereby reducing neuronal apoptosis and promoting neurite outgrowth [ 11 ]. However, despite these findings, the specific role and mechanisms of miR-99b-5p in the macrophage/microglia-centered immune microenvironment following SCI remain unclear. In particular, there is a lack of direct evidence regarding its cell type-specific regulatory functions, especially its ability to intervene in the polarization of macrophages/microglia directly. This knowledge gap limits the potential for developing this molecule into a precise therapeutic target for SCI. To address this gap, this study aims to systematically elucidate the specific role and molecular mechanisms of miR-99b-5p in remodeling the immune microenvironment after SCI, with a focus on its regulatory effects on macrophage/microglia polarization. The findings are expected to provide new insights into the immunopathological mechanisms of SCI and offer a potential therapeutic target for developing immune modulation-based neural repair strategies. Materials and methods 2.1 Study participants In this study, 90 individuals with SCI admitted to Hankou Hospital of Wuhan City from June 2022 to June 2024 were enrolled as the study group. Additionally, 70 healthy individuals with routine physical examinations during the same period were selected as controls, excluding those with neurological disorders, inflammatory conditions, or organic lesions. This study was approved by the Hospital Ethics Committee, adhering to the Declaration of Helsinki. 2.2 Main Reagents and Instruments Cell lines (HMC3, THP-1, SH-SY5Y, HEK293T) were from the Cell Bank of the Chinese Academy of Sciences. miR-99b-5p mimics/inhibitors (RiboBio), AKT1 overexpression plasmid (GeneChem), ELISA kits (Boster), qRT-PCR kits (TaKaRa), and the Dual-Luciferase® System (Promega) were used. Key instruments included an ABI 7500 qPCR system and a ChemiDoc XRS + imager. 2.3 Clinical Sample Detection and Functional Evaluation 2.3.1 Serum Analysis Serum was isolated and stored at -80°C. Inflammatory cytokines (TNF-α, IL-1β, IL-6) and polarization markers (M1 markers: TNF-α, iNOS; M2 markers: CD206, Arg-1) were quantified by ELISA. Serum miR-99b-5p levels were quantified by qRT-PCR. 2.3.2 Neurological and Functional Recovery Assessment Evaluations were conducted using standardized scales. The severity of neurological impairment was graded using the American Spinal Injury Association Impairment Scale (AIS), while functional recovery was comprehensively assessed through the Functional Independence Measure (FIM), the Spinal Cord Independence Measure III (SCIM-III), and the Walking Index for SCI II (WISCI-II). All assessments were independently conducted by two experienced clinicians who were blinded to the laboratory results. 2.4 In Vitro Cell Experiments 2.4.1 Cell Culture and Group Treatment All cell lines were cultured in DMEM supplemented with 10% fetal bovine serum. THP-1 cells were differentiated into macrophages using 100 nM PMA for 24 hours. For functional studies, cells were treated with LPS (5 µg/mL, 24 h) to induce M1 polarization, following transfection with miR-99b-5p mimics, inhibitors, or the AKT1 overexpression plasmid. 2.4.2 CCK-8 Assay for Cytotoxicity HMC3 and THP-1 cells were seeded in 96-well plates, treated with miR-99b-5p mimic for 24 h, and 10 µL of CCK-8 reagent was added. Absorbance at 450 nm was measured, and cell viability was calculated to determine a non-cytotoxic working concentration. 2.4.3 qRT-PCR for Polarization Marker Expression Total RNA from cells was reverse-transcribed. mRNA levels of M1 markers (iNOS, TNF-α) and M2 markers (CD206, Arg-1) were measured by qRT-PCR, with GAPDH as the endogenous control. Relative expression was calculated using the 2^(-ΔΔCt) method. 2.4.4 Microglia-Neuron Co-culture A non-contact co-culture system was established using Transwell chambers. HMC3 microglia (upper chamber) were stimulated with LPS and transfected. SH-SY5Y neurons (lower chamber) were cultured separately. Conditioned media and cells from both compartments were collected after 48 hours for downstream analysis. 2.4.5 Western Blot for Protein Expression Proteins from SH-SY5Y cells were extracted, separated by SDS-PAGE, and transferred to PVDF membranes. Membranes were incubated with primary antibodies against Bax, Bcl-2, and β-actin, followed by HRP-conjugated secondary antibodies. Protein bands were visualized using an ECL substrate. 2.4.6 Dual-Luciferase Reporter Assay AKT1 wild-type (WT) and mutant (MUT) 3′-UTR fragments were cloned into luciferase reporter plasmids. HEK293T cells were co-transfected with reporter plasmids and NC mimic/miR-99b-5p mimic. Luciferase activities were measured 48 h post-transfection, with relative activity normalized to Renilla luciferase luminescence. 2.5 Bioinformatics Analysis The genes associated with SCI were retrieved from the GeneCards database ( https://www.genecards.org/ ), using a relevance score cutoff of ≥ 20. This threshold is commonly applied to prioritize genes that have direct functional links to SCI pathophysiology [ 12 ]. Predicted targets of miR-99b-5p were obtained from the miRWalk database ( http://mirwalk.umm.uni-heidelberg.de/ ), with the following filtering criteria: (a) predicted by at least 3 of the 12 integrated algorithms (including TargetScan, miRanda, RNAhybrid, etc.) to ensure reliability; (b) conserved binding sites between miR-99b-5p and the 3′-UTR of target genes (as defined by TargetScan conservation score ≥ 0.5); (c) excluding genes with predicted binding free energy > -20 kcal/mol (indicating unstable binding interactions). 2.6 Statistical Analysis Statistical analyses were performed using SPSS 26.0, and graphs were generated with GraphPad Prism 8.0. Continuous data were expressed as mean ± standard deviation (x̄ ± s). Between-group comparisons for continuous data were analyzed by Student’s t-test (two groups) or one-way analysis of variance (ANOVA) with LSD post-hoc test (multiple groups). Categorical data were compared using the chi-square test. Correlations between serum miR-99b-5p levels and neurological/clinical variables were evaluated by Spearman’s rank correlation analysis. A two-tailed P value < 0.05 was considered statistically significant. Results High miR-99b-5p Expression Correlates with Improved Functional Recovery in Individuals with SCI Baseline demographic characteristics, including gender, age, and body mass index (BMI), were comparable between the SCI group and healthy controls (Table 1 ), which helped minimize potential confounding effects. Table 1 Comparison of Baseline Data between Healthy Control Individuals and SCI Individuals Control group n = 70 SCI group n = 90 P value Basic information Age (years, mean ± SD) 39.77 ± 12.58 41.77 ± 12.29 0.366 BMI (kg/m², mean ± SD ) 22.47 ± 0.93 22.47 ± 0.84 0.925 Gender (Male/Female, n) 34/36 47/43 0.647 Cause of damage Traffic accidents (n, %) - 31/33.3 - Falls from height (n, %) - 21/22.50 - Injury from a heavy object (n, %) - 22/23.6 - Other (n, %) - 16/17.20 - Injured segments Cervical segment (n, %) - 27/29.0 - Thoracolumbar segment (n, %) - 30/32.2 - Waist section (n, %) - 33/35.4 - Note: SCI, spinal cord injury; BMI, body mass index (unit: kg/m²). "-" indicates data not applicable to the corresponding group. While miR-99b-5p upregulation has been documented in murine SCI models, its serum expression profile in humans with SCI remains largely uncharacterized [ 13 ]. Given the well-recognized role of miRNAs in regulating the pathophysiological processes of SCI [ 14 ], serum miR-99b-5p levels were quantified initially. Consistent with trends reported in preclinical animal studies, serum miR-99b-5p expression was significantly higher in the SCI group than in the healthy control group (Fig. 1 A). Receiver operating characteristic (ROC) curve analysis was further performed to evaluate its discriminative ability, showing an area under the curve (AUC) of 0.872 (95% CI, 0.820 to 0.923, Fig. 1 B), indicating that serum miR-99b-5p has promising diagnostic potential for SCI. To evaluate the associations between serum miR-99b-5p and clinical characteristics in individuals with SCI, Spearman correlation analysis was conducted (Fig. 1 C). The results showed no significant correlations between miR-99b-5p levels and non-neurological variables (age, BMI, gender, cause of damage, injured segments; all P > 0.05). In contrast, miR-99b-5p was positively correlated with key neurological indices: the correlation coefficient (Spearman’s ρ) was 0.344 ( P < 0.001) for AIS grade, 0.551 ( P < 0.001) for FIM score, 0.527 ( P < 0.001) for SCIM-III score, and 0.724 ( P < 0.001) for WISCI-II score. To further explore the diagnostic value of serum miR-99b-5p for neurological outcomes in SCI, ROC curve analysis was performed (Fig. 1 D). The results revealed that miR-99b-5p had good discriminatory ability: the AUC was 0.817 (95% CI, 0.731 to 0.902, P < 0.001) for AIS grade, 0.789 (95% CI, 0.694 to 0.884, P < 0.001) for FIM score, 0.853 (95% CI, 0.769 to 0.937, P < 0.001) for SCIM-III score, and 0.639 (95% CI, 0.524 to 0.754, P = 0.018) for WISCI-II score. In