Use of FOXO3 as a biomarker of skeletal muscle aging in primates

Abstract

This invention discloses the application of FOXO3 as a biomarker for skeletal muscle aging in primates. Specifically, it discloses the application of the biomarker and / or substances that detect the biomarker in identifying or assisting in identifying the degree of aging of skeletal muscle cells, wherein the biomarker is the FOXO3 gene. Experiments show that inhibiting or knocking out the FOXO3 gene induces cellular aging phenotypes in human myotubular cells, such as reduced diameter and increased SA-β-gal activity, while enhanced FOXO3 gene inheritance delays myotubular cell aging. This indicates that FOXO3 is crucial for the homeostasis and functional maintenance of human myotubular cells. The biomarker of this invention provides important molecular indicators for the research and intervention of skeletal muscle aging-related diseases, particularly for the assessment of human skeletal muscle function and disease diagnosis. It plays a vital role in the early identification, early diagnosis, prevention, and delay of skeletal muscle aging, and has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of FOXO3 as a biomarker of skeletal muscle aging in primates. Background Technology

[0002] Skeletal muscle, a type of striated muscle, attaches to bones and provides the power for the human musculoskeletal system. The state of skeletal muscle is closely related to the prevention and maintenance of systemic diseases. One of the most significant characteristics of skeletal muscle aging is muscle weakness and atrophy (sarcopenia). Studies show that older adults experience a faster loss of muscle mass and strength, and muscle aging and atrophy greatly increase the risk of fractures and joint injuries. Sarcopenia can also affect organ function, potentially leading to heart and lung failure, and even death. Besides reduced skeletal muscle mass (decreased muscle fiber volume), skeletal muscle aging is also accompanied by inflammation, adipose tissue infiltration, fibrosis, and a decrease in capillary skeletal muscle formation. Furthermore, with age, the cyclical changes in denervation and renervation eventually lead to changes in fiber type composition, with an increase and accumulation of type I fibers and loss of sarcomere tissue and structure. Skeletal muscle aging may be related to skeletal muscle stem cell aging, reduced skeletal muscle fiber repair capacity, mitochondrial dysfunction, and loss of genomic protein homeostasis, but the specific mechanisms require further investigation.

[0003] Therefore, identifying key factors in skeletal muscle cell aging and developing biomarkers for skeletal muscle aging will provide important molecular indicators for the research and intervention of skeletal muscle aging-related diseases, especially for the assessment of human skeletal muscle function and the diagnosis of diseases. These indicators will play a vital role in the early identification, early diagnosis, prevention, and delay of skeletal muscle aging, and are of great significance to human health, especially the health of the elderly. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to evaluate or assist in evaluating the degree of aging of skeletal muscle cells, and / or how to prevent, intervene in, improve, delay, or treat skeletal muscle cell aging. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To address the aforementioned technical problems, the present invention first provides any of the following applications of biomarkers and / or substances for detecting said biomarkers:

[0006] A1) Application in identifying or assisting in identifying the degree of aging of animal skeletal muscle cells;

[0007] A2) Application in the preparation of products for identifying or assisting in the identification of the degree of aging of animal skeletal muscle cells;

[0008] A3) Application in evaluating or assisting in the evaluation of the aging degree of animal skeletal muscle cells;

[0009] A4) Application in the preparation of products for evaluating or assisting in the evaluation of the aging degree of animal skeletal muscle cells;

[0010] A5) Application in regulating the diameter of animal skeletal muscle cells or in the preparation of products that regulate the diameter of animal skeletal muscle cells;

[0011] A6) Application in regulating SA-β-gal activity in animal skeletal muscle cells or in the preparation of products that regulate SA-β-gal activity in animal skeletal muscle cells;

[0012] The biomarker may be the FOXO3 gene.

[0013] The animal in question may be a primate.

[0014] The biomarkers can be used to identify, assist in identifying, evaluate, or assist in evaluating skeletal muscle aging in primates.

[0015] Furthermore, the senescence level of the tested skeletal muscle cells with low FOXO3 gene expression was higher than that of the tested skeletal muscle cells with high FOXO3 gene expression.

[0016] The FOXO3 gene has a Gene ID of 2309 (updated on 6-Sep-2022) and a Genbank Accession of NM_001455 (Update Date 18-SEP-2022). The FOXO3 gene includes the FOXO3 gene and any polynucleotides of the FOXO3 gene that have functional equivalents.

[0017] The protein encoded by the FOXO3 gene is the FOXO3 protein, whose protein ID is O43524 (updated on 6-Sep-2022) and Genbank Accession is NP_001446 (Update Date 18-SEP-2022).

[0018] The SA-β-gal is an aging-related β-galactosidase.

[0019] In the above applications, the substance used to detect the biomarker can be any of the following:

[0020] B1) A reagent used to detect the mRNA expression level of the FOXO3 gene;

[0021] B2) A reagent used to detect the protein expression level of the FOXO3 gene.

[0022] Specifically, the mRNA expression level refers to the abundance of mRNA transcribed from a gene at the transcriptional level; the protein expression level refers to the abundance of protein encoded by a gene at the translational level.

[0023] In the above applications, the substances used to detect the biomarkers include reagents for detecting the biomarkers by reverse transcription-polymerase chain reaction, real-time quantitative PCR, transcriptome sequencing, Northern blot, in situ hybridization, gene chip technology, Nanopore sequencing, PacBio sequencing, Western blotting, immunohistochemistry, immunofluorescence, radioimmunoassay, immunoprecipitation, enzyme-linked immunosorbent assay, enzyme immunoassay, flow cytometry, high-performance liquid chromatography, capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunochromatography, fluorescence immunochromatography, surface plasmon resonance, immuno-PCR, or biotin-avidin technology.

[0024] In the above applications, the product includes a substance for detecting the biomarker.

[0025] In the above applications, the products may include reagent kits, gene chips, protein chips, immunochromatographic diagnostic strips, high-throughput sequencing platforms, or biosensors.

[0026] In the above applications, the test sample for the product can be a skeletal muscle tissue sample or a myotubular cell sample.

[0027] The kit may be a gene detection kit or a protein immunoassay kit; the gene detection kit includes reagents for detecting the transcriptional level of the FOXO3 gene; the protein immunoassay kit includes specific antibodies against the FOXO3 protein.

