Use of enolase 1 (ENO1) as RNA binding protein in myoblast differentiation

Abstract

The invention discloses an application of enolase 1 (ENO1) as RNA (Ribonucleic Acid) binding protein in myoblast differentiation. Specifically, the invention discloses application of ENO1 protein as a negative regulatory factor in regulating myoblast differentiation. Different types of ENO1 inhibitors are developed, including siRNA, shRNA and sgRNA for silencing or knocking out ENO1 genes, and the different types of inhibitors can remarkably promote differentiation and fusion of myoblasts. Results prove that the ENO1 as an RNA binding protein directly regulates the expression of HDAC2, by reducing the expression of ENO1 protein and increasing the expression of HDAC2, the expression of ALT1 and ALT2 is promoted, the metabolic level of alanine is improved, the differentiation and fusion of myoblasts are promoted, and the growth and development of skeletal muscles are further promoted. The method has wide application prospects in the fields of animal husbandry industry muscle variety improvement and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of enolase 1 (ENO1) as an RNA-binding protein in myoblast differentiation. Background Art

[0002] Skeletal muscle is the largest organ in the animal body, accounting for 40%-50% of the total body mass. Skeletal muscle is not only an important locomotor organ, but also a very important metabolic organ as one of the main protein pools in the body, regulating blood glucose homeostasis and energy metabolism in the animal body, and playing an indispensable role in metabolism, locomotion and body health. Therefore, the maintenance of skeletal muscle function is of great significance to body health. In addition, in livestock production, the growth and development of skeletal muscle is one of the main factors determining the meat production performance of meat animals. The growth and development of skeletal muscle is inseparable from meat yield and meat quality. Revealing the regulatory mechanism of livestock skeletal muscle growth and development is the basis for understanding skeletal muscle development and livestock meat production traits. Therefore, the research on skeletal muscle development is of great significance in livestock production. The growth and development of skeletal muscle mainly include processes such as myoblast proliferation, differentiation and fusion. The coordinated progress of this process depends on a highly complex molecular regulatory network. The biological process of myoblast differentiation is the key core link controlling skeletal muscle growth and development. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of ENO1 protein and / or ENO1 gene as an RNA-binding protein in promoting myoblast differentiation. The technical problems to be solved are not limited to the described technical topics, and those skilled in the art can clearly understand other technical topics not mentioned herein through the following description.

[0004] In order to achieve the above purpose, the present application first provides the application of ENO1 protein and / or ENO1 gene in any of the following:

[0005] A1) Application in regulating myoblast differentiation;

[0006] A2) Application in regulating myoblast fusion;

[0007] A3) Application in regulating the number of myotubes of myoblasts;

[0008] A4) Application in regulating the expression level of HDAC2 protein or interacting with HDAC2 protein;

[0009] A5) Application in regulating the expression level of ALT1 and / or ALT2.

[0010] The present invention also provides the application of an ENO1 inhibitor in any of the following:

[0011] Use in promoting myoblast differentiation;

[0012] Use in promoting myoblast fusion;

[0013] Use in increasing the number of myotubes of myoblasts;

[0014] Use in increasing the expression level of HDAC2 protein;

[0015] Use in increasing the expression level of ALT1 and / or ALT2;

[0016] Use in the preparation of products for promoting myoblast differentiation and / or fusion;

[0017] Use in promoting animal muscle development or in the preparation of products for promoting animal muscle development.

[0018] In the above applications, the ENO1 inhibitor includes any one of the following:

[0019] C1) Substances that inhibit the replication, transcription, translation, post-transcriptional modification, and / or post-translational modification of the ENO1 gene;

[0020] C2) Substances that inhibit or reduce the content, activity, and / or function of the ENO1 protein.

[0021] The substance can be any substance that inhibits the activity of the ENO1 protein and / or reduces the content of the ENO1 protein through regulation at the gene level or protein level.

[0022] The regulation of gene expression at the gene level may include regulation of expression at the chromatin level (such as histone modification, chromatin remodeling), transcriptional level (such as regulation of promoters, transcription factors, co-regulatory factors), post-transcriptional level (such as RNA splicing, microRNA regulation), and post-translational level (such as ubiquitination, SUMOylation, acetylation, glycosylation, phosphorylation, methylation, NEDD8 modification, etc.).

[0023] The regulation at the protein level may include regulating the activity and / or content of the protein through protein degradation, protein interaction, or other methods capable of regulating protein activity.

[0024] Furthermore, the substance includes substances that cause the deletion or inactivation of the ENO1 gene through site-directed mutagenesis technology, gene knockdown technology, gene editing technology, and / or gene knockout technology, or substances that specifically bind to the ENO1 protein to reduce its content or inactivate its function.

[0025] It is well-known to those skilled in the art to inhibit gene expression, silence or knockout genes by using site-directed mutagenesis techniques (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis, cassette mutagenesis, etc.), gene knockdown techniques (including RNA interference technique, Morpholino interference technique, antisense nucleic acid technique, ribozyme technique, etc.), gene editing techniques (including zinc finger nuclease gene editing technique, TALEN gene editing technique, CRISPR gene editing technique, etc.) or gene knockout techniques (including complete gene knockout and conditional gene knockout). For example, shRNA, siRNA or miRNA targeting the ENO1 gene can be used to inactivate gene expression or silence the gene at the post-transcriptional or translational level. The CRISPR-Cas system containing sgRNA and Cas protein can also be used to knockout the ENO1 gene. Or site-directed mutagenesis technique can be used to mutate the ENO1 gene to generate frameshift mutations or premature translation termination, thereby inactivating or weakening the function of the ENO1 gene.

[0026] Furthermore, the substance includes nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, polypeptides, proteins, recombinant vectors (such as gene editing vectors), recombinant cells or viral vectors (such as lentivirus and adeno-associated virus vectors).

[0027] Furthermore, the nucleic acid molecule may include (1) double-stranded RNA (dsRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), etc. used in RNA interference technique; (2) antisense RNA (asRNA) and antisense oligonucleotide (AON) used in antisense nucleic acid technique; (3) gRNA and sgRNA used in gene editing technique; (4) aptamer and ribozyme, etc.

[0028] Those skilled in the art know that target sequences can be selected according to the sequence of the ENO1 gene or the mRNA transcribed therefrom to design nucleic acid molecules such as siRNA, miRNA, shRNA or dsRNA. The nucleic acid molecule can interfere with gene transcription, translation or post-transcriptional and post-translational modifications, thereby affecting protein expression.

[0029] Methods of knocking out genes using gene editing technology are also well-known to those skilled in the art. The Cas protein (such as Cas9 protein) that matches (or binds) to the designed guide RNA (sgRNA) can be selected, as long as the purpose of knocking out can be achieved. For example, the sgRNA and the Cas protein can be ligated into the same vector and driven by a dual promoter, or the sgRNA and the Cas protein can be ligated onto different vectors respectively, or a backbone vector already containing the Cas protein gene (such as PX459 vector, PX458 vector, PX461 vector, PX462 vector, PX551 vector, PX552 vector, pGK1.1 vector, PX330 vector, PX335 vector, PX165 vector, eSpCas9(1.1) vector, etc.) can be selected as the expression vector for the sgRNA, and the DNA molecule encoding the sgRNA is cloned into this backbone vector to construct a gene editing vector targeting the target gene.

