Spinal cord injury (SCI) is a complex central nervous system disease that has emerged as a major global health issue. The global incidence of SCI is estimated to be around 500,000 cases annually, and this number continues to rise (Karsy and Hawryluk, 2019). The primary function of the spinal cord is to transmit sensory and motor signals between the brain and other parts of the body (Akinrodoye and Lui, 2021). As such, SCI is one of the most severe types of injury that can occur to the central nervous system. Various factors, including trauma, falls, collisions, spinal cord infections, or tumors, can cause spinal cord injuries, and it results in motor, sensory, and sphincter disorders below the level of the injury (Patil et al., 2015; Hamid et al., 2018). SCI can also lead to pressure ulcers, neuropathic pain, and a range of complications related to urinary dysfunction. Due to the loss of nerve tissue, the recovery of nerve function is limited, which negatively affects synaptic remodeling and neural circuit regeneration. Following an SCI, cavities or scars are formed by an influx of inflammatory cells at the injury site, which can impair the healing of the SCI. Despite intense research efforts and significant interest in the treatment of SCI, there have been relatively few significant breakthroughs that have translated into clinical applications (Freund et al., 2019). As such, the prevention, treatment, and cure of SCI have become a key concern for contemporary society, given the burden it places on families and communities.

Spinal cord injuries are typically classified into two stages: acute and chronic (Anjum et al., 2020). Cell-based therapy, particularly in the subacute stage, has emerged as an effective treatment for promoting neuronal recovery and nerve regeneration (Nishimura et al., 2013). Menstrual blood-derived stem cells (MenSCs) are a new type of mesenchymal stem cells with promising therapeutic effects in animal models and clinical trials (Chen et al., 2019; Zhu et al., 2019). In a previous study, we found that MenSCs transplantation in rat SCI models resulted in significant functional recovery and reduced inflammation and lesion cavities (Wu et al., 2018). However, the underlying mechanisms of MenSC therapy remain unknown. Recent developments in bioinformatics have shed new light on protein-coding mRNAs and non-coding RNAs (ncRNAs) and their role in SCI (Li et al., 2019; Peng et al., 2020; Salah et al., 2020; Wang J. et al., 2020). Molecular changes, including microRNAs (miRNAs), long ncRNAs (lncRNAs), and circular RNAs (circRNA), have been reported to play important roles in the pathophysiology of SCI, affecting various physiological processes such as cell proliferation, differentiation, apoptosis, and synaptic plasticity (Li M. et al., 2018; Zhang S. B. et al., 2019). Understanding these molecular changes may help in elucidating the therapeutic mechanisms of MenSCs and inform the development of more effective treatments for SCI (Chen and Schuman, 2016).

Non-coding RNAs (ncRNAs) are a crucial type of RNA that are involved in various genetic and epigenetic processes such as transcriptional and post-transcriptional regulation (Geng et al., 2018; Panni et al., 2020). Interestingly, despite the fact that only 10% of the genome codes for proteins, most of it is transcribed into functional non-coding RNAs (Zhao et al., 2020). NcRNAs can be classified into basic structure and regulatory types, and they play a significant role in both physiological and pathological processes (Li M. et al., 2018), including the regulation of neuropathic pain and activation of Wnt/β-catenin signaling in the spinal cord (Sun et al., 2018; Cui et al., 2019; Zhang S. B. et al., 2019). Due to their potential as diagnostic biomarkers and therapeutic targets for various diseases, ncRNAs are currently a popular research area.

In our study, we utilized RNA-seq technology to analyze the differentially expressed genes (DEGs) in the epicenter of the spinal cord in a hemisection rat SCI model, and predicted their functions in order to better understand the potential diagnostic, prognostic, and therapeutic value of these DEGs. Notably, lncRNAs and circRNAs can contain common miRNA response elements (MREs), and thus act as sponges that competitively bind with miRNAs through these MREs, thereby regulating the expression of target gene transcripts and affecting each other. These RNAs can form competing endogenous RNA (ceRNA) networks in human diseases, mediated by miRNAs. Given the potential roles of ceRNA networks in SCI progression, it is important to comprehensively elucidate the interactions between lncRNA/circRNA and miRNA, which may lead to promising biomarkers for the diagnosis and treatment of SCI (Hansen et al., 2013).