[0028] The gene chip includes a solid support and oligonucleotide probes immobilized on the solid support, the oligonucleotide probes including oligonucleotide probes for detecting the FOXO3 gene.

[0029] The protein chip includes a solid support and a specific antibody against FOXO3 protein immobilized on the solid support.

[0030] The immunochromatographic diagnostic test strip contains a specific antibody against the FOXO3 protein.

[0031] The high-throughput sequencing platform includes reagents for detecting the FOXO3 gene.

[0032] The biosensor includes reagents for detecting the FOXO3 gene.

[0033] In the above applications, the substance used to detect the biomarker may include at least one of the following:

[0034] C1) Primers for specific amplification of the FOXO3 gene;

[0035] C2) Probes that specifically recognize the FOXO3 gene;

[0036] C3) A substance that specifically binds to the FOXO3 gene;

[0037] C4) A substance that specifically binds to the FOXO3 protein.

[0038] Furthermore, the primers may include a forward primer that specifically amplifies the FOXO3 gene with the nucleotide sequence of SEQ ID No. 1 and a reverse primer that specifically amplifies the FOXO3 gene with the nucleotide sequence of SEQ ID No. 2.

[0039] The forward primer (SEQ ID No. 1) and reverse primer (SEQ ID No. 2) are used to detect the mRNA expression level of the FOXO3 gene.

[0040] Furthermore, the substance that specifically binds to the FOXO3 gene can be a nucleic acid molecule or a protein.

[0041] Furthermore, the substance that specifically binds to the FOXO3 protein may be an antibody.

[0042] The antibodies described in this article can be monoclonal antibodies, polyclonal antibodies, genetically engineered antibodies, or antibody variable regions Fv, single-chain antibody ScFv, antigen-binding fragments Fab or Fab', F(ab')2, Fab'-SH, and other antibody fragments, as well as antibody derivatives.

[0043] Furthermore, the antibody may be a FOXO3 antibody. In one or more embodiments of the present invention, the FOXO3 antibody is a rabbit anti-FOXO3 monoclonal antibody (FoxO3a(75D8)Rabbit mAb#2497), purchased from Cellsignaling Technology, catalog number: 2497.

[0044] The present invention also provides a method for constructing recombinant skeletal muscle cells, the method comprising introducing a substance that upregulates, enhances, or increases the expression of the FOXO3 gene into recipient skeletal muscle cells and / or introducing a substance that upregulates, enhances, or increases the activity and / or content of the FOXO3 protein into recipient skeletal muscle cells to obtain recombinant cells, wherein the aging level (degree) of the recombinant skeletal muscle cells is lower than that of the recipient skeletal muscle cells.

[0045] The recombinant skeletal muscle cells provide a cell model for aging studies of the body and / or organs and / or tissues and / or cells.

[0046] The present invention also provides a kit, which may be any of the following:

[0047] D1) The kit contains reagents for detecting the mRNA expression level of the FOXO3 gene;

[0048] D2) The kit contains reagents for detecting the protein expression level of the FOXO3 gene;

[0049] The kit may have at least one of the following uses:

[0050] E1) To identify or assist in identifying the degree of aging of animal skeletal muscle cells;

[0051] E2) Prepare products for identifying or assisting in identifying the degree of aging of animal skeletal muscle cells;

[0052] E3) Evaluate or assist in the evaluation of the aging degree of animal skeletal muscle cells;

[0053] E4) Prepare products for evaluating or assisting in the evaluation of the aging degree of animal skeletal muscle cells;

[0054] E5) Regulating the diameter of animal skeletal muscle cells or preparing products that regulate the diameter of animal skeletal muscle cells;

[0055] E6) Regulate the SA-β-gal activity of animal skeletal muscle cells or prepare products that regulate the SA-β-gal activity of animal skeletal muscle cells.

[0056] The various reagent components of the kit may be present in separate containers, or may be pre-assembled into a reagent mixture, either wholly or partially.

[0057] Furthermore, the kit may be a gene detection kit or a protein immunoassay kit.

[0058] Furthermore, the kit may be an ELISA kit, a qPCR kit, an electrochemiluminescence detection kit, an immunoblotting detection kit, an immunochromatographic detection kit, a flow cytometry analysis kit, or an immunohistochemistry detection kit, but is not limited to these.

[0059] Furthermore, the gene detection kit may contain a forward primer that specifically amplifies the FOXO3 gene with the nucleotide sequence of SEQ ID No. 1 and a reverse primer that specifically amplifies the FOXO3 gene with the nucleotide sequence of SEQ ID No. 2.

[0060] Furthermore, the gene detection kit may also include Taq DNA polymerase, dNTPs, PCR buffer, and Mg2+ required for PCR amplification. 2+ One or more of them.

[0061] Furthermore, the gene detection kit may also include an internal reference gene detection reagent, which may be the GAPDH gene and / or the β-actin gene, but is not limited thereto.

[0062] The protein immunoassay kit may contain an antibody that binds to the FOXO3 protein.

[0063] The present invention also provides any of the following applications of the kit:

[0064] Application of H1 in identifying or assisting in identifying the degree of aging of animal skeletal muscle cells;

[0065] Application of H2 in the preparation of products for identifying or assisting in the identification of the degree of aging of animal skeletal muscle cells;

[0066] Application of H3 in evaluating or assisting in the evaluation of the aging degree of animal skeletal muscle cells;

[0067] Application of H4 in the preparation of products for evaluating or assisting in the evaluation of the aging degree of animal skeletal muscle cells;

[0068] H5) Application in regulating the diameter of animal skeletal muscle cells or in the preparation of products that regulate the diameter of animal skeletal muscle cells;

[0069] Application of H6 in regulating SA-β-gal activity in animal skeletal muscle cells or in the preparation of products that regulate SA-β-gal activity in animal skeletal muscle cells.

[0070] This invention also provides the use of a FOXO3 gene promoter in the preparation of a medicament for the prevention, intervention, improvement, delay, and / or treatment of skeletal muscle cell aging, wherein the FOXO3 gene promoter may include increasing FOXO3 protein activity and / or increasing FOXO3 S253A\S315A Reagents for protein activity, namely FOXO3 S253A\S315A Proteins can be any of the following:

[0071] F1) The amino acid sequence is that of the protein in SEQ ID No. 3;

[0072] F2) A protein that has more than 80% identity with and has the same function as the protein shown in F1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 3.

