KR102279751B1 - Composition for diagnosis of myopathy comprising an agent for determining the level of NOGO-A gene or myogenic factor and method for diagnosing myopathy using the same - Google Patents

Abstract

The present invention relates to a composition for diagnosing myopathy comprising a NOGO-A measuring agent and a method for diagnosing myopathy using the same, and a composition for diagnosing myopathy comprising an agent for measuring the expression level of NOGO-A gene or the amount of NOGO-A protein, Provided are kits used and a method for providing information for diagnosing myopathy.

Description

Composition for diagnosis of myopathy comprising an agent for determining the level of NOGO-A gene or myogenic factor and method for diagnosing myopathy using the same }

It relates to a composition for diagnosing myopathy, including a myogenic factor or NOGO-A measuring agent, and a method for diagnosing myopathy using the same.

Skeletal muscles make up 40% of the total body tissue, and this dynamic tissue is involved in the body's metabolism, respiration, postural support, and body movement. Muscle fibers are multinucleated muscle cells that make up myofibrils and contractile units that stimulate movement through repeated contraction and relaxation. The performance of body muscles can be affected by physiological changes such as loss of motor units, muscle fiber atrophy, ischemia, heat stress, and decreased neuromuscular activation. Muscular dystrophy is a group of inherited muscle disorders that result in impaired muscle fiber function due to occluded muscle regeneration, as well as weakening and destruction of muscle fibers through inflammation. Duchenne muscular dystrophy (DMD) is a severe myopathy associated with an occluded dystrophin gene and is characterized by muscle membrane defects associated with increased pro-inflammatory cytokines, mitochondrial dysfunction, and compromised satellite cell (muscle stem cell) polarity. cause

Reticulon 4 (RTN4) is a gene encoding the NOGO protein and produces three isoforms called NOGO-A, NOGO-B and NOGO-C. Among them, NOGO-A is well known as a neurite growth inhibitor, and there is a report that NOGO-A is increased in skeletal muscle of patients with amyotrophic lateral sclerosis (ALS). This suggests the involvement of NOGO-A in the pathophysiology of ALS. Also, NOGO-C is known to be the predominant subtype in skeletal muscle, and it has been reported that it functions as an apoptosis inducer of myocardial cells during myocardial infarction.

Under this technical background, studies on NOGO isoforms have been actively conducted (Korean Patent No. 10-15748140000), but the mechanism of NOGO-A skeletal muscle activation in relation to muscle regeneration and development has not yet been elucidated.

One aspect is to provide a composition for diagnosing myopathy comprising an agent for measuring the expression level of the NOGO-A gene or the amount of the NOGO-A protein.

Another aspect is to provide a composition kit for diagnosing myopathy comprising the composition.

Another aspect is a method comprising: measuring an expression level of a NOGO-A gene or an amount of a NOGO-A protein in a biological sample isolated from a subject suspected of having myopathy; and

It is to provide an information providing method for diagnosing myopathy comprising the step of comparing the measured gene expression level or protein amount with the control gene expression level or protein amount.

One aspect is to provide a composition for diagnosing myopathy comprising an agent for measuring the expression level of the NOGO-A gene or the amount of the NOGO-A protein.

As used herein, the term “diagnosis” refers to confirming the presence or characteristics of a pathological condition, and may mean including confirming whether or not a myopathy-related disease develops or is likely to develop.

As used herein, the term “measuring the expression or activity level” may include measuring the level of expression or activity of a myopathy-related disease-related marker in a biological sample in order to diagnose a myopathy-related disease.

The NOGO-A is a neurite outgrowth inhibitor, and may be named as reticulon 4 isoform A, or RTN4-A. The NOGO-A gene may encode the polypeptide of SEQ ID NO: 1. The NOGO-A gene may include the polynucleotide of SEQ ID NO: 2 as a transcript.

In one embodiment, the agent for measuring the expression or activity of the gene is an agent for checking the presence or level of mRNA, and the like, and may be for measuring the amount of mRNA. As an analysis method for this, for example, reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction, real-time reverse transcription polymerase reaction, RNase protection assay, Northern blotting, DNA chip may be used, and the mRNA It may be an antisense oligonucleotide, a primer pair, or a probe that specifically binds to.

The antisense oligonucleotide is a sequence of nucleotide bases and sub-nucleotides, wherein the antisense oligomer hybridizes to a target sequence in RNA by Watson-Crick base pairing, allowing the formation of an oligomeric heteroduplex, typically mRNA and RNA, within the target sequence. It may refer to an oligomer having an inter-unit backbone. An oligomer may have exact sequence complementarity or approximate complementarity to a target sequence. These antisense oligomers can block or inhibit the translation of mRNA and alter the processing of mRNA to produce splice variants of the mRNA.

The primer is a nucleic acid sequence having a short free 3-terminal hydroxyl group, and may refer to a short nucleic acid sequence capable of forming a base pair with a complementary template and serving as a starting point for template strand copying. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature.

The probe may refer to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as long as possible to achieve specific binding to mRNA, and is labeled to determine the presence or absence of a specific mRNA. can The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. Selection of suitable probes and hybridization conditions can be modified based on those known in the art.

The antisense oligonucleotide, primer or probe may be chemically synthesized using a phosphor amidite solid support method or other well-known methods. Such nucleic acid sequences may also be modified using a number of means known in the art. Examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides (eg, uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) or phospho transformation into charged linkages such as phosphorothioate, phosphorodithioate, etc.).

In another embodiment, the agent for measuring the expression or activity of the protein is an agent for checking the presence or level of the protein and the like, and may be for measuring the amount of the protein. For this, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometry, and protein chip are used. It may be an oligopeptide, monoclonal antibody, polyclonal antibody, chimeric antibody, ligand, PNA or aptamer that specifically binds to the protein.

The antibody is a term known in the art and refers to a specific protein molecule directed to an antigenic site. Each gene can be cloned into an expression vector according to a conventional method to obtain a protein encoded by the marker gene, and can be prepared from the obtained protein by a conventional method. The antibody may be a monoclonal antibody, a polyclonal antibody, or a chimeric antibody, and optionally, a special antibody such as a humanized antibody may also be included.

In one embodiment, the NOGO-A gene or protein thereof may be excessively increased in expression or activity in muscle tissue or cells of a patient with a myopathy-related disease compared to a normal person.

The NOGO-A gene or other regulatory genes related to sarcopenia are fibroblast growth factor 23 (FGF23), secreted protein acidic and rich in cysteine (SPARC), macrophage migration inhibitory factor (MIF) and insulin-like (IGF-1). growth factor-1) may be included.

The term "muscle damage disease" refers to any disease in which muscle tissue or muscle tissue is damaged by damage to the muscle tissue. The muscle damage disease may be muscular dystrophy, muscular atrophy, myositis, polymyositis, peripheral vascular disease, sarcopenia, or fibrosis. there is. The muscular dystrophy is, for example, Becker's muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dreyfuss muscular dystrophy (Emery-Dreifusstrophy) muscular dystrophy, facial scapulohumeral muscular dystrophy, zone muscular dystrophy, myotonic muscular dystrophy, oropharyngeal muscular dystrophy, myotonic muscular dystrophy, sarcoglycanosis, spinal muscular atrophy, inflammatory myopathy, immune necrotizing myopathy myopathy) or Brown-Vialetto-Van Laere syndrome (BVVL). The Nogo-A gene and the agent are the same as described above.

Another aspect is to provide a composition kit for diagnosing myopathy comprising an agent for measuring the expression level of the NOGO-A gene or the amount of the NOGO-A protein.

The preparation for measuring the expression level of NOGO-A gene or the amount of NOGO-A protein, and myopathy-related diseases are as described above.

The kit has the effect of early diagnosis of myopathy-related diseases by measuring the expression or activity level of the NOGO-A gene or its protein. The kit may further include one or more other component compositions, solutions, or devices suitable for the analysis method, as well as primer pairs, probes, and antibodies for the diagnosis of myopathy-related diseases, RT-PCR kit, micro It may include an array chip kit, a DNA chip kit, and a protein chip kit.

In one embodiment, the kit for measuring the mRNA expression level of the NOGO-A gene may be a kit including elements necessary for performing RT-PCR. In addition to each primer pair specific for a marker gene, the RT-PCR kit includes a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC -Water (DEPC-water), sterile water, etc. may be included.

In another embodiment, the kit may be a kit including essential elements necessary for performing a microarray. The microarray kit includes a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof. It can be easily manufactured by the manufacturing method used as In order to fabricate a microarray, a micropipetting method using a piezoelectric method or a pin type to immobilize the detected marker on a substrate of a DNA chip using the probe DNA molecule A method using a spotter or the like can be used. The substrate of the microarray chip may be coated with an active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde. In addition, the substrate may be selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane, and nitrocellulose membrane.

In another embodiment, the kit may be a kit including essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, and the like for preparing a fluorescent probe. In addition, the substrate may include cDNA or oligonucleotide corresponding to a control gene or a fragment thereof.