[0030] In the above application, the ENO1 inhibitor includes nucleic acid molecules for silencing or knocking out the ENO1 gene (such as siRNA or shRNA targeting and interfering with the expression of the ENO1 gene, or sgRNA targeting and knocking out the ENO1 gene, etc.).

[0031] In the above application, the ENO1 inhibitor includes any one of the following:

[0032] D1) siRNA: The nucleotide sequence of the sense strand of the siRNA is the 1st - 19th position of SEQ ID NO:1 or SEQ ID NO:1; the nucleotide sequence of the antisense strand of the siRNA is the 1st - 19th position of SEQ ID NO:2 or SEQ ID NO:2;

[0033] D2) A DNA molecule encoding the siRNA described in D1);

[0034] D3) An expression cassette, recombinant vector or recombinant microorganism containing the siRNA described in D1) or the DNA molecule described in D2);

[0035] D4) shRNA: The target sequence of the shRNA is as shown in SEQ ID NO:3;

[0036] D5) A DNA molecule encoding the shRNA described in D4);

[0037] D6) An expression cassette, recombinant vector or recombinant microorganism containing the shRNA described in D4) or the DNA molecule described in D5);

[0038] D7) sgRNA1 and sgRNA2: The target sequence of the sgRNA1 is shown as SEQ ID NO:4; the target sequence of the sgRNA2 is shown as SEQ ID NO:5;

[0039] D8) DNA molecule 1 encoding the sgRNA1 described in D7) and DNA molecule 2 encoding the sgRNA2 described in D7);

[0040] D9) An expression cassette, recombinant vector or recombinant microorganism containing the sgRNA1 and sgRNA2 described in D7), or containing the DNA molecule 1 and DNA molecule 2 described in D8);

[0041] D10) A CRISPR / Cas9 system containing the sgRNA1 and sgRNA2 described in D7).

[0042] One or both ends of an siRNA known to those skilled in the art may have overhangs (at least one unpaired nucleotide extending from the end of the RNA strand). The overhangs of the siRNA may be present at the 5' end or 3' end of the siRNA, and the number of nucleotides constituting the overhangs may be 1, 2, 3, 4, 5 or 6. The lengths of the overhangs of the two strands may be the same or different. The overhangs may be TT, TU, UT or UU, but are not limited thereto. In one or more embodiments of the present application, both strands of the siRNA contain 3' overhangs. Specifically, a "TT" overhang is added to the 3' end of the siRNA to stabilize the siRNA, which does not affect the specificity and function of the siRNA. The "TT" part of the 3' overhang is optional and non-limiting.

[0043] The CRISPR / Cas9 system described in D10 further includes a Cas9 protein. The Cas9 protein is not limited to a specific protein, as long as it can be used in combination with the sgRNA of the present invention. For example, the Cas9 protein may include Streptococcus pyogenes Cas9 (spCas9, subtype II-A), spCas9 HF (high-fidelity), nickase Cas9 (nCas9), Staphylococcus aureus Cas9 (saCas9, subtype II-A), Neisseria meningitidis Cas9 (NmCas9, subtype II-C), Francisella novicida Cas9 (FnCas9, subtype II-B), Streptococcus thermophilus Cas9 (St1Cas9, St3Cas9), Campylobacter jejuni Cas9 (CjCas9), and Treponema sp. Cas9, as well as Cas9 orthologs from other organisms but not limited thereto. The Cas9 protein may also include high-fidelity Cas9 mutants (such as SpCas9-HF1, eSpCas9-1.1, and TrueCut TM HiFi Cas9 protein), etc.

[0044] In the above application, the recombinant microorganism may be a recombinant virus (such as recombinant lentivirus, recombinant adenovirus, or recombinant adeno-associated virus, etc.).

[0045] Any ENO1 inhibitor described herein is also within the scope of protection of the present invention.

[0046] The present invention also provides a method for promoting myoblast differentiation, the method comprising reducing the content and / or activity of ENO1 protein in myoblasts.

[0047] In the above method, the reduction of the content and / or activity of ENO1 protein in myoblasts can be achieved by reducing the expression level of the encoding gene of ENO1 protein in myoblasts.

[0048] The present invention also provides a product for promoting animal muscle development or skeletal muscle development, the product containing any ENO1 inhibitor described herein.

[0049] Furthermore, the product may be feed, feed additive, muscle growth promoter, muscle enhancement supplement, etc.

[0050] Furthermore, the animals include, but are not limited to: deer, sheep, chickens, ducks, fish, humans, rats, mice, guinea pigs, hamsters, nude mice, rabbits, pigs, dogs, monkeys, horses, cows, etc.

[0051] The myoblasts described in this article include skeletal muscle myoblasts.

[0052] Through extensive and in-depth research, the present invention for the first time discovers the application of ENO1 as an RNA-binding protein in regulating myoblast differentiation. The inventors further designed and developed different types of ENO1 inhibitors, including siRNA and shRNA for silencing the ENO1 gene, and sgRNA for knocking out the ENO1 gene. The experimental results show that different types of ENO1 inhibitors can significantly promote the differentiation and fusion of myoblasts, specifically manifested in that ENO1 inhibitors can increase the number of myotubes of myoblasts, improve the expression levels of myogenic differentiation factors MyoD and MyoG, increase the expression levels of ALT1 and ALT2, and increase the expression level of HDAC2 protein. It has not been envisioned in the art to use the ENO1 protein as an RNA-binding protein to regulate the differentiation and fusion of myoblasts. Unexpectedly, the inventors of this application have proven through different means that the ENO1 protein participates in the differentiation of myoblasts as a negative regulator, and as an RNA-binding protein, ENO1 regulates the expression of HDAC2, and regulates the expression of ALT1 and ALT2 through HDAC2, thereby realizing the function of regulating the differentiation of myoblasts. By reducing the expression of the ENO1 protein, the differentiation and fusion of myoblasts can be significantly promoted, and thus the growth and development of skeletal muscle can be promoted. The present invention has broad application prospects in the fields of livestock industry muscle variety improvement, livestock breeding, muscle disease treatment, muscle regeneration and repair, etc.

[0053] Term Definitions

[0054] In this application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, to better understand this application, the following provides the definitions and explanations of related terms.

[0055] The term "HDAC2" refers to histone deacetylase 2.

[0056] The term "ALT1" refers to Alanine aminotransferase 1, and the term "ALT2" refers to Alanine aminotransferase 2. "ALT1" and "ALT2" are two isoenzymes of Alanine aminotransferase (ALT).

[0057] The term "expression cassette" generally refers to a nucleic acid construct containing nucleic acid elements sufficient to express a gene of interest. A typical expression cassette contains a promoter, an MCS (multiple cloning site), and a terminator. The expression cassette may also include a gene of interest, a marker gene (such as the TK gene, DHFR gene, CAT gene, and NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly(A) signal sequence, and / or an mRNA splicing signal sequence, etc. The elements in the expression cassette can be directly linked or indirectly linked through a linker.