summary, the findings demonstrate that elevated serum miR-99b-5p levels are strongly correlated with favorable functional recovery in individuals with SCI. High Expression of miR-99b-5p Significantly Inhibits M1 Polarization of Microglia Within the complex microenvironment of SCI, the polarization states of macrophages and microglia have been established as pivotal regulators of secondary injury and repair processes, whose dynamic balance critically influences neuronal survival, axonal regeneration, and functional recovery outcomes [ 15 , 16 ]. To investigate whether miR-99b-5p is involved in regulating this balance, serum levels of polarization markers were measured initially. Individuals with SCI were stratified into low expression and high expression groups based on the median serum miR-99b-5p level. Compared with the low expression group, individuals in the high miR-99b-5p group exhibited significantly lower levels of M1 markers (iNOS and TNF-α) and higher levels of M2 markers (CD206 and Arg-1) (Fig. 2 A), suggesting a potential role for miR-99b-5p in suppressing M1 polarization. To further verify this regulatory effect and assess its cell-type specificity, in vitro M1 polarization models were established using HMC3 microglia and THP-1 macrophages. The miR-99b-5p mimic showed no significant cytotoxicity in either cell line as determined by the CCK-8 assay (Fig. 2 B-C), and a concentration of 10 µM was therefore selected for subsequent experiments. In LPS-stimulated HMC3 microglia, the miR-99b-5p mimic significantly suppressed the upregulation of M1 markers (iNOS and TNF-α) (Fig. 2 D). In contrast, in THP-1 macrophages, the miR-99b-5p mimic did not consistently reverse LPS-induced M1 polarization but significantly attenuated the LPS-mediated downregulation of the M2 marker Arg-1 (Fig. 2 E), highlighting the complexity and cell-context dependency of macrophage polarization regulation. Together, these results indicate that miR-99b-5p selectively inhibits M1 polarization in microglia while exerting a distinct regulatory effect on macrophages. miR-99b-5p Attenuates Microglial M1 Polarization by Directly Targeting AKT1 To systematically identify the downstream targets and signaling pathways of miR-99b-5p in SCI, two gene sets were integrated for analysis: (1) SCI-associated genes with high relevance (GeneCards score ≥ 20) to ensure functional relevance to SCI; (2) high-confidence targets of miR-99b-5p (predicted by ≥ 3 algorithms, conserved binding sites, and binding free energy < -20 kcal/mol) to reduce false-positive predictions. This integration yielded 933 overlapping candidate genes (Fig. 3 A), which were hypothesized to mediate the biological effects of miR-99b-5p in SCI. KEGG pathway enrichment analysis identified 15 significantly enriched pathways. The top 10 pathways (Fig. 3 B) included multiple signaling cascades and biological processes closely related to SCI pathophysiology: (1) Pathways in cancer; (2) MAPK signaling pathway; (3) Pathways of neurodegeneration – multiple diseases; (4) Neuroactive ligand-receptor interaction; (5) AGE-RAGE signaling pathway in diabetic complications; (6) Colorectal cancer; (7) cAMP signaling pathway; (8) Cell adhesion molecules; (9) Apoptosis; (10) Cholinergic synapse (Fig. 3 B). Among these, the MAPK signaling pathway was prioritized for further validation. This pathway is a well-characterized core regulator of microglial polarization, neuroinflammation, and neuronal survival—key pathological processes driving secondary injury after SCI [ 17 , 18 ]. Consistent with this prediction, LPS stimulation significantly upregulated the expression of key MAPK members (p38 MAPK, ERK1/2, and JNK), and miR-99b-5p mimic transfection significantly reversed this upregulation (Fig. 3 C), confirming that miR-99b-5p suppresses microglial M1 polarization at least partially via modulating the MAPK pathway. To identify the direct upstream target of miR-99b-5p mediating this effect, a protein-protein interaction (PPI) network was constructed from the intersecting genes. Among top-ranked hub genes, AKT serine/threonine kinase 1 (AKT1) was a promising candidate. Although its pivotal role in modulating microglial polarization through the PI3K/AKT pathway is well-recognized [ 19 ], whether it serves as a functional target for miR-99b-5p in the context of SCI remained unknown. In silico analysis predicted a high-probability binding site for miR-99b-5p within the 3′-UTR of AKT1, featuring canonical seed region complementarity (Fig. 3 D). This interaction was experimentally confirmed by dual-luciferase reporter assay, where the miR-99b-5p mimic significantly suppressed luciferase activity of the wild-type (WT) AKT1 3'-UTR reporter but not the mutant (MUT) construct (Fig. 3 E), confirming AKT1 as a direct target of miR-99b-5p. Rescue experiments verified the functional relevance of the miR-99b-5p-AKT1 interaction. miR-99b-5p mimic effectively inhibited LPS-induced upregulation of M1 markers (iNOS, TNF-α), while AKT1 co-overexpression partially but significantly reversed this suppression (Figs. 3 F-G). Collectively, these findings demonstrate that miR-99b-5p directly targets AKT1, modulates downstream pathways including MAPK, and thereby attenuates microglial M1 polarization, providing novel insights into the molecular mechanism of miR-99b-5p in regulating microglial polarization during SCI. miR-99b-5p Alleviates Neuroinflammation and Neuronal Apoptosis by Suppressing Microglial M1 Polarization To link the observed clinical associations with the proposed cellular mechanisms, the correlation between serum miR-99b-5p levels and systemic inflammation was investigated. The results showed that individuals with high serum miR-99b-5p expression exhibited significantly lower levels of key pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) compared with those with low expression (Fig. 4 A). To verify whether miR-99b-5p exerts neuroprotection by regulating microglial paracrine signaling, a controlled in vitro model was established using a non-contact Transwell co-culture system to isolate the effects of soluble factors secreted by HMC3 microglia on SH-SY5Y neurons. Transfection of miR-99b-5p mimic into LPS-stimulated microglia significantly downregulated M1 marker secretion (iNOS, TNF-α) and reduced neuroinflammatory factors (IL-6, IL-1β) in the neuronal compartment compared to the LPS control. Conversely, the miR-99b-5p inhibitor exacerbated these inflammatory responses (Figs. 4 B-E). These results confirm that miR-99b-5p mitigates neuroinflammation via a microglia-dependent paracrine mechanism. The impact of attenuated inflammation on neuronal survival was subsequently explored. KEGG analysis implicated the apoptosis pathway, prompting an examination of key apoptotic regulators. In neurons co-cultured with miR-99b-5p-overexpressing microglia, pro-apoptotic Bax protein expression was significantly decreased, while anti-apoptotic Bcl-2 levels were increased (Figs. 4 F-H). This anti-apoptotic effect was validated using an independent pre-miR-99b-5p overexpression system with consistent results. Collectively, data from the clinical cohort and reductionist co-culture model demonstrate that miR-99b-5p dually attenuates neuroinflammation and inhibits neuronal apoptosis, dependent on its ability to suppress microglial M1 polarization. Discussion This study delineates a neuroprotective role for miR-99b-5p in SCI by demonstrating its capacity to mitigate neuroinflammation and neuronal apoptosis through the specific suppression of microglial M1 polarization via the AKT1 pathway. These findings integrate miR-99b-5p into the immunoregulatory network of the injured spinal cord, underscore the significance of miRNA-mediated regulation in neural injury, and offer novel insights into the underlying pathophysiological mechanisms. Clinically, serum levels of miR-99b-5p were found to be significantly elevated in individuals following SCI. Notably, higher circulating levels of this microRNA correlated with less severe neurological impairment and more favorable functional recovery, reinforcing the concept that the upregulation of specific protective microRNAs post-injury is associated with improved clinical outcomes [ 20 , 21 ]. While prior investigations have largely centered on other microRNAs such as miR-21 and miR-124, the present study is the first to establish a direct link between miR-99b-5p and clinical prognosis in human SCI, thereby expanding the scope of potential prognostic biomarkers. Collectively, the results suggest that miR-99b-5p functions not merely as a passive injury marker but as an endogenous protective factor that actively mitigates secondary pathological processes. This aligns with the broader theoretical framework wherein microRNAs are recognized as key regulators of cellular homeostasis within the injury microenvironment, critically influencing the ultimate course of tissue repair and recovery [ 22 ]. A pivotal mechanistic finding is the specific inhibition of microglial M1 polarization by miR-99b-5p, supported by both clinical biomarker correlations and direct in