Another aspect is a method comprising: measuring an expression level of a NOGO-A gene or an amount of a NOGO-A protein in a biological sample isolated from a subject suspected of having myopathy; and

It is to provide an information providing method for diagnosing myopathy comprising the step of comparing the measured gene expression level or protein amount with the control gene expression level or protein amount.

As used herein, the term "individual" refers to an individual who wants to determine whether or not a degenerative cranial nerve disease develops or the possibility of the onset or predicts the risk of onset. The subject may be a vertebrate, specifically mammals, amphibians, reptiles, birds, etc., may be more specifically a mammal, for example, a human (Homo sapiens).

As used herein, the term "biological sample" may include whole blood, serum, plasma, saliva, urine, sputum, lymph, cells, tissues, etc. isolated from an individual, preferably a sample secreted from a living body, more specifically , runny nose, saliva, urine, sputum, or lymph fluid isolated from the subject.

The "method for measuring expression or activity level" is a reverse transcription polymerase reaction, a competitive reverse transcription polymerase reaction, a real-time reverse transcription polymerase reaction, an RNase protection assay, Northern blotting, DNA chip, Western blot, ELISA, radioimmunoassay, radiation It may be at least one selected from the group consisting of an immunodiffusion method, an Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometry, and protein chip.

In the above method, when the expression or activity level of the NOGO-A gene or its protein measured in the tissue or cells of the individual is increased compared to the normal control, it can be determined as a degenerative cranial nerve disease.

According to one embodiment, the subject diagnosed with the myopathy exhibits significantly higher expression levels of NOGO-A, MyoD and myogenin mRNA.

According to one embodiment, in the circadian clock model of NOGO-A KO muscle, the down-regulation of BMAL1 and NPAS2 and the up-regulation of C/EBPa are more significant in NOGO KO muscle, and genes involved in increased muscle fat accumulation. increased expression and decreased the expression of cytoskeletal formation-related genes.

According to one embodiment, NOGO-A silenced cells are accompanied by a decreased p-AKT/AKT ratio, thereby blocking signal transduction of the AKT-mediated differentiation process and inhibiting muscle formation.

According to one embodiment, the NOGO-A gene regulates circadian rhythm regulation-related genes, thereby promoting the production of fat in the muscle when it is deficient, and preventing myogenesis differentiation, making it difficult to maintain muscle homeostasis. It also causes disturbances in sleep control.

According to a myopathy-related disease comprising an agent for measuring the expression or activity of NOGO-A gene or myogenic factor or protein thereof according to an aspect, a kit for diagnosing myopathy-related disease including the same, and a method, more conveniently and accurately related to myopathy It is effective in diagnosing diseases.

1 and 2 are results showing changes in the expression levels of NOGO and myogenic factors in the muscle in a pathological state.
3 is a result showing the change in the expression level of NOGO and myogenic factors in an animal model with muscle in a pathological state.
4 and 5 show the results of intracellular metabolic processes affected by NOGO-A deficiency.
6 is a result of analysis of the histological characteristics of the muscle induced by ^{myotoxic damage and the expression level of myogenic factors in NOGO +/+} and NOGO ^{−/- mice.}
7 is a result showing the change in the expression level of the protein indicating abnormal homeostasis in NOGO KO muscle.
8 is a result showing changes in the expression levels of NOGO-A and myogenic factors during the differentiation process of myoblasts.
9 and 10 are results showing changes in the expression level of myogenic factors in NOGO-A-silenced C2C12 cells.
FIG. 11 shows the results of imaging the co-expression of NOGO-A and MYH2 in the mature gastrocnemius of FIG. 1 by immunofluorescence.
12 is a result showing the change of fatty liver index in ALD mice by the HCFD diet + alcohol administration of FIG. 2 .
FIG. 13 is a result showing the expression level of IL-6 and the expression level of inflammatory cytokines in BMDM isolated from NOGO KO mice of FIG. 4 .
14 is a result showing the TF related to the NOGO-A expression process of FIG.
FIG. 15 is a result showing the absence of membrane-receptor interaction between SIPR2 and NOGO-A in C2C12 cells of FIG. 6 .
Figure 16 shows the relationship between NOGO, a circadian rhythm regulatory gene and muscle homeostasis.

Hereinafter, the present invention will be described in more detail through experimental examples. However, these examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these examples.

In the present invention, 12-week-old male wild-type (WT, C57BL / 10J) and MDX (C57BL / 10ScSn-Dmdmdx / J) mice were used as animal models for DMD experiments. MDX mice were provided by Jacques P. Tremblay (CHUQ Research Center, Quebec City, Canada) and wild-type mice were provided by SLC, Inc., Japan (Hamamatsu, Japan). NOGO KO mice in which the NOGO gene was knocked out were provided by Binhai Zheng (University of California, San Diego, CA, USA). The above animal experiments and protocols were approved by the Animal Experimental Ethics Committee (IAUCC) of Kyungpook National University (Daegu, KNU 2014-0167, KNU 2018-0105) and complied with the guidelines for animal testing of the National Institutes of Health (NIH). An intragastric (iG) ethanol feeding model was provided by the Animal Core Tissue Sharing Program of the Southern California Research Center (P50AA011999).

Statistical differences between the control group and the experimental group were evaluated by student's unpaired t- test (two-tailed). All results are expressed as mean SEM. P values less than 0.05 were considered statistically significant.

Experimental Example 1. Changes in the expression of NOGO and myogenic factors in muscle tissue in a pathological state

In this experimental example, by examining muscle tissue, which is a pathological condition including DMD and inflammatory myopathy, it was attempted to confirm the relationship between NOGO gene expression and myogenic factor expression at the transcriptional level. The muscle tissue section was approved by the medical ethics committee in accordance with Pusan National University Yangsan Hospital (Yangsan, Institutional Ethics Committee No. 05-2018-045) and Yonsei University School of Medicine (Seoul, Institutional Inspection Board No. 3-2018-0060). got it Skeletal muscle tissue was collected from 10 myopathy and DMD patients (D1-D10). Five age-matched control samples were collected (N1 to N5). The disease information of each patient is shown in Table 1 below.

# gender age (y) medical diagnosis D1 M 5 inflammatory myopathy D2 M 5.6 inflammatory myopathy D3 M 1.83 DMD (exon 45 deletion) D4 M 5.1 inflammatory myopathy D5 M 2 Miyoshi-type myopathy D6 F 81 inflammatory myopathy D7 F 46 inflammatory myopathy D8 F 57 inflammatory myopathy D9 M 15 DMD (exon45-52 deletion) D10 M 20 DMD (exon 50 deletion) N1 F 42 normal N2 F 26 normal N3 F 41 normal N4 F 18 normal N5 F 1.5 normal

The NOGO gene has three isoforms (A, B, C) through selective splicing and different initiation codons, with NOGO-C being the predominant isoform in skeletal muscle. The expression of NOGO and myogenic factors in patient muscle tissues in pathological conditions including DMD and inflammatory myopathies was evaluated at the transcriptional level.

RNA isolation and quantitative PCR analysis were performed as follows. Total RNA was isolated from mouse and human cells and muscle tissue using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from 2 μg of total RNA using RT-qPCR (Thermo Fisher Scientific) Maxima First Strand cDNA Synthesis Kit according to the manufacturer's instructions. Expression levels of various genes were analyzed by qRT-PCR using SYBR green (TOPreal qPCR premix, enzynomics) and Rotor-Gene Q (Qiagen). Expression of gene transcripts was normalized to GAPDH (human) or 18S rRNA (mouse), and the results were evaluated with Rotor-Gene Q series software (Qiagen).

Preparation of muscle tissue sections and hematoxylin and eosin staining were performed as follows. Muscle tissues of humans and mice were fixed in 4% paraformaldehyde in PBS for 16 hours, and then immersed in 30% and 35% sucrose solutions in PBS for 6 hours and 16 hours, respectively. The muscle tissue was then blocked in optimal cleavage temperature compound (OCT, Leica) and stored at -70 °C. They were cut into 4-5 µm sizes on a Leica cryostat, placed on slides, and dried in room air for 30 min. Prepared sections were stored at -70 °C until staining. For H (hematoxylin) & E (eosin) staining, muscle tissue sections were rehydrated with PBS _{, washed with H 2} O, and stained with a hematoxylin solution. After washing with H ₂ O, counterstained with eosin and dehydrated with xylene. Stained sections were mounted and images were captured in digital format using a Leica DM5000B.

Immunofluorescence staining of cells was performed as follows. After removal from the culture medium, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min. For permeabilization, cells were incubated in very cold methanol for 10 min and washed with PBS. Cells were blocked using a mixture of 5% donkey serum in PBS and primary antibody diluted with blocking solution overnight at 4°C. The fluorescence-conjugated secondary antibody mixture was then applied to the cells for 1 h. Coverslips were mounted with DAPI and anti-discoloration mounting solutions and images were visualized with a confocal laser scanning microscope. Images were analyzed using the manufacturer-supplied ZEN 2.1 Lite software.