[0058] The term "vector" generally refers to a vector that can carry foreign DNA or a gene of interest into a host cell for amplification and / or expression. The vector can be a cloning vector or an expression vector. The vector can be introduced into the host cell by transformation, transduction, or transfection, so that the genetic material elements it carries can be amplified and / or expressed in the host cell. Those skilled in the art can select a suitable vector according to the purpose of genetic engineering and the nature of the recipient cell. The vectors include but are not limited to: plasmids, phages (such as λ phage or M13 phage), cosmids (i.e., cosmid plasmids), phagemids, shuttle vectors (such as yeast expression vectors), Ti plasmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 artificial chromosomes (PAC), or Ti plasmid artificial chromosomes (TAC)), viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, polyomaviruses (such as SV40), herpesviruses (such as herpes simplex virus)). A vector can contain various elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.

[0059] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasmas, chlamydiae, spirochetes, algae, etc. For example, the bacteria may be from the genus Corynebacterium sp. (such as Corynebacterium glutamicum, Corynebacterium pekinense, Corynebacterium crenatum, etc.), the genus Brevibacterium sp. (such as Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium ammoniagenes, etc.), the genus Escherichia sp. (such as Escherichia coli), the genus Erwinia sp., the genus Agrobacterium sp. (such as Agrobacterium tumefaciens), the genus Flavobacterium sp., the genus Alcaligenes sp., the genus Pseudomonas sp., and the genus Bacillus sp. (such as Bacillus subtilis), etc. The viruses may include rotavirus, baculovirus, retrovirus (such as lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papovavirus (such as SV40), and herpesvirus (such as herpes simplex virus), etc. The fungi may be from the genus Saccharomyces sp. (such as Saccharomyces cerevisiae, Candida sp., Pichia methanolica, Pichia pastoris), the genus Fusarium sp., the genus Rhizoctonia sp., the genus Verticillium sp., the genus Penicillium sp., the genus Aspergillus sp., and Cephalosporium sp., etc. The actinomycetes may be from the genus Streptomyces sp. (such as Streptomyces). The algae may be from the phylum Cyanophyta (such as Cyanobacteria), the genus Fucus sp., the genus Achnanthes sp., the genus Amphiprora sp., the genus Amphora sp., the genus Ankistrodesmus sp., the genus Asteromonas sp., and the genus Boekelovia sp., etc.

[0060] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by ligating an exogenous target gene and a vector in vitro, which can be constructed in any suitable manner as long as the constructed recombinant vector can carry the exogenous target gene into a recipient cell and provide the ability for the exogenous target gene to replicate, integrate, amplify, and / or express in the recipient cell.

[0061] The term "recombinant microorganism" generally refers to a microorganism whose genes have been manipulated and modified to obtain a recombinant microorganism with changed functions. For example, introducing an exogenous target gene or recombinant vector into the target microorganism, or directly performing gene editing on the endogenous genes of the target microorganism.

[0062] The term "inhibitor (also known as antagonist)" has the meaning well-known in the art and can refer to any substance that reduces (downregulates) the level and / or activity of a target protein or gene.

[0063] The term "ENO1 inhibitor" in the present application can be a substance that inhibits the replication, transcription, translation, post-transcriptional modification, and / or post-translational modification of the ENO1 gene, or can also be a substance that inhibits or reduces the content, activity, and / or function of the ENO1 protein. The ENO1 inhibitors include substances that cause the deletion or inactivation of the ENO1 gene through techniques such as site-directed mutagenesis, gene knock-down, gene editing, and gene knock-out. The ENO1 inhibitors also include substances that can target and bind to the ENO1 protein, inhibit the activity of the ENO1 protein, or prevent the ENO1 protein from performing its function.

[0064] The term "site-directed mutagenesis" generally refers to changing one or several bases in a certain gene through site-directed mutagenesis, resulting in a change in the amino acid composition of the corresponding protein. Site-directed mutagenesis methods include oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis, and cassette mutagenesis, etc.

[0065] The term "gene knock-down" also known as gene knockdown generally refers to a technique that inactivates gene expression or causes gene silencing at the post-transcriptional level or translational level, while the DNA sequence of the gene remains unchanged. Gene knock-down includes RNA interference technology, Morpholino interference technology, antisense nucleic acid technology, and ribozyme technology, etc.

[0066] The term "gene editing" generally refers to a technique that can complete the change of a specific gene sequence in any cell including somatic cells, and can cause base deletion, duplication, insertion, frameshift mutation, and replacement and knockout of the target gene in the genome, achieving replacement, deletion, cleavage, and single-base change of the genomic sequence, that is, the technique of arbitrarily "editing" the sequence of the genome or a specific gene. Gene editing includes zinc finger nuclease gene knockout technology, TALEN gene editing technology, and CRISPR gene editing technology.

[0067] The term "gene knock-out" generally refers to a technique that uses an exogenous mutated gene to replace the endogenous normal homologous gene through homologous recombination, thereby inactivating the endogenous gene, including complete gene knock-out (such as complete mutation of the target gene based on a replacement-type targeting vector or an insertion-type targeting vector) and conditional gene knock-out (such as tissue-specific knock-out based on the Cre-LoxP recombinase system or the FLP-FRT recombinase system).

[0068] The term "RNA interference (RNAi)" generally refers to a technique that uses double-stranded RNA (dsRNA) to induce the degradation of the mRNA of a target gene that is homologous and complementary to it, silence the expression of the gene, thereby triggering post-transcriptional gene silencing (PTGS), and achieving the purpose of preventing gene expression. Double-stranded RNA (dsRNA) is the trigger for RNAi, which triggers the degradation of the complementary single-stranded RNA (ssRNA). Long dsRNA can be cleaved into small fragments of dsRNA, namely small interfering RNA (siRNA), by Dicer enzyme in cells, and siRNA mediates mRNA cleavage. Therefore, RNAi can also be achieved by introducing siRNA corresponding to the target gene. In addition, RNAi induced by introducing dsRNA into mammalian cells is transient. A gene encoding an RNA with inverted repeats that can form a hairpin (i.e., short hairpin RNA, shRNA) can be introduced, and this gene continuously provides dsRNA in the form of a hairpin structure to make RNAi proceed continuously.

[0069] The term "siRNA (small interfering RNA)" generally refers to a class of double-stranded RNA molecules that can mediate the cleavage, degradation or silencing of target RNA (such as mRNA, tRNA and viral RNA, etc.) that is complementary to it. siRNA is usually double-stranded, including an antisense strand complementary to the target RNA and a sense strand complementary to the antisense strand. The preparation methods of siRNA are well-known to those skilled in the art, such as chemical synthesis method, in vitro transcription method, in vitro preparation method by degrading long dsRNAs with RNaseIII (or Dicer), and the method of expressing and preparing by transfecting host cells with an siRNA expression vector, etc.

[0070] The term "DNA encoding siRNA" generally refers to a DNA molecule capable of transcribing to generate the siRNA. As is well-known to those skilled in the art, if the sequence of a given siRNA is known, the U in the siRNA sequence can be changed to T while other nucleotides remain unchanged to obtain the DNA sequence encoding the siRNA.

[0071] The term "comprising" is not intended to be restrictive, is intended to be inclusive and means that there may be other elements in addition to the listed elements, and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of". In this article, the terms "comprising" and "including" are used interchangeably. Brief Description of the Drawings

[0072] Figure 1 It shows the expression change of ENO1 during the differentiation of ovine skeletal muscle satellite cells in Example 1.

[0073] Figure 2 It shows the expression change of ENO1 during the differentiation of mouse C2C12 cells in Example 1.

[0074] Figure 3 It shows the detection of the expression of ENO1 after interfering with the expression of ENO1 in ovine skeletal muscle satellite cells in Example 2.