In this study, we conducted a comprehensive analysis of the transcriptome of the injured spinal cord epicenter in MenSCs-treated SCI rat models and control groups. We examined the expression profiles of various genes, including lncRNAs, circRNAs, miRNAs, and mRNAs, and identified DEGs through heatmap and volcano plot analyses. Additionally, the biological functions of DEGs by GO and KEGG pathway analyses. We also established PPI and ceRNA networks to investigate potential mechanisms underlying the therapeutic effects of MenSCs in SCI. These findings provide valuable insights into the pathophysiology of SCI and suggest potential novel targets for SCI treatment.

Complex pathogenic processes, including acute and chronic phases, are activated by SCI, resulting in a succession of damaging events, such as inflammation, oxidative stress, ischemia, apoptosis, and motor dysfunction (Anjum et al., 2020). As regenerative medicine has advanced in recent years, stem cell transplantation has become a popular treatment for spinal cord injuries. The transplanted stem cells are capable of secreting nutritional factors, therefore stimulating vascular regeneration, cell differentiation, anti-inflammatory and anti-apoptosis (Gao et al., 2020; Gong et al., 2020; Yamazaki et al., 2020). Nonetheless, the underlying molecular mechanism by stem cell therapy has not yet been well understood.

In this study, we performed RNA-seq to investigate the gene expression pattern in rats with spinal cord injury (SCI) and MenSCs-treated rats 21 days after SCI. The T10 hemisection model was used to induce SCI in the rats, and MenSCs treatment was initiated 7 days after the operation. RNA sequencing was performed on collected specimens. To ensure the reliability and reproducibility of the injury model, we strictly followed a previously established protocol (Wu et al., 2018). Notably, MenSCs treatment resulted in a reduction in the volume of spinal cord lesions, as well as enhanced neuron survival, outgrew axon fibers, decreased CCR7 expression, and improved recovery of motor function. The use of RNA-seq enabled us to comprehensively evaluate the RNAs involved in the injury and treatment processes.

Genome-wide association studies (GWAS) offer a novel perspective on the molecular pathophysiology of diseases (Wang M. H. et al., 2019). Non-coding RNAs (ncRNAs) are enriched in the central nervous system and play a crucial role in growth and development, making them excellent candidates for disease biomarkers and therapeutic targets (Guennewig and Cooper, 2014). ncRNAs are also known to be involved in the key processes of SCI pathophysiology (Ding et al., 2016). In our RNA sequencing analysis, we identified 89 upregulated and 65 downregulated lncRNAs, as well as 65 upregulated and 72 downregulated circRNAs. Additionally, 120 miRNAs were upregulated, and 74 miRNAs were downregulated, while 422 mRNAs were upregulated, and 190 mRNAs were downregulated. These sequencing results were validated by qRT-PCR and were consistent with each other.

To investigate the potential functional implications of the differentially expressed (DE) mRNAs in spinal cord injury (SCI) and MenSCs treatment groups, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. These analyses were conducted to further elucidate the biological functions of the DE mRNAs. Molecular function analysis revealed that the DE mRNAs were involved in various functions, such as protein binding, signaling receptor binding, antigen binding, molecular function regulator, cytokine receptor activity, chemokine activity, and actin binding. Biological pathway analysis identified cell adhesion and immune system involvement in the process, which supported the construction of cell-cell interaction after injury, promoted axon growth, and reduced growth inhibitors near the injury site (Li R. et al., 2018; Platek et al., 2020). The ceRNA theory was utilized to analyze the regulatory network related to the DE ncRNAs and mRNAs in spinal cord tissue after traumatic injury (Wang N. et al., 2020). Significant changes in the expression of related ncRNAs and mRNAs were observed, and the structure and potential function of the regulatory network were predicted. Among the DE ncRNAs, the eight highest expressed node lncRNAs were selected for further analysis, which included MSTRG.17099.1, MSTRG.116.1, MSTRG.13543.1, MSTRG.11909.1, MSTRG.18736.1, MSTRG.12598.1, MSTRG.14094.1, and MSTRG.17976.1. Furthermore, the target miRNAs of these lncRNAs were predicted through the database. The results showed that MSTRG.17099.1/MSTRG.116.1/MSTRG.13543.1 targeted miR-741-3p, MSTRG.11909.1/MSTRG.18736.1 targeted miR-3084a-5p, and MSTRG.12598.1/MSTRG.14094.1 targeted miR-6318. The RNA sequencing data was verified by quantitative real-time PCR (qRT-PCR) and found to be consistent.