[0073] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of amino acid sequences, then the identity value (%) can be obtained.

[0074] In this document, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0075] In the above applications, the FOXO3 gene promoter may have at least one of the following functions:

[0076] G1) increases or upregulates skeletal muscle cell diameter;

[0077] G2) reduces or downregulates SA-β-gal activity in skeletal muscle cells.

[0078] In the above applications, the FOXO3 gene promoter may include a FOXO3 gene editing vector, the FOXO3 protein, or the FOXO3 gene itself. S253A\S315A protein.

[0079] The FOXO3 S253A\S315A The protein or the FOXO3 gene editing vector is also within the scope of protection of this invention.

[0080] The present invention also provides the aforementioned FOXO3 S253A\S315A The following applications of the protein or the FOXO3 gene editing vector:

[0081] N1) Application in identifying or assisting in identifying the degree of aging of animal skeletal muscle cells;

[0082] Application of N2 in the preparation of products for identifying or assisting in the identification of the degree of aging of animal skeletal muscle cells;

[0083] Application of N3 in evaluating or assisting in the evaluation of the aging degree of animal skeletal muscle cells;

[0084] N4) is used in the preparation of products for evaluating or assisting in the evaluation of the aging degree of animal skeletal muscle cells;

[0085] N5) Application in regulating the diameter of animal skeletal muscle cells or in the preparation of products that regulate the diameter of animal skeletal muscle cells;

[0086] N6) is used in regulating the SA-β-gal activity of animal skeletal muscle cells or in the preparation of products that regulate the SA-β-gal activity of animal skeletal muscle cells.

[0087] N7) increases FOXO3 protein activity and / or increases FOXO3 S253A\S315A Applications in protein activity;

[0088] N8) In the preparation of FOXO3 protein activity and / or increase FOXO3 S253A\S315A Applications in products containing protein activity.

[0089] The FOXO3 gene editing vector may be rpAMHDAdGT8-4.

[0090] The recombinant vector rpAMHDAdGT8-4 is a recombinant vector obtained by inserting the DNA molecule shown in SEQ ID No. 4 into the pAMHDAdGT8-4 vector (Addgene#26421).

[0091] The FOXO3 gene editing vector can be used to mutate the FOXO3 gene in primates, thereby reducing the aging of primate iliac muscle cells. The mutation can be to mutate the serine residue (S) at position 253 of the FOXO3 protein to an alanine residue (A) and the serine residue (S) at position 315 to an alanine residue (A).

[0092] The FOXO3 gene editing vector can be used to enhance the activity of the FOXO3 protein.

[0093] The present invention also provides any of the following applications of the said biomarker as a target:

[0094] The application of M1 in the preparation of drugs for the prevention, intervention, improvement, delay and / or treatment of skeletal muscle cell aging;

[0095] The application of M2 in screening drugs for the prevention, intervention, improvement, delay, and / or treatment of skeletal muscle cell aging, wherein the screening involves targeting the FOXO3 gene with drugs or preparations that can enhance FOXO3 protein activity or FOXO3 S253A\S315A Drugs or formulations with protein activity are considered as drug candidates.

[0096] The regulation of skeletal muscle cell diameter described in this article can be achieved by increasing (upregulating) or decreasing (downregulating) the diameter of skeletal muscle cells.

[0097] The regulation of SA-β-gal activity in skeletal muscle cells described in this article can increase (upregulate) or decrease (downregulate) SA-β-gal activity in skeletal muscle cells.

[0098] The skeletal muscle cells described in this article may be at least one of the following: fast-twitch type IIA, fast-twitch type IIX, slow-twitch type I, postsynaptic muscle fibers of the neuromuscular junction, terminal Schwann cells, skeletal muscle stem cells, tendon fibroblasts, or myotubular cells differentiated from human embryonic stem cells.

[0099] The animals described in this article may be primates.

[0100] The primates mentioned in this article may be cynomolgus monkeys or humans.

[0101] In this document, the products may include reagent kits, gene chips, protein chips, immunochromatographic diagnostic strips, high-throughput sequencing platforms, or biosensors.

[0102] This invention can detect the expression level of the FOXO3 gene based on cDNA and RNA sequencing technologies.

[0103] This invention can use second-generation sequencing and third-generation sequencing to detect the transcriptome in cDNA, and then detect the expression level of the FOXO3 gene. This invention can also use Nanopore sequencing to directly detect RNA expression levels, thereby quantifying the expression level of the FOXO3 gene.

[0104] The applications described herein may be for disease diagnosis, disease prognosis, and / or disease treatment purposes, or they may be for non-disease diagnosis, non-disease prognosis, and non-disease treatment purposes; their direct purpose may be to obtain information on intermediate results of disease diagnosis, disease prognosis, and / or disease treatment, or their direct purpose may be for non-disease diagnosis, non-disease prognosis, and / or non-disease treatment purposes.

[0105] This invention first constructed a single-cell nuclear sequencing library of cynomolgus monkey skeletal muscle and obtained the FOXO3 gene, a biomarker of skeletal muscle cell aging, through bioinformatics analysis and screening. Furthermore, the function of the FOXO3 gene was verified by inhibiting FOXO3 gene expression with small interfering RNA (siRNA), knocking out the FOXO3 gene, and genetically enhancing the FOXO3 gene using gene editing technology. Experimental results showed that inhibiting or knocking out FOXO3 gene expression induces senescent phenotypes in human myotubular cells, such as reduced diameter and increased SA-β-gal activity, while genetic enhancement of the FOXO3 gene delays myotubular cell aging. This indicates that the FOXO3 gene is crucial for the homeostasis and functional maintenance of human myotubular cells, confirming that the FOXO3 gene is a novel biomarker for aging skeletal muscle cells.