Extraction of muscle protein and detection by Western blotting and ELISA analysis were performed as follows. In the muscle protein extraction step, Human and mouse muscles were lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 5 mM Na3VO4, 20 mM NaF, 10 mM sodium pyrophosphate, protease inhibitor mixture (Roche), 1 mM PMSF, 1% Triton-X, 0.1% SDS). Homogenization was performed using a homogenizer (IKA). After centrifugation (15,000 x g for 15 minutes at 4 °C) to remove debris, the total protein concentration of the supernatant was measured by BCA Protein Assay Kit (Pierce BCA Protein Assay Kit).

To extract cellular proteins to be used for western blotting, Cells in 6-well plates are lysed in 200 μl of cell lysis buffer (1X RIPA buffer (cell signaling), 1 mM PMSF, protease inhibitor cocktail (Roche)) and then rinse the cells with PBS. Lysate was incubated at 4 °C with shaking for 30 min to aid protein extraction. After centrifugation (15,000 x g for 15 min at 4 °C) to remove debris, the total protein concentration of the supernatant was measured by BCA Protein Assay Kit (Pierce BCA Protein Assay Kit, Thermo Scientific).

Western blotting was performed as follows. Samples are adjusted for protein concentration (2-3 μg/μl) and an appropriate volume of 5X SDS loading buffer (10% SDS, 500 mM DTT, 50% glycerol, 250 mM Tris-HCl and 0.5% bromophenol blue dye, pH 6.8), boiled at 95 °C for 10 min and electrophoresed on SDS polyacrylamide gel (30% Acrylamnide/Bis Solution, 29:1, 10 or 12% using Bio-rad). Proteins were transferred to nitrocellulose membranes (0.2 μm, Amersham), stained with Ponso solution (Translab), _{washed with H 2} O, and in blocking buffer (1% BSA in TBS containing 0.1% Tween 20) for 1 h. incubated. The membranes were then incubated with primary antibody diluted 1:1000 in blocking buffer overnight at 4 °C, followed by incubation with an appropriate secondary HRP-conjugated antibody for 1.5 h. Protein levels were determined by visualization using ECL reagent (Thermo Fisher Scientific). Bands were imaged with a chemiluminescence imager (Amersham) and band intensities were analyzed using ImageJ software.

ELISA analysis was performed as follows. The isolated muscle was transferred to lysis buffer (ProEX CETi lysis buffer), homogenized with a homogenizer (IKA), and the supernatant was collected after centrifugation at 15,000 x g for 15 min at 4 °C. The amount of IL-6 was measured using a mouse IL-6 platinum ELISA kit (Life Technologies Corporation) according to the manufacturer's instructions.

As a result, as shown in FIG. 1A , the expression level of the NOGO isoform in the muscle of patients with myopathy was significantly altered. Levels of NOGO-B were comparable to those in healthy muscle tissue, but NOGO-C was significantly lower and NOGO-A was significantly increased than normal. In addition, the transcripts of MyoD and myogenin were significantly elevated in the muscle in the pathological state compared to the normal muscle. As shown in FIGS. 2A and 2B , the increased NOGO-A and myogenin transcripts increased the levels of NOGO-A and myogenin in the pathologically damaged muscle. As shown in FIG. 1B , the muscles of patients with DMD and other myopathy including inflammatory myopathies showed typical histological features of degenerative muscles, including damaged myofibrillar structures, inflammatory cell infiltration, and fibrosis due to failure of muscle regeneration. As shown in FIG. 2C, DMD muscle tissue had high levels of NOGO-A in regenerated muscle fibers, and the increase in myosin heavy chain 2 (MYH2) expression was also significant.

Through the above results, it was found that NOGO isoforms are relevant in regulating the muscle regeneration process in patients with genetic and inflammatory muscle disease through changes in the expression levels of NOGO isoforms and myogenic factors.

Experimental Example 2. NOGO-A and MYH2 (myosin heavy chain 2) co-expression (Co-localize)

In this experimental example, the expression patterns of MYH and NOGO-A in mature skeletal muscle were observed, and it was attempted to determine whether the two proteins were co-expressed.

MYH expression is involved in the structural and functional properties of skeletal muscle fibers. NOGO-A and MYH2 were visualized in the gastrocnemius tissue of Experimental Example 1, and whether mature skeletal muscle expressed NOGO-A was observed by immunofluorescence.

Immunofluorescence staining of muscle tissue sections was performed as follows. The prepared muscle tissue sections were boiled in 10 mM citrate buffer (pH 6.0) for 10 minutes to remove antigen, cooled at room temperature for 30 minutes, washed with PBS, and infiltrated with 0.1% Triton-X in PBS for 5 minutes. The muscle tissue sections were then blocked with 5% donkey serum in PBS for 1 h and incubated with the primary antibody mixture in blocking buffer overnight at 4 °C. After washing with PBS, the fluorescently-conjugated secondary antibody was allowed to bind at room temperature for 1 hour. After washing with PBS, sections or cells were mounted using an anti-discoloration mounting solution with DAPI (Cell signaling) and images were visualized on a confocal laser scanning microscope (Carl Zeiss). Images were analyzed using the manufacturer-supplied ZEN 2.1 Lite software.

As a result, as shown in FIG. 11 , mature muscle showed strong MYH2 and NOGO-A expression, which were present under the same location.

From the above results, it can be seen that these molecules are co-localized.

Experimental Example 3. Changes in expression levels of NOGO and myogenic factors in a mouse model of muscle disease

In this experimental example, the expression of the transcriptional levels of NOGO and myogenic factors in a mouse model with muscles of various pathological conditions was investigated, and the increased NOGO-A expression level and the common expression pattern of myogenic factors regulating muscle differentiation were investigated. wanted to confirm.

In this experimental example, the pathological conditions are diverse, such as MDX, a genetic disease model of human DMD, a myotoxin-mediated acute muscle injury model, and alcoholic liver disease (ALD), a chronic inflammatory myopathy model, and the function of NOGO in muscle disease was observed. was analyzed to Although ALD is not a direct inducer of muscle damage, a relationship between skeletal muscle loss and ALD has been established. Skeletal muscle from ALD mice was also analyzed to elucidate the effect of hepatic dysfunction on chronic muscle injury via NOGO.

As a method for preparing a mouse model with abnormal muscle, to induce myotoxin-induced myotoxicity-mediated myopathy, 8-week-old WT male (C57BL / 6J, NOGO ^+/+ ) and NOGO KO (NOGO ^−/- ) mice were treated with notexin. (1 μg/mL in PBS, Latosan) was intramuscularly injected in a single 50 μl into gastrocnemius muscle. Mice were sacrificed 3 days or 2 weeks after injection and the gastrocnemius muscle was harvested. In addition, tunicamycin was intraperitoneally injected into 10-week-old WT males (n = 4) at a dose of 1 ug/kg. The gastrocnemius was harvested 24 hours after injection.

To integrate the Western HCFD (high-calorie/high-fat diet) feeding model with the iG ethanol feeding model, WT male mice were fed a solid HCFD (Dyets Inc., Catalog # 180724) or regular diet for 2 weeks. After feeding, iG ethanol was supplied through an iG catheter, and 60% of total daily caloric intake was supplied through a high-fat liquid diet. Mice consumed 40% of the remaining calories through free intake of HCFD or chow. The ethanol dose was gradually increased to 27 g/kg/day for 8 weeks, and the control mice were fed only the same calorie high-fat liquid diet to form the following two groups: Chow + Cont (control group), HCFD + Alc (experimental group). ). In addition, HCFD + Alc mice received weekly additional alcohol administration starting from the second week of iG feeding. In this case, ethanol injection was stopped for 5 to 6 hours, and an additional dose of 4 to 5 g / kg was administered to ethanol (HCFD + Alc + Binge). Mice were sacrificed, and gastrocnemius muscle was harvested from each mouse.

As a procedure for preparing muscle tissue sections, the mouse muscle tissue was fixed in 4% paraformaldehyde in PBS for 16 hours, and then immersed in 30% and 35% sucrose solutions in PBS for 6 hours and 16 hours, respectively. . The muscle tissue was then blocked in optimal cleavage temperature compound (OCT, Leica) and stored at -70 °C. They were cut into 4-5 µm sizes on a Leica cryostat, placed on slides, and dried in room air for 30 min. Prepared sections were stored at -70 °C until staining.

Western blot and ELISA analysis for protein extraction and detection were performed as described in Experimental Example 1 above.