vitro evidence. In serum from individuals with SCI, elevated miR-99b-5p levels were inversely correlated with classic M1 polarization markers, including iNOS and TNF-α, reinforcing the well-established link between balanced microglial polarization and functional recovery [ 23 , 24 ]. Notably, this regulatory effect exhibited distinct cell-type specificity, being pronounced in HMC3 microglia but minimal in THP-1-derived macrophages. This distinction advances the understanding of lineage-selective miRNA actions in immune regulation and highlights a potential avenue for precise therapeutic intervention in SCI that may spare systemic immunity [ 25 , 26 ]. Bioinformatic analysis implicated the MAPK pathway, a central regulator of microglial activation and SCI progression. As AKT1 is a known upstream modulator of MAPK signaling, a coherent regulatory axis is proposed whereby miR-99b-5p targets AKT1 to modulate MAPK activity, thereby suppressing microglial M1 polarization. The translational potential of miR-99b-5p is underscored by its dual role, serving both as a quantifiable serum biomarker and as a functional regulator of the local spinal cord microenvironment. Its serum levels correlate with both the extent of systemic inflammation and the degree of neurological recovery, suggesting its potential utility as an auxiliary, non-invasive prognostic tool for the early clinical assessment of SCI. To investigate the direct neuroimmune interaction underlying this clinical correlation, an in vitro co-culture model was employed to isolate and examine the paracrine axis between microglia and neurons [ 27 ]. The experimental results established a clear causal relationship: overexpression of miR-99b-5p in microglia directly suppressed their polarization toward the M1 phenotype and the secretion of pro-inflammatory factors, which was sufficient to attenuate neuronal apoptosis. This finding provides direct mechanistic evidence for the therapeutic strategy of achieving neuroprotection by targeting the regulation of microglial polarization [ 28 ]. Nevertheless, the limitations inherent in this simplified in vitro model are acknowledged. While invaluable for dissecting specific cellular interactions, it cannot replicate the full pathophysiological complexity of the SCI microenvironment, which includes dynamic crosstalk among astrocytes, oligodendrocytes, infiltrating peripheral immune cells, and the evolving glial scar, all critical factors influencing repair and regeneration [ 29 ]. Most importantly, the anti-inflammatory and neuroprotective efficacy observed in vitro requires validation in a living organism. Therefore, future investigations utilizing established in vivo rodent spinal SCI models are essential for verifying therapeutic efficacy, defining pharmacokinetic profiles and optimal delivery strategies, and evaluating the long-term functional impact of miR-99b-5p modulation within an integrated physiological context [ 30 ]. In summary, this work establishes miR-99b-5p as a multifaceted candidate in SCI: a serologically accessible prognostic biomarker and a cell-type-specific molecular regulator that alleviates neuroinflammation and neuronal apoptosis by targeting AKT1-mediated microglial polarization. These insights offer a novel therapeutic target and a refined conceptual framework for mitigating the detrimental neuroimmune cascade following SCI. Declarations Acknowledgments We would like to acknowledge all patients with SCI and healthy volunteers who participated in this study, as well as the medical team of the Orthopedics Department, Hankou Hospital, Wuhan City, Hubei Province, for their support in sample recruitment and clinical data collection. CRediT authorship contribution statement Deming Chen: Data curation, Methodology, Writing – review & editing. Shibo Feng: Formal analysis, Writing – review & editing. Chenge Xian: Investigation, Writing – review & editing. Ruye Li: Resources, Conceptualization, Project administration , Supervision, Writing – review & editing. Funding This research received no external funding. Declaration of competing interest No known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper exist. Acknowledgments We sincerely thank all patients with SCI and healthy volunteers for their participation in this study. We also acknowledge the Orthopedics Medical Team of Hankou Hospital, Wuhan, Hubei Province, for their valuable support in sample collection and clinical data acquisition. Additionally, we are grateful to the Scientific Research Department of Hankou Hospital for providing the experimental platform and technical assistance. Ethics declarations Competing interests No known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper exist. Ethical approval The study was carried out following the Declaration of Helsinki and applicable national and international guidelines for research involving human participants. It received ethical approval from the Ethics Committee of Hankou Hospital in Wuhan City (Ethics Approval Number: Hanyilunshen [2022]-RC-012). Before starting the study, the research protocol, informed consent form, and other relevant materials were reviewed and approved by the ethics committee to ensure participants' safety and adherence to research ethics. Informed consent was obtained from all participants involved in the study. References Zheng B, Tuszynski MH. Regulation of axonal regeneration after mammalian spinal cord injury. Nat Rev Mol Cell Biol. 2023;24(6):396–413. Hellenbrand DJ, Quinn CM, Piper ZJ, Morehouse CN, Fixel JA, Hanna AS, et al. Inflammation after spinal cord injury: a review of the critical timeline of signaling cues and cellular infiltration. J Neuroinflammation. 2021;18(1):284. 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Bellver-Landete V, Bretheau F, Mailhot B, Vallières N, Lessard M, Janelle ME, et al. Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury. Nat Commun. 2019;10(1):518. Hutson TH, Di Giovanni S. The translational landscape in spinal cord injury: focus on neuroplasticity and regeneration. Nat Rev Neurol. 2019;15(12):732–745. Additional Declarations There is no duality of interest Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 27 Feb, 2026 Review # 3 received at journal 15 Feb, 2026 Review # 2 received at journal 02 Feb, 2026 Review # 1 received at journal 29 Jan, 2026 Reviewer # 3 agreed at journal 22 Jan, 2026 Reviewer # 2 agreed at journal 19 Jan, 2026 Reviewer # 1 agreed at journal 15 Jan, 2026 Reviewers invited by journal 14 Jan, 2026 Editor assigned by journal 12 Jan, 2026 Submission checks completed at journal 12 Jan, 2026 First submitted to journal 11 Jan, 2026 Unknown event 06 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8525596","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":574877207,"identity":"d9873207-56c8-49c9-9a54-b921ff2a4c61","order_by":0,"name":"Ruye Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYBACA2YwJSHHz97YcOCDgY0dsVosjCV7Dh88OKMgLZmwFghVkbjhhlvyYZ4PhxgbCGph530mzVMhkThzBo/BYRuDA8wM7IePbsDvMHYzaZ4zEsb90j0Gh3MM7vAx8KSl3cCvhY1NmrdNQnbmnDMgLc+YGSR4zIjQ8k+CccONHIPDFgaHGRuI09IgobjhRlrCYQYitTBbzjkmAQrkAwd7DNKS2Qj5xb7/GOONNzV1oKhs/vDjj40dP/vhY3i1gAATDzKPjZByEGD8QYyqUTAKRsEoGLkAANI+R/dqzc8rAAAAAElFTkSuQmCC","orcid":"","institution":"Wuhan Hankou hospital","correspondingAuthor":true,"prefix":"","firstName":"Ruye","middleName":"","lastName":"Li","suffix":""},{"id":574877208,"identity":"6b5fc5d6-f580-428a-8629-d099b22cd6d3","order_by":1,"name":"Deming Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Deming","middleName":"","lastName":"Chen","suffix":""},{"id":574877209,"identity":"ebb0c036-c698-4c6d-942e-fee2b3f588dc","order_by":2,"name":"Shibo Feng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shibo","middleName":"","lastName":"Feng","suffix":""},{"id":574877210,"identity":"28284bbb-c68d-496a-b4d1-3ebf990cedc1","order_by":3,"name":"Chenge Xian","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chenge","middleName":"","lastName":"Xian","suffix":""}],"badges":[],"createdAt":"2026-01-06 01:51:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8525596/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8525596/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100607524,"identity":"520ecf85-008b-4ed8-a0bb-517973db9fc8","added_by":"auto","created_at":"2026-01-19 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16:01:32","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1582082,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8525596/v1/8b431c2db412765877d23746.tif"},{"id":100607420,"identity":"8dba9c36-77a6-4a7c-a3c0-b3577bccb7a6","added_by":"auto","created_at":"2026-01-19 16:01:48","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1879354,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8525596/v1/06119366e00b80f47eaa591f.tif"},{"id":100607545,"identity":"eafeadd0-7136-4fd4-880a-889bbd17e9b6","added_by":"auto","created_at":"2026-01-19 16:02:29","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":88234,"visible":true,"origin":"","legend":"","description":"","filename":"SC202600130structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8525596/v1/3b142de243849babfda507b7.xml"},{"id":100607624,"identity":"e45ec0cd-e7db-47e7-9741-82932c75204e","added_by":"auto","created_at":"2026-01-19 