[0106] Through extensive and in-depth research, the inventors unexpectedly discovered a significant difference in FOXO3 gene expression in skeletal muscle cells of young and old cynomolgus monkeys. The expression level of FOXO3 gene in the young group was significantly higher than that in the old group. Based on this, a biomarker for identifying or evaluating skeletal muscle aging in primates was developed. The FOXO3 gene, as a biomarker, can be used to identify, evaluate, or assess the degree of skeletal muscle aging or atrophy in primates. Furthermore, the FOXO3 gene can also serve as a drug target for screening and developing drugs to prevent, intervene in, improve, delay, and / or treat skeletal muscle cell aging.

[0107] The skeletal muscle aging biomarkers of this invention play an important role in the screening and identification of skeletal muscle cells. They can assess skeletal muscle aging-related diseases such as sarcopenia, detect human skeletal muscle function, and evaluate the degree of human skeletal muscle aging. They provide important molecular indicators for the research and intervention of skeletal muscle aging-related diseases, especially for the assessment of human skeletal muscle function and the diagnosis of diseases. They play an important role in the early identification, early diagnosis, prevention, and delay of skeletal muscle aging, and have significant application value. They are of great significance to human health, especially the health of the elderly. Attached Figure Description

[0108] Figure 1 A schematic diagram illustrating the construction and analysis of a single-cell nuclear sequencing library for cynomolgus monkey skeletal muscle.

[0109] Figure 2 To analyze the skeletal muscle cell clusters of cynomolgus monkeys using bioinformatics.

[0110] Figure 3 To analyze the number of downregulated transcription factors in differentially expressed genes related to aging in different types of skeletal muscle cells using bioinformatics.

[0111] Figure 4This shows the expression of FOXO3 gene mRNA in different types of skeletal muscle in young and old groups, based on single-cell nuclear sequencing data.

[0112] Figure 5 In Example 2, the expression of FOXO3 pre-mRNA in different types of skeletal muscle in the young and old groups was detected by RNA-FISH technology.

[0113] Figure 6 For the quantitative PCR in Example 2 ( Figure 6 (A) and protein blotting ( Figure 6 (B) Identify the expression of FOXO3 in skeletal muscle of young and old groups.

[0114] Figure 7 In Example 2, protein imprinting was used to identify the age-related downregulation of FOXO3 expression in human skeletal muscle.

[0115] Figure 8 The protein imprinting method used in Example 2 was used to identify the downregulation of FOXO3 expression in human myotubular cells with aging.

[0116] Figure 9 This represents the result of verifying the function of FOXO3 using small interfering RNA (siRNA) in Example 3. Figure 9 A diagram shows human myotubular cells with FOXO3 expression suppressed by small interfering RNA; Figure 9 In section B, protein imprinting was used to identify FOXO3 protein expression in human myotubular cells that inhibit FOXO3 expression. Figure 9 In the middle, C represents the diameter of human myotubular cells with inhibited FOXO3 expression as determined by immunofluorescence assay; Figure 9 In the middle D, the galactosidase assay was used to identify the degree of senescence in human myotubular cells with inhibited FOXO3 expression.

[0117] Figure 10 This is the result of verifying the function of FOXO3 through FOXO3 gene knockout in Example 3. Figure 10 A in the diagram is a schematic diagram of FOXO3 knockout of human myotubular cells; Figure 10 In B, protein imprinting was used to identify FOXO3 protein expression in FOXO3 knockout human myotubular cells. Figure 10 In the middle C, the diameter of human myotubular cells after FOXO3 knockout was determined by immunofluorescence assay. Figure 10 In the middle D, the galactosidase assay is used to identify the degree of aging of human myotubular cells after FOXO3 knockout.

[0118] Figure 11 This represents the results of verifying the function of FOXO3 through FOXO3 genetic enhancement gene editing in Example 3. Among them, Figure 11A diagram of human myotubular cells with genetically enhanced FOXO3 expression is shown in Figure A. Figure 11 B represents the identification of FOXO3 protein expression in human myotubular cells with genetically enhanced FOXO3 expression using protein imprinting. Figure 11 In the middle, C represents the diameter of human myotubular cells with genetically enhanced FOXO3 expression, as determined by immunofluorescence assay. Figure 11 In the middle D, the galactosidase assay was used to identify the degree of aging in human myotubular cells with genetically enhanced FOXO3 expression. Detailed Implementation

[0119] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0120] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0121] The following examples used Graphpad 8.0 statistical software to process the data. Experimental results are expressed as mean ± standard deviation. The t-test method was used, with P < 0.05 (*) indicating statistical significance, P < 0.01 (**) indicating statistically significant difference, and P < 0.001 (***) indicating extremely significant difference. Unless otherwise specified, all quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0122] The biological materials and reagents involved in the following embodiments are:

[0123] The cynomolgus monkeys were purchased from the Beijing Xieerxin Institute of Biological Resources. The cynomolgus monkey experiments in the following examples were conducted in accordance with the "Ethical Guidelines for the Treatment of Non-Human Primates" and have been approved by the Institute of Zoology, Chinese Academy of Sciences.

[0124] The FOXO3 antibody was a rabbit anti-FOXO3 monoclonal antibody (FoxO3a(75D8)Rabbit mAb#2497), purchased from Cellsignaling Technology, catalog number: 2497.

[0125] The internal control antibody was a mouse anti-GAPDH monoclonal antibody (GAPDH(G-9) mouse monoclonal antibody) purchased from Santa Cruz Biotechnology, catalog number sc-365062.

[0126] Human myotubular cells were derived from directed differentiation of human embryonic stem cells. The human embryonic stem cells were purchased from WiCell Research, catalog number WA09(H9)-DL-7.

[0127] The human skeletal muscle tissue was obtained from Peking University First Hospital.

[0128] Example 1: Obtaining a single-cell nuclear sequencing library of cynomolgus monkey skeletal muscle

[0129] Based on the average lifespan of cynomolgus monkeys and its correlation with human age, four female and four male cynomolgus monkeys aged 4-5 years were selected to form a young group, while four female and four male cynomolgus monkeys aged 18-20 years were selected to form an old group. All cynomolgus monkeys underwent physical examinations, including measurements of basic parameters (such as body mass index, BMI), detection of biochemical indicators (such as complete blood count and urinalysis), brain MRI, and ultrasound examinations of important internal organs such as the heart, to ensure that all cynomolgus monkeys in both the young and old groups were free of major diseases.