As a result, as shown in A and B of FIG. 3 , the muscle of the MDX mouse showed significantly higher levels of NOGO-A, MyoD, and myogenin mRNA expression levels compared to the WT mouse (control group). Also, as shown in Fig. 3C and D, the up-regulated transcription levels of NOGO-A and MyoD were accompanied by an increase in the expression levels of these proteins. As shown in E, NOGO-A increased and NOGO-C decreased in skeletal muscle tissue of control and ALD mice, and as shown in F, increased myogenin and Pax7 were committed Pax7 + satellite cells and myogenin This indicates an increase in benign myoblasts. As shown in G and H, acute muscle injury induced by notexin was associated with a sharp increase in NOGO-A, a decrease in NOGO-C, and an increase in MyoD and myogenin. In addition, the number of Pax7 + committed satellite cells was not affected in this muscle injury, but MYH2 expression was significantly down-regulated, indicating structural muscle damage. Also, as shown in Fig. I, similarly to the notexin-induced damage, the tunicamycin-injured muscle also showed a dramatic increase in NOGO-A expression.

As shown in FIG. 12A , ALD mice fed high cholesterol and high saturated fat diet (HCFD) + alcohol for 10 weeks exhibited hepatomegaly (liver weight to body weight ratio). As shown in B, liver tissue analysis results of control and ALD mice. Pathological liver conditions were observed with increased macro and microvesicular steatosis. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) produced by the liver and muscle tissue are important indicators of liver dysfunction. AST is also considered a marker of skeletal muscle damage. As shown in C, the concentrations of plasma ALT and AST were upregulated in ALD mice compared to controls, suggesting both liver dysfunction and muscle damage. Also, as shown in D, impaired fat homeostasis was shown in the liver of ALD mice, which was associated with increased expression of adipogenic factors including PPDγ and C/EBPa.

Through the above results, it was possible to observe MyoD and myogenin as common expression patterns of NOGO-A and myogenic factors even in muscle tissues of different pathological conditions.

Experimental Example 4. Whether or not to prevent muscle differentiation, enhance adipocyte differentiation and enhance lipid metabolism gene expression in NOGO KO mice

In this experimental example, mRNA sequencing of gastrocnemius tissue obtained from WT and NOGO KO (knock-out) mice was performed, and the molecular function of NOGO was confirmed in the regulation of myogenesis.

mRNA-sequencing and data analysis were performed as follows. RNA was isolated from abdominal muscle tissue of WT and NOGO KO mice (3 biological replicates per group) using the Qiagen RNeasy mini Kit. The mRNA was then isolated from total RNA, ^{fragmented using Illumina Truseq™} Stranded mRNA LT sample preparation kit with poly-T oligo magnetic beads, and reverse transcription of the mRNA fragment was performed with Superscript III reverse transcriptase (Life Technologies). Adapter ligated libraries were generated according to the manufacturer's protocol and sequenced using a HiSeq 2500 system. Adapter sequences (TruSeq universal and indexed adapters) were trimmed with cutadapt to preprocess the read sequence data. Trimmed data were aligned to the reference mouse genome (GRCm38.p6) using the STAR aligner, and gene characterization in pieces per kilobase of transcription per million pieces of value (FPKM) mapped using Cufflinks software (GRCm38.95. gtf) was quantified.

Recognition of differentially expressed genes (DEGs) was performed as follows. Genes with FPKM greater than or equal to 1 for at least one of the six samples were defined as expressed genes. For these expressed genes, we normalized the log2 (FPKM) values using the quantile normalization method. To identify DEGs among expressed genes, adjusted p-values were calculated as previously described. For each gene, t-statistics were calculated using Student's t-test and log2-median-ratio (log2-fold-change) by first comparing log2 (FPKM) values between NOGO KO and WT samples. Six samples were then randomly repeated 1000 times to generate empirical KO distributions of t-statistic values and log2-fold changes. Based on the two empirical KO distributions for each gene, corrected p-values for Student's t-test and median-ratio test were calculated, and these adjusted p-values were combined by Stouffer's method. Genes with combined p-values of less than 0.05 and log2-fold combined were selected as DEGs with a cutoff (absolute mean of the 2.5th and 97.5th percentile values in the empirical distribution of log2-fold change, 1.3-fold change).

Functional enrichment analysis (GOBP and GOMF) was performed as follows. To identify cellular processes and pathways represented by DEG, enrichment analysis of GOBP and molecular function GOMF was performed and the KEGG pathway was determined using DAVID software. GO conditions and KEGG pathways with p-values less than 0.05 were designated as enriched analysis by DEG.

The recognition of key transcription factors (TFs) was performed as follows. To identify the major TFs responsible for DEG, TRED, Amadeus, HTRIdb, MSigDB, EEDB, bZIPDB and MetaCore™ (GeneGo, St. Joseph, MI, USA) should be collected. Human TF-target relationships were converted to mouse TF-target relationships using MGI human-mouse homolog mapping. Using these TF-target relationships, we finally selected major TFs as modulating significant (p < 0.05) DEGs (at least 5) based on Fisher's exact test.

Network analysis was performed as follows. To build a network model to explain the effects of NOGO on myotrophic or cellular differentiation, we first selected DEGs involved in these processes-related GOBP, GOMF and KEGG pathways. Protein-protein interactions for DEGs were collected, selected from the following protein-protein interaction databases: BioGrid, ConcensusPathDB, DIP, IntAct, MINT and STRING. Using protein-protein interactions for selected DEGs, a network model was built using Cytoscape, and then nodes were arranged according to the pathway information in the KEGG pathway database.

Transcription factor (TF) prediction was performed as follows. To predict TFs potentially regulating NOGO expression, the NOGO TF binding site sequence (ENSMUSR00000513840) was analyzed through the JASPAR database with reference to the Ensembl database. Probable factors are listed based on relative scores greater than 0.9.

When comparing the mRNA expression patterns of the mice in Tables 2 and 3, 703 differentially expressed genes (DEGs) were listed among them. Of these, 355 up-regulated genes are listed in Table 2, and 348 down-regulated genes in NOGO KO muscle are listed in Table 3.

Tables 4 and 5 below show the gene ontology biological process (GOBP) and gene ontology molecular function (GOMF) using DAVID software to investigate potential cellular processes affected by NOGO deficiency. The results of performing the concentration analysis are shown.

Genes upregulated in NOGO KO muscle tissue were mainly involved in adipocyte development. As a result, as shown in A and B of FIG. 4 , brown and white adipocyte differentiation and lipid metabolism proceed through fatty acid metabolism, insulin receptor signaling pathway, and PPAR signaling pathway. In addition, downregulated genes were mainly involved in actin filament-based processes, actin cytoskeletal formation, actin binding, muscle development via regulation of muscle hypertrophy, and cytoskeletal formation via regulation of skeletal muscle adaptation and cell differentiation. GOMF, enhanced by downregulated genes, contained S1P receptor activity known to induce myogenic formation.

To reveal the potential transcription factor (TF) regulated by NOGO, TF enrichment analysis of muscle tissue was performed, and as a result, 17 major TFs were found as shown in FIG. 4C . Among them, NOGO KO increased four TFs (Cebpa, Cebpb, Cebpd, Pparg) known to regulate adipocyte development, but downregulated Myod1, a key TF for muscle development.

Through the above results, both up-regulated and down-regulated genes contained significant numbers related to transcription factor (TF) binding, suggesting that NOGO regulates TF function. These data suggest that a significant number of TFs are genes downstream of NOGO, contributing to the attenuation of myotube development.

Fig. 4D shows a network model explaining the GOBP gene and major TFs to understand the functions of differentially expressed genes (DEGs) and five functions important for the deterioration of myotube development. In the left part, we show that NOGO KO down-regulates Wnt signaling in Wnt5a, Fzd9 and Six1, thereby reducing the activation of Myod1. Also in the lower part, we show that negative regulators of Myod1 are upregulated by NOGO KO, including Id3/3 and the upstream TFs Cebpa/b/d and Pparg. In the right part, we show that NOGO KO altered the mRNA expression levels of Slc8a3, Cacna1g and Sphk2 molecules involved in reducing the amount of cytosolic calcium. In the middle part, NOGO KO further down-regulated Calm1 and Adcy9a components in downstream calcium signaling and sphingosine-1 phosphate (S1P)-PI3K (S1pr3/4, Pik3ca and Prkcd) pathways, leading to decreased cytoskeletal reorganization activation resulted (Marcks, Ppp1r14a and Acta2). These data imply that NOGO-A actively upregulates Wnt, calcium, Myod1 and S1P-PI3K signaling pathways, and downregulates insulin signaling and Cebpa/b/d and Pparg to regulate myogenesis.

According to a previous study, NOGO-A-deficient transgenic mice displayed altered circadian activity patterns, indicating that NOGO-A is involved in the maintenance of the circadian clock. Figure 5A shows muscle attenuation through 24-hour period control of an alternative network model of NOGO KO. Interestingly, the circadian rhythm-related genes shown in Fig. 5A are the groups whose up-regulation and down-regulation were measured in the present invention.

To elucidate potential circadian clock activity by NOGO KO, the identified DEGs were mapped to the KEGG (Kyoto and Encyclopedia of Genes and Genomes) pathway database, and key clock elements Cry1, Cry2, Per1, Per2 , Per3, Bhlhe40, Bmal1, Npas2 and Clock were identified as DEGs in the circadian clock pathway.