[0075] Figure 4 It shows the statistical result of the fusion rate of satellite cells after 48 hours of differentiation after interfering with the expression of ENO1 in ovine skeletal muscle satellite cells in Example 2.

[0076] Figure 5 It shows the mRNA expression changes of myogenic differentiation factors MyoD and MyoG after interfering with the expression of ENO1 in ovine skeletal muscle satellite cells in Example 2.

[0077] Figure 6 It shows the expression of ENO1 after interfering with the expression of ENO1 in mouse C2C12 cells in Example 3.

[0078] Figure 7 It shows the cell fusion condition after 48 hours of differentiation after interfering with the expression of ENO1 in mouse C2C12 cells in Example 3.

[0079] Figure 8 It is the gene sequence diagram of ENO1.

[0080] Figure 9 It is the PCR identification of positive cells with the ENO1 gene knocked out in Example 3: the fragment size after knockout is 901 bp.

[0081] Figure 10 It is the sequencing analysis of the PCR product in Example 3.

[0082] Figure 11 To detect the expression of positive cell ENO1 at the protein level in Example 3.

[0083] Figure 12 To show the fusion of mouse C2C12 cells at 48 h of differentiation after knocking out the ENO1 gene in Example 3.

[0084] Figure 13 To compare the expression of MyoD and MyoG proteins between knockout cells (ENO1-KO cells) and WT cells at 48 h of differentiation in Example 3.

[0085] Figure 14 To show the expression of ALT1 and ALT2 in knockout cells (ENO1-KO) and WT cells in Example 4.

[0086] Figure 15 To show the change in ALT activity in knockout cells (ENO1-KO) and WT cells in Example 4.

[0087] Figure 16 To detect the expression of ALT1 and ALT2 after interfering with ALT1 and ALT2 in Example 4.

[0088] Figure 17 To show the expression of myogenic differentiation factors in mouse C2C12 after interfering with ALT1 and ALT2 in Example 4.

[0089] Figure 18 To analyze the difference in RIP-QPCR results during the cell proliferation period of WT cells in Example 4.

[0090] Figure 19 To analyze the difference in RIP-QPCR results at 48 h of cell differentiation of WT cells in Example 4.

[0091] Figure 20 To analyze the difference in HDAC2 expression between the ENO1-KO and WT cell groups at the mRNA level in Example 4.

[0092] Figure 21 To compare and analyze the difference in HDAC2 expression between the ENO1-KO and WT cell groups at the protein level in Example 4.

[0093] Figure 22 To show the interaction between HDAC2 and ALT1 during the cell proliferation period of WT cells in Example 4.

[0094] Figure 23 To show the interaction between HDAC2 and ALT1 at 48 h of cell differentiation of WT cells in Example 4.

[0095] Figure 24For the WT cell proliferation period in Example 4, the interaction between HDAC2 and ALT2.

[0096] Figure 25 For the WT cells differentiated for 48 h in Example 4, the interaction between HDAC2 and ALT2.

[0097] Figure 26 For Example 4, after adding the HDAC2 inhibitor to WT cells, the expression changes of ALT1 and ALT2.

[0098] Figure 27 For Example 4, after transfecting WT cells with HDAC2 siRNA, the expression changes of ALT1 and ALT2. Detailed implementation mode

[0099] The present invention will be further described in detail below in combination with the specific implementation mode. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0100] The experimental methods in the following embodiments, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0101] The following embodiments use SPSS statistical software to process the data. The experimental results are expressed as the mean ± standard deviation. The independent sample T-test method is used. P < 0.05 (*) indicates a statistically significant difference, P < 0.01 (**) indicates a significant statistical difference, and P < 0.001 (***) indicates an extremely significant statistical difference. For the quantitative experiments in the following embodiments, unless otherwise specified, three biological replicate experiments are set, and the results are averaged.

[0102] In the following embodiments, "ENO1" is fully named enolase 1, also known as α-enolase. The term "ENO1" herein is intended to include enolase 1, as well as its variants, orthologs or paralogs. The variants, orthologs or paralogs substantially retain the biological functions of the sequences from which they are derived; the variants can be naturally occurring variants (such as allelic variants or splicing variants), or non-naturally occurring variants. The variants include proteins that have more than 80% identity with the ENO1 protein and have the same function obtained by substituting, deleting and / or adding amino acid residues to the amino acid sequence of the ENO1 protein; the variants can also include fragments of the ENO1 protein that have the same or substantially the same function as the ENO1 protein. The sequences of the ENO1 protein and the ENO1 gene are well known in the art and are publicly available from the NCBI website. Exemplarily, in the following embodiments, the GenBank accession number of the amino acid sequence of the Ovis aries ENO1 protein can be XP_014954720.2 (Update Date 30-OCT-2023); the nucleotide sequence of the coding sequence (CDS) of the Ovis aries ENO1 gene can be positions 147-1451 of GenBank Accession No. XM_015099234.3 (Update Date 30-OCT-2023). The GenBank accession number of the amino acid sequence of the Mus musculus ENO1 protein can be NP_001366056.1 (Update Date 15-OCT-2024); the nucleotide sequence of the coding sequence (CDS) of the Mus musculus ENO1 gene can be positions 44-1348 of GenBank Accession No. NM_001379127.2 (Update Date 15-OCT-2024). In other species other than Ovis aries and Mus musculus, the amino acid sequences and nucleotide sequences of the ENO1 protein are also well known.

[0103] In the following embodiments, Ovis aries skeletal muscle satellite cells were isolated from Hu sheep about 1 day after birth. Mouse C2C12 cells were purchased from the Cell Bank of the Shanghai Institute of Life Sciences, Chinese Academy of Sciences. The siRNA sequences were synthesized by Jiangsu Saisuofei Biotechnology Co., Ltd. The sgRNA was synthesized by Genewiz Suzhou. Antibodies ENO1, Myf5, MyoD, ALT1, HDAC2 were purchased from Abcam, USA; antibody ALT2 was purchased from Wuhan Sanying Proteintech Co., Ltd.; antibody MyoG was purchased from the Developmental Studies Hybridoma Bank (DSHB), USA; α-Tubulin was purchased from Cell Signaling Technology, Inc (CST), USA; LaminB1 was purchased from Beyotime, China, and small molecule inhibitors were purchased from MedChemExpress (MCE), USA.

[0104] Example 1: ENO1 protein is highly expressed in skeletal muscle myoblasts

[0105] To explore the role of ENO1 in the differentiation process of skeletal muscle myoblasts, the expression changes of ENO1 in sheep skeletal muscle myoblasts and mouse C2C12 cells at different differentiation times were first detected. The specific steps are as follows:

[0106] Transfer the sheep skeletal muscle myoblasts stored in liquid nitrogen to a 37°C water bath for resuscitation. After resuscitation, spread them in culture dishes, a total of 3 dishes, labeled as P, D24h, and D48h. Add proliferation medium (containing 10% FBS, 1% PS) and place them in a 37°C constant temperature incubator for culture. When the cell density of the 3 dishes reaches 90%, digest and collect the cells labeled as P. Use RIPA lysis buffer (Aidlab, PP1101) to extract the total protein of the cells, and store the total protein in an -80°C refrigerator. For the other two dishes of cells, change to induction differentiation medium (containing 5% HS) for induction differentiation, labeled as D0h. When induced to D24h, collect the cells labeled as D24h using the same method. When induced to D48h, collect the cells labeled as D48h. After extracting the total protein, use a BCA kit (Aidlab, PP01) to detect the concentration of the total protein in the three groups. Use Western Blot to detect the expression changes of ENO1 in sheep skeletal muscle myoblasts.

