Lncrna snhg8 encoding polypeptide and application thereof in spinal cord injury repair

Abstract

The application discloses a kind of lncRNA SNHG8 coding polypeptide and its application in spinal cord injury repair, belong to biological medicine field.The application first confirms that SNHG8 encodes two functional small peptides by two start codons, and based on this provides a kind of nucleotide comprising the nucleotide sequence of the two small peptides coding nucleotide sequence, overall nucleotide sequence is shown as SEQ ID NO.3, and the recombinant vector, recombinant lentivirus and recombinant bone marrow mesenchymal stem cell etc.of nucleotide sequence comprising this.Then it is shown by functional verification experiment that SNHG8 overexpression promotes BMSCs neurospheres formation and nerve marker expression, and BMSCs carrying SEQ ID NO.3 can significantly improve spinal cord injury model movement, sensation and electrophysiological function, reduce histopathological injury, promote axon regeneration, and its mechanism is related to activating MAPK pathway.The application provides a new strategy for spinal cord injury treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a lncRNA SNHG8 encoded polypeptide and its application in spinal cord injury repair. Background Technology

[0002] Spinal cord injury is a common and serious traumatic disease of the central nervous system, often caused by external factors such as traffic accidents, falls from heights, and violent impacts, as well as non-traumatic factors such as spinal degeneration, tumor compression, and vascular lesions. It is characterized by high disability and mortality rates and low cure rates, severely reducing patients' quality of life and imposing a heavy burden on families and society. The pathological process of spinal cord injury is divided into primary and secondary injuries. Primary injury is caused directly by external force, resulting in spinal cord tissue tearing, hemorrhage, and acute neuronal necrosis, which is instantaneous and irreversible. Secondary injury is a persistent pathological cascade reaction induced after primary injury, mainly including inflammatory storms, oxidative stress, neuronal apoptosis, excessive glial scar proliferation, and neurovascular microcirculatory disorders, which are the core causes of the continuous deterioration of neurological function.

[0003] Currently, spinal cord injury repair programs are broadly categorized into two types: routine clinical interventions and regenerative repair techniques. Routine clinical interventions primarily focus on symptomatic treatment and functional compensation, including acute-phase hormone pulse therapy, surgical decompression and fixation, rehabilitation physiotherapy, nerve electrical stimulation, and anti-inflammatory and antioxidant drugs. These can only alleviate the progression of secondary injury, reduce tissue edema, and prevent complications, but cannot achieve the regeneration of damaged neurons or the reconstruction of neural conduction pathways. Regenerative and functional reconstruction techniques are the core direction for overcoming the bottleneck of intractable spinal cord injury, encompassing stem cell regeneration and repair, intelligent neural regulation, biomaterial remodeling, and gene molecular targeted intervention. Among these, stem cell transplantation is the mainstream repair method with the largest volume of basic research and the most extensive clinical exploration. Relying on the dual effects of cell replacement and paracrine signaling, it can achieve in situ replenishment of nerve cells. However, in vitro expansion and culture still commonly suffer from problems such as stem cell loss, disordered differentiation, low in vivo survival rate, and insufficient targeted homing, resulting in significant individual differences in repair effects and unstable regeneration efficiency, which urgently require further optimization. Therefore, exploring novel molecular targets and precise intervention methods with high specificity that can regulate the pathological process of spinal cord injury and stem cell regeneration in multiple ways is a core research hotspot for overcoming existing repair bottlenecks.

[0004] Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs longer than 200 nucleotides that lack complete protein-coding functions. Recent studies have confirmed that they participate in various pathophysiological processes through ceRNA sponge adsorption, gene transcription regulation, epigenetic modification, and protein-protein interactions. With the advancement of transcriptomics research, the open reading frames of some lncRNAs have been shown to encode functional small polypeptides. These lncRNA-derived polypeptides can independently exert biological functions and synergistically participate in disease regulation with lncRNA transcripts, forming a dual regulatory system of "lncRNA-polypeptide".

[0005] Studies have shown that the expression of numerous lncRNAs fluctuates significantly after complete spinal cord transection injury in rats, influencing the degree of spinal cord injury and repair prognosis by regulating downstream inflammation, apoptosis, and oxidative stress-related pathways. lncRNA Malat1 can regulate astrocyte function by modulating the miR-124-3p axis; various exosome-derived lncRNAs can regulate microglia polarization and inhibit post-injury inflammatory storms; protective lncRNAs can inhibit M1-type pro-inflammatory polarization in microglia and alleviate oxidative stress damage, creating favorable conditions for axonal myelin regeneration and neural circuit reconstruction. Furthermore, lncRNAs can target and intervene in stem cell differentiation pathways such as BMP and Smad, regulating stem cell stemness maintenance and directed differentiation capacity. lncRNAs participate in mediating the entire process of secondary injury, neural microenvironment remodeling, and neural regeneration, and are key endogenous molecules regulating neuronal survival, glial cell activation, axonal regeneration, and blood-spinal cord barrier homeostasis.

[0006] Although existing research has clearly established the important regulatory role of lncRNAs in spinal cord injury, significant shortcomings remain. Most studies remain focused on expression profiling and single ceRNA pathway phenotypic validation, concentrating primarily on passive protective mechanisms such as inflammation and apoptosis. Research into the deeper mechanisms by which lncRNAs regulate the directed differentiation of neural stem cells, inhibit glial scar proliferation, and promote functional neurogenesis is weak. Furthermore, current research is largely limited to the non-coding regulatory functions of lncRNAs, with very little attention paid to novel regulatory modes involving open reading frames encoding functional peptides. The lack of systematic research on RNA-peptide dual regulatory networks severely restricts the clinical translation of lncRNA-targeted therapies for precise repair of spinal cord injury.

[0007] SNHG8 is a widely reported central nervous system protective lncRNA that is stably expressed in the central nervous system and exhibits specific low expression after nerve injury. Its expression level is closely related to the degree of nerve injury, the intensity of the inflammatory response, and the prognosis of nerve repair. Studies have shown that upregulation of SNHG8 can significantly inhibit excessive microglial activation and the release of pro-inflammatory factors, block the neuroinflammatory cascade, and reduce secondary damage. Simultaneously, it can antagonize neuronal apoptosis and improve neuronal survival, making it a promising novel molecular target for precise targeted repair of spinal cord injury. However, current research focuses only on basic phenotypic validation in a few models such as cerebral ischemia, and its specific expression characteristics and molecular regulatory mechanisms in spinal cord injury have not been systematically explored. Furthermore, its regulatory effects on stem cell differentiation, axonal myelin regeneration, and glial scar remodeling remain unclear.

[0008] Furthermore, relying solely on lncRNA for injury repair has inherent limitations: as nucleic acid molecules, lncRNAs are easily degraded by nucleases in vivo, have poor stability, and are difficult to maintain effective concentrations in the lesion area; moreover, they rely on indirect pathways to function, resulting in limited regulatory specificity and a tendency to produce off-target effects. They also suffer from weak tissue penetration and difficulty in targeted delivery, significantly limiting their clinical application value. Currently, there are no reports on the use of functional peptides derived from lncRNA SNHG8 in spinal cord injury repair to promote stem cell-directed differentiation, regulate axonal myelin regeneration, or improve the injury microenvironment. Summary of the Invention

[0009] To address the problems existing in the prior art, this invention provides a lncRNA SNHG8-encoded peptide and its application in spinal cord injury repair. This invention is the first to identify two functional small peptides derived from lncRNA SNHG8, with amino acid sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively. Verification showed that these peptides can effectively promote the neural differentiation of bone marrow mesenchymal stem cells, and after transplantation, they can significantly improve motor, sensory, and electrophysiological functions in spinal cord injury models, reduce tissue pathological damage, and promote axonal regeneration. Compared with single lncRNA nucleic acid molecules, the small peptides of this invention have advantages such as small molecular size, high in vivo stability, strong tissue penetration, high specificity, and ease of artificial synthesis and modification. This invention provides novel peptide drug candidates and stem cell modified formulations for spinal cord injury repair, with promising prospects for clinical translation and application.