[0130] A schematic diagram of the construction and analysis of cynomolgus monkey single-cell sequencing libraries is shown below. Figure 1 The specific steps are as follows:

[0131] I. Obtaining a suspension of cynomolgus monkey skeletal muscle cell nuclei

[0132] Preparation of single-cell nucleus samples from cryopreserved skeletal muscle tissue of cynomolgus monkeys: The cryopreserved tissues were taken out from liquid nitrogen, ground into powder in a mortar pre-cooled by liquid nitrogen, transferred to a grinding tube, homogenized liquid and steel balls were added, and ground in a tissue homogenizer.

[0133] After the tissue was ground, it was filtered twice through a cell sieve. The liquid was collected, centrifuged at 4°C and the supernatant was discarded. The precipitate was resuspended in PBS solution containing 0.3% bovine serum albumin (BSA) and then screened by flow cytometry for Hoechst 33342 and PI double positive cell nuclei, thus obtaining a suspension of cynomolgus monkey skeletal muscle single cell nuclei.

[0134] II. Preparation of Single-Cell Nucleus Transcriptome Libraries

[0135] The single-cell nuclear suspension obtained in step one was used to construct a single-cell 3' RNA-seq library by using the Chromium Single Cell 3' GEM Library and Gel Bead Kit v3 (10×Genomics).

[0136] Cell nuclei were captured in droplet emulsions, and libraries were constructed according to the instructions of the 10×Genomics Chromium Single Cell 3' GEM Library and Gel Bead Kit v3. Using 7000 nuclei captured per sample as a standard, all reactions were performed in a Bio-Rad C1000 thermal cycler equipped with 96-well deep-well plates. After reverse transcription to cDNA, amplification was performed. The cDNA fragments and the average fragment length of the libraries were evaluated using a fragment analyzer. Library construction was performed using the Chromium Single Cell 3' GEM Library and Gel Bead Kit v3 to obtain single-nucleus sequencing libraries from cynomolgus monkey skeletal muscle cells of young and old groups. Sequencing was performed using a NovaSeq 6000 Sequencing System after library preparation.

[0137] The primer names and nucleotide sequences used to construct the single-cell nuclear sequencing library of cynomolgus monkey skeletal muscle are shown in Table 1.

[0138] Table 1. Primer names and nucleotide sequences for constructing a single-cell nuclear sequencing library of cynomolgus monkey skeletal muscle.

[0139]

[0140] Note: In the barcode primers, "XXXXXXXX" is an 8bp barcode sequence, "NNNNNNNN" is an 8bp random unique molecular identifier (UMI) sequence, and "T25" represents 25 Ts; in the TSO primers, "rG" is riboguanosine, and "+G" is guanosine modified with locked nucleic acid (LNA); in the biotin primers, " / biotin / " indicates that the 5' end of the primer is modified with biotin, and " / index / " is a 6bp library barcode sequence.

[0141] Example 2: Obtaining and detecting skeletal muscle cell aging biomarkers

[0142] I. Bioinformatics analysis to obtain skeletal muscle cell aging biomarkers

[0143] Based on the single-cell nuclear sequencing libraries of cynomolgus monkey skeletal muscle from the young and old groups in Example 1, the FOXO3 gene, a biomarker of skeletal muscle cell aging, was obtained using the find_all_markers function of the Seurat software package. The screening thresholds were |LogFC|>0.5 and the corrected P value<0.05.

[0144] Bioinformatics analysis results such as Figure 2 and Figure 3 As shown. Among them. Figure 2The analysis of cynomolgus monkey skeletal muscle cell clusters was performed using bioinformatics. Figure 3 This study aimed to analyze the number of downregulated transcription factors in differentially expressed genes related to aging in different types of skeletal muscle cells using bioinformatics. Figure 2 It can be seen that single-cell nuclear transcriptomics technology can obtain various types of skeletal muscle cells, including various types of myofibrils, postsynaptic myofibrils at the neuromuscular junction, terminal Schwann cells, skeletal muscle stem cells, fibroblasts / fibroblast adipocyte progenitors, tendon fibroblasts, smooth muscle cells, pericytes, endothelial cells, adipocytes, macrophages, and T cells, indicating that this technology can comprehensively display the molecular characteristics of multiple skeletal muscle cell types. In addition, from Figure 3 It can also be seen that FOXO3 occupies a central position among the downregulated transcription factors in differentially expressed genes related to aging in different types of skeletal muscle cells, indicating that FOXO3 may be an important core transcription factor affecting skeletal muscle aging.

[0145] The expression of the FOXO3 gene in different types of skeletal muscle cells in young and old groups is as follows: Figure 4 The results showed that there were significant differences in the expression levels of FOXO3 gene in fast-twitch muscle fibers (Type IIA, Type IIIX, Type I, and Type I), postsynaptic muscle fibers at the neuromuscular junction, skeletal muscle stem cells (muscle stem cells), endothelial cells, smooth muscle cells, pericytes, fibroblasts / fibroblast adipocyte progenitors, terminal Schwann cells, and T cells between the young and old groups. The expression level of FOXO3 gene in the young group was significantly higher than that in the old group. Therefore, FOXO3 gene is a biomarker for the aging degree of skeletal muscle cells; that is, skeletal muscle cells with low FOXO3 gene expression levels are more aged than those with high FOXO3 gene expression levels. The FOXO3 gene has the following gene IDs: Gene ID: 2309 (updated on 6-Sep-2022), Protein ID: O43524 (updated on 6-Sep-2022).

[0146] II. RNA-fluorescence in situ hybridization (RNA-FISH)

[0147] OCT-embedded cynomolgus skeletal muscle tissue was sectioned into 8 μm thick slices using a cryostat and immediately placed on polylysine-coated coverslips. 119 visualization probes for detecting cynomolgus FOXO3 pre-mRNA were designed using PaintSHOP. Slices were fixed in 4% PFA for 15 min and then treated with 0.5% Triton X-100 in 1×PBS (PBST) for 30 min. After treatment with 10 μg / ml proteinase K (diluted with PBST) for 10 min and pre-hybridization at 43°C for 30 min, the slices were incubated overnight at 43°C with a hybridization mixture containing probes targeting cynomolgus FOXO3 pre-mRNA. The slides were washed several times and incubated with a hybridization mixture containing Alexa488-labeled fluorescent probes at 37°C for 1 h. After incubation for 10 minutes in Hoechst 33342 (Thermo Fisher Scientific, H3570) (diluted with 1×PBS) and washing several times, sections were mounted with VECTASHIELD anti-quenching mounting medium (Vector Laboratories, H-1000) and analyzed using a confocal laser capture imaging-scanning microscope (Zeiss 900) and ImageJ software.