16:03:19","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102013,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8525596/v1/2a89ce37639c00c2891737b6.html"},{"id":100607623,"identity":"2f38416c-80dc-46e8-bfa3-f6bc2d268f91","added_by":"auto","created_at":"2026-01-19 16:03:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":901419,"visible":true,"origin":"","legend":"\u003cp\u003eAssociations of serum miR-99b-5p with functional recovery in individuals with SCI. (A) Relative expression of serum miR-99b-5p was measured by qRT-PCR in healthy controls (n=70) and in individuals with SCI (n=90). Compared with the control group, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (B) The discriminative ability of serum miR-99b-5p between the two groups was evaluated by ROC curve analysis. The diagonal reference line (AUC = 0.5) is shown, with the calculated AUC and 95% CI indicated. (C) Spearman rank correlation analysis between serum miR-99b-5p expression and clinical indicators. (D) ROC curve analysis of serum miR-99b-5p for predicting neurological outcomes in individuals with SCI, including neurological impairment severity (assessed by AIS grade) and functional recovery (evaluated by FIM, SCIM-III, and WISCI-II scores).\u003c/p\u003e","description":"","filename":"Figure125.png","url":"https://assets-eu.researchsquare.com/files/rs-8525596/v1/ed1030d65f9db5d45bc7c996.png"},{"id":100607469,"identity":"c9e2c69a-a695-4da8-897c-d2f3b70a788c","added_by":"auto","created_at":"2026-01-19 16:02:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":656153,"visible":true,"origin":"","legend":"\u003cp\u003emiR-99b-5p specifically inhibits microglial M1 polarization. (A) Serum concentrations of M1 markers (iNOS, TNF-α) and M2 markers (CD206, Arg-1) were measured by ELISA in individuals with SCI, who were stratified into low and high miR-99b-5p expression groups according to the median serum miR-99b-5p level of the SCI cohort. (B-C) Cytotoxicity of the miR-99b-5p mimic on HMC3 microglia and PMA-differentiated THP-1 macrophages was assessed using the CCK-8 assay after 24 hours of treatment. (D-E) The mRNA expression levels of M1 markers (iNOS, TNF-α) and M2 markers (CD206, Arg-1) in LPS-stimulated HMC3 cells and PMA- and LPS-co-stimulated THP-1 cells were determined by qRT-PCR following transfection with miR-99b-5p mimic or NC mimic. Data are presented as mean ± SD. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"Figure224.png","url":"https://assets-eu.researchsquare.com/files/rs-8525596/v1/ccbaf77ae881b2c3152b8e0f.png"},{"id":100607466,"identity":"50bb3201-a27b-4a68-b4df-7fd943b93f88","added_by":"auto","created_at":"2026-01-19 16:02:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":932914,"visible":true,"origin":"","legend":"\u003cp\u003emiR-99b-5p targets AKT1 to inhibit microglial M1 polarization. (A) Venn diagram showing the intersection of SCI-associated genes (from GeneCards) and predicted target genes of miR-99b-5p (from miRWalk). (B) KEGG pathway enrichment analysis of the intersecting genes. (C) mRNA expression levels of key MAPK pathway genes (p38 MAPK, ERK1/2, JNK) in HMC3 cells, measured by qRT-PCR. (D) Schematic of the predicted binding site of miR-99b-5p within the 3′-UTR of AKT1. (E) Validation of direct targeting of AKT1 by miR-99b-5p via dual-luciferase reporter assay. Luciferase activity was measured in HEK293T cells co-transfected with miR-99b-5p mimic and a reporter plasmid containing either the wild-type (WT) or mutant (MUT) AKT1 3′-UTR. (F-G) The mRNA expression levels of M1 markers (iNOS and TNF-α) in HMC3 cells were determined by qRT-PCR after treatment with LPS, miR-99b-5p mimic, and AKT1 overexpression (AKT1-OE). Data are presented as mean ± SD. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"Figure317.png","url":"https://assets-eu.researchsquare.com/files/rs-8525596/v1/67e3fa5a2c388f08b35c6800.png"},{"id":100607661,"identity":"89730a85-956a-4a95-a4a8-e9cba37a98dd","added_by":"auto","created_at":"2026-01-19 16:03:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1052746,"visible":true,"origin":"","legend":"\u003cp\u003emiR-99b-5p attenuates neuroinflammation and neuronal apoptosis by suppressing microglial M1 polarization in a co-culture system. (A) Serum levels of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were measured by ELISA in SCI individuals stratified by low and high serum miR-99b-5p expression. (B-C) Concentrations of the M1 polarization markers iNOS and TNF-α in the upper chamber (microglial compartment) were quantified by ELISA. (D-E) Levels of the neuroinflammatory factors IL-6 and IL-1β in the lower chamber (neuronal compartment) were determined by ELISA. (F-H) Expression of the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 in SH-SY5Y cells from the lower chamber was analyzed by Western Blot. Representative blots (F) and quantitative analysis (G, H) are shown. Data are presented as mean ± SD. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure415.png","url":"https://assets-eu.researchsquare.com/files/rs-8525596/v1/a15b52c0780133975d049c88.png"},{"id":100610349,"identity":"ee57d38a-5706-4f12-b3f1-2cbc10b62b36","added_by":"auto","created_at":"2026-01-19 16:32:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3676720,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8525596/v1/8927a029-1080-43f2-8348-eb4771f61de0.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"miR-99b-5p Targets AKT1 to Modulate Microglial Polarization: A Prognostic Biomarker for Spinal Cord Injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFollowing spinal cord injury (SCI), the complex pathological cascade is central to the development of long-term neurological deficits, with immune and inflammatory responses driving secondary injury being particularly critical [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In this process, the dynamic polarization of microglia and infiltrating macrophages profoundly influences the homeostasis of the local microenvironment and the outcomes of neural repair [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, elucidating the key molecular mechanisms that regulate the fate of these immune cells is an important direction for exploring novel therapeutic strategies for SCI.\u003c/p\u003e \u003cp\u003eIn recent years, microRNAs (miRNAs), as key post-transcriptional regulators of gene expression, have garnered increasing attention for their roles in the pathological progression of SCI [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. miR-99b-5p has emerged as a molecule of particular interest due to its diverse regulatory roles across multiple disease contexts. Current research highlights its core function in modulating immune-inflammatory pathways and cell survival, with distinct roles reported in different pathological settings. In cancer, for example, miR-99b-5p acts as a tumor suppressor by targeting the mTOR/AR axis, thereby inhibiting the proliferation of prostate cancer cells and inducing autophagy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. miR-99b-5p can alleviate apoptosis and pyroptosis of neurons after paclitaxel chemotherapy by inhibiting the activation of NLRP3 and improving oxidative stress [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Beyond these, it participates in immune regulation in osteoarthritis and schizophrenia-like models, where it modulates macrophage polarization or microglial inflammatory responses via pathway-specific mechanisms [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Collectively, these findings establish miR-99b-5p as a key regulator of cell fate and immune homeostasis, with potential as a biomarker and therapeutic target across various diseases.\u003c/p\u003e \u003cp\u003eStudies have shown that miR-99b-5p expression is upregulated in mice after SCI and that it can target the mTOR signaling pathway [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Inhibition of miR-99b-5p can reverse the injury-induced downregulation of mTOR, thereby reducing neuronal apoptosis and promoting neurite outgrowth [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, despite these findings, the specific role and mechanisms of miR-99b-5p in the macrophage/microglia-centered immune microenvironment following SCI remain unclear. In particular, there is a lack of direct evidence regarding its cell type-specific regulatory functions, especially its ability to intervene in the polarization of macrophages/microglia directly. This knowledge gap limits the potential for developing this molecule into a precise therapeutic target for SCI.\u003c/p\u003e \u003cp\u003eTo address this gap, this study aims to systematically elucidate the specific role and molecular mechanisms of miR-99b-5p in remodeling the immune microenvironment after SCI, with a focus on its regulatory effects on macrophage/microglia polarization. The findings are expected to provide new insights into the immunopathological mechanisms of SCI and offer a potential therapeutic target for developing immune modulation-based neural repair strategies.