[0107] Transfer the mouse C2C12 cells stored in liquid nitrogen to a 37°C water bath for resuscitation. After resuscitation, spread them in culture dishes, a total of 6 dishes, labeled as P, D3h, D5h, D12h, D24h, and D48h. Add proliferation medium (containing 10% FBS, 1% PS) and place them in a 37°C constant temperature incubator for culture. When the cell density of the 6 dishes reaches 90%, digest and collect the cells labeled as P. Use RIPA lysis buffer (Aidlab, PP1101) to extract the total protein of the cells, and store the total protein in an -80°C refrigerator. For the other 5 dishes of cells, change to induction differentiation medium (containing 1 μM dexamethasone, 1 μg / mL linoleic acid, 1 μM insulin) for induction differentiation, labeled as D0h. When induced to D3h, D5h, D12h, D24h, and D28h, collect the cells at the corresponding time points using the same method. After extracting the total protein, use a BCA kit (Aidlab, PP01) to detect the concentration of the total protein in the three groups. Use Western Blot to detect the expression changes of ENO1 in mouse C2C12 cells.

[0108] The results are as Figure 1 shown. During the differentiation process of sheep skeletal muscle myoblasts, ENO1 always maintains a stable state of expression and does not change significantly with the progress of differentiation. Similarly, as Figure 2As shown, ENO1 also remained stably expressed throughout the differentiation of mouse C2C12 cells.

[0109] Example 2: ENO1 participates in the differentiation of ovine skeletal muscle myoblasts as a negative regulator

[0110] Use interfering RNA (siRNA) to reduce the expression of ENO1 in ovine skeletal muscle myoblasts, the steps are as follows:

[0111] (1) Experimental grouping:

[0112] Experimental group (ENO1-si): Transfected with the ENO1-siRNA designed in this application;

[0113] Negative control (NC): Transfected with NC-siRNA.

[0114] (2) Design and sequence of siRNA

[0115] For the coding sequence (CDS) of the ovine ENO1 gene: positions 147 - 1451 of GenBank Accession No. XM_015099234.3 (Update Date 30 - OCT - 2023), the ENO1-siRNA sequence designed in this application is shown as follows:

[0116] ENO1-siRNA sense strand: 5’-GGAGCUCCGAGACAAUGAUTT-3’ (SEQ ID NO:1),

[0117] ENO1-siRNA antisense strand: 5’-AUCAUUGUCUCGGAGCUCCTT-3’ (SEQ ID NO:2).

[0118] The NC-siRNA sequence of the control group is shown as follows:

[0119] NC-siRNA sense strand: 5’-UUCUCCGAACGUGUCACGUTT-3’,

[0120] NC-siRNA antisense strand: 5’-ACGUGACACGUUCGGAGAATT-3’.

[0121] (3) Transfection

[0122] The siRNA was synthesized by the company and transfected into ovine skeletal muscle myoblasts through the Gencefe Super Trans transfection reagent (purchased from Jiangsu Saisuofei Biotechnology Co., Ltd., product number TRS002), and the transfection steps are as follows:

[0123] One day before transfection, 0.5 - 2.0×10 5 cells per well were seeded in 500 μL of proliferation medium (containing 10% FBS, 1% PS). When transfected, the cells grew to a density of approximately 60% - 80%.

[0124] Place the GencefeSuperTrans transfection reagent at room temperature and gently mix it before use; add 50 μL of serum-free medium to a sterile tube, add 2 μL of transfection reagent, gently mix with a pipette, and let it stand at room temperature for 5 min;

[0125] Add 50 μL of serum-free medium to another sterile tube, add 2 μL of siRNA, gently mix with a pipette, and let it stand at room temperature for 5 min;

[0126] Drop the GencefeSuperTrans medium mixture into the siRNA medium mixture, gently mix with a pipette, and let it stand at room temperature for 15 - 20 min, then immediately transfect.

[0127] Add 100 μL of transfection complex to each well of cells and gently shake. After 6 - 8 hours of transfection, the medium can be replaced with complete medium.

[0128] (4) Differentiation

[0129] After 24 hours of transfection, when the cell density reached 90%, the two groups of cells with ENO1-siRNA and NC-siRNA were respectively replaced with induction differentiation medium (containing 5% HS) and induced to differentiate for 48 hours. The total RNA of the cells was extracted using a kit (Adlai, RN28).

[0130] (5) Detection of mRNA levels of ENO1, MyoD, and MyoG by real-time quantitative PCR

[0131] Use the HiScript II 1st Strand cDNA Synthesis Kit from Vazyme Company for in vitro reverse transcription of RNA. The reverse transcription system is shown in Table 1.

[0132] Table 1 RNA reverse transcription system

[0133]

[0134]

[0135] Reverse transcription program: 25°C for 5 min, 50°C for 15 min, 85°C for 2 min. After the reaction, the samples were placed at -20°C for later use.

[0136] Quantitative real-time PCR was performed using the SYBR Green PCR Kit from QIAGEN. The reaction system is shown in Table 2. Reaction procedure: 95°C for 2 min; 95°C for 5 s, 60°C for 10 s, 40 cycles. Melting curve program: 95°C for 1 min; 65°C for 30 s, 95°C for 30 s. The relative expression levels of the target genes were calculated using the 2 -ΔΔCT method, and the internal reference gene was β-actin. The primers used in the quantitative real-time PCR experiment are as follows:

[0137] ENO1: F: 5’-GGTGAGAACTCGAGGAAGGGC-3’,

[0138] R: 5’-ACAGCAGCTCTGAAGAGACCTTT-3’,

[0139] β-actin: F: 5’-CGGGACCTGACAGACTACCT-3’,

[0140] R: 5’-TTGGCATACAGGTCCTTTCG-3’,

[0141] MyoD: F: 5’-AGGCGCCCAATGAACCCC-3’,

[0142] R: 5’-GAAACACAACAGTTCCCTCGC-3’,

[0143] MyoG: F: 5’-GCGCCATCCAGTACATAGAG-3’,

[0144] R: 5’-TTGTGGGCATCTGTAGGGTC-3’.

[0145] Table 2 Quantitative real-time PCR reaction system

[0146]

[0147] (6) Detection of the fusion rate of skeletal muscle myoblasts

[0148] When the cells were induced to differentiate to D48h, DIL (Beyotime, C1415S) was used for cell membrane staining, and DAPI was used for nuclear staining. Photos were taken at wavelengths of 594 nm and 405 nm respectively under a confocal laser microscope, and the number of fused nuclei and the total number of nuclei were counted to calculate the fusion index.

[0149] Calculation formula for the fusion rate: the number of nuclei in myotubes / the total number of nuclei × 100%.