[0148] The results are as follows Figure 5 As shown, the expression level of FOXO3 pre-mRNA in the skeletal muscle tissue of older cynomolgus monkeys was significantly lower than that in younger monkeys.

[0149] III. Real-time quantitative PCR (RT-qPCR) detection

[0150] The mRNA expression level of the FOXO3 gene in the skeletal muscle tissue of eight cynomolgus monkeys from both young and old groups was statistically analyzed (i.e., the mRNA expression level of the FOXO3 gene was detected).

[0151] 1. Locate the primer sequence for the target gene in PrimerBank and synthesize the primers. Dissolve the primers in ddH2O. Take out the cDNA template after reverse transcription and dilute it 10-fold with ddH2O. Then prepare the Toyobo SYBR qPCR Mix (QPS-201C) RT-qPCR system.

[0152] 2. Prepare the reaction system, mix it thoroughly, and place it in a real-time PCR instrument to carry out the reaction according to the program.

[0153] 3. The primer sequences for real-time quantitative PCR are shown below:

[0154] Forward primer: 5'-TCACGCACCAATTCTAACGC-3' (SEQ ID No. 1);

[0155] Reverse primer: 5'-CATGGCTTGTTCACTGAAGG-3' (SEQ ID No. 2).

[0156] Forward primer (SEQ ID No. 1) and reverse primer (SEQ ID No. 2) were used to detect the mRNA expression level of the FOXO3 gene.

[0157] Real-time quantitative PCR detection results are as follows Figure 6 As shown in Figure A. The results indicate that the mRNA expression level of the FOXO3 gene in the skeletal muscle tissue of older cynomolgus monkeys was significantly lower than that in younger monkeys.

[0158] IV. Western blot detection

[0159] The expression levels of FOXO3 protein were statistically analyzed in eight cynomolgus monkey skeletal muscle tissues from young and old groups, seven human skeletal muscle tissues (aged 54-81 years), and human myotubular cells cultured for a long period (gradually aging).

[0160] The skeletal muscle tissue was placed in 1% SDS solution and homogenized using a tissue homogenizer. The human myotubular cells were repeatedly mixed and lysed using a pipette in 1% SDS solution. The mixture was heated to 105°C for 10 minutes to allow for complete protein denaturation. Then, equal amounts of protein were taken and subjected to Western blot analysis (using GAPDH expression level as a reference) to detect the FOXO3 protein content in the skeletal muscle tissue of young and old cynomolgus monkeys.

[0161] The specific steps for Western blot are as follows:

[0162] 1. Add an appropriate amount of concentrated protein loading buffer (Guangzhou Dingguo Biotechnology Co., Ltd., catalog number: WB-0091) to an equal volume of protein sample. Load the prepared protein sample into the SDS-PAGE gel wells for protein electrophoresis.

[0163] 2. Stop electrophoresis when the bromophenol blue in the protein loading buffer reaches near the bottom of the gel.

[0164] 3. After completing step 2, use a transfer apparatus to transfer the protein for 2 hours.

[0165] 4. After completing step 3, immediately seal the container with skim milk powder at room temperature for two hours.

[0166] 5. After completing step 4, incubate with primary antibodies using FOXO3 antibody and internal reference protein antibody GAPDH at 4°C overnight.

[0167] 6. After completing step 5, incubate with the appropriate secondary antibodies (goat anti-mouse secondary antibody, ZB-2305; goat anti-rabbit secondary antibody, ZB-2301) at room temperature for 1 hour. Develop immediately after washing.

[0168] Western blot results are as follows Figure 6 B, Figure 7 and Figure 8 As shown in the figure. The results indicate that the protein expression level of the FOXO3 gene in the skeletal muscle cells of the older group of cynomolgus monkeys was significantly reduced compared with that in the younger group.

[0169] The above results once again confirm that FOXO3 is a novel biomarker for the degree of skeletal muscle cell aging.

[0170] Example 3: Functional verification of FOXO3, a biomarker of skeletal muscle cell aging.

[0171] I. Verification of FOXO3 function using small interfering RNA (siRNA).

[0172] (a) Human myotubular cells were divided into two groups: the si-FOXO3 group and the si-NC group. The expression of FOXO3 gene inhibition in human myotubular cells was detected by small interfering RNA (siRNA). Figure 9 (A)

[0173] 1. Using the Lipofectamine 3000 kit (Invitrogen), two small interfering RNAs targeting FOXO3 (si-FOXO3: CAACCTGTCACTGCATAGT) and a negative control small interfering RNA (si-NC) were transfected into human myotubular cells of the si-FOXO3 group and the si-NC group, respectively. After culturing for 48 hours, the cells were harvested to obtain the cells of the si-FOXO3 group and the si-NC group.

[0174] 2. The expression level of FOXO3 protein in cells was detected by Western blot.

[0175] Protein sample preparation:

[0176] The aforementioned human myotubular cells were lysed by repeatedly pipetting and mixing with a pipette in 1% SDS solution, and then heated to 105°C for 10 min to fully denature the proteins. Equal amounts of protein were then subjected to Western blot analysis (using GAPDH expression levels as a reference).

[0177] The Western blot detection method is the same as the steps in Example 2, Section 4.

[0178] Western blot results are as follows Figure 9 As shown in Figure B, the expression level of FOXO3 protein in cells of the si-FOXO3 group was significantly lower than that of the si-NC group, indicating that the expression of the FOXO3 gene was successfully suppressed.

[0179] (ii) Experimental detection of the diameter of human myotubular cells after inhibiting FOXO3 gene expression.