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Study participants\u003c/h2\u003e \u003cp\u003eIn this study, 90 individuals with SCI admitted to Hankou Hospital of Wuhan City from June 2022 to June 2024 were enrolled as the study group. Additionally, 70 healthy individuals with routine physical examinations during the same period were selected as controls, excluding those with neurological disorders, inflammatory conditions, or organic lesions. This study was approved by the Hospital Ethics Committee, adhering to the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Main Reagents and Instruments\u003c/h2\u003e \u003cp\u003eCell lines (HMC3, THP-1, SH-SY5Y, HEK293T) were from the Cell Bank of the Chinese Academy of Sciences. miR-99b-5p mimics/inhibitors (RiboBio), AKT1 overexpression plasmid (GeneChem), ELISA kits (Boster), qRT-PCR kits (TaKaRa), and the Dual-Luciferase\u0026reg; System (Promega) were used. Key instruments included an ABI 7500 qPCR system and a ChemiDoc XRS\u0026thinsp;+\u0026thinsp;imager.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Clinical Sample Detection and Functional Evaluation\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Serum Analysis\u003c/h2\u003e \u003cp\u003eSerum was isolated and stored at -80\u0026deg;C. Inflammatory cytokines (TNF-α, IL-1β, IL-6) and polarization markers (M1 markers: TNF-α, iNOS; M2 markers: CD206, Arg-1) were quantified by ELISA. Serum miR-99b-5p levels were quantified by qRT-PCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Neurological and Functional Recovery Assessment\u003c/h2\u003e \u003cp\u003eEvaluations were conducted using standardized scales. The severity of neurological impairment was graded using the American Spinal Injury Association Impairment Scale (AIS), while functional recovery was comprehensively assessed through the Functional Independence Measure (FIM), the Spinal Cord Independence Measure III (SCIM-III), and the Walking Index for SCI II (WISCI-II). All assessments were independently conducted by two experienced clinicians who were blinded to the laboratory results.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 In Vitro Cell Experiments\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Cell Culture and Group Treatment\u003c/h2\u003e \u003cp\u003eAll cell lines were cultured in DMEM supplemented with 10% fetal bovine serum. THP-1 cells were differentiated into macrophages using 100 nM PMA for 24 hours. For functional studies, cells were treated with LPS (5 \u0026micro;g/mL, 24 h) to induce M1 polarization, following transfection with miR-99b-5p mimics, inhibitors, or the AKT1 overexpression plasmid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 CCK-8 Assay for Cytotoxicity\u003c/h2\u003e \u003cp\u003eHMC3 and THP-1 cells were seeded in 96-well plates, treated with miR-99b-5p mimic for 24 h, and 10 \u0026micro;L of CCK-8 reagent was added. Absorbance at 450 nm was measured, and cell viability was calculated to determine a non-cytotoxic working concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 qRT-PCR for Polarization Marker Expression\u003c/h2\u003e \u003cp\u003eTotal RNA from cells was reverse-transcribed. mRNA levels of M1 markers (iNOS, TNF-α) and M2 markers (CD206, Arg-1) were measured by qRT-PCR, with \u003cem\u003eGAPDH\u003c/em\u003e as the endogenous control. Relative expression was calculated using the 2^(-ΔΔCt) method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4 Microglia-Neuron Co-culture\u003c/h2\u003e \u003cp\u003eA non-contact co-culture system was established using Transwell chambers. HMC3 microglia (upper chamber) were stimulated with LPS and transfected. SH-SY5Y neurons (lower chamber) were cultured separately. Conditioned media and cells from both compartments were collected after 48 hours for downstream analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.5 Western Blot for Protein Expression\u003c/h2\u003e \u003cp\u003eProteins from SH-SY5Y cells were extracted, separated by SDS-PAGE, and transferred to PVDF membranes. Membranes were incubated with primary antibodies against Bax, Bcl-2, and β-actin, followed by HRP-conjugated secondary antibodies. Protein bands were visualized using an ECL substrate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.4.6 Dual-Luciferase Reporter Assay\u003c/h2\u003e \u003cp\u003eAKT1 wild-type (WT) and mutant (MUT) 3\u0026prime;-UTR fragments were cloned into luciferase reporter plasmids. HEK293T cells were co-transfected with reporter plasmids and NC mimic/miR-99b-5p mimic. Luciferase activities were measured 48 h post-transfection, with relative activity normalized to Renilla luciferase luminescence.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Bioinformatics Analysis\u003c/h2\u003e \u003cp\u003eThe genes associated with SCI were retrieved from the GeneCards database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), using a relevance score cutoff of \u0026ge;\u0026thinsp;20. This threshold is commonly applied to prioritize genes that have direct functional links to SCI pathophysiology [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Predicted targets of miR-99b-5p were obtained from the miRWalk database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mirwalk.umm.uni-heidelberg.de/\u003c/span\u003e\u003cspan address=\"http://mirwalk.umm.uni-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with the following filtering criteria: (a) predicted by at least 3 of the 12 integrated algorithms (including TargetScan, miRanda, RNAhybrid, etc.) to ensure reliability; (b) conserved binding sites between miR-99b-5p and the 3\u0026prime;-UTR of target genes (as defined by TargetScan conservation score\u0026thinsp;\u0026ge;\u0026thinsp;0.5); (c) excluding genes with predicted binding free energy \u0026gt; -20 kcal/mol (indicating unstable binding interactions).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS 26.0, and graphs were generated with GraphPad Prism 8.0. Continuous data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (x̄ \u0026plusmn; s). Between-group comparisons for continuous data were analyzed by Student\u0026rsquo;s t-test (two groups) or one-way analysis of variance (ANOVA) with LSD post-hoc test (multiple groups). Categorical data were compared using the chi-square test. Correlations between serum miR-99b-5p levels and neurological/clinical variables were evaluated by Spearman\u0026rsquo;s rank correlation analysis. A two-tailed \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eHigh miR-99b-5p Expression Correlates with Improved Functional Recovery in Individuals with SCI\u003c/em\u003e \u003c/p\u003e \u003cp\u003eBaseline demographic characteristics, including gender, age, and body mass index (BMI), were comparable between the SCI group and healthy controls (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which helped minimize potential confounding effects.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of Baseline Data between Healthy Control Individuals and SCI Individuals\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl group\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;70\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSCI group\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;90\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBasic information\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39.77\u0026thinsp;\u0026plusmn;\u0026thinsp;12.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.77\u0026thinsp;\u0026plusmn;\u0026thinsp;12.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.366\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI (kg/m\u0026sup2;, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003csup\u003e)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.925\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender (Male/Female, n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34/36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47/43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.647\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCause of damage\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTraffic accidents (n, %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31/33.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFalls from height (n, %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21/22.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInjury from a heavy object (n, %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22/23.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOther (n, %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16/17.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eInjured segments\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCervical segment (n, %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27/29.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThoracolumbar segment (n, %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30/32.