In particular, CLOCK: the BMAL1 inhibitors Cry2, Per1, Per2, Per3 and Bhlhe40 were up-regulated, and the CLOCK: BMAL1 elements Bmal1, Npas2 and Clock were down-regulated. These clock components are known to regulate myogenesis. As a result, as shown in the left side of FIG. 5A, CLOCK: BMAL1 elements Bmal1, Npas2 and Clock bind to Myod1 enhancers to induce Myod1 expression. In contrast, Bhlhe40 in the stomach interacts with Cebpb to regulate adipogenic differentiation. This model proposes that NOGO-A regulates myogenesis through regulation of circadian clock gene expression.

To establish the role of NOGO in circadian regulation and muscle adipocyte deposition, we analyzed the muscles of 70-week-old mice. As a result, as shown in B and C of FIG. 5 , downregulation of BMAL1 and NPAS2 was enhanced in NOGO KO muscle, and the increase in C/EBPa was more significant in NOGO KO muscle. Therefore, it was found that NOGO regulates fat accumulation in muscle through regulation of the circadian clock.

Experimental Example 5. Increase in MyoD expression level and increase in inflammatory cell infiltration due to notexin damage in NOGO KO mice

NOGO-A is the longest of the NOGO isoforms and contains the sequences NOGO-B and -C. In this experimental example, the skeletal muscle of NOGO KO mice was investigated, and the function of NOGO-A was investigated in muscle development and maintenance of muscle integrity. As shown in Fig. 6A, the muscles of the WT mice showed well-structured muscle fibers and no immune cell infiltration, whereas the muscles of the NOGO KO mice showed defective muscle fibers and immune cell infiltration.

Myotoxicity-mediated injury has broadened our understanding of the activity of myogenic factors and inflammation in the myogenesis process of skeletal muscle. In this experimental example, to investigate the role of NOGO-A in regulating myogenesis, a mouse model in which muscle damage was induced by notexin was used. In the muscle damage induced by myotoxicity, inflammation appears 3 days after notexin injection, and muscle regeneration is completed for 28 days after injection.

Preparation and staining of muscle tissue sections, Western blotting and ELISA analysis were performed as described in Experimental Example 1.

As shown in Fig. 6B, exposure to notexin showed severe inflammation and typical histological results of destroyed muscle fibers. NOGO KO enhanced inflammatory cell infiltration to the site of injury compared with the WT control. As shown in Fig. 6C, during muscle regeneration, new muscle fibers grew by proliferation and differentiation of myoblasts; NOGO KO increased the number of regenerated central core muscle fibers compared to the control group.

As shown in G and H of FIG. 3 reviewed above, Notexin damage resulted in a significant increase in NOGO-A mRNA level as well as an increase in myogenic factors including MyoD and myogenin 3 days after notexin injection.

As shown in Fig. 6D and E, as the mRNA level increased in notexin injury-induced muscle, the NOGO-A protein level also increased; The level of this increase decreased slightly during the 2 weeks of muscle regeneration after notexin injury. As shown in D and F, the effect of NOGO KO on Pax7 level was minimally decreased the cell number at 8 weeks of committed satellite cells, but the decrease in cell number increased at 10 weeks. As shown in D and G, the level of Pax7-positive progenitor cells in NOGO KO muscle in the inflammatory state on the 3rd day after notexin injection was lower than that of the control group, but recovered to the control level at 2 weeks after injection. MyoD is a myogenic factor regulating muscle fiber proliferation, and the level of MyoD was increased in NOGO KO muscle regardless of notexin damage. As shown in D and H, NOGO deficiency was associated with a decrease in myogenin, a differentiation inducer that forms myotubes, and the decrease was increased in the inflammatory state.

Through the above results, it was found that NOGO KO muscle, along with a decrease in myogenin-inducing ability, continued to differentiate excessively myoblasts to induce defective regeneration.

Experimental Example 6. Whether ER stress and apoptosis increase in NOGO KO mice

Fig. 6A shows the defect features of ^{NOGO −/−} muscle, including irregular myofiber structure and inflammatory cell deposition. In this experimental example, by analyzing the apoptotic state using the ER stress marker caspase 3 and the apoptosis inducer CHOP in the abnormal muscle structure of ^{NOGO -/- mice, it was determined whether the destructive signal was enhanced.} wanted to confirm.

Western blotting and ELISA analysis was performed as described in Experimental Example 1.

Caspase 3 (35 kDa) activates the death signal after cleavage into two fragments (15 and 17 kDa) by active caspase 8. 7A and B, the muscles of WT mice showed similar expression levels of procaspase 3, whereas muscles of NOGO KO mice showed increased expression levels of procaspase 3. NOGO KO is associated with a further increase in procaspase 3 upon notexin damage. No cleaved caspase 3 was detected (data not shown). As shown in C and D, CHOP expression was enhanced in NOGO KO muscle compared with the control group, and the degree of increase was strongest in the muscle regeneration phase 2 weeks after muscle injury.

Activation of AKT mediates signal transduction-promoting protein synthesis and inhibits proteolysis, but inhibition of AKT activation leads to FOXO1-mediated lysosomal proteolysis and proteasome degradation. As shown in E to G of Figure 7, NOGO KO mice significantly increased the expression levels of AKT and phosphorylated AKT (p-AKT), WT mice did not show a significant increase. As shown in H, the ratio of p-AKT/AKT did not change significantly, the enhanced AKT signaling pathway exhibited persistent hypertrophic signals affected by the absence of NOGO, and young muscles were more sensitive to this phenomenon.

Experimental Example 7. Whether the IL-6 expression level of NOGO KO mice increased

Interleukin-6 (IL-6) is a myokin produced by inflammatory cells that promotes muscle development after muscle injury. In this experimental example, muscle damage was investigated, and it was attempted to determine whether the IL-6 expression level was increased accordingly.

WT and NOGO KO (n = 3 for each group) mice were sacrificed and the tibia and femur were isolated. Joints were sterilized with 70% ethanol, washed with PBS, cut with sterile scissors, and bone marrow was isolated using a syringe filled with sterile PBS. Bone marrow was dispersed in 15 mL tubes, and cells were collected after centrifugation at 1,500 rpm at 4 degrees for 5 min. Cells were resuspended in R10 medium (RPMI1640, 10% FBS, 20 mM HEPES, 100 U/mL penicillin-streptomycin and 15% L929 conditioned medium), transferred to a culture dish, and medium changed every 2 days for 7 days. incubated during To induce inflammation, LPS (100 ng/mL) was added to the culture medium and cultured for 24 hours.

Western blotting and ELISA analysis was performed as described in Experimental Example 1, and immunofluorescence staining was performed as described in Experimental Examples 1 and 2.

As shown in Figures 6A and B, NOGO KO mice displayed high levels of inflammatory cell infiltration in muscle damage and muscle dysregulation.

The effect of muscle damage on IL-6 expression was evaluated by measuring the expression levels of IL-6 transcripts and proteins. As shown in FIG. 13A , the expression level of the IL-6 transcript was lower in the NOGO ^{−/− muscle than in the NOGO +/+} muscle, and the increase in IL-6 in the ^{NOGO −/− muscle upon injection of notexin was NOGO + //} less than in the muscle. As shown in FIG. 13B, IL-6 production in NOGO KO muscle was confirmed as a result of analyzing IL-6 in muscle tissue by ELISA.

Since macrophages are a major source of IL-6 during inflammation, bone marrow-derived macrophages (BMDM) were isolated from ^{NOGO +/+} and NOGO ^{−/- mice.} Expression of inflammatory cytokines such as IL-6, interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in the presence or absence of lipopolysaccharide (LPS), a potent inflammatory inducing factor analyzed. As shown in Fig. 13C, LPS increased the expression of inflammatory cytokines and NOGO ^−/− BMDM expressed a low level of cytokine compared with ^{NOGO +/+ BMDM.}

These results suggest that the absence of NOGO down-regulates the levels of IL-6, an inflammatory cytokine, in muscle tissue and macrophages.

Experimental Example 8. Relationship between NOGO-A and myogenic factor expression in late differentiation of C2C12 cells

Based on the observation that NOGO-A levels increase under pathological conditions in this experimental example, the role of NOGO-A on myogenesis in C2C12 mouse myoblasts was evaluated.

C2C12 source cells, immortalized mouse myoblast cell line a in humidified atmosphere containing 5% CO ₂ at 37 ° C 10% fetal bovine serum (FBS) and antibiotic-a is added (100 U / mL Penicillin Streptomycin) Dulbecco's modified Eagle's Cultured in medium (DMEM). Culture iMSCs and sort-iMSCs in DMEM containing 10% FBS, 10% HS, antibiotics and fibroblast growth factor (FGF, 5 ng/mL), and in low glucose DMEM medium containing 5% HS and antibiotics 3 Differentiation was induced by incubation for days.