[0150] (7) Results and analysis

[0151] Figure 3 showed that after transfection with siRNA, the expression of ENO1 was significantly decreased. The results of fusion rate detection were as Figure 4 shown. After reducing the expression of ENO1, the fusion rate of ovine skeletal muscle satellite cells was significantly increased. The fusion rate of skeletal muscle satellite cells refers to the proportion of skeletal muscle satellite cells that fuse to form multinucleated myotubes during development, and is an important index for evaluating the differentiation efficiency of satellite cells. Further detection of the expression of myogenic differentiation factors MyoD and MyoG showed results as Figure 5 shown. The qPCR results showed that the expression of myogenic differentiation factors MyoD and MyoG was also significantly increased compared with the control group. The above results indicate that by reducing the expression of ENO1 in skeletal muscle satellite cells, the differentiation level of skeletal muscle satellite cells can be significantly improved.

[0152] Example 3: ENO1 is involved in the differentiation of mouse C2C12 cells

[0153] (1) Use lentiviral interfering RNA to reduce the expression of ENO1 in mouse C2C12 cells (the lentivirus was synthesized by Shanghai GeneChem Co., Ltd.)

[0154] For the coding sequence (CDS) of mouse ENO1 gene: positions 44 - 1348 of GenBank Accession No. NM_001379127.2 (Update Date 15 - OCT - 2024), design shRNA targeting ENO1. The target sequence of shRNA is as follows:

[0155] 5’-GCCCTAGAACTCCGAGACAATGATAAGAC-3’ (SEQ ID NO:3).

[0156] Design primers according to the target sequence of shRNA and the destination vector to be inserted as follows:

[0157] Forward primer: 5’-CCGGGCCCTAGAACTCCGAGACAATGATAAGACCTCGAGGTCTTATCATTGTCTCGGAGTTCTAGGGCTTTTTG-3’,

[0158] Reverse primer: 5’-AATTCAAAAAGCCCTAGAACTCCGAGACAATGATAAGACCTCGAGGTCTTATCATTGTCTCGGAGTTCTAGGGC-3’.

[0159] Anneal the above single-stranded primers to form double-stranded DNA fragments, incubate in a water bath at 90 °C for 15 min, and allow to cool naturally to room temperature to obtain the annealing product (double-stranded DNA fragments with sticky ends). Clone the annealing product into the lentiviral expression vector GV112, transform Escherichia coli, and incubate with shaking at 37 °C for 1 h. Take an appropriate amount of the bacterial solution and spread it evenly on a plate containing the corresponding antibiotic, and incubate it upside down in a constant temperature incubator for 12 - 16 h. Pick a single colony, confirm the colony with correct ligation through screening and identification (PCR), extract the plasmid to obtain the recombinant lentiviral vector expressing shRNA, and obtain the recombinant lentivirus expressing shRNA after packaging. Measure the virus activity and transfect it into mouse C2C12 cells according to MOI = 1.

[0160] Carry out lentiviral infection of mouse C2C12 cells (myoblasts) carrying the interference target at a ratio of lentivirus:myoblasts = 1:1. After 48 h of infection, change to the proliferation culture medium, and then perform flow sorting on the infected cells to screen for successfully infected positive cells. Detect the expression of ENO1 by qPCR technology, and the results are as Figure 6 shown. The expression of ENO1 in the infected group cells was significantly decreased. Compare the fusion of mouse C2C12 cells in the knockdown group and the control group at 48 h of differentiation. At 48 h of differentiation, the number of myotubes in mouse C2C12 cells in the knockdown group was significantly higher than that in the control group ( Figure 7 ). The results indicate that by reducing the expression of ENO1 in mouse myoblasts, the number of myotubes in myoblasts can be significantly increased, promoting the differentiation of myoblasts.

[0161] (2) By searching the ensemble database, it was found that there are 5 alternative splicings of the ENO1 gene in mice. To inactivate its function, as Figure 8 shown, select the exons shared by the 5 alternative splicings: exon 2 and exon 3, design targets on both sides of these two exons (denoted as target 1 and target 2), input the intron sequences on both sides of exons 2 and 3 of the mouse ENO1 gene on NCBI into the CRISPOR website, and respectively take the sgRNA with the highest score (except for sgRNAs with too high GC content), and then use the BLAST function of NCBI to detect the uniqueness of the targeting sites for the 2 obtained sgRNAs. For the mouse ENO1 gene sequence: GenBank Accession No. NC_000070.7 (REGION: 150321165 - 150333336) (07 - FEB - 2024), the target sequences of the 2 designed sgRNAs are as follows:

[0162] Target sequence of sgRNA1: 5’-CGGATGATCGCGGCCGTGGA-3’ (SEQ ID NO: 4),

[0163] Target sequence of sgRNA2: 5'-TACTGTAACCAATAGTGGCG-3' (SEQ ID NO:5).

[0164] The sequences of two single-stranded oligos designed for sgRNA1 are as follows:

[0165] sgRNA1-F: 5'-CACCGCGGATGATCGCGGCCGTGGA-3',

[0166] sgRNA1-R: 5'-AAACTCCACGGCCGCGATCATCCGC-3'.

[0167] The sequences of two single-stranded oligos designed for sgRNA2 are as follows:

[0168] sgRNA2-F: 5'-CACCGTACTGTAACCAATAGTGGCG-3',

[0169] sgRNA2-R: 5'-AAACCGCCACTATTGGTTACAGTAC-3'.

[0170] (3) Construction of knockout vector

[0171] Digest the px458 vector at 37°C for 30 min according to the following enzyme digestion system in the table.

[0172] Table 3. px458-BbsI enzyme digestion system

[0173]

[0174] Anneal the two pairs of single-stranded oligos for sgRNA1 and sgRNA2 according to the following system in the table.

[0175] Table 4. sgRNA-oligo annealing system

[0176]

[0177] The annealing program is: metal bath at 95°C for 10 min, take out after turning off the metal bath for 20 min. The annealing products are double-stranded DNAs, which are the double-stranded DNA formed by annealing sgRNA1-F and sgRNA1-R (named DNA1), and the double-stranded DNA formed by annealing sgRNA2-F and sgRNA2-R (named DNA2).

[0178] Dilute the above annealing products 50 times and perform ligation according to the following ligation system in the table.

[0179] Table 5. Ligation system

[0180]

[0181] React at 16°C for 30 min.

[0182] Take 2 μL of the above ligation product and transform it with competent cells. Sequence the selected clones and choose the correctly ligated ones for plasmid extraction. Two gene editing vectors for knocking out the ENO1 gene were obtained and named px458-ENO1-1 and px458-ENO1-2.

[0183] Both recombinant vectors contain 1 editing target (SEQ ID NO: 4 or SEQ ID NO: 5) and the coding gene of Cas9 protein. After being introduced into recipient cells, the two guide RNAs (sgRNA1 and sgRNA2) transcribed can target the ENO1 gene in the recipient cells through base complementary pairing. The Cas9 protein causes double-strand breaks in the DNA at two target sites of the ENO1 gene, and through the organism's own DNA damage repair response mechanism, the sequences on the left side of target 1 and the right side of target 2 are ligated together, thus achieving the knockout of the ENO1 gene.