[0010] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a nucleotide comprising nucleotide sequences encoding a first small peptide and a second small peptide derived from the long non-coding RNA SNHG8, wherein the amino acid sequence of the first small peptide is shown in SEQ ID NO.5, the amino acid sequence of the second small peptide is shown in SEQ ID NO.6, and the start codon located in open reading frame 1 of the nucleotide sequence is mutated from ATG to ATT, wherein open reading frame 1 is the open reading frame encoding the first small peptide.

[0011] Preferably, the sequence of the nucleotide is as shown in SEQ ID NO.3.

[0012] A second objective of this invention is to provide a recombinant vector carrying the nucleotide sequence.

[0013] Preferably, the vector is a lentiviral vector.

[0014] The third objective of this invention is to provide a recombinant lentivirus, which is obtained by viral packaging of the recombinant vector.

[0015] The fourth objective of this invention is to provide a recombinant bone marrow mesenchymal stem cell containing the recombinant lentivirus.

[0016] The fifth objective of this invention is to provide a method for preparing the recombinant bone marrow mesenchymal stem cells, comprising the following steps: (1) The nucleotide sequence is cloned into a lentiviral vector to obtain a recombinant lentiviral vector; (2) The recombinant lentiviral vector from step (1) and the viral packaging helper plasmid were co-transfected into HEK293T cells to package and obtain recombinant lentivirus; (3) Infect bone marrow mesenchymal stem cells with the recombinant lentivirus from step (2) to obtain recombinant bone marrow mesenchymal stem cells.

[0017] The sixth objective of this invention is to provide the application of the nucleotide sequence, the recombinant lentivirus, or the recombinant bone marrow mesenchymal stem cells in the preparation of a medicament for treating spinal cord injury.

[0018] The seventh objective of this invention is to provide a polypeptide, which is the first small peptide, and its amino acid sequence is shown in SEQ ID NO.5.

[0019] The eighth objective of this invention is to provide another polypeptide, namely the second small peptide, whose amino acid sequence is shown in SEQ ID NO.6.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention reveals for the first time that lncRNA SNHG8 has the ability to encode two functional small peptides, filling the gap in the lack of peptide coding function research of SNHG8 in the field of spinal cord injury, and expanding a new paradigm for lncRNA functional research.

[0021] (2) This invention confirms that SNHG8 and its encoded small peptide can significantly promote the neural differentiation of bone marrow mesenchymal stem cells, providing a novel active molecule for stem cell modification.

[0022] (3) This invention is the first to use SNHG8-encoded small peptides for spinal cord injury repair. In vivo experiments have confirmed that it can significantly improve the motor, sensory and electrophysiological functions of spinal cord injury model animals, reduce tissue pathological damage, and promote axon regeneration, providing a new drug candidate molecule for the treatment of spinal cord injury.

[0023] (4) Compared with complete lncRNA nucleic acid molecules, the small peptides of the present invention have the advantages of small molecular size, high stability, strong tissue penetration, low immunogenicity, and easy artificial synthesis and modification. They effectively avoid the technical bottlenecks of low in vivo delivery efficiency and easy degradation of nucleic acid drugs and have good prospects for clinical translation. Attached Figure Description

[0024] Figure 1 The experimental results of SNHG8 overexpression promoting neurogenic differentiation of BMSCs are shown. A shows bright-field morphological images of the neurospheres in the SNHG8-NC and SNHG8-OE groups on days 3, 6, and 9 (scale bar: 1 mm); B shows immunofluorescence staining images of Nestin (green) and βIII-Tubulin (red) and DAPI (blue) counterstaining images in the SNHG8-NC and SNHG8-OE groups (scale bar: 1 mm); C shows quantitative statistical analysis of neurosphere diameter on days 3, 6, and 9; D shows quantitative analysis of Nestin / βIII-Tubulin double-positive cell counts; E shows RT-qPCR results of NCAM mRNA expression level; F shows RT-qPCR results of NeuroD mRNA expression level; GAPDH was used as an internal control gene.

[0025] Figure 2The experimental results of knocking down SNHG8 to inhibit the neurogenic differentiation of BMSCs are shown. A shows bright-field morphological images of the neurosphere in the sh-Control and sh-SNHG8 groups on days 3, 6, and 9 (scale bar: 1 mm); B shows the quantitative statistical analysis of neurosphere diameter on days 3, 6, and 9; C shows images of Nestin (red) immunofluorescence staining and DAPI (blue) counterstaining in the neurosphere after induction (scale bar: 1 mm); D shows the quantitative analysis of Nestin positive expression; E shows images of βIII-Tubulin (red) immunofluorescence staining and DAPI (blue) counterstaining in the neurosphere after induction (scale bar: 1 mm); F shows the quantitative analysis of βIII-Tubulin positive expression; G shows the RT-qPCR results of NCAM mRNA expression level; H shows the RT-qPCR results of NeuroD mRNA expression level; GAPDH was used as an internal control gene.

[0026] Figure 3 The validation results of lncRNA SNHG8 encoding two small peptides are shown. A is a schematic diagram of the construction of the SNHG8 ORF and GFPmut fusion expression vector, where the start codon ATGGTG of the GFP gene is mutated to ATTGTT (GFPmut), and the start codons (ATG) of SNHG8 ORF1 and ORF2 are site-directed mutated to ATT (ORF1mut and ORF2mut); B shows the results of Western blot detection of SNHG8-GFP fusion protein expression level using an anti-GFP antibody after transfection with the specified vector, with GAPDH as an internal control; C shows GFP fluorescence and corresponding bright-field cell images after transfection with the specified vector, with a scale bar of 0.5 mm.

[0027] Figure 4 The experimental results show that the small peptide encoded by SNHG8 promotes the neural differentiation of BMSCs. In the figures, A shows bright-field morphological images of the neurosphere on days 3, 6, and 9 after transfection with NC, ORF-GFPmut, 5'UTR-ORF-GFPmut, and 5'UTR-ORF1mut-GFPmut vectors, with a scale bar of 1 mm; B shows the quantitative statistical analysis of neurosphere diameter on days 3, 6, and 9; C shows Nestin (red) immunofluorescence staining and DAPI (blue) counterstaining images in each group of cells after induction, with a scale bar of 1 mm; D shows the quantitative analysis of Nestin-positive expression units; E shows βIII-Tubulin (red) immunofluorescence staining and DAPI (blue) counterstaining images in each group of cells after induction, with a scale bar of 1 mm; and F shows the quantitative analysis of βIII-Tubulin-positive expression units.

[0028] Figure 5The experimental results of BMSCs-10559 transplantation promoting motor, sensory, and electrophysiological recovery after spinal cord injury are presented. A shows the BBB scores at weeks 0, 1, 2, 3, 4, and 5; B shows the quantitative analysis results of grip force per unit body weight; C shows the quantitative analysis results of claw withdrawal pain threshold; D shows the footprint distribution map of CatWalk gait analysis; E shows the 3D footprint pressure intensity map of CatWalk gait analysis; F shows the gait sequence phase dispersion map of CatWalk gait analysis; G shows the quantitative analysis of the right hind limb (RH) imprint area; H shows the quantitative analysis of RH stride length; I shows the quantitative analysis of RH swing velocity; J shows the representative waveforms of motor evoked potentials for each group; K shows the quantitative analysis of root mean square (RMS); L shows the quantitative analysis of area under the curve (AUC); and M shows the quantitative analysis of peak-to-peak amplitude.

[0029] Figure 6 The experimental results of BMSCs-10559 transplantation in alleviating spinal cord tissue pathological damage and bladder wall remodeling are shown. In the figures, A shows gross morphological images of the damaged spinal cord segments in each group of rats; B shows representative H&E staining images of longitudinal sections of the spinal cord injury area in each group; C shows representative Nissl staining images of spinal cord tissue in each group; and D shows representative H&E staining images of bladder tissue in each group.

[0030] Figure 7 Immunofluorescence staining results of BMSCs-10559 transplantation promoting axonal regeneration in spinal cord tissue are shown. In the figures, A shows 5-HT (red) and DAPI (blue) staining images of spinal cord tissue in each group; B shows co-stained images of Tubb3 (red), GFAP (green), and DAPI (blue) in each group of spinal cord tissue; C shows multiple staining images of Nestin (red), GAP43 (green), MAP2 (magenta), and DAPI (blue) in each group of spinal cord tissue; scale bars are shown in each figure.