Immunoprecipitation (IP) of C2C12 cells was performed as follows. C2C12 cells were plated in 100 mm tissue culture dishes and cultured with appropriate culture medium. Then, the cells were washed with PBS, lysed with 400 μl of Pierce IP lysis buffer (Thermo Fisher Scentific), homogenized for 4 minutes and 30 seconds, and then shaken. The lysate was centrifuged at 15,000 g for 5 min at 4 °C and the supernatant was collected. Total protein concentration was determined by BCA protein assay (Thermo Scientific).

Cell lysates (300 µg of total protein in a 250 µl volume) were pre-washed with protein G-coated dynabeads (Invitrogen, 5 µl/sample) at 4 °C for 1 h, followed by 2 µg of S1PR2 antibody overnight at 4 °C with rotation. was immunoprecipitated with

Immune complexes were collected by spinning with protein G-coated dynabeads (1 μL/sample) by incubation at 4 °C for 3 h. The collected beads were washed with 1 ML of IP buffer (3 times for 10 min each wash), added 50 μl of 1X SDS loading buffer, and boiled at 95 °C for 10 min. Western blotting was performed to detect NOGO-A S1PR2 as a lysate control. The lysate control contained 30 μl of the IP sample and 30 μl of the unbound solution under reducing conditions of 30 μl.

Western blotting and ELISA analysis was performed as described in Experimental Example 1, and immunofluorescence staining was performed as described in Experimental Examples 1 and 2.

According to A showing the initial differentiation of C2C12 myoblasts in FIG. 8 , the expression levels of the satellite cell marker Pax7 and the initial myogenic differentiation marker MyoD were significantly high during the initial differentiation, and NOGO-A was expressed in the Pax7 negative and MyoD positive cells. This appeared. According to B showing the late differentiation of C2C12 myoblasts in FIG. 8, myogenin and MYH2, which are late differentiation markers, also appeared in NOGO-A-positive cells. 8C is a graph showing the expression levels of NOGO-A and myogenic factors during the differentiation of myoblasts, and it can be observed that the expression levels of both MyoD and myogenin increase as the expression level of NOGO-A increases. . Myotube formation was induced by inducing myoblast differentiation and fusion by culturing the cells with a differentiation medium (DM), and as shown in Fig. 8D to F, the differentiated cells showed a dramatic decrease in Pax7 and MYH2 and NOGO- represents an increase in A. Analysis of the expression levels of NOGO-A and myogenic factors was in good agreement with these observations. During differentiation, NOGO-A expression and its cytoplasmic localization appeared corresponding to the localization of MYH2.

Induced myogenic stem cells (iMSCs) with strong myogenic differentiation capacity were extracted from mouse embryonic fibroblasts (MEFs), and iMSCs classified as having stem cell markers (sort-iMSCs) have been shown to have enhanced myogenic potential. showed In FIG. 8, H shows the differentiated iMSCs, I shows that Pax7 or MYH2 is stained in green, and NOGO-A is stained in red in sort-iMSCs. As shown in Fig. 8G to I, differentiated iMSCs and sort-iMSCs were analyzed to confirm the expression of NOGO-A during differentiation, and enhanced NOGO-A expression and co-expression with MYH2 during differentiation in both populations. (co-localization) appeared.

From the above results, it was found that NOGO-A co-expressed with MYH2 in C2C12 mouse myoblasts and increased the expression of myogenic factors.

Experimental Example 9. Whether or not myoD and myogenin regulation of NOGO-A regulates myogenesis

This experimental example was to investigate whether NOGO-A performs the completion of myotube formation for muscle functional activity, and to confirm that it is important for later muscle differentiation. To this end, the function was confirmed by reducing NOGO-A expression in C2C12 cells by using small interfering RNA (si-RNA) targeting NOGO-A (si-NOGO-A) and then inducing muscle differentiation.

Immunofluorescence staining of muscle tissue sections and cells was performed in the same manner as in Experimental Example 1.

Analysis of the immunofluorescent staining images was performed as follows. Stained cells and muscle sections were imaged with a confocal laser scanning microscope LSM700 (Carl Zeiss). Five non-overlapping images at 100X, 200X, or 400X magnification were acquired from each slide. Pinhole size, image resolution, color depth, scan rate and average (number of scans per image) were identical in all experiments. Image adjustments and analysis were performed using Zen 2.1 Lite (Carl Zeiss). For each field, the length of the root canal and the total number of nuclei were recorded. The fusion index was calculated as the percentage of total nuclei contained in the root canal.

As shown in FIG. 9A , the NOGO-A silencing effect by si-NOGO-A at the NOGO-B and NOGO-C levels was insignificant. Down-regulated NOGO-A simultaneously reduced myogenin and MYH2 transcript levels as shown in Fig. 9B, and protein expression levels of MyoD and myogenin as shown in C and D. The activity of NOGO-A during myoblast differentiation was shown by visualizing MyoD and myogenin in NOGO-A-silenced C2C12 cells as shown in FIGS. 9E to I. Differentiated NOGO-A-silenced cells significantly reduced the number of MyoD and myogenin-expressing cells, as shown in E, G, H, and I of FIG. 9, suggesting that NOGO-A is an important regulator of myogenic factors. do. As shown in FIG. 10A , myotube development was attenuated by NOGO-A silencing, and as shown in B, myotube fusion index was significantly reduced in NOGO-A silencing cells. After that, as shown in C and D of FIG. 10 , as a result of the decrease in NOGO-A expression, small diameter and immature muscle fibers were formed.

AKT mediates signal transduction to promote muscle regeneration. As shown in E and F of FIG. 10 , during the differentiation process, the expression level of AKT increased compared to the proliferation process. However, NOGO-A-silenced cells showed a greater increase in AKT expression level, compared with a slight increase in p-AKT and a decrease in the p-AKT/AKT ratio. Increased AKT concomitantly with reduced p-AKT/AKT ratio in NOGO-A-silenced cells blocks signaling of AKT-mediated differentiation processes and prevents them from regulating myogenesis.

Through the above results, it was found that NOGO-A plays a role as a myogenic regulator by participating in the number of myogenin and MyoD-expressing cells and in the AKT-mediated differentiation process.

Experimental Example 10. Binding of ERR-gamma (ERRg, ERRγ) to the NOGO-A transcription factor binding site

In this experimental example, it was attempted to determine whether ERR-gamma (ERRg, ERRγ) was bound to the NOGO-A transcription factor binding site. In Fig. 14A, we analyzed NOGO TF binding sites and presented a list of TFs expected to bind to those sites. Differentiated C2C12 cells were analyzed to evaluate the involvement of predicted TFs in myoblast differentiation. As shown in Fig. 14B, the expression levels of Arid3b, Arnt, Mlxip, Klf1, Mycn and ERRg among the predicted TFs were significantly increased.

Western blotting and ELISA analysis was performed as described in Experimental Example 1.

Gene silencing was achieved by transfection of specific siRNAs against NOGO-A and ERRγ into C2C12 myoblasts. siRNAs for mouse NOGO-A (si-NOGO-A) and mouse ERR (si-ERR) were chemically synthesized (Bioneer) and pre-made si-negative control RNAs (si-scrambled, Bioneer) were purchased. C2C12 cells were ^{plated in 6-well plates at 1 x 10 5} cells per well, incubated to a density of 40%, and incubated with antibiotic-free culture medium for 2 h before transfection. Lipofectamine 2000 reagent (Invitrogen, 1:100) and si-RNA (50 nM) were diluted with opti-MEM according to the manufacturer's instructions, and the mixture was added to the cells for 6 h. After removing the si-RNA mixture from the cells, the cells were incubated with growth medium for 2 days; The medium was changed to DM to induce culture differentiation and cells were cultured for 3 days.

After ERRg (ERR-gamma) showed the most significant change, the involvement of ERRg in NOGO-A expression was further investigated using the gene silencing method. As shown in FIG. 14C , the decrease in ERRγ lowered the NOGO-A transcription level, indicating that ERRγ plays an important role in NOGO-A expression. As shown in D and E of FIG. 14 , the expression level of ERRγ in muscle was increased in the tissues of patients with myopathy and MDX mice compared to those in normal or WT muscles.

From the above results, it was found that NOGO-A can be induced by ERRγ expression during muscle regeneration.

Experimental Example 11. Independent of NOGO-A and membrane receptor interaction during myogenesis

In this experimental example, by examining NOGO-A activity in the plasma membrane, it was attempted to determine whether it was induced through binding to the NOGO receptor NgR in the NOGO-66 region and S1PR2 in the NOGO-A-specific region 20.

NgR is a common receptor for all three NOGO isomers, and S1PR2 is specific for NOGO-A only. The interaction between NOGO-A and S1PR2 was analyzed by differentiated C2C12 cells to investigate whether NOGO participates in myoblast differentiation in a membrane receptor-dependent manner.