[0184] (4) Cell transfection

[0185] Transfer healthy C2C12 cells into a 10-cm culture dish and culture until the cell confluence is about 80%. Digest the cells with trypsin and collect them into a centrifuge tube. Resuspend the cells with 100 μL of electroporation buffer and mix well. Add 10 μg of px458-ENO1-1 and 10 μg of px458-ENO1-2 respectively, mix well and then add them to an electroporation cuvette. Place it in a Lonza Amaxa Nucleofector 2B cell nucleus transfection instrument, adjust to program A-033 for electroporation. Immediately after electroporation, add 500 μL of DMEM medium and place it in a 37°C cell incubator for 10 min. Seed the cells into a 6-well plate with complete medium containing 20% FBS. After the cells adhere, change to complete medium containing 10% FBS. Observe the fluorescence of the cells 48 h after electroporation and perform flow sorting to facilitate the identification of knockout cells.

[0186] (5) Identification of positive cells

[0187] The electrically transfected cells were sorted by flow cytometry and collected into 96-well plates to obtain monoclonal cells. After the cells grew for about two weeks, the monoclonal cells were verified at the genomic level. Since the knockout fragment was relatively large, about 4 kb, it was difficult to amplify the complete fragment. Therefore, when verifying positive clones, two pairs of primers were used to perform PCR at both ends of the knockout target. Theoretically, no fragment could be amplified from positive clones, while a fragment could be amplified from negative clones, and the product lengths were 612 bp and 658 bp respectively. Six clones were identified according to this identification method, and no fragment could be amplified on both sides of the two knockout targets. Subsequently, an upstream primer was designed on the left side of target 1 and a downstream primer was designed on the right side of target 2, and the six obtained clones were amplified for the full length. Theoretically, the length amplified from positive clones was 901 bp. As Figure 9 shown, fragments of 901 bp were amplified from all six clones, while WT cells could not amplify fragments of 901 bp. Subsequently, the PCR products were sequenced. As Figure 10 shown, a fragment deletion containing the second exon and the third exon occurred in the ENO1 gene. Finally, verification was performed by Western blot immunoblotting to detect the expression of ENO1 at the protein level in these six monoclonal cells. As Figure 11 shown, ENO1 was no longer expressed in the knockout cells. Therefore, this indicated that the construction of ENO1 knockout cells was successful, and C2C12 cells with ENO1 gene knockout (named: ENO1-KO cells) were obtained. The primer sequences used to identify positive clone cells are as follows:

[0188] ENO1 target 1 primer sequence (612 bp) F: 5’-TGCTTTGCAGCGATCCTACT-3’

[0189] R: 5’-TTGCCTAATTGCGTCGAGGT-3’

[0190] ENO1 target 2 primer sequence (658 bp) F: 5’-CCTACCAACACCCACCTTCC-3’

[0191] R: 5’-GGGTGCTTTGAGGGGTGTTA-3’

[0192] Knockout positive cell full-length amplification identification primer (901 bp): F: 5’-CCGCCTCGGTCACGCTCTTCTA-3’

[0193] R: 5’-AGCCCTACCCACTTCCCCCACA-3’.

[0194] Next, it was further explored whether the differentiation ability of the knockout cells (ENO1-KO) was affected. As Figure 12As shown, at 48 h of differentiation, the differentiation and fusion ability of ENO1-KO cells was significantly higher than that of WT cells. Subsequently, the expression of myogenic differentiation factors at the protein level was further detected, and the results were as Figure 13 shown. At 48 h of differentiation, the expression of myogenic differentiation factors MyoD and MyoG in the ENO1-KO group of cells was significantly higher than that of WT cells, which further demonstrated that ENO1 has the function of regulating the differentiation of myoblasts. By knocking out the ENO1 gene, the differentiation ability of myoblasts can be significantly improved.

[0195] Example 4: ENO1 initiates the reprogramming of cell metabolism as an RNA-binding protein (RBP)

[0196] (1) Alanine transaminase (ALT) plays a key role in amino acid metabolism, catalyzing the conversion of pyruvate and glutamate into alanine and α-ketoglutaric acid (α-KG). α-KG can promote the differentiation of myoblasts. Alanine transaminase (ALT) is mainly divided into ALT1 and ALT2. The expression of the two genes and the changes in enzyme activity were compared between the knockout cells (ENO1-KO) and WT cells. Western Blot was used to detect the expression of ALT1 and ALT2, and a kit (Nanjing Jiancheng, C009-2-1) was used to detect the ALT enzyme activity. The results were as Figure 14 and Figure 15 shown. After knocking out the ENO1 gene, the expression of ALT1 and ALT2 increased significantly, and the ALT enzyme activity increased significantly, indicating that knocking out the ENO1 gene significantly improved the differentiation ability of myoblasts.

[0197] Subsequently, we also explored the roles of ALT1 and ALT2 in the differentiation process of mouse C2C12 cells. The expression of ALT1 and ALT2 was simultaneously interfered with using siRNA (the siRNA transfection method is shown in Example 2). As Figure 16 shown, the expression of ALT1 and ALT2 decreased significantly. The expression of myogenic differentiation factors was detected. As Figure 17 shown, the differentiation of mouse C2C12 cells was blocked, and the expression of myogenic differentiation factors decreased significantly, indicating that ALT1 and ALT2 play important roles in the differentiation process.

[0198] The siRNAs used to interfere with ALT1 and ALT2 are as follows:

[0199] ALT1-siRNA sense strand: 5’-GGCUGAUGAGGUAUACCAATT-3’,

[0200] ALT1-siRNA antisense strand: 5’-UUGGUAUACCUCAUCAGCCTT-3’,

[0201] ALT2-siRNA sense strand: 5’-GGACAUUGUUGUGAAUCCATT-3’

[0202] ALT2-siRNA antisense strand: 5’-UGGAUUCACAACAAUGUCCTT-3’

[0203] Negative control-siRNA sense strand: 5’-UUCUCCGAACGUGUCACGUTT-3’

[0204] Negative control-siRNA antisense strand: 5’-ACGUGACACGUUCGGAGAATT-3’

[0205] (2) To further explore the mechanism of ENO1 regulating the expression changes of ALT1 and ALT2, the RNA-binding protein immunoprecipitation (RIP) kit (product number Bes5101) from Boxin Biology was used to screen the target genes of ENO1 in mouse C2C12 cells. First, after collecting 2×10 7 cells, the cells were lysed thoroughly, and then the DNA was removed. In the experimental group, 3 μg of the primary antibody was added, and in the control group, an equal amount of isotype control antibody was added for immunoprecipitation. Incubate overnight at 4°C. The next day, magnetic beads were added for adsorption. After washing three times, RNA extraction was performed. Finally, the obtained RNA was reverse transcribed (Novizan, product number: R323) to obtain cDNA, and finally qPCR was carried out. As Figure 18 shown, during the proliferation period of WT cells, HDAC2 was significantly enriched in the RIP group. Similarly, during the differentiation period of WT cells, as Figure 19 , HDAC2 was also significantly enriched. As Figure 20 、 21As shown, in the ENO1 knockout group cells, the mRNA and protein levels of HDAC2 were significantly increased. This result indicates that ENO1, as an RNA-binding protein, regulates the expression of HDAC2D. To verify that HDAC2 regulates the expression of ALT1 and ALT2 as a transcription factor, the CUT&Tag Assay Kit (TD904) from Novizan was used for the experiment to verify the interaction between HDAC2 and ALT1, ALT2 in the verification group. First, 80,000 - 100,000 cells were collected and incubated with activated ConABeads. Subsequently, the cell-bead complex was incubated with the HDAC2 antibody at 4°C overnight. The next day, the secondary antibody was incubated with the cell-bead complex at room temperature for 1 h, and the transposon was added and incubated at room temperature for 1 h. Then, the enriched DNA was fragmented, extracted, and purified to obtain DNA fragments. The interaction between HDAC2 and ALT1 and ALT2 was verified by qPCR using the designed primers. Among them, two binding sites were designed near 1 kb downstream of the transcription start site of ALT1, namely 1# and 2#, and four binding sites were designed in the promoter region of ALT2, namely 1#, 2#, 3#, and 4#, as Figure 22 , Figure 23 shown, HDAC2 had a significant interaction with the genomic region of ALT1 during the proliferation period and 48 h of differentiation in WT cells. Similarly, as Figure 24 , Figure 25 shown, HDAC2 had a significant interaction with the promoter region of ALT2 during the proliferation period and 48 h of differentiation in WT cells.