[0031] Figure 8Experimental results of SNHG8-10559 activating the downstream MAPK signaling pathway are shown. In this diagram, A is a volcano plot of differentially expressed genes between the SNHG8-10559 and SNHG8-NC groups, with red dots representing significantly upregulated genes (276), blue dots representing significantly downregulated genes (117), and gray dots representing genes with no significant differences; B is a heatmap of differentially expressed genes by hierarchical clustering; C is the RT-qPCR validation results for NGF mRNA expression levels; D is the RT-qPCR validation results for RHBG mRNA expression levels; E is the RT-qPCR validation results for BEST3 mRNA expression levels; F is the RT-qPCR validation results for BMX mRNA expression levels; G is the RT-qPCR validation results for FGB mRNA expression levels; H is the RT-qPCR validation results for FSTL5 mRNA expression levels; I is a bar chart of KEGG pathway enrichment analysis for differentially expressed genes; J is the RT-qPCR detection results for MAPK14 mRNA expression levels; K is the RT-qPCR detection results for MAPK8 mRNA expression levels; L is the RT-qPCR detection results for JUN mRNA expression levels; M is the RT-qPCR detection results for MAPK7 mRNA expression levels; and M is the RT-qPCR detection results for MAPK8 mRNA expression levels. RT-qPCR results for mRNA expression levels; N represents RT-qPCR results for MAPK1 mRNA expression levels; O represents RT-qPCR results for MAP2K7 mRNA expression levels.

[0032] In the above figures, data are expressed as mean ± standard deviation (Mean ± SD); Student's t test, one-way ANOVA, or repeated measures ANOVA were used to determine statistical significance. P≤0.05, P≤0.01, P≤0.001, P≤0.0001; ns represents no significant difference. Detailed Implementation

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0034] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0035] Example 1: Construction of LncRNA SNHG8-encoded related virus To investigate whether the start codons of the hypothesized open reading frames (ORFs) within the lncRNA SNHG8 are active, this invention constructed a series of fusion expression vectors. Specifically, GFPmut (in which the original classical start codon ATGGTG of GFP is mutated to ATTGTT to eliminate background translation) is fused into the C-terminus of the SNHG8 ORFs and the ORF sequence containing the 5' untranslated region (5'UTR). Figure 3 A).

[0036] 1. Basic methods for constructing lentiviruses: 1.1 Preparation of overexpression lentiviral clones: 1.1.1 Vector linearization. Lentiviral vectors are obtained by digesting them with restriction endonucleases.

[0037] 1.1.2 Obtaining the target gene fragment. The target gene fragment is prepared by amplification using PCR technology. When designing amplification primers, homologous recombination sequences need to be added to their 5' ends to ensure that the sequences at both ends of the amplification product are completely identical to the sequences at both ends of the linearized vector described above.

[0038] 1.1.3 Homologous recombination. The linearized vector and the amplified target gene fragment are prepared into a recombination reaction system and subjected to in vitro recombination reaction to achieve in vitro circularization of the linearized vector and the target gene fragment.

[0039] 1.1.4 Transformation and Identification. The recombinant product was directly transformed into competent cells, and single clones on the plates were picked for PCR identification. Positive clones were sequenced and the results were analyzed.

[0040] 1.1.5 Plasmid extraction. The cloned bacterial culture that has been verified by sequencing is expanded and cultured, followed by plasmid extraction to obtain high-purity plasmids, which are used as tool vectors in downstream virus packaging.

[0041] 1.2 Packaging and Harvesting of Lentivirals 1.2.1 Cell preparation. HEK293T cells were used as packaging cells for lentiviruses for culture.

[0042] 1.2.2 Co-transfection with three plasmids. The three plasmids—the tool vector plasmid carrying the target gene, the viral packaging helper plasmid Helper 1.0, and the viral packaging helper plasmid Helper 2.0—were mixed with the transfection reagent to form a transfection system, which was then co-transfected into HEK293T cells.

[0043] 1.2.3 Virus harvest. After transfection and 48 hours of culture, the unpurified cell supernatant containing lentivirus was collected.

[0044] 1.2.4 Virus Concentration and Purification. The collected cell supernatant was centrifuged to remove cell debris and impurities, and then filtered. Subsequently, the lentivirus was concentrated and purified using ultracentrifugation. After centrifugation, the supernatant was discarded, and an appropriate amount of virus preservation solution was added to resuspend the precipitate, finally obtaining a high-titer lentivirus preservation solution.

[0045] 1.3 Lentiviral quality testing 1.3.1 Physical index testing. The color and state of the lentivirus preservation solution were judged by visual inspection, and the liquid was slowly aspirated using a pipette to determine whether it had obvious viscosity or lag in aspiration.

[0046] 1.3.2 Safety Testing. Virus samples shall be tested for mycoplasma, chlamydia, bacteria, fungi, and endotoxins. All test results must be negative or below the specified threshold.

[0047] 1.3.3 Titer Detection. Based on actual experimental requirements, the final titer of the lentivirus sample is calculated and determined using either a fluorescence method (by observing the fluorescence ratio of infected cells) or an absolute quantitative qPCR method (by detecting viral characteristic single-copy genes and host characteristic single-copy genes in the cell genome).

[0048] 2. Virus sequence SNHG8(ORF-GFPmut), SEQ ID NO.1: GCCGTTTTTGGCTTTTTTGTTAGACGAAGCTTGGGCTGCAGGTCGACTCTAGAGGATCCAACTTTGTGCCA ACCGGT CGCCACCATGATAATTGGGCCGAAACTTACTGCCTTACCTAAAAGGCAGCGCAGTCAGGATATTGGTAGGTCGGGGGCGGCTTTGGAAACCCTTAAGTTTACAAGCATGCGCGGACTTGAGTGCTCATTAGGTCGCCGGGCGTCCACGTGCAGCCCTGGACCC ATTGTTAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGAC GGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAA GTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCT TCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGC ACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCG CATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCC ACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGAC GGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA CTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGA CCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTGA GAATTCCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACC In this text, the underlined parts are enzyme cleavage sites, the bold parts are ORF regions, and the italic parts are GFPmut regions.

[0049] SNHG8 (5'UTR-ORF-GFPmut), SEQ ID NO.2: CGACTCTAGAGGATCCAACTTTGTGCCA ACCGGT( CACATTCGGGAAGCGTCGGGATTAGGTGAAAGTACGTAGTTGTCTTTCGTAAGTTAAA)ATGATAATTGGGCCGAAACTTACTGCCTTACCTAAAAGGCAGCGCAGTCAGGATATTGGTAGGTCGGGGGCGGCTTTGGAAACCCTTAAGTTTACAAGCATGCGCGGACTTGAGTGCTCATTAGGTCGCCGGGCGTCCACGTGCAGCCCTGGACCC ATTGTTAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTG GTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAA GCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACG GCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTAC GTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACAC CCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACA ACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGACGGCATCAAGGTGAACTTCCAAGATCCGCCAC AACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGCCCGTGCTGCT GCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGC TGGAGTTCGTGACCGCCGCCGGGATCACTCTCGCATGGACGAGCTGTACAAGTGA GAATTC CTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGC In this diagram, the underlined part represents the restriction enzyme site, the part inside parentheses represents the 5'-UTR region, the bold part represents the ORF region, and the italic part represents the GFPmut region.

[0050] SNHG8(5'UTR-ORF1mut-GFPmut), SEQ ID NO.3: CTGGCCGTTTTTGGCTTTTTTGTTAGACGAAGCTTGGGCTGCAGGTCGACTCTAGAGGATCCAACTTTGTGCCA ACCGGT( CACATTCGGGAAGCGTCGGGATTAGGTGAAAGTACGTAGTTGTCTTTCGTAAGTTAAA) TO ATAATTGGGCCGAAACTTACTGCCTTACCTAAAAGGCAGCGCAGTCAGGATATTGGTAGGTCGGGGGCGGCTTTGGAAACCCTTAAGTTTACAAGCATGCGCGGACTTGAGTGCTCATTAGGTCGCCGGGCGTCCACGTGCAGCCCTGGACCC AT TGTTAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACA AGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGC AAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCCGCTACCCCGACCA CATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACG ACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATC GACTTCAGGAGGACGGCAACATCCTGGGGCACCAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGC CGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCG ACCACTACCAGCAGAACACCCCCCATCGGCGACGGCCCCGTGCTGCTCGCCGCAACCACTACCTGAGCACCCAGTCC GCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCT CGGCATGGACGAGCTGTACAAGTGA GAATTC CTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCG In this diagram, the underlined portion represents the restriction enzyme site, the portion inside parentheses represents the 5'-UTR region, the bold portion represents the ORF region (the bold and underlined portion indicates that the first start codon in the ORF region has been mutated from ATG to ATT), and the italic portion represents the GFPmut region.