[0180] 1. Human myotubular cells from the si-FOXO3 and si-NC groups were fixed with 4% paraformaldehyde for 30 minutes, washed three times with PBS for 5 minutes each time. Then, they were permeabilized with 0.4% Triton X-100 (Sigma-Aldrich, catalog number: T9284) solution for 15 minutes. After washing three more times with PBS for 5 minutes each time, they were blocked with 10% donkey serum at room temperature for 1 hour. Primary antibody incubation: The blocking solution was discarded, and the cells were covered with a diluted primary antibody (MyHC antibody, DSHB, MF20) and incubated overnight at 4°C. Secondary antibody incubation: On the second day, the cells were washed three times with PBS for 5 minutes each time, followed by the addition of the corresponding fluorescent secondary antibody (Alexa). 568 donkey anti-mouse IgG (H+L), Invitrogen (A10037) and Hoechst 33342 were incubated at room temperature for 1 hour. The mixture was then washed three times with PBS on a shaker for 10 minutes each time.

[0181] Test results as follows Figure 9 As shown in Figure C, the diameter of human myotubular cells in the si-FOXO3 group was significantly lower than that in the si-NC group. This indicates that inhibiting FOXO3 gene expression significantly reduces the diameter of human myotubular cells. This suggests that inhibiting FOXO3 gene expression increases the aging of myotubular cells.

[0182] (iii) Experimental detection of SA-β-gal activity in human myotubular cells after inhibition of FOXO3 gene expression.

[0183] Human myotubular cells were removed, the culture medium was discarded, and the cells were washed twice with PBS. Fixation was then performed with fixative at room temperature for 5 min. The cells were then washed three times with PBS, and 2 mL of staining solution was added to each well of a 6-well plate. The plates were incubated overnight at 37°C in the dark. The next day, the staining solution was discarded, and the cells were washed twice with PBS. Then, Hoechst 33342 was diluted 1:1000 with PBS for nuclei staining (5 min). After washing twice with PBS, images were taken using a microscope.

[0184] Fixative formulation: PBS containing 0.2% glutaraldehyde and 2% formaldehyde.

[0185] Staining solution formulation: 40mM citrate-sodium phosphate buffer (pH=6.0), 100mM K4[Fe(CN)6]·3H2O, 100mM K3[Fe(CN)6], 2mM MgCl2·6H2O, 150mM NaCl and X-gal to a final concentration of 1mg / mL.

[0186] The results are as follows Figure 9 As shown in Figure D, the results indicate that inhibiting the expression of the FOXO3 gene in human myotubular cells significantly increased the activity of SA-β-gal. This further suggests that inhibiting the expression of the FOXO3 gene increases the senescence of myotubular cells.

[0187] II. Verification of FOXO3 function through FOXO3 gene knockout.

[0188] 1) Human myotubular cells were divided into two groups: wild-type group (FOXO3) + / + ) and FOXO3 knockout group (FOXO3 - / - ()( Figure 10 (A)

[0189] FOXO3 - / - Methods for obtaining myotubular cells:

[0190] 1.1-kb and 1.3-kb homologous arms were amplified from the genomic DNA of human embryonic stem cells and cloned into a donor plasmid containing a drug resistance gene cassette. The primers used in the construction were:

[0191] FOXO3 LArm Primer F: 5'-ATAGGGCCCGACGCCTCCTCTCGCTGGCAGACCCAC-3';

[0192] FOXO3 LArm Primer R: 5'-CCGCTCGAGTAGAACGCGACACCAGGTAACACCCGAGA-3';

[0193] FOXO3 RArm Primer F: 5'-CGCGGATCCATGTTGCGTCGTCACTAGCTGAATGAGAACCT-3';

[0194] FOXO3 RArm Primer R: 5'-CGGGGTACCCCTGTCCGCTTACGCCTTCCTGATACTATACACT-3'.

[0195] Donor plasmids and TALEN vectors (TAL2268, Addgene, Plasmid #36705; TAL2269, Addgene, Plasmid #36706) were electroporated into H9 human embryonic stem cells using electroporation. After approximately two weeks of culture and selection, G418-resistant human embryonic stem cell clones were selected and expanded for further differentiation into human myotubular cells.

[0196] Human embryonic stem cell clones in good condition, uniform size, and without spontaneous differentiation tendency were selected and digested with 0.5 mg / mL Dispase at 37°C for 30 min to form embryoid EB spheres. These spheres were then cultured in suspension in hESC medium with 5 ng / mL FGF2 for two days before being transferred to Matrigel-coated cell culture plates. The medium was then replaced with MEMα medium containing 10% FBS, 10 ng / mL bFGF, and 5 ng / mL TGFβ for approximately 10 days. TGFβ was removed, and the cells were passaged into 10 cm cell culture dishes coated with 0.1% Gelatin for amplification. Cells showing triple-positive CD73 / CD90 / CD105 were selected by flow cytometry as myotubular progenitor cells. The MyoD-ER(T) plasmid was packaged into a virus and used to infect early-stage myotubular progenitor cells. On the second day after viral infection, 1 μM 4-OH-Tamoxifen was added, and the cells were then incubated at 37°C in a 5% CO2, 3%-5% O2 incubator; this marks the first day of initial differentiation. After 24 hours, the myotubular cell differentiation medium was changed to DMEM medium containing 2% horse serum, and 1 μM 4-OH-Tamoxifen was added to continue induction for two more days. Thereafter, the myotubular cell differentiation medium was changed every two days.

[0197] Protein sample preparation:

[0198] The aforementioned human myotubular cells were lysed by repeatedly pipetting and mixing with a pipette in 1% SDS solution, and then heated to 105°C for 10 min to fully denature the proteins. Equal amounts of protein were then subjected to Western blot analysis (using GAPDH expression levels as a reference).

[0199] The expression level of FOXO3 protein in cells was detected using Western blot (same as the steps in Example 2, Part 4).

[0200] Test results as follows Figure 10 As shown in Figure B, the FOXO3 knockout group showed no FOXO3 protein expression, indicating that the FOXO3 gene was successfully knocked out.

[0201] (ii) Experimental detection of the diameter of human myotubular cells after FOXO3 gene knockout

[0202] The experimental method is the same as the detection method in steps one and two of Example 3 for the diameter of human myotubular cells after inhibiting FOXO3 gene expression.