As shown in Fig. 15, the expression of S1PR2 did not change during the differentiation process and no interaction with NOGO-A occurred in the differentiated myoblasts, and NOGO-A functions due to an intracytoplasmic mechanism independent of S1PR2 in myoblast differentiation. appeared to do

Experimental Example 12. Functions of NOGO-A related to circadian rhythm regulation during muscle regeneration of myoblasts

NOGO KO muscle exhibits defects in the differentiation process for myofibrils to form myofibrils due to the deficiency of NOGO, and has limitations in maintaining muscle homeostasis.

As shown in Fig. 16, NOGO expressed in myoblasts increases NOGO-A production to form functional muscles and then is involved in proper differentiation. A well-controlled 24-hour rhythm helps maintain muscle homeostasis. However, NOGO KO myoblasts perform the defective myogenesis associated with NOGO-A deficiency, resulting in impaired muscle structure and function. The expression of NOGO KO-associated circadian rhythm regulatory genes is altered to downregulate myogenesis as well as upregulate the adipogenic signaling pathway, thereby increasing adipocyte deposition.

As reviewed above, NOGO KO fails to maintain the homeostasis and integrity of the damaged muscle, and NOGO-A is very important in this process.

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Thr 545 550 555 560 Ser Glu Ala Ile Gln Glu Ser Ile Tyr Pro Thr Ala Gln Leu Cys Pro 565 570 575 Ser Phe Glu Glu Ala Glu Ala Thr Pro Ser Pro Val Leu Pro Asp Ile 580 585 590 Val Met Glu Ala Pro Leu Asn Ser Leu Leu Pro Ser Thr Gly Ala Ser 595 600 605 Val Ala Gln Pro Ser Ala Ser Pro Leu Glu Val Pro Ser Pro Val Ser 610 615 620 Tyr Asp Gly Ile Lys Leu Glu Pro Glu Asn Pro Pro Pro Tyr Glu Glu 625 630 635 640 Ala Met Ser Val Ala Leu Lys Thr Ser Asp Ser Lys Glu Glu Ile Lys 645 650 655 Glu Pro Glu Ser Phe Asn Ala Ala Ala Gln Glu Ala Glu Ala Pro Tyr 660 665 670 Ile Ser Ile Ala Cys Asp Leu Ile Lys Glu Thr Lys Leu Ser Thr Glu 675 680 685 Pro Ser Pro Glu Phe Ser Asn Tyr Ser Glu Ile Ala Lys Phe Glu Lys 690 695 700 Ser Val Pro Asp His Cys Glu Leu Val Asp Asp Ser Ser Pro Glu Ser 705 710 715 720 Glu Pro Val Asp Leu Phe Ser Asp Asp Ser Ile Pro Glu Val Pro Gln 725 730 735 Thr Gln Glu Glu Ala Val Met Leu Met Lys Glu Ser Leu Thr Glu Val 740 745 750 Ser Glu Thr Val Thr Gln His Lys His Lys Glu Arg Leu Ser Ala 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Val 965 970 975 Asp Leu Leu Tyr Trp Arg Asp Ile Lys Lys Thr Gly Val Val Phe Gly 980 985 990 Ala Ser Leu Phe Leu Leu Leu Ser Leu Thr Val Phe Ser Ile Val Ser 995 1000 1005 Val Thr Ala Tyr Ile Ala Leu Ala Leu Leu Ser Val Thr Ile Ser Phe 1010 1015 1020 Arg Ile Tyr Lys Gly Val Ile Gln Ala Ile Gln Lys Ser Asp Glu Gly 1025 1030 1035 1040 His Pro Phe Arg Ala Tyr Leu Glu Ser Glu Val Ala Ile Ser Glu Glu 1045 1050 1055 Leu Val Gln Lys Tyr Ser Asn Ser Ala Leu Gly His Val Asn Ser Thr 1060 1065 1070 Ile Lys Glu Leu Arg Arg Leu Phe Leu Val Asp Asp Leu Val Asp Ser 1075 1080 1085 Leu Lys Phe Ala Val Leu Met Trp Val Phe Thr Tyr Val Gly Ala Leu 1090 1095 1100 Phe Asn Gly Leu Thr Leu Leu Ile Leu Ala Leu Ile Ser Leu Phe Ser 1105 1110 1115 1120 Ile Pro Val Ile Tyr Glu Arg His Gln Ala Gln Ile Asp His Tyr Leu 1125 1130 1135 Gly Leu Ala Asn Lys Ser Val Lys Asp Ala Met Ala Lys Ile Gln Ala 1140 1145 1150 Lys Ile Pro Gly Leu Lys Arg Lys Ala Glu 1155 1160 <210> 2 <211> 6165 <212> RNA <213> Mus musculus <400> 2 cccctgccgg gaaggtgagt cacgccaaac tgggcggaga gtctgccctc ctctctcagt 60 tgctcatctg ggcggcggcg gctgctgcaa ctgaggacag ggcgggtggc gcatctcgag 120 cgcggaggca ggaggagaag tcttattgtt cctggagctg tcgcctttgc gggttcctcg 180 gcttggttcg gccagcccgg cctctgccag tcctgcccaa cccccacaac cgcccgcggc 240 tctgaggaga agtggcccgc ggcggcagta gctgcagcat catcgccgac catggaagac 300 atagaccagt cgtcgctggt ctcctcgtcc gcggatagcc cgccccggcc cccgcccgct 360 ttcaagtacc agttcgtgac ggagcccgag gacgaggagg acgaggaaga cgaggaggag 420 gaggaggacg acgaggacct ggaggaattg gaggtgctgg agaggaagcc cgcagccggg 480 ctgtccgcgg ctccggtgcc ccccgccgcc gcaccgctgc tggacttcag cagcgactcg 540 gtgccccccg cgccccgcgg gccgctgccg gccgcgcccc ccaccgcccc tgagaggcag 600 ccgtcctggg aacgcagccc cgcggcgtcc gcgccatccc tgccgcccgc tgccgcagtc 660 ctgccctcca agctcccgga ggacgacgag cctccagcgc ggcctccggc gccagccggc 720 gcgagccccc tagcggagcc cgccgcgccc ccttccacgc cggccgcgcc caagcgcagg 780 ggctcgggct cagtggatga gacccttttt gctcttcctg ctgcatctga gcctgtgata 840 ccctcctctg cagaaaaaat tatggatttg aaggagcagc caggtaacac tgtttcgtct 900 ggtcaagagg atttcccatc tgtcctgttt gaaactgctg cctctcttcc ttctctatct 960 cctctctcaa ctgtttcttt taaagaacac ggataccttg gtaacttatc agcagtggca 1020 tccacagaag gaactattga agaaacttta aatgaagctt ctagagaatt gccagagagg 1080 gcaacaaatc catttgtaaa tagagagtca gcagagtttt cagtattaga atactcagaa 1140 atgggatcat ctttcaatgg ctccccaaaa ggagagtcag ccatgttagt agaaaacact 1200 aaggaagaag taattgtgag gagtaaagac aaagaggatt tagtttgtag tgcagccctt 1260 cataatccac aagagtcacc tgcgaccctt actaaagtgg ttaaagaaga cggagttatg 1320 tctccagaaa agacaatgga catttttaat gaaatgaaaa tgtcagtggt agcacctgtg 1380 agggaagagt atgcagattt taagccattt gaacaagcat gggaagtgaa agatacttat 1440 gagggaagta gggatgtgct ggctgctaga gctaatatgg aaagtaaagt ggacaaaaaa 1500 tgctttgaag atagcctgga gcaaaaaggt catgggaagg atagtgaaag cagaaatgag 1560 aatgcttctt tccccaggac cccagaactt gtgaaggacg gctccagagc gtacatcacc 1620 tgtgattcct ttagctcagc aaccgagagt actgcagcaa acattttccc tgtgctagaa 1680 gatcacactt cagaaaacaa aacagatgaa aaaaaaatag aagaaaggaa ggcccaaatt 1740 ataacagaga agactagccc caaaacgtca aatcctttcc ttgtagcaat acatgattct 1800 gaggcagatt atgtcacaac agataattta tcaaaggtga ctgaggcagt agtggcaacc 1860 atgcctgaag gtctaacgcc agatttagtt caggaagcat gtgaaagtga actgaacgaa 1920 gccacaggta caaagattgc ttatgaaaca aaagtggact tggtccagac atcagaagct 1980 atacaagagt caatttaccc cacagcacag ctttgcccat catttgagga agctgaagca 2040 actccgtcac cagttttgcc tgatattgtt atggaagcgc cattaaattc tctccttcca 2100 agcactggtg cttctgtagc gcagcccagt gcatccccac tagaagtacc gtctccagtt 2160 agttatgacg gtataaagct tgagcctgaa aatcccccac catatgaaga agccatgagt 2220 gtagcactaa aaacatcgga ctcaaaggaa gaaattaaag agcctgaaag ttttaatgca 2280 gctgctcagg aagcagaagc tccttatata tccattgcat gtgatttaat taaagaaaca 2340 aagctctcca ctgagccaag tccagagttc tctaattatt cagaaatagc aaaatttgag 2400 aagtcggtgc ctgatcactg tgagctcgtg gatgattcct cacccgaatc tgaaccagtt 2460 gacttattta gtgatgattc aattcctgaa gtcccacaaa cacaagagga ggctgtgatg 2520 ctaatgaagg agagtctcac tgaagtgtct gagacagtaa cacaacacaa acataaggag 2580 agacttagtg cttcacctca ggaggtagga aagccatatt tagagtcttt tcagcccaat 2640 ttacatatta caaaagatgc tgcatctaat gaaattccaa cattgaccaa aaaggagaca 2700 atttctttgc aaatggaaga gtttaatact gcaatttatt ccaatgatga cttactttct 2760 tctaaggaag acaaaatgaa agaaagtgaa acattttccg attcatctcc cattgagata 2820 atagatgagt ttcccacatt tgtcagtgct aaagatgatt ctcctaagga gtacactgac 2880 ctagaagtat ccaacaaaag tgaaattgct aatgtccaga gcggggccaa ttcgttgcct 2940 tgctcagaat tgccctgtga cctttctttc aagaatacat atcctaaaga tgaagcacat 3000 gtctcagatg aattctccaa aagtaggtcc agtgtatcta aggtgccctt attgcttcca 3060 aatgtttctg ctttggaatc tcaaatagaa atgggcaaca tagttaaacc caaagtactt 3120 acgaaagaag cagaggaaaa acttccttct gatacagaga aagaggacag atccctgaca 3180 gctgtattgt cagcagagct gaataaaact tcagttgttg acctcctgta ctggagagac 3240 attaagaaga ctggagtggt gtttggtgcc agcttattcc tgctgctgtc tctgacagtg 3300 ttcagcattg tcagtgtaac ggcctacatt gccttggccc tgctctctgt gactatcagc 3360 tttaggatat ataagggtgt gatccaagct atccagaaat cagatgaagg ccacccattc 3420 agggcatatt tggaatctga agttgccata tcagaggaat tggttcagaa atatagtaat 3480 tctgctcttg gtcatgtgaa cagcacaata aaagaattga ggcgtctctt cttagttgat 3540 gatttagttg attccctgaa gtttgcagtg ttgatgtggg tatttactta cgttggtgcc 3600 ttgttcaatg gtttgacact actgatttta gctctgatct cactcttcag tattcctgtt 3660 atatatgaac ggcatcaggc gcagatagat cattatctag gacttgcaaa caagagcgtt 3720 aaggatgcca tggccaaaat ccaagcaaaa atccctggat tgaagcgcaa agcagaatga 3780 aaaggcccca aacagtagac attcatcttt aaaggggaca ctcccttggt tacggggtgg 3840 gcgggtcagg ggtgagccct gggtggccgt gcagtttcag ttatttttag cagtgcactg 3900 tttgaggaaa aattacctgt cttgacttcc tgtgtttatc atcttaagta ttgtaagctg 3960 ctgtgtatgg atctcattgt agtcatactt gttttcccca gatgaggcac ttggtgaata 4020 aaggatgctg ggaaaactgt gtgttatatt ctgttgcagg tagtctggct gtatttggaa 4080 agttgcaaag aaggtagatt tgggggcagg aaaaacaacc cttttcacag tgtactgtgt 4140 ttggttggtg taaaactgat gcagattttt ctgaaatgag atgtttagat gagcatacta 4200 ctaaagcaga gtggaaaaat ctgtctttat ggtatgttct aggtgtattg tgatttactg 4260 ttagattgcc aatataagta aatatagaca taatctatat atagtgtttc acaaagctta 4320 gatctttaac cttgcagctg ccccacagtg cttgacctct gagtcattgg ttatacagtg 4380 tagtcccaag cacataaact aggaagagaa tgtatttgta ggagcgctac cacctgtttt 4440 caagagaaca tagaactcca acgtaaccgt catttcaaag atttactgta tgtatagttg 4500 attttgtgga ctgaatttaa tgcttccaaa tgtttgcagt taccaaacat tgttatgcaa 4560 gaaatcataa aatgaagact tataccattg tgtttaagct gtattgaatt atctgtggaa 4620 tgcattgtga actgtaaagc aaagtatcaa taaagcttat agacttacct ttgtgtttag 4680 tgttttagtt tcatgaatgc acagcaaaaa cacggtggta ggcttagaga gtggacacat 4740 ggtaacatgc ttttagaaag gttttagttc atgaaacagc ttaagaacaa agaatatatt 4800 tacatagtga gatttatttg actcataaca aaaggtttta aattatttta tactttgaaa 4860 ataaattcat gcaccaatat tttaacagaa tacagtgcaa gattatgaa tatacataaa 4920 attacaccat ataaatttta caaataagac tttcaaagtc tttataacag acactattgc 4980 tcttcaaata tatacatata tcattgatta gtcagttgtt catccacatg gttacttaat 5040 gcaagatctg tctgaatgaa atgtcagtag tacaagacag gcagacacag tgatcactca 5100 gcatcaccag gtacagaaaa cagaatcagg gctgcatagg gctctactga ggacccagca 5160 acctgctagt gggttgatgt aaggcattaa taagttggcg tgtaaaatag cttaatgtgt 5220 aatctaattc ttttagaatg ctgaagcact tctgtggtaa atgttgataa tctattcttt 5280 aactgaaaat gcttatttca acctttctct aaaatggcaa cttcatataa ctagaaactc 5340 aaggtctaga attttagtgc acagactgga aggactcggt ggtgtgtgta ctcacgactc 5400 caactcccat cagccttctt aactaatagt cgtcaagtca cattctgtcc gacaactgac 5460 tggagaaact caaatactcc ttacagtggg gaatatgttc aagaggtttt taaaaatctg 5520 aatttaccct gcattaatca tctgaaatga gcagagccaa gccagtccta cccaagaggg 5580 tgtaaactaa acagcaagac agctcactcg tcacactgga gctttccctg ctttccgtgt 5640 tgctactttc tgtggagctg gactcttctt tgctgctcac cttataactg ctttcctaga 5700 gcaggacata gtggtgaatt tgctaatccc atagccctcc tcagttttga agttttagca 5760 ccatgtgaag gaagaagacg acatgggagg tgaggcagca gcccacacaa ctgggaactt 5820 tgaaggcact taatggatat aaaactgcaa atgaaacttt taaaaattaa cattttaaaa 5880 cttgtttata catggctcat ttagttttga aagctaaatg tggcaaacag ggtatttctg 5940 tacttaagtg cttccaactt acattgtgtc cagtgaacat tcttaaaata catagaaaca 6000 gaatagcaaa acacctttgt aaaagtctta atgcaaatta aacgctacat attggttagg 6060 gtaattgagg atgtaaattt tatctgtggg ctacatcatc ccaattttgc cgttagtgaa 6120 gttacaataa gtataggttt tagtctccag aacagaagca aacag 6165