[0051] SNHG8 (5'UTR-ORF1mut-ORF2mut-GFPmut), SEQ ID NO.4: CTGGCCGTTTTTGGCTTTTTTGTTAGACGAAGCTTGGGCTGCAGGTCGACTCTAGAGGATCCAACTTTGTGCCA ACCGGT9( CACATTCGGGAAGCGTCGGGATTAGGTGAAAGTACGTAGTTGTCTTTCGTAAGTTAAA) TO ATAATTGGGCCGAAACTTACTGCCTTACCTAAAAGGCAGCGCAGTCAGGATATTGGTAGGTCGGGGGCGGCTTTGGAAACCCTTAAGTTTACAAGC TO CGCGGACTTGAGTGCTCATTAGGTCGCCGGGCGTCCACGTGCAGCCCTGGACCC A TTGTTAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCAC AAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGG CAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCCGCTACCCCGACC ACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGAC GACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCAT CGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGG CCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCCAAGATCCCGCCAACATCGAGGACGGCAGCGTGCAGCTCGCC GACCACTACCAGCAGAACACCCCCCATCGGCGACGCCCGTGCTGCTCGCCGCAACCACTACCTGAGCACCCAGTC CGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTC TCGGCATGGACGAGCTGTACAAGTGA GAATTC CTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGA AGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTAT In this diagram, the underlined portion represents the restriction site, the portion inside parentheses represents the 5'-UTR region, the bold portion represents the ORF region (the bold and underlined portion indicates that the first and second start codons of the ORF region have been mutated from ATG to ATT), and the italic portion represents the GFPmut region.

[0052] 3. Specific experimental procedures for the construction, packaging, infection, and expression verification of SNHG8-related viruses. 3.1 Vector Design and Target Fragment Acquisition. Using the lncRNA SNHG8 open reading frame (ORF), 5'UTR-ORF, 5'UTR-ORF1mut, and 5'UTR-ORF1mut-ORF2mut fragments as target sequences, the target fragments were synthesized or amplified by PCR according to the nucleotide sequences listed in Section 2 of Example 1. The expression vector used to verify ORF translational activity preferably employs a strategy of fusing the C-terminus of the SNHG8 fragment with GFPmut. In this strategy, the classic start codon ATGGTG of GFPmut is mutated to ATTGTT to reduce background translation; in the ORF mutant vector, the corresponding start codon ATG is mutated to ATT to verify the actual translational contribution of different ORFs. Lentiviral vectors used for stable expression or cell modification can be GV series overexpression vectors, preferably the GV492 vector. The vector element sequence is Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin, and the cloning site can be BamHI / AgeI or a restriction endonuclease site matching the target fragment.

[0053] 3.2 Vector Linearization and Homologous Recombination. The lentiviral tool vector was digested with the corresponding restriction endonuclease to obtain a linearized vector. When amplifying the SNHG8 target fragment by PCR, homologous arm sequences identical to those at both ends of the linearized vector were added to the 5' ends of the upstream and downstream primers, respectively. The PCR product was purified after confirming the band size by agarose gel electrophoresis. The linearized vector and the target fragment were added to the homologous recombination system at an approximately 1:2 molar ratio, reacted at 37°C for approximately 30 min, and then cooled on ice. Subsequently, the mixture was transformed into TOP10, JM109, or Stbl3 competent cells.

[0054] 3.3 Screening and Sequencing of Positive Clones. Transformation products were plated on LB agar plates containing the appropriate antibiotics and incubated upside down at 37°C for 12-16 h. Single clones were picked for colony PCR identification. Positive clones were expanded and then plasmids were extracted. After confirming the correct insertion sequence, mutation site, reading frame, and GFPmut fusion direction by enzyme digestion and Sanger sequencing, the extracted products were used as downstream viral packaging tool plasmids.

[0055] 3.4 Lentiviral Packaging and Harvesting. HEK293T cells were used as packaging cells and cultured in DMEM medium containing 10% fetal bovine serum (FBS). 48 h before transfection, logarithmically growing HEK293T cells were digested and seeded into 10 cm culture dishes. Transfection was performed when the cell density reached 70%-80%. One h before transfection, the medium was replaced with DMEM containing 2% FBS. 20 μg of the GV tool vector plasmid carrying the SNHG8 target sequence, 15 μg of pHelper 1.0 helper plasmid, and 10 μg of pHelper 2.0 helper plasmid were mixed with the transfection reagent and brought to approximately 1 mL. After incubation at room temperature for 15 min, the mixture was evenly added to the HEK293T cell culture medium and cultured at 37°C with 5% CO2. 6-8 h after transfection, the transfection system was discarded, the cells were gently washed once with PBS, and cultured again in medium containing 2% FBS. Collect cell supernatant 48 h after transfection, centrifuge at 4000 g for 10 min at 4 °C to remove cell debris, and filter through a 0.45 μm filter. If a high titer of virus is required, it can be concentrated by ultracentrifugation at 25000 rpm for 2 h at 4 °C. After discarding the supernatant, gently resuspend the virus pellet in virus preservation solution, PBS or serum-free medium, aliquot and store at -80 °C to avoid repeated freeze-thaw cycles.

[0056] 3.5 Virus Quality Control and Titer Determination. Virus samples should be tested for appearance, mycoplasma, chlamydia, bacteria, fungi, and endotoxins. For viruses carrying the GFP reporter gene, the titer can be determined using a fluorescence method: HEK293T cells are seeded in 24-well plates, approximately 1.5 × 10^5 cells per well. The virus is then serially diluted 10-fold to infect the cells. The proportion of GFP-positive cells is observed 72 h post-infection, and the titer is calculated using the formula: "Titer (TU / mL) = Total number of cells × Proportion of fluorescently positive cells × 1000 / Infected virus volume (μL)". Alternatively, absolute quantitative qPCR can be used to detect the viral characteristic single-copy gene and the host single-copy gene, and the functional viral titer can be calculated using the qPCR titer formula.

[0057] 3.6 BMSCs Infection and Establishment of Stable Cell Lines. Human-derived BMSCs were seeded at a density of 30%-50%. The following day, SNHG8-related lentivirus and 5-8 μg / mL polybrene were added for infection, with an optimal MOI of 20-50. The medium was replaced with complete medium 12-24 h after infection. GFP expression was observed 48-72 h post-infection. If necessary, selection was performed with 1-2 μg / mL puromycin for 3 days to obtain stable cells containing the above sequence. Infection efficiency was determined by RT-qPCR detection of SNHG8 or target fragment expression.

[0058] Example 2: SNHG8 overexpression promotes neurosphere formation in BMSCs and increases the expression of neural markers. To investigate the role of SNHG8 in BMSCs neurogenesis, this invention observed morphological changes in BMSCs during neurogenesis to indicate their differentiation into neuron-like cells.

[0059] 1. Methods for inducing neural differentiation of BMSCs and in vitro detection 1.1 Cell Preparation. Healthy BMSCs with consistent passage counts were selected and divided into SNHG8-NC and SNHG8-OE experimental groups. Cells were washed with PBS before induction, digested with trypsin, and counted to ensure consistent cell numbers across groups.

[0060] 1.2 Neural induction culture. Each group was cultured with 1×10⁻⁶ cells. 6 Cells were seeded in ultra-low adsorption culture dishes for neurosphere culture and induced for 9 consecutive days. The preferred neurosphere induction medium was DMEM / F12 based, supplemented with B27, EGF, bFGF, NGF, and 1% penicillin and streptomycin; EGF and bFGF were used in the range of 10-20 ng / mL. During induction, half of the medium was changed every 3 days, and neurosphere formation, cell process extension, and neuron-like morphological changes were recorded under a microscope on days 3, 6, and 9.