[0203] Test results as follows Figure 10 As shown in Figure C, the diameter of human myotubular cells in the FOXO3- / - group was significantly lower than that in the FOXO3 group. + / + The results showed that knocking out the FOXO3 gene significantly reduced the diameter of human myotubular cells. This indicates that inhibiting FOXO3 gene expression increases the aging of skeletal muscle cells.

[0204] (iii) Experimental detection of SA-β-gal activity in human myotubular cells after FOXO3 gene knockout.

[0205] The experimental method is the same as the detection method in steps one and three of Example 3 for SA-β-gal activity in human myotubular cells after inhibiting FOXO3 gene expression.

[0206] Test results as follows Figure 10 As shown in Figure D, knocking out the FOXO3 gene in human myotubular cells significantly increased the activity of SA-β-gal. This further indicates that inhibiting the expression of the FOXO3 gene increases the aging of skeletal muscle cells.

[0207] III. Verification of FOXO3 function through FOXO3 genetic enhancement gene editing.

[0208] Using gene editing technology and a FOXO3 promoter (FOXO3 gene editing vector), we verified that promoting FOXO3 gene expression can reduce or delay the aging of skeletal muscle cells.

[0209] 1) Human myotubular cells were divided into two groups: wild-type group (FOXO3) + / + ) and FOXO3 genetic enhancement group (FOXO3 2SA / 2SA ()( Figure 11 (A). Detection was performed using PCR genotyping.

[0210] FOXO3-enhanced embryonic stem cells refer to the FOXO3 mentioned in the literature "FOXO3-Engineered Human ESC-Derived Vascular Cells Promote Vascular Protection and Regeneration. Cell StemCell. 2019; 24(3):447-461". 2SA / 2SA hESCs (i.e., FOXO3-enhanced embryonic stem cells). FOXO3 2SA / 2SAhESCs were obtained from the human embryonic stem cell line H9 ESC via recombinant adenovirus gene-targeted editing, FOXO3 2SA / 2SA hESCs are obtained by mutating the T at positions 757 and 943 of the FOXO3 gene (denoted as the wild-type FOXO3 gene) in the human embryonic stem cell H9 ESC cell line to G, respectively. The mutated FOXO3 gene is designated as the FOXO3 enhancer gene. The wild-type FOXO3 protein has serine at positions 253 and 315, while the FOXO3 enhancer protein has both positions mutated to alanine. Compared to the wild-type FOXO3 protein, the activity of the FOXO3 enhancer protein is enhanced. The human embryonic stem cell H9 ESC cell line is abbreviated as H9 cells and is a product of WiCell, catalog number WA09(H9)-DL-7.

[0211] The FOXO3 gene editing vector used to enhance FOXO3 protein activity is rpAMHDAdGT8-4 (a recombinant vector obtained by inserting the DNA molecule shown in SEQ ID No. 4 into the pAMHDAdGT8-4 vector (Addgene#26421)).

[0212] Test results as follows Figure 11 As shown in Figure B, the FOXO3 genetic enhancement group exhibits mutations at two phosphate sites, S253A and S315A. The mutated FOXO3 protein (named FOXO3) S253A\S315A The amino acid sequence of ) is SEQ ID No. 3.

[0213] (ii) Experimental detection of the diameter of human myotubular cells after FOXO3 genetic enhancement

[0214] The experimental method is the same as the detection method in steps one and two of Example 3 for the diameter of human myotubular cells after inhibiting FOXO3 gene expression.

[0215] Test results as follows Figure 11 As shown in C, FOXO3 2SA / 2SA The diameter of myotubular cells in group A was significantly higher than that in group FOXO3. + / + The results showed that gene editing technology to enhance the genetic expression of FOXO3 promoted the expression of the FOXO3 gene, and the expression product FOXO3... S253A\S315A Increased protein activity led to a significant increase in the diameter of human myotubular cells. This indicates that promoting FOXO3 gene expression can significantly reduce or delay the aging process of skeletal muscle cells.

[0216] (iii) Experimental detection of SA-β-gal activity in human myotubular cells after FOXO3 genetic enhancement

[0217] The experimental method is the same as the detection method in steps one and three of Example 3 for SA-β-gal activity in human myotubular cells after inhibiting FOXO3 gene expression.

[0218] Test results as follows Figure 11 As shown in Figure D, genetic enhancement of the FOXO3 gene in human myotubular cells using gene editing technology promoted the expression of the FOXO3 gene, and the expression product FOXO3... S253A\S315A Increased protein activity and significantly decreased SA-β-gal activity in cells further indicate that increased expression of the FOXO3 gene can significantly reduce or delay the aging process of skeletal muscle cells.

[0219] All the above results demonstrate that inhibiting or knocking out FOXO3 gene expression induces cellular senescence phenotypes in human myotubular cells, such as reduced diameter and increased SA-β-gal activity, while enhanced FOXO3 gene inheritance delays myotubular cell senescence. This indicates that the FOXO3 gene is crucial for the homeostasis and functional maintenance of human myotubular cells, thus further confirming that the FOXO3 gene is a novel biomarker for aging skeletal muscle cells.

[0220] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for constructing recombinant skeletal muscle cells, the method comprising introducing the FOXO3 gene and / or FOXO3 into recipient skeletal muscle cells. S253A\S315A The protein-coding gene was used to obtain recombinant cells, the recombinant skeletal muscle cells having a lower senescence level than the recipient skeletal muscle cells; the FOXO3 S253A\S315A The amino acid sequence of the protein is SEQ ID No.

3.

2. The use of a FOXO3 gene promoter in the preparation of a medicament for the prevention, intervention, improvement, delay, and / or treatment of skeletal muscle cell aging, wherein the FOXO3 gene promoter is used to increase the activity of FOXO3 protein and / or increase the FOXO3 as described in claim 1. S253A\S315A Reagents for protein activity: The reagent that increases the activity of FOXO3 protein is the FOXO3 gene or a FOXO3 gene expression vector. The FOXO3 in claim 1 is added. S253A\S315A The reagent for protein activity is the aforementioned FOXO3. S253A\S315A The gene encoding the protein or expressing the FOXO3 S253A\S315A Protein carrier.

3. The application according to claim 2, characterized in that, The FOXO3 gene promoter has at least one of the following functions: G1) increases or upregulates skeletal muscle cell diameter; G2) reduces or downregulates SA-β-gal activity in skeletal muscle cells.

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