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWaist section (n, %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33/35.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: SCI, spinal cord injury; BMI, body mass index (unit: kg/m\u0026sup2;). \"-\" indicates data not applicable to the corresponding group.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhile miR-99b-5p upregulation has been documented in murine SCI models, its serum expression profile in humans with SCI remains largely uncharacterized [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Given the well-recognized role of miRNAs in regulating the pathophysiological processes of SCI [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], serum miR-99b-5p levels were quantified initially. Consistent with trends reported in preclinical animal studies, serum miR-99b-5p expression was significantly higher in the SCI group than in the healthy control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Receiver operating characteristic (ROC) curve analysis was further performed to evaluate its discriminative ability, showing an area under the curve (AUC) of 0.872 (95% CI, 0.820 to 0.923, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), indicating that serum miR-99b-5p has promising diagnostic potential for SCI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the associations between serum miR-99b-5p and clinical characteristics in individuals with SCI, Spearman correlation analysis was conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The results showed no significant correlations between miR-99b-5p levels and non-neurological variables (age, BMI, gender, cause of damage, injured segments; all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast, miR-99b-5p was positively correlated with key neurological indices: the correlation coefficient (Spearman\u0026rsquo;s ρ) was 0.344 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) for AIS grade, 0.551 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) for FIM score, 0.527 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) for SCIM-III score, and 0.724 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) for WISCI-II score. To further explore the diagnostic value of serum miR-99b-5p for neurological outcomes in SCI, ROC curve analysis was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The results revealed that miR-99b-5p had good discriminatory ability: the AUC was 0.817 (95% CI, 0.731 to 0.902, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) for AIS grade, 0.789 (95% CI, 0.694 to 0.884, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) for FIM score, 0.853 (95% CI, 0.769 to 0.937, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) for SCIM-III score, and 0.639 (95% CI, 0.524 to 0.754, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018) for WISCI-II score.\u003c/p\u003e \u003cp\u003eIn summary, the findings demonstrate that elevated serum miR-99b-5p levels are strongly correlated with favorable functional recovery in individuals with SCI.\u003c/p\u003e \u003cp\u003e \u003cem\u003eHigh Expression of miR-99b-5p Significantly Inhibits M1 Polarization of Microglia\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWithin the complex microenvironment of SCI, the polarization states of macrophages and microglia have been established as pivotal regulators of secondary injury and repair processes, whose dynamic balance critically influences neuronal survival, axonal regeneration, and functional recovery outcomes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To investigate whether miR-99b-5p is involved in regulating this balance, serum levels of polarization markers were measured initially. Individuals with SCI were stratified into low expression and high expression groups based on the median serum miR-99b-5p level. Compared with the low expression group, individuals in the high miR-99b-5p group exhibited significantly lower levels of M1 markers (iNOS and TNF-α) and higher levels of M2 markers (CD206 and Arg-1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), suggesting a potential role for miR-99b-5p in suppressing M1 polarization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further verify this regulatory effect and assess its cell-type specificity, in \u003cem\u003evitro\u003c/em\u003e M1 polarization models were established using HMC3 microglia and THP-1 macrophages. The miR-99b-5p mimic showed no significant cytotoxicity in either cell line as determined by the CCK-8 assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C), and a concentration of 10 \u0026micro;M was therefore selected for subsequent experiments. In LPS-stimulated HMC3 microglia, the miR-99b-5p mimic significantly suppressed the upregulation of M1 markers (iNOS and TNF-α) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In contrast, in THP-1 macrophages, the miR-99b-5p mimic did not consistently reverse LPS-induced M1 polarization but significantly attenuated the LPS-mediated downregulation of the M2 marker Arg-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), highlighting the complexity and cell-context dependency of macrophage polarization regulation. Together, these results indicate that miR-99b-5p selectively inhibits M1 polarization in microglia while exerting a distinct regulatory effect on macrophages.\u003c/p\u003e \u003cp\u003e \u003cem\u003emiR-99b-5p Attenuates Microglial M1 Polarization by Directly Targeting AKT1\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo systematically identify the downstream targets and signaling pathways of miR-99b-5p in SCI, two gene sets were integrated for analysis: (1) SCI-associated genes with high relevance (GeneCards score\u0026thinsp;\u0026ge;\u0026thinsp;20) to ensure functional relevance to SCI; (2) high-confidence targets of miR-99b-5p (predicted by \u0026ge;\u0026thinsp;3 algorithms, conserved binding sites, and binding free energy \u0026lt; -20 kcal/mol) to reduce false-positive predictions. This integration yielded 933 overlapping candidate genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), which were hypothesized to mediate the biological effects of miR-99b-5p in SCI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKEGG pathway enrichment analysis identified 15 significantly enriched pathways. The top 10 pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) included multiple signaling cascades and biological processes closely related to SCI pathophysiology: (1) Pathways in cancer; (2) MAPK signaling pathway; (3) Pathways of neurodegeneration \u0026ndash; multiple diseases; (4) Neuroactive ligand-receptor interaction; (5) AGE-RAGE signaling pathway in diabetic complications; (6) Colorectal cancer; (7) cAMP signaling pathway; (8) Cell adhesion molecules; (9) Apoptosis; (10) Cholinergic synapse (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Among these, the MAPK signaling pathway was prioritized for further validation. This pathway is a well-characterized core regulator of microglial polarization, neuroinflammation, and neuronal survival\u0026mdash;key pathological processes driving secondary injury after SCI [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Consistent with this prediction, LPS stimulation significantly upregulated the expression of key MAPK members (p38 MAPK, ERK1/2, and JNK), and miR-99b-5p mimic transfection significantly reversed this upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), confirming that miR-99b-5p suppresses microglial M1 polarization at least partially via modulating the MAPK pathway.\u003c/p\u003e \u003cp\u003eTo identify the direct upstream target of miR-99b-5p mediating this effect, a protein-protein interaction (PPI) network was constructed from the intersecting genes. Among top-ranked hub genes, AKT serine/threonine kinase 1 (AKT1) was a promising candidate. Although its pivotal role in modulating microglial polarization through the PI3K/AKT pathway is well-recognized [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], whether it serves as a functional target for miR-99b-5p in the context of SCI remained unknown. In silico analysis predicted a high-probability binding site for miR-99b-5p within the 3\u0026prime;-UTR of AKT1, featuring canonical seed region complementarity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). This interaction was experimentally confirmed by dual-luciferase reporter assay, where the miR-99b-5p mimic significantly suppressed luciferase activity of the wild-type (WT) AKT1 3'-UTR reporter but not the mutant (MUT) construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), confirming AKT1 as a direct target of miR-99b-5p.\u003c/p\u003e \u003cp\u003eRescue experiments verified the functional relevance of the miR-99b-5p-AKT1 interaction. miR-99b-5p mimic effectively inhibited LPS-induced upregulation of M1 markers (iNOS, TNF-α), while AKT1 co-overexpression partially but significantly reversed this suppression (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G).