Claims (14)

A composition for diagnosing myopathy, comprising an agent for measuring the increase in the expression level of NOGO-A, MyoD, myogenin and IL-6 genes, proteins or fragments thereof, and the increase in the ratio of p-AKT / AKT,
Wherein the myopathy is inflammatory myopathy or myotoxic mediated myopathy. The composition of claim 1, wherein the agent for measuring the expression level of the NOGO-A gene comprises a primer, a probe or an antisense oligonucleotide that specifically binds to the NOGO-A gene. The composition of claim 1, wherein the agent for measuring the amount of the NOGO-A protein is an antibody or antigen-binding fragment thereof that specifically binds to the NOGO-A protein or a fragment thereof. delete delete delete delete The composition according to claim 1, wherein the NOGO-A gene promotes adipogenic differentiation and inhibits muscle production by regulating circadian rhythm regulation genes when deficient. As a composition kit for diagnosing myopathy, comprising an agent for measuring an increase in the expression level of NOGO-A, MyoD, myogenin and IL-6 genes, proteins or fragments thereof, and an increase in the ratio of p-AKT/AKT,
The composition kit, wherein the myopathy is inflammatory myopathy or myotoxic mediated myopathy. The composition kit for diagnosing myopathy according to claim 9, wherein the kit is any one selected from the group consisting of an RT-PCR kit, a microarray chip kit, a DNA kit, and a protein chip kit. measuring the expression level of NOGO-A, MyoD, myogenin and IL-6 genes, proteins or fragments thereof and the ratio of p-AKT/AKT in a biological sample isolated from muscle tissue of an individual suspected of having myopathy; and
The measured NOGO-A, MyoD, myogenin and IL-6 gene, protein, or increase in the expression level of a fragment thereof and the increase in the ratio of p-AKT / AKT were compared to NOGO-A, MyoD, myogenin in the control group. And an information providing method for diagnosing myopathy comprising the step of comparing the increase in the expression level of the gene, protein or fragment thereof of IL-6 and the increase in the ratio of p-AKT / AKT,
The method of providing information, wherein the myopathy is inflammatory myopathy or myotoxic mediated myopathy. delete delete delete

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