[0061] 1.3 Immunofluorescence detection. Neurospheres or adherent cells were collected on day 9 of induction, fixed with 4% paraformaldehyde for 15-30 min, permeabilized with 0.1%-0.3% Triton X-100, and blocked with 5% normal serum or 1%-5% BSA for 1 h. The cells were then incubated overnight at 4°C with primary antibody Nestin (a marker for neural stem / progenitor cells) and βIII-Tubulin / Tubb3 (a marker for early neurons). After washing with PBS, the cells were incubated with fluorescent secondary antibody at room temperature in the dark for 1 h. Cell nuclei were counterstained with DAPI, and images were taken using a fluorescence microscope or confocal microscope. At least three fields of view were randomly selected from each group to determine the proportion of Nestin, βIII-Tubulin-positive cells, or positive neurospheres.

[0062] 1.4 Molecular Validation. Total RNA was extracted after induction, and RT-qPCR was used to detect neural differentiation-related genes such as NCAM and NeuroD, with GAPDH as an internal control.

[0063] The results of this invention show that, compared with the SNHG8-NC group, the neurosphere volume of the SNHG8-OE group was significantly increased ( Figure 1 A, C). Furthermore, this invention used immunofluorescence staining to assess the expression of neurospecific markers, and quantitative analysis showed that the number of Nestin and βIII-Tubulin double-positive cells was significantly increased in the SNHG8-OE group compared to the SNHG8-NC group. Figure 1B, D). Simultaneously, this invention utilized real-time quantitative RT-PCR to detect the levels of neural differentiation-related genes, finding that the expression of NCAM and NeuroD in the SNHG8-OE group was significantly higher than that in the SNHG8-NC group (B, D). Figure 1 E, F). These results indicate that SNHG8 overexpression promotes the neurogenic differentiation potential of BMSCs.

[0064] Example 3: SNHG8 knockdown inhibits BMSCs neurosphere formation and reduces the expression of neural markers. To investigate the role of SNHG8 in the neural differentiation of BMSCs, this invention observed morphological changes in BMSCs during neural induction to indicate their differentiation process into neuron-like cells.

[0065] The methods for inducing BMSCs to differentiate into neurons and for in vitro detection are the same as those in Sections 1.1-1.4 of Example 2. The experimental groups were the sh-Control group and the sh-SNHG8 group. The nucleotide sequences of the specific shRNAs used to knock down SNHG8 are shown in Table 1 below.

[0066] Table 1. Details of SNHG8 knockdown sequences

[0067] The results showed that, compared with the sh-Control group, the diameter of the neurospheres formed in the sh-SNHG8 group was significantly smaller at each time point. Figure 2 A, B). Furthermore, this invention used immunofluorescence staining to assess the expression of neuronal-specific markers, and quantitative analysis revealed that Nestin (A, B) was significantly higher in the sh-SNHG8 group compared to the sh-Control group. Figure 2 C, D) and βIII-Tubulin (C, D) and βIII-Tubulin Figure 2 The positive expression of E and F was significantly reduced. Simultaneously, this invention utilized real-time quantitative RT-PCR to detect the expression levels of neural differentiation-related genes, finding that NCAM (E, F) expression was significantly reduced in the sh-SNHG8 group. Figure 2 G) and NeuroD ( Figure 2 SNHG8 expression was significantly lower in the sh-Control group than in the sh-Control group. These results collectively indicate that knockdown of SNHG8 can inhibit the neurogenic differentiation potential of BMSCs.

[0068] Example 4: LncRNA SNHG8 encodes two small peptides To investigate whether the start codons of the hypothesized open reading frames (ORFs) inside lncRNA SNHG8 are active, this invention constructed a series of fusion expression vectors.

[0069] This invention fuses GFPmut (in which the original classical start codon ATGGTG of GFP is mutated to ATTGTT to eliminate background translation) into SNHG8 ORFs and the C-terminus of the ORF sequence containing the 5' untranslated region (5'UTR). Figure 3 A). Notably, Western blot analysis confirmed that in cells transfected with both ORF-GFPmut and 5'UTR-ORF-GFPmut, the SNHG8-GFP fusion protein clearly produced two independent bands, indicating that lncRNA SNHG8 has the ability to encode two small peptides (A). Figure 3 B). When the start codon of ORF1 (5'UTR-ORF1mut-GFPmut) is mutated alone, the band signal intensity of the second peptide is significantly enhanced. Figure 3 B). This phenomenon strongly suggests that there may be translational competition, leaky scanning, or some kind of synergistic regulatory mechanism between ORF1 and ORF2. Conversely, when both start codons (5'UTR-ORF1mut-ORF2mut-GFPmut) are mutated simultaneously, the expression signals of both small peptide bands completely disappear. Figure 3 B). Consistent with the Western blot results, fluorescence microscopy showed strong GFP signals in cells transfected with wild-type SNHG8 ORF, 5'UTR-ORF, and the single mutant ORF1 vector, while the fluorescence signal in cells transfected with the double mutant vector decreased sharply to background levels. Figure 3 C). In summary, these data directly demonstrate that the hypothesized long non-coding RNA SNHG8 actually encodes two novel small peptides through a finely regulated translation mechanism. The method for constructing the SNHG8 open reading frame translation activity using the fusion expression vector is described in Example 1.

[0070] Example 5: Small peptides encoded by LncRNA SNHG8 promote neural differentiation. To investigate the functional role of small peptides encoded by lncRNA SNHG8 in neural differentiation, this invention evaluated neurosphere formation and neural marker expression in cells transfected with different SNHG8 vectors.

[0071] The methods for inducing neural differentiation of BMSCs and for in vitro detection are the same as those in Sections 1.1-1.3 of Example 2. The experimental groups are NC group, ORF-GFPmut group, 5'UTR-ORF-GFPmut group and 5'UTR-ORF1mut-GFPmut group.

[0072] Bright-field microscopy revealed that, compared to the negative control (NC) group, cells transfected with ORF-GFPmut and 5'UTR-ORF-GFPmut (exogenously expressing SNHG8 peptide) formed significantly larger neurospheres on days 3, 6, and 9 after induction. Figure 4 A, B). Notably, transfection with the 5'UTR-ORF1mut-GFPmut vector (previous results have confirmed that mutations in this ORF1 significantly enhance the translational expression of the second polypeptide) resulted in a further significant increase in neurosphere diameter compared to the unmutated 5'UTR-ORF-GFPmut group. Figure 4 A, B). Furthermore, immunofluorescence staining results showed that, compared to the NC group, the neural stem cell marker Nestin (… Figure 4 C, D) and early neuronal marker βIII-Tubulin (C, D) Figure 4 E and F showed highly significant increases in all three peptide expression groups. Notably, there was no statistically significant difference in the number of positive fluorescence markers among the three peptide expression groups (ORF-GFPmut, 5'UTR-ORF-GFPmut, and 5'UTR-ORF1mut-GFPmut). These findings confirm that the small peptide encoded by SNHG8 promotes neurogenic differentiation, with the second peptide potentially playing a more prominent role in driving neurosphere differentiation.

[0073] Example 6: BMSCs-10559 transplantation promotes motor and sensory function recovery in a spinal cord injury model. To investigate whether the peptide encoded by lncRNA SNHG8 promotes spinal cord injury repair by facilitating neural differentiation of stem cells, the 5'UTR-ORF1mut-GFPmut group was subsequently named 10559.

[0074] 1. Rat T10 spinal cord injury model and in situ reimplantation method of BMSCs-10559 1.1 Experimental Animals and Grouping. Six- to eight-week-old female Sprague-Dawley rats, preferably weighing 180-220g, were selected. Animals were randomly divided into four groups: Sham, SCI, SCI+BMSCs-NC, and SCI+BMSCs-10559, with n=6 animals in each group. All animal experiments were approved by an ethics committee and conducted under aseptic conditions.

[0075] 1.2 Anesthesia and Model Establishment. Rats were fasted preoperatively but allowed free access to water. After weighing, they were anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg / kg). Rats were fixed in a prone position. After skin preparation and disinfection, the skin and muscles were incised along the midline of the back to expose the T9-T10 lamina. Laminectomy was performed under a microscope or magnified view to fully expose the T10 segment of the spinal cord. In the Sham group, only laminectomy was performed without spinal cord injury; in the SCI group and cell therapy group, a spinal cord hemisection method was used to establish an injury model at the T10 segment to ensure consistent injury location, extent, and depth.