In previous studies, down-regulation of miR-741-3p has been shown to reduce neuronal apoptosis and decrease the expression of pro-inflammatory factors such as IL-6 and TNF-α (Ou et al., 2021). These findings suggest that MSTRG.17099.1/MSTRG.116.1/MSTRG.13543.1-regulated miR-741-3p may have far-reaching significance in SCI, and may represent a potential target for lncRNA-based therapies. By targeting WT1, miR-743a suppresses the proliferation of MM cells in vitro, and probably possesses vital functions in kidney development and kidney-associated diseases (Xue et al., 2016). As shown in the gene expression data in the MGI database, miRNA-743 is highly expressed in the head and craniocervical region of mice at embryonic days 12.5–14. However, the expression is diminished afterward. Subsequently, it is highly expressed in the head and craniocervical region of mice from postnatal days 4 to adulthood. Further exploration identified the central nervous system (CNS) of the brain and spinal cord as the main expression sites for miRNA-743, with no expression detected in the peripheral nervous system.⁶ This specific expression pattern, along with its potential involvement in cell proliferation, suggests a potential role in the treatment of spinal cord injury. However, to the best of our knowledge, the potential functions of miRNA-743, miR-3084a-5p and miR-6318 in the context of SCI have not yet been investigated. Further studies are needed to elucidate the potential role of these miRNAs in SCI and their interactions with the identified lncRNAs.

MicroRNAs (miRNAs) play a crucial role in the regulation of gene expression and provide valuable insights into the pathogenesis of various diseases. It has been reported that miRNAs regulate up to 60% of protein-coding genes and participate in various pathological processes (Friedman et al., 2009). For instance, miR-871-3p is involved in regulating cellular signaling and metabolic processes and may affect signaling pathways relevant to insulin secretion and some age-related diseases (Zhang et al., 2020). Similarly, miR-410-5p is involved in the development of diabetic cardiomyopathy and regulates cardiomyocyte apoptosis by targeting PIM1 protein and its downstream protein Bcl-2/Bax (Xia et al., 2020). This miRNA also acts as an endocrine regulator and promotes heart remodeling mediated by metabolic diseases (Zou et al., 2018). Additionally, miR-410-5p is a potential biomarker for prostate cancer and down-regulation of this miRNA can inhibit tumor angiogenesis and tumor growth (Wang et al., 2016, 2017). In the context of stroke, miR-331-5p has been found to mediate the neuroprotective effect of valproic acid-induced middle cerebral artery occlusion in rats after injury (Hunsberger et al., 2012). This miRNA regulates the NLRP3 inflammasome by targeting TRAF6 and may become a novel therapeutic target for ischemic stroke (Zhang H. S. et al., 2019). Furthermore, down-regulation of miR-741-3p can reduce neuronal apoptosis, and therefore, targeting this miRNA may offer a promising strategy for the treatment of SCI.

The analysis of mRNA molecules within the ceRNA network revealed that a majority of the genes had associations with regulating NK cell activity. NK cells serve an “effector” role by releasing cytotoxic granules against xenogeneic cells and an “affecter” role on other immune cells by secreting cytokines. The ceRNA network in current study was enriched with NK inhibitory receptors (KIRs) such as KIR2DL1, KIR2DL3, KIR2DL4, KIR2DS2, and KIR2DS4, as well as NK-activating receptor NKG2D (Liu et al., 2011; Sivori et al., 2019). This gene set is known to be associated with tolerance in xenotransplantation, immunocompetency in endometriosis, reproductive failure, malignant mesothelioma, and acute myeloid leukemia. Additionally, genes such as LILRB1 and Fc alpha receptor (CD89) have also been reported to play a role in immune response as vasculitis (Liu et al., 2022; Woon et al., 2022; Xu et al., 2022). The collection of mRNAs enlisted in the ceRNA network primarily focuses on regulating the inflammatory response function.