\u003c/p\u003e \u003cp\u003eCollectively, these findings demonstrate that miR-99b-5p directly targets AKT1, modulates downstream pathways including MAPK, and thereby attenuates microglial M1 polarization, providing novel insights into the molecular mechanism of miR-99b-5p in regulating microglial polarization during SCI.\u003c/p\u003e \u003cp\u003e \u003cem\u003emiR-99b-5p Alleviates Neuroinflammation and Neuronal Apoptosis by Suppressing Microglial M1 Polarization\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo link the observed clinical associations with the proposed cellular mechanisms, the correlation between serum miR-99b-5p levels and systemic inflammation was investigated. The results showed that individuals with high serum miR-99b-5p expression exhibited significantly lower levels of key pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) compared with those with low expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify whether miR-99b-5p exerts neuroprotection by regulating microglial paracrine signaling, a controlled in \u003cem\u003evitro\u003c/em\u003e model was established using a non-contact Transwell co-culture system to isolate the effects of soluble factors secreted by HMC3 microglia on SH-SY5Y neurons. Transfection of miR-99b-5p mimic into LPS-stimulated microglia significantly downregulated M1 marker secretion (iNOS, TNF-α) and reduced neuroinflammatory factors (IL-6, IL-1β) in the neuronal compartment compared to the LPS control. Conversely, the miR-99b-5p inhibitor exacerbated these inflammatory responses (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-E). These results confirm that miR-99b-5p mitigates neuroinflammation via a microglia-dependent paracrine mechanism.\u003c/p\u003e \u003cp\u003eThe impact of attenuated inflammation on neuronal survival was subsequently explored. KEGG analysis implicated the apoptosis pathway, prompting an examination of key apoptotic regulators. In neurons co-cultured with miR-99b-5p-overexpressing microglia, pro-apoptotic Bax protein expression was significantly decreased, while anti-apoptotic Bcl-2 levels were increased (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-H). This anti-apoptotic effect was validated using an independent pre-miR-99b-5p overexpression system with consistent results.\u003c/p\u003e \u003cp\u003eCollectively, data from the clinical cohort and reductionist co-culture model demonstrate that miR-99b-5p dually attenuates neuroinflammation and inhibits neuronal apoptosis, dependent on its ability to suppress microglial M1 polarization.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study delineates a neuroprotective role for miR-99b-5p in SCI by demonstrating its capacity to mitigate neuroinflammation and neuronal apoptosis through the specific suppression of microglial M1 polarization via the AKT1 pathway. These findings integrate miR-99b-5p into the immunoregulatory network of the injured spinal cord, underscore the significance of miRNA-mediated regulation in neural injury, and offer novel insights into the underlying pathophysiological mechanisms.\u003c/p\u003e \u003cp\u003eClinically, serum levels of miR-99b-5p were found to be significantly elevated in individuals following SCI. Notably, higher circulating levels of this microRNA correlated with less severe neurological impairment and more favorable functional recovery, reinforcing the concept that the upregulation of specific protective microRNAs post-injury is associated with improved clinical outcomes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. While prior investigations have largely centered on other microRNAs such as miR-21 and miR-124, the present study is the first to establish a direct link between miR-99b-5p and clinical prognosis in human SCI, thereby expanding the scope of potential prognostic biomarkers. Collectively, the results suggest that miR-99b-5p functions not merely as a passive injury marker but as an endogenous protective factor that actively mitigates secondary pathological processes. This aligns with the broader theoretical framework wherein microRNAs are recognized as key regulators of cellular homeostasis within the injury microenvironment, critically influencing the ultimate course of tissue repair and recovery [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA pivotal mechanistic finding is the specific inhibition of microglial M1 polarization by miR-99b-5p, supported by both clinical biomarker correlations and direct in \u003cem\u003evitro\u003c/em\u003e evidence. In serum from individuals with SCI, elevated miR-99b-5p levels were inversely correlated with classic M1 polarization markers, including iNOS and TNF-α, reinforcing the well-established link between balanced microglial polarization and functional recovery [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Notably, this regulatory effect exhibited distinct cell-type specificity, being pronounced in HMC3 microglia but minimal in THP-1-derived macrophages. This distinction advances the understanding of lineage-selective miRNA actions in immune regulation and highlights a potential avenue for precise therapeutic intervention in SCI that may spare systemic immunity [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Bioinformatic analysis implicated the MAPK pathway, a central regulator of microglial activation and SCI progression. As AKT1 is a known upstream modulator of MAPK signaling, a coherent regulatory axis is proposed whereby miR-99b-5p targets AKT1 to modulate MAPK activity, thereby suppressing microglial M1 polarization.\u003c/p\u003e \u003cp\u003eThe translational potential of miR-99b-5p is underscored by its dual role, serving both as a quantifiable serum biomarker and as a functional regulator of the local spinal cord microenvironment. Its serum levels correlate with both the extent of systemic inflammation and the degree of neurological recovery, suggesting its potential utility as an auxiliary, non-invasive prognostic tool for the early clinical assessment of SCI. To investigate the direct neuroimmune interaction underlying this clinical correlation, an in vitro co-culture model was employed to isolate and examine the paracrine axis between microglia and neurons [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The experimental results established a clear causal relationship: overexpression of miR-99b-5p in microglia directly suppressed their polarization toward the M1 phenotype and the secretion of pro-inflammatory factors, which was sufficient to attenuate neuronal apoptosis. This finding provides direct mechanistic evidence for the therapeutic strategy of achieving neuroprotection by targeting the regulation of microglial polarization [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNevertheless, the limitations inherent in this simplified in vitro model are acknowledged. While invaluable for dissecting specific cellular interactions, it cannot replicate the full pathophysiological complexity of the SCI microenvironment, which includes dynamic crosstalk among astrocytes, oligodendrocytes, infiltrating peripheral immune cells, and the evolving glial scar, all critical factors influencing repair and regeneration [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Most importantly, the anti-inflammatory and neuroprotective efficacy observed in vitro requires validation in a living organism. Therefore, future investigations utilizing established in vivo rodent spinal SCI models are essential for verifying therapeutic efficacy, defining pharmacokinetic profiles and optimal delivery strategies, and evaluating the long-term functional impact of miR-99b-5p modulation within an integrated physiological context [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn summary, this work establishes miR-99b-5p as a multifaceted candidate in SCI: a serologically accessible prognostic biomarker and a cell-type-specific molecular regulator that alleviates neuroinflammation and neuronal apoptosis by targeting AKT1-mediated microglial polarization. These insights offer a novel therapeutic target and a refined conceptual framework for mitigating the detrimental neuroimmune cascade following SCI.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge all patients with SCI and healthy volunteers who participated in this study, as well as the medical team of the Orthopedics Department, Hankou Hospital, Wuhan City, Hubei Province, for their support in sample recruitment and clinical data collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDeming Chen: Data curation, Methodology, Writing \u0026ndash; review \u0026amp; editing. Shibo Feng: Formal analysis, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eChenge Xian: Investigation, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eRuye Li: Resources, Conceptualization, Project administration , Supervision, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank all patients with SCI and healthy volunteers for their participation in this study. We also acknowledge the Orthopedics Medical Team of Hankou Hospital, Wuhan, Hubei Province, for their valuable support in sample collection and clinical data acquisition. Additionally, we are grateful to the Scientific Research Department of Hankou Hospital for providing the experimental platform and technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eNo known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was carried out following the Declaration of Helsinki and applicable national and international guidelines for research involving human participants. It received ethical approval from the Ethics Committee of Hankou Hospital in Wuhan City (Ethics Approval Number: Hanyilunshen [2022]-RC-012). Before starting the study, the research protocol, informed consent form, and other relevant materials were reviewed and approved by the ethics committee to ensure participants\u0026apos; safety and adherence to research ethics. Informed consent was obtained from all participants involved in the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZheng B, Tuszynski MH. Regulation of axonal regeneration after mammalian spinal cord injury. Nat Rev Mol Cell Biol. 2023;24(6):396\u0026ndash;413.