[0076] 1.3 Cell-hydrogel preparation and administration. BMSCs-NC or BMSCs-10559 cells were collected and resuspended in PBS or serum-free DMEM to prepare a 5 μL cell suspension with a cell count of 2 × 10^6. The cell suspension was thoroughly mixed with 30 μL of ECM hydrogel (Sigma E1270) on ice to form a cell-hydrogel complex. The cell suspension to hydrogel volume ratio was approximately 1:6, with a total volume of approximately 35 μL. Immediately after modeling, the complex was implanted in situ into the spinal cord hemisection defect. If a microinjection method was used, injections were administered at multiple points in the injury center and adjacent areas on the head and tail sides, with the needle tip penetrating approximately 0.8-1.0 mm into the spinal cord parenchyma. The injection rate was controlled at 0.5-1.0 μL / min, and the needle was left in place for approximately 3 minutes before slowly withdrawing to reduce reflux. After treatment, the muscle and skin were sutured layer by layer.

[0077] 1.4 Postoperative care. After surgery, place the animal on a warm mat or in a warm environment until it is fully awake, keeping it in a single cage. Administer analgesia for 3 consecutive days, using buprenorphine 0.05 mg / kg subcutaneously twice daily. Simultaneously, administer anti-infective treatment according to animal ethics requirements, such as penicillin or cephalosporin antibiotics for 3 consecutive days. Observe the animal's weight, mental status, wound healing, and hind limb movement daily postoperatively. If urinary retention occurs, artificially empty the bladder twice daily until the bladder reflex returns.

[0078] This invention assessed motor behavior deficits in experimental animals over a 5-week period using the Basso-Beattie-Bresnahan (BBB) ​​rating scale.

[0079] 2. Methods for detecting BBB score, grip strength, and claw withdrawal pain threshold 2.1 BBB Motor Score. The Basso-Beattie-Bresnahan (BBB) ​​score was performed at 0, 1, 2, 3, 4 and 5 weeks postoperatively. Rats were allowed to move freely in an open enclosure. Two researchers, unaware of their assigned groups, observed hind limb joint movement, weight-bearing, gait coordination and paw strike, and scored the rats independently. The average scores were used for statistical analysis.

[0080] 2.2 Grasping Force Test. Adaptation training was conducted preoperatively. During the test, a digital gripping force meter or SDI GripStrength System was used. The rat was instructed to grasp a metal rod / grid with its forelimbs, hindlimbs, or all four limbs. The experimenter smoothly pulled the animal's tail horizontally and recorded the maximum pulling force before the animal released its grip. The test was repeated 3-5 times per animal, with intervals of at least 30 seconds between each measurement. After removing obvious outliers, the maximum value or average maximum value was taken and normalized to body weight, expressed as gripping force / body weight.

[0081] 2.3 Withdrawal Pain Threshold. The mechanical withdrawal pain threshold was determined using von Frey fibers. Before testing, rats were placed in a transparent observation cage or on a metal mesh platform for 15-30 minutes to acclimatize. Using the Touch Test Sensory Kit, preferably with fibers of 5.18, 5.46, 5.88, and 6.10 specifications, the fibers were vertically contacted from low to high to the corresponding dermal area on the sole or dorsal side of the hind paw, held for 1-2 seconds. Each fiber was stimulated 10 times consecutively, and the number of positive responses (withdrawal, licking, and shaking) was recorded. The positive response rate was calculated, and the minimum stimulation intensity required to induce a stable positive response for the first time was taken as the withdrawal pain threshold. If an up-down method was used, the next fiber was selected according to the Dixon sequential method, and the 50% response threshold was calculated.

[0082] All behavioral tests were conducted in a quiet environment by experimenters who were unaware of their assigned groups.

[0083] Following injury, the animals exhibited significant motor dysfunction. From week 1 post-cell transplantation until week 5, the SCI+BMSCs-10559 group showed progressive and statistically significant improvement in motor function compared to the SCI group and the SCI+BMSCs-NC group. Figure 5 A).

[0084] Furthermore, this invention evaluated the effects of this intervention on overall physical performance and sensory responses. Compared to the untreated SCI group, the SCI+BMSCs-10559 group showed a significant increase in grip strength (force / body weight). Figure 5 B). The paw withdrawal threshold improved slightly in all treatment groups, with the paw withdrawal threshold in the SCI+BMSCs-NC group and the SCI+BMSCs-10559 group essentially returning to normal levels. Figure 5 C).

[0085] To analyze gait and fine motor coordination, this invention performs CatWalk gait analysis.

[0086] 3. CatWalk gait analysis method Animals underwent preoperative adaptation training using the CatWalk XT system. During testing, rats were placed at one end of a CatWalk glass track and allowed to spontaneously walk to the target box in a dark environment. Each animal had at least three qualified running records, with the criteria being continuous, uninterrupted, stable speed, and clear footprints. The software automatically identified footprints on all four limbs and output parameters such as the right hind limb (RH) imprint area, stride length, swing speed, stance phase, maximum contact intensity, gait regularity index, and left-right coordination. Visual analysis was performed on footprint patterns, 3D footprint pressure intensity maps, and gait phase dispersion. If necessary, principal component analysis (PCA) and cluster analysis could be performed on all gait parameters to differentiate the motor recovery characteristics of different treatment groups.

[0087] Visualization mapping of footprint patterns and 3D footprint intensity maps showed that animals in the BMSCs-10559 treatment group exhibited improvements in both body weight support and postural coordination. Figure 5 D, E, F). Quantitative spatiotemporal gait parameters further confirmed this recovery: compared with the SCI group and the NC control group, the right hind limb (RH) imprint area was significantly increased and the RH stride length was significantly prolonged in the SCI+BMSCs-10559 group. Figure 5 G, H). The RH oscillation speed also changes ( Figure 5 I) indicates that the hindlimb kinematics has been fully restored.

[0088] To evaluate the impact of BMSCs-10559 transplantation on signal transduction in the damaged spinal cord, electrophysiological recordings were performed in this invention.

[0089] 4. Electrophysiological detection methods Motor evoked potentials (MEPs) or compound electromyography (EMG) signals were detected at week 5 post-surgery or at the endpoint. Rats were anesthetized with ketamine or an equivalent regimen and then fixed prone in a stereotactic apparatus. Stimulating electrodes were placed in the corresponding region of the cranial sensorimotor cortex or at a proximal stimulation point in the spinal cord. Recording electrodes were placed in target muscles such as the gastrocnemius, tibialis anterior, or forearm extensors. A reference electrode was placed in adjacent subcutaneous tissue. The stimulation intensity could be set to 1-15 mA, with a pulse width of 0.1-0.5 ms; preferably, 10 mA, 0.3 ms stimulation was used, and the average waveform was recorded after 10 consecutive pulses. Waveforms were acquired and analyzed using pClamp, Clampex / Clampfit, or similar systems. Key parameters included latency, peak-to-peak amplitude, root mean square (RMS), area under the curve (AUC), and stimulus-response curve.

[0090] Severe disruption of signal transduction was observed in the SCI group, and treatment with BMSCs-10559 significantly improved neurophysiological outcomes. Representative electrophysiological waveforms visually demonstrate the effective recovery of signal amplitude in the BMSCs-10559 treatment group. Figure 5J). Quantitative analysis showed that, compared with the SCI group alone and the SCI+BMSCs-NC group, the root mean square (RMS) of the SCI+BMSCs-10559 group was significantly lower. Figure 5 K) and the area under the curve (AUC) Figure 5 The peak-to-peak amplitude (PPA) was significantly higher after BMSCs-10559 treatment. Consistent with these findings, the PPA was significantly increased after BMSCs-10559 treatment. Figure 5 M) reflects the restoration of spinal cord excitability and the improvement of functional conduction within spinal cord neural circuits.

[0091] Example 7: Transplantation of BMSCs-10559 alleviates spinal cord histopathological damage and improves neurogenic bladder remodeling. To evaluate the histological repair effect of cell transplantation on the injured spinal cord, this invention conducted gross morphological observation and histopathological examination.