In the PPI network, the three hub genes CD74, RT1 class II locus DA (RT1-Da), and RT1-A2 were found. CD74 (Ii) was a type II transmembrane protein that regulated protein transport (Borghese and Clanchy, 2011). Leng et al. (2003) revealed in 2003 that CD74 is a receptor for the cytokine macrophage migration inhibitory factor (MIF). Numerous studies have demonstrated that MIF can regulate the immune system and neural stem cell regeneration via the CD74 pathway (Su et al., 2017; Farr et al., 2020), MIF-CD74 signal can promote the release of chemokine CCL5 from astrocytes, CCL5 promoted the migration of M2-macrophages, and regulated the inflammatory microenvironment in order to maintain homeostasis (Zhang Y. et al., 2019). MIF-CD74 stimulated macrophages by increasing MAPK phosphorylation and increased macrophage phagocytosis to govern regeneration of damaged spinal cord regions (Wang Y. et al., 2019). MIF-CD74 protected against intestine, liver, kidney, and cardiac injury (Farr et al., 2020). However, its involvement in spinal cord damage must be investigated further. RT1-Da contributed to the immune response through antigen processing and presentation of endogenous peptide antigen via MHC class II (Duyar et al., 2005), and was a prototypical MHC class II gene (Ettinger et al., 2004). Under healthy and pathological situations, RT1-A2 regulated the molecular pathways of brain processes such as learning, memory, and synaptic plasticity, according to bioinformatics analyses (VanGuilder Starkey et al., 2012; Fernandes et al., 2018). To confirm the significance of these genes in regulating the positive effects of SCI, however, more experimental verification and modification was required.

Another hub gene, CCR7, was identified to be involved in SCI. CCR7, a member of the G protein-coupled receptor family, is found in lymphoid organs and activates B and T cells. It regulates the migration of memory T lymphocytes to inflamed tissues and promotes dendritic cell maturation. CCR7, functioning as a chemokine receptor, significantly influences Tfh cell differentiation, lymphocyte homing, and germinal center formation (Arnold et al., 2007). Markedly upregulated expression of CCR7 was observed in patients suffered with pancreatic cancer, contributing to the enhancement of angiogenesis via chemotactically binding with CCL21 (Zhao et al., 2011). The regulation of Tfh cell differentiation by CCR7 significantly impacts multiple sclerosis relapse (Fan et al., 2015). The expression of CCR7 was found to be decreased in acute SCI patients. In the current study, an upregulation in the transcriptional level and a downregulation in the translational level of CCR7 were observed at day 21 post-surgery. These differences in mRNA and protein levels may be attributed to variances in post-translational modifications. Our research has unveiled the substantial role of CCR7 in the recovery of SCI patients.

In conclusion, based on our research, we have proposed fresh insights that can inspire the creation of novel therapeutic procedures. It should be mentioned, however, that our research is still preliminary, and due to the low number of samples from the sequencing results, additional research and validation are still required.

The data presented in the study are deposited in the NCBI SRA repository, accession numbers SRR17599937, SRR17599938, SRR17599939 and NCBI BioProject repository, accession number PRJNA1067278.

The animal study was approved by the Institutional Animal Care and Use Committee of Nantong University. The study was conducted in accordance with the local legislation and institutional requirements.

LQ: Funding acquisition, Investigation, Project administration, Resources, Writing—original draft. WJ: Data curation, Investigation, Methodology, Writing—original draft. WH: Formal analysis, Investigation, Methodology, Software, Writing—original draft. XL: Formal analysis, Methodology, Project administration, Visualization, Writing—original draft. JW: Data curation, Formal analysis, Methodology, Validation, Writing—original draft. SC: Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing—original draft. ZL: Formal analysis, Investigation, Validation, Visualization, Writing—original draft. SY: Formal analysis, Methodology, Software, Validation, Writing—original draft. JL: Methodology, Software, Validation, Writing—original draft. YS: Formal analysis, Methodology, Project administration, Writing—original draft. QFW: Conceptualization, Funding acquisition, Resources, Supervision, Writing—review and editing. CD: Conceptualization, Funding acquisition, Project administration, Supervision, Writing—original draft, Writing—review and editing. QHW: Conceptualization, Funding acquisition, Project administration, Supervision, Writing—original draft, Writing—review and editing.