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHellenbrand DJ, Quinn CM, Piper ZJ, Morehouse CN, Fixel JA, Hanna AS, et al. Inflammation after spinal cord injury: a review of the critical timeline of signaling cues and cellular infiltration. 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Brain Res. 2025;1849:149381.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCianciulli A, Porro C, Calvello R, Trotta T, Lofrumento DD, Panaro MA. Microglia Mediated Neuroinflammation: Focus on PI3K Modulation. Biomolecules. 2020;10(1):137.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi YJ, Wang Y, Wang YY. MicroRNA-99b suppresses human cervical cancer cell activity by inhibiting the PI3K/AKT/mTOR signaling pathway. J Cell Physiol. 2019;234(6):9577\u0026ndash;9591.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi D, Dai Y, Li Z, Bi H, Li H, Wang Y, et al. Resveratrol Upregulates miR-124-3p Expression to Target DAPK1, Regulating the NLRP3/Caspase-1/GSDMD Pathway to Inhibit Pyroptosis and Alleviate Spinal Cord Injury. J Cell Mol Med. 2025;29(2):e70338.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRupp R, Biering-S\u0026oslash;rensen F, Burns SP, Graves DE, Guest J, Jones L, et al. International Standards for Neurological Classification of Spinal Cord Injury: Revised 2019. Top Spinal Cord Inj Rehabil. 2021;27(2):1\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Z, Zhang X, Lin X, Wang Y, Han C, Wang S. Research Advances and Application Progress on miRNAs in Exosomes Derived From M2 Macrophage for Tissue Injury Repairing. Int J Nanomedicine. 2025;20:1543\u0026ndash;1560.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu C, Jin X, Ji K, Lan K, Mao X, Huang Z, et al. Macrophage-targeted Mms6 mRNA-lipid nanoparticles promote locomotor functional recovery after traumatic spinal cord injury in mice. Sci Adv. 2025;11(13):eads2295.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan Z, Jia M, Zhou J, Zhu Z, Wu Y, Lin X, et al. Pharmacological targeting cGAS/STING/NF-κB axis by tryptanthrin induces microglia polarization toward M2 phenotype and promotes functional recovery in a mouse model of spinal cord injury. Neural Regen Res. 2025;20(11):3287\u0026ndash;3301.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao J, He Z, Wang J. MicroRNA-124: A Key Player in Microglia-Mediated Inflammation in Neurological Diseases. Front Cell Neurosci. 2021;15:771898.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao X, Bie B, Wang Z, Chen Y, Sheng L, Li M. MiR-223-3p promote microglia \"M2\" polarization by targeting FOXO3a in subarachnoid hemorrhage. J Neuroimmunol. 2025;406:578677.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, He X, Kawaguchi R, Zhang Y, Wang Q, Monavarfeshani A, et al. Microglia-organized scar-free spinal cord repair in neonatal mice. Nature. 2020;587(7835):613\u0026ndash;618.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang YP, Qin ZH, Zhang Y, Ning B. Implications of microglial heterogeneity in spinal cord injury progression and therapy. Exp Neurol. 2023;359:114239.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBellver-Landete V, Bretheau F, Mailhot B, Valli\u0026egrave;res N, Lessard M, Janelle ME, et al. Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury. Nat Commun. 2019;10(1):518.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHutson TH, Di Giovanni S. The translational landscape in spinal cord injury: focus on neuroplasticity and regeneration. Nat Rev Neurol. 2019;15(12):732\u0026ndash;745.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"spinal-cord","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"sc","sideBox":"Learn more about [Spinal Cord](http://www.nature.com/sc/)","snPcode":"41393","submissionUrl":"https://mts-sc.nature.com/cgi-bin/main.plex","title":"Spinal Cord","twitterHandle":"@journalsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Spinal cord injury, miR-99b-5p, microglial polarization, AKT1","lastPublishedDoi":"10.21203/rs.3.rs-8525596/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8525596/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eStudy design:\u003c/h2\u003e \u003cp\u003eCross-sectional study.\u003c/p\u003e\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eThis study aimed to investigate the mechanism by which miR-99b-5p regulates microglial polarization via targeting AKT1, and to evaluate its potential as a prognostic biomarker and therapeutic target for spinal cord injury (SCI).\u003c/p\u003e\u003ch2\u003eSetting:\u003c/h2\u003e \u003cp\u003eThe study was conducted at Wuhan Hankou Hospital and Nedong District People's Hospital, China.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 90 individuals with SCI and 70 healthy controls were enrolled. Serum levels of miR-99b-5p, inflammatory cytokines, and microglial polarization markers were quantified. Functional outcomes were assessed using the AIS, FIM, SCIM-III, and WISCI-II scales. In \u003cem\u003evitro\u003c/em\u003e investigations included cell transfection, LPS-induced polarization, co-culture, and dual-luciferase reporter assays.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSerum miR-99b-5p was significantly elevated in SCI individuals (AUC\u0026thinsp;=\u0026thinsp;0.872) and positively correlated with AIS grade and functional outcomes (FIM, SCIM-III, WISCI-II). The miR-99b-5p mimic selectively inhibited M1 polarization in microglia, downregulating markers such as iNOS and TNF-α, while exhibiting no significant effect on macrophages. AKT1 was identified as a direct target of miR-99b-5p, and its overexpression reversed the inhibitory effect of miR-99b-5p on microglial polarization. Furthermore, miR-99b-5p alleviated neuronal inflammation and apoptosis, as evidenced by decreased Bax and increased Bcl-2 expression, through its regulatory action on microglia.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003emiR-99b-5p targets AKT1 to suppress microglial M1 polarization, thereby alleviating neuroinflammation and neuronal damage in SCI. Its serum level serves as a promising prognostic biomarker and potential therapeutic target.\u003c/p\u003e","manuscriptTitle":"miR-99b-5p Targets AKT1 to Modulate Microglial Polarization: A Prognostic Biomarker for Spinal Cord Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 15:00:07","doi":"10.21203/rs.3.rs-8525596/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-02-27T15:29:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-15T19:26:15+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-02T08:44:57+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-01-30T00:55:43+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-22T16:48:02+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-19T14:13:08+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-15T08:53:53+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-01-15T04:34:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-12T22:51:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-12T22:49:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Spinal Cord","date":"2026-01-12T02:52:27+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-01-06T15:59:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"spinal-cord","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"sc","sideBox":"Learn more about [Spinal Cord](http://www.nature.com/sc/)","snPcode":"41393","submissionUrl":"https://mts-sc.nature.com/cgi-bin/main.plex","title":"Spinal Cord","twitterHandle":"@journalsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f24e3c72-ebcd-4ed3-b533-3d0c006f164d","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61172609,"name":"Health sciences/Medical research/Biomarkers/Prognostic markers"},{"id":61172610,"name":"Biological sciences/Neuroscience/Spine regulation and structure/Spine motility"}],"tags":[],"updatedAt":"2026-04-21T03:20:07+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-19 15:00:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8525596","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8525596","identity":"rs-8525596","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}