[0092] 1. Sampling and Gross Observation. At the endpoint, animals were euthanized due to excessive anesthesia, and the heart was perfused with PBS followed by perfusion with 4% paraformaldehyde. Spinal cord tissue containing approximately 0.5 cm from the injury center and the head and tail sides was collected, and the gross morphology of the injured segment, such as necrosis, collapse, cavitation, and tissue continuity, was recorded. Bladder tissue could also be collected for pathological evaluation of neurogenic bladder.

[0093] 2. HE and Nissl staining. Spinal cord tissue was fixed in 4% paraformaldehyde for 24 h, dehydrated and protected with 30% sucrose, and then sectioned after paraffin or cryopreservation. The section thickness for HE and Nissl staining can be set to 5 μm, and the frozen section thickness for cell-free immunofluorescence can be set to 30 μm. HE staining is used to assess syringomyelia, glial scars, and tissue continuity; Nissl staining is used to assess neuronal survival and Nissl body structure; Nestin immunohistochemistry is used to evaluate the response of neural progenitor cells in the injury area.

[0094] 3. Immunofluorescence. Sections were blocked with blocking buffer (5% normal serum, 1% BSA, 0.3% Triton X-100 / PBS) at room temperature for 1 h, then incubated overnight at 4°C with primary antibody. 5-HT, Tubb3 / βIII-Tubulin, GFAP, Nestin, GAP43, and MAP2 were detected. The following day, after washing with PBS, the sections were incubated with the corresponding fluorescent secondary antibody at room temperature in the dark for approximately 60 min, and the cell nuclei were counterstained with DAPI.

[0095] 4. Image Acquisition. Using a fluorescence microscope or confocal microscope, preferably with a 20× or 40× objective lens, representative images for each group were acquired under the same exposure, laser intensity, and gain parameters. For each animal, consecutive sections from adjacent areas at the lesion center, head, and tail were selected for observation. Each group of images was used to demonstrate the fluorescence staining results and the distribution of markers in the lesion area.

[0096] Gross observation showed that the surface of the damaged spinal cord segment in the SCI group exhibited extensive necrosis and severe tissue collapse. After transplantation of BMSCs-NC and BMSCs-10559, the necrosis of the damaged spinal cord improved in both groups; among them, the SCI+BMSCs-10559 group showed the most significant reduction in gross necrosis area and the best restoration of tissue continuity. Figure 6 A). H&E staining results further confirmed that the SCI group had huge tissue cavities and structural damage, with a large amount of collagen scarring around the damage; the tissue cavities in the SCI+BMSCs-NC group were smaller than those in the SCI group, and the structural damage was alleviated to some extent; while the SCI+BMSCs-10559 group showed the largest reduction in cavity volume, with the lesion area filled by a large amount of new tissue, and the continuity of the spinal cord tissue structure was most significantly improved. Figure 6 B).

[0097] Neuronal survival was further assessed using Nissl staining. Results showed extremely severe neuronal loss and necrosis around the lesions in the SCI group. A small number of surviving neurons were observed in the SCI+BMSCs-NC group, indicating some degree of recovery. In contrast, the SCI+BMSCs-10559 group showed a significantly increased number of surviving neurons within the field of view, and the Nissl body structure remained intact. Figure 6 (C) indicates that BMSCs-10559 transplantation has a better protective and repairing effect on damaged neurons.

[0098] Spinal cord injury often leads to neurogenic bladder dysfunction, manifested as compensatory hypertrophy of the bladder wall smooth muscle. H&E staining of bladder tissue showed a highly significant increase in bladder wall thickness in the SCI group compared to the Sham group. After intervention, the bladder wall thickening in the SCI+BMSCs-NC group was alleviated, while the bladder muscle layer thickness in the SCI+BMSCs-10559 group further decreased significantly, approaching the normal level of the Sham group. Figure 6 D). This result indicates that although BMSCs-NC intervention can bring about some improvement, BMSCs-10559 transplantation shows superior efficacy in reversing SCI-induced distal organ pathological remodeling.

[0099] Example 8: BMSCs-10559 transplantation significantly promoted axonal regeneration after spinal cord injury. To evaluate the specific impact of specific cell transplantation on nerve regeneration in the damaged spinal cord microenvironment, this invention performed multiplex immunofluorescence staining analysis on the lesion area. The descending projection of serotonergic (5-HT) nerve fibers is crucial for the recovery of motor function. Immunofluorescence results showed a significant loss of 5-HT-positive fibers in the lesion area of ​​the SCI group, indicating severe conduction tract disruption. After transplantation of BMSCs-NC, a small amount of 5-HT fibers were observed to be preserved in localized areas. In contrast, the SCI+BMSCs-10559 group showed the most significant increase in the density of 5-HT-positive fibers in and around the lesion area, indicating that this intervention effectively promoted the protection or budding regeneration of descending motor conduction tracts. Figure 7 A).

[0100] Co-staining with Tubb3 (a marker of neuronal and axonal structure) and GFAP (a marker of astrocytes) was used to assess neuronal network integrity and reactive gliosis. In the SCI group, large areas of Tubb3 expression were interrupted and lost, accompanied by strong GFAP fluorescence accumulation at the cavity edges, suggesting typical and dense glial scar formation. Although tissue destruction was slowed in the SCI+BMSCs-NC group, the continuity and distribution area of ​​Tubb3-positive structures were most significantly improved in the SCI+BMSCs-10559 group, indicating that neurons and axonal networks had more space for remodeling. Figure 7 B).

[0101] Furthermore, this invention systematically detected the expression abundance of Nestin (a marker of neural stem / progenitor cells), GAP43 (a marker of axonal growth cones), and MAP2 (a marker of mature dendrites / neurons). In the SCI group lesions, the expression of these markers was extremely weak and structurally disordered. BMSCs-NC intervention induced a certain degree of recovery in the expression of these markers. Significantly, in the SCI+BMSCs-10559 group, the fluorescence signal intensities of Nestin, GAP43, and MAP2 all reached the highest levels. This result indicates that BMSCs-10559 transplantation not only effectively stimulated the activation of local neural progenitor cells and the growth of new axons, but also effectively promoted the maintenance of the mature neuronal cytoskeleton and the reconstruction of neural networks. Figure 7 C).

[0102] Example 9: SNHG8-10559 alters downstream gene expression and activates key signaling pathways such as MAPK. To explore the potential molecular mechanism by which SNHG8-10559 functions, this invention performed transcriptome sequencing (RNA-seq) analysis on the SNHG8-10559 group and the SNHG8-NC group.

[0103] 1. RNA-seq and RT-qPCR validation methods 1.1 Sample Preparation and RNA Quality Control. Cells from the SNHG8-10559 and SNHG8-NC groups were used, with at least three biological replicates for each group. RNA was extracted using TRIzol or a total RNA extraction kit. A260 / A280 and A260 / A230 ratios were detected using NanoDrop, and integrity was assessed using Bioanalyzer or agarose gel electrophoresis. Samples with RIN ≥ 7.0 and A260 / A280 of 1.8–2.1 were preferred for library construction.

[0104] 1.2 Library Construction, Sequencing, and Differential Analysis. Libraries were constructed using either poly(A) enrichment or rRNA removal strategies based on sample type, and paired-end sequencing was performed on the Illumina platform. Raw data underwent FastQC quality control, adapter and low-quality sequence removal, and alignment to the corresponding reference genome. Gene expression levels were expressed as read count, FPKM, or TPM. Differential expression analysis was performed using DESeq2 or similar software, with selection criteria of p < 0.05 and |FoldChange| > 2, or using corrected p-values ​​(padj) to control multiple tests. Differentially expressed genes were used for volcano plots, heatmaps, GO functional enrichment, KEGG pathway enrichment, and GSEA analysis.

[0105] 1.3 RT-qPCR Validation. Representative differentially expressed genes were selected for validation based on the RNA-seq results, with NGF, RHBG, BEST3, BMX, FGB, FSTL5, and core MAPK pathway genes MAPK14, MAPK8, JUN, MAPK7, MAPK1, and MAP2K7 being the preferred candidates. Approximately 1 μg of total RNA from each sample was reverse transcribed into cDNA, and qPCR was performed using a SYBR Green or TaqMan system, with three technical replicates per sample. GAPDH or PPIA was used as an internal control, and relative expression levels were calculated using the 2^-ΔΔCt method. If the RT-qPCR trend was consistent with the RNA-seq trend, the sequencing results were considered reliable.

[0106] 2. Statistical Analysis All experimental data are expressed as mean ± standard deviation (Mean ± SD) or mean ± standard error (Mean ± SEM). Student's t-test or Welch-corrected t-test was used for comparisons between two groups; one-way ANOVA or Kruskal-Wallis test was used for comparisons among multiple groups; repeated measures ANOVA was used for repeated measures data such as BBB scores over time. A p-value < 0.05 was considered statistically significant. Behavioral, image acquisition, and quantitative analyses were performed by researchers unaware of the group assignments.

[0107] Volcano plot results showed a large number of differentially expressed genes (DEGs) between the two groups. Among them, 276 genes were significantly upregulated and 117 genes were significantly downregulated in the SNHG8-10559 group (screening criteria were p<0.05 and |FoldChange|>2). Figure 8 A). Hierarchical clustering heatmaps further confirmed that the two groups of samples had significant differences in gene expression patterns, and good intra-group repeatability. Figure 8 B).

[0108] To verify the reliability of the RNA-seq sequencing results, this invention selected some representative differentially expressed genes (NGF, RHBG, BEST3, BMX, FGB, FSTL5) for RT-qPCR verification. The results showed that, compared with the SNHG8-NC group, the relative mRNA expression levels of the above genes were significantly upregulated in the SNHG8-10559 group, which is highly consistent with the trend observed in transcriptome sequencing. Figure 8 CH).

[0109] Subsequently, this invention performed KEGG pathway enrichment analysis on differentially expressed genes to clarify their biological functions. The results showed that these differentially expressed genes were significantly enriched in several important signaling pathways. Notably, neuroactive ligand-receptor interaction, the calcium signaling pathway, the cAMP signaling pathway, and the MAPK signaling pathway were significantly enriched. Figure 8 I).

[0110] Given the crucial regulatory role of the MAPK signaling pathway in cell proliferation, differentiation, and neural regeneration and repair, this invention further used RT-qPCR to detect the expression of key genes in this pathway. The results confirmed that, compared with the control group, SNHG8-10559 significantly upregulated the expression levels of MAPK14, MAPK8, JUN, MAPK7, MAPK1, and MAP2K7. Figure 8 These data suggest that SNHG8-10559 may exert its biological functions of tissue repair and neuroprotection by activating MAPK and its associated signaling pathway networks.

[0111] The small peptide 1 encoded by LncRNA SNHG8 in this invention (SEQ ID NO.5): MIIGPKLTALPKRQRSQDIGRSGAALETLKFTSMRGLECSLGRRASTCSPGP In this invention, the small peptide 2 encoded by LncRNA SNHG8 (SEQ ID NO. 6) is: MRGLECSLGRRASTCSPGP Other relevant primer sequences used can be found in the following literature: 1-MAPK1: Yuan W, Ferreira LAQ, Khade R, et al. Enhanced OsteogenicDifferentiation of hMSCs Using BMP@ZIF-8-Loaded GelMA Nanocomposite Hydrogels with Controlled BMP-2 Release. ACS Omega, 2025, 10(11):10826-10834. 2-MAP2K7: Cao J, Yang Y, Duan B, et al. LncRNA PCED1B-AS1 mediates miR-3681-3p / MAP2K7 axis to promote metastasis, invasion and EMT in gastric cancer. Biol Direct. 2024;19(1):34. 3-MAPK14: Liu L, Rezvani HR, Back JH, et al. Inhibition of p38 MAPKsignaling augments skin tumorigenesis via NOX2 driven ROS generation. PLoSOne, 2014, 9(5):e97245. 4-MAPK8: Desideri E, Ciriolo MR. Inhibition of JNK increases the sensitivity of hepatocellular carcinoma cells to lysosomotropic drugs via LAMP2A destabilization. Cell Death Discov. 2021 Feb 8;7(1):29. 5-JUN:Su H,Liang L,Wang J,Yuan X,Zhao B. ZFP36,an RNA-binding proteinpromotes hBMSCs osteogenic differentiation via binding with JUN. J OrthopSurg Res. 2024;19(1):758. 6-MAPK7:Li M,Liu X. Pitavastatin maintains MAPK7 expression andalleviates angiotensin II-induced vascular endothelial cell inflammation andinjury. Exp Ther Med. 2022;23(2):132. 7-BMX:Sundaramoorthy S,Colombo DF,Sanalkumar R,et al. Preclinicalspheroid models identify BMX as a therapeutic target for metastatic MYCNnonamplified neuroblastoma. JCI Insight. 2024;9(14):e169647. 8-BEST3:Song W,Yang Z,He B. Bestrophin 3 ameliorates TNFα-inducedinflammation by inhibiting NF-κB activation in endothelial cells. PLoS One.2014;9(10):e111093. 9-RHBG:Merhi A,De Mees C,Abdo R,Victoria Alberola J,Marini AM. Wnt / β-Catenin Signaling Regulates the Expression of the Ammonium Permease Gene RHBGin Human Cancer Cells. PLoS One. 2015;10(6):e0128683. 10-FSTL5: Ma S, Chen F, Lin C, et al. MiR-186-5p prevents hepatocellularcarcinoma progression by targeting methyltransferase-like 3 that regulatesm6A-mediated stabilization of follistatin-like 5. Heliyon. 2024;10(5):e26767. 11-FGB: Jin T, Chen G, An Q, et al. miR-139-5p Suppresses Proliferation and Angiogenesis of Intracranial Aneurysm via FGB. J Healthc Eng. 2022;2022:5824327. 12-NGF: Liu J, Kang J, Zou T, et al. Functional cobalt-doped hydrogelscaffold enhances concurrent vascularization and neurogenesis. JNanobiotechnology. 2025;23(1):179. 13-NeuroD, 14-NCAM: Zhang C, Ye W, Zhao M, et al. MLL1 inhibits the neurogenic potential of SCAPs by interacting with WDR5 and repressing HES1. Int J Oral Sci, 2023,15:48. The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A nucleotide, characterized in that, The nucleotides comprise nucleotide sequences encoding a first small peptide and a second small peptide derived from the long non-coding RNA SNHG8. The amino acid sequence of the first small peptide is shown in SEQ ID NO.5, and the amino acid sequence of the second small peptide is shown in SEQ ID NO.

6. Furthermore, the start codon located in open reading frame 1 of the nucleotide sequences is mutated from ATG to ATT, and the open reading frame 1 is the open reading frame encoding the first small peptide.

2. The nucleotide according to claim 1, characterized in that, The sequence of the nucleotides is shown in SEQ ID NO.

3.

3. A recombinant vector, characterized in that, The recombinant vector carries the nucleotide sequence as described in claim 1 or 2.

4. The recombinant vector according to claim 3, characterized in that, The vector is a lentiviral vector.

5. A recombinant lentivirus, characterized in that, The recombinant lentivirus is obtained by viral packaging of the recombinant vector as described in claim 3 or 4.

6. A recombinant bone marrow mesenchymal stem cell, characterized in that, The bone marrow mesenchymal stem cells contain the recombinant lentivirus as described in claim 5.

7. A method for preparing the recombinant bone marrow mesenchymal stem cells of claim 6, characterized in that, Includes the following steps: (1) Cloning the nucleotide sequence described in claim 1 or 2 into a lentiviral vector to obtain a recombinant lentiviral vector; (2) The recombinant lentiviral vector from step (1) and the viral packaging helper plasmid were co-transfected into HEK293T cells to package and obtain recombinant lentivirus; (3) Infect bone marrow mesenchymal stem cells with the recombinant lentivirus from step (2) to obtain recombinant bone marrow mesenchymal stem cells.

8. The use of the nucleotide sequence of claim 1 or 2, the recombinant lentivirus of claim 5, or the recombinant bone marrow mesenchymal stem cells of claim 6 in the preparation of a medicament for treating spinal cord injury.

9. A polypeptide, characterized in that, The polypeptide is the first small peptide described in claim 1, and its amino acid sequence is shown in SEQ ID NO.

5.

10. A polypeptide, characterized in that, The polypeptide is the second small peptide described in claim 1, and its amino acid sequence is shown in SEQ ID NO. 6.

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