[0206] The primer sequences for verifying the binding of HDAC2 to ALT1 and ALT2 are as follows:

[0207] ALT1-1#F: 5’-GCCCTCGAGTACTATGCGTC-3’

[0208] R: 5’-GAGCTGATAGGTTTACCCGTGGG-3’

[0209] ALT1-2#F: 5’-TGCCCTTGCTCTCCAACCC-3’

[0210] R: 5’-CTGACTGGAAAGTCAGCCGC-3’

[0211] ALT2-1#F: 5’-GGTGTGCGAATACAGGGAGCC-3’

[0212] R: 5’-CGCTCCGGTTAAGGTGAGCCT-3’

[0213] ALT2-2#F: 5’-GAGGGTGGGAATACAGCCCTG-3’

[0214] R: 5’-GCAAGACGTCACGCACAGC-3’

[0215] ALT2-3#F: 5’-CGTTGCATACCCACATTAGGCG-3’

[0216] R: 5’-ATTCCCACCCTCTGGTTACTGG-3’

[0217] ALT2-4#F: 5’-GAGGGTGGGAATACAGCCCT-3’

[0218] R: 5’-GCAAGACGTCACGCACAG-3’.

[0219] (3) To further explore the regulatory effect of HDAC2 on the expression of ALT1 and ALT2, first, prepare two groups of WT cells with the same density (two dishes of cells in each group), denoted as P and D48h. When the cell density of the P group reaches 60%, add the HDAC2 inhibitor (MCE, USA, HY-12163, 1 μM) to one dish, and the other dish of cells is the control group. After adding for 48 h, collect the cells and extract the total protein. When the cell density of the D48h group reaches 90%, add the HDAC2 inhibitor (MCE, HY-12163, 1 μM) to one dish, and the other dish of cells is the control group. After adding for 48 h, collect the cells and extract the total protein. Use Western Blot to detect the protein expression of ALT1 and ALT2 in the two groups of cells. The results are as Figure 26 shown. After inhibiting the expression of HDAC2, the expression of ALT1 and ALT2 decreased significantly. Secondly, the siRNA was also used to interfere with the expression of HDAC2 (see Example 2 for the siRNA transfection method). First, prepare two groups of WT cells with the same density (two dishes of cells in each group), denoted as P and D48h. When the cell density of the P group reaches 60%, transfect siRNA into one dish, and transfect negative control siRNA into the other dish of cells. After transfecting for 48 h, collect the cells and extract the total protein. When the cell density of the D48h group reaches 90%, transfect siRNA into one dish, and transfect negative control siRNA into the other dish of cells. After transfecting for 48 h, collect the cells and extract the total protein. Use Western Blot to detect the protein expression of ALT1 and ALT2 in the two groups of cells. As Figure 27 shown, the expression of HDAC2 decreased significantly. Moreover, the expression of ALT1 and ALT2 also decreased significantly. In summary, this indicates that HDAC2 regulates the expression of ALT1 and ALT2 as a transcription factor.

[0220] The HDAC2 siRNA sequences are as follows:

[0221] HDAC2-siRNA sense strand: 5’-CCAUUCGAGCAUCAGACAATT-3’,

[0222] HDAC2-siRNA antisense strand: 5’-UUGUCUGAUGCUCGAAUGGTT-3’,

[0223] Negative control-siRNA sense strand: 5’-UUCUCCGAACGUGUCACGUTT-3’,

[0224] Negative control-siRNA antisense strand: 5’-ACGUGACACGUUCGGAGAATT-3’.

[0225] The above results indicate that as an RNA-binding protein, ENO1 regulates the expression of HDAC2, and HDAC2, as a transcription factor, regulates the expression of ALT1 and ALT2. Therefore, as an RNA-binding protein (RBP), ENO1 initiates the reprogramming of cell metabolism, thereby realizing the function of regulating the differentiation of myoblasts.

[0226] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. Use of ENO1 protein and / or ENO1 gene in any of the following: A1) Use in regulating myoblast differentiation; A2) Use in regulating myoblast fusion; A3) Use in regulating the number of myotubes of myoblasts; A4) Use in regulating the expression level of HDAC2 protein or in interacting with HDAC2 protein; A5) Use in regulating the expression level of ALT1 and / or ALT2.

2. Use of an ENO1 inhibitor in any of the following: B1) Use in promoting myoblast differentiation; B2) Use in promoting myoblast fusion; B3) Use in increasing the number of myotubes of myoblasts; B4) Use in increasing the expression level of HDAC2 protein; B5) Use in increasing the expression level of ALT1 and / or ALT2; B6) Use in preparing a product for promoting myoblast differentiation and / or fusion; B7) Use in promoting animal muscle development or in preparing a product for promoting animal muscle development.

3. The application according to claim 2, characterized in that The ENO1 inhibitor includes any of the following: C1) A substance that inhibits the replication, transcription, translation, post-transcriptional modification, and / or post-translational modification of the ENO1 gene; C2) A substance that inhibits or reduces the content, activity, and / or function of ENO1 protein.

4. The application according to claim 2 or 3, characterized in that, The ENO1 inhibitor includes a nucleic acid molecule for silencing or knocking out the ENO1 gene.

5. The application according to any one of claims 2-4, characterized in that The ENO1 inhibitor includes any of the following: D1) siRNA: The nucleotide sequence of the sense strand of the siRNA is the 1st - 19th position of SEQ ID NO:1 or SEQ ID NO:1; the nucleotide sequence of the antisense strand of the siRNA is the 1st - 19th position of SEQ ID NO:2 or SEQ ID NO:2; D2) A DNA molecule encoding the siRNA described in D1); D3) An expression cassette, recombinant vector, or recombinant microorganism containing the siRNA described in D1) or the DNA molecule described in D2); D4) shRNA: The target sequence of the shRNA is as shown in SEQ ID NO:3; D5) A DNA molecule encoding the shRNA described in D4); D6) An expression cassette, recombinant vector, or recombinant microorganism containing the shRNA described in D4) or the DNA molecule described in D5); D7) sgRNA1 and sgRNA2: The target sequence of sgRNA1 is as shown in SEQ ID NO:4; the target sequence of sgRNA2 is as shown in SEQ ID NO:5; D8) A DNA molecule 1 encoding sgRNA1 described in D7) and a DNA molecule 2 encoding sgRNA2 described in D7); D9) An expression cassette, recombinant vector, or recombinant microorganism containing sgRNA1 and sgRNA2 described in D7), or containing the DNA molecule 1 and DNA molecule 2 described in D8); D10) A CRISPR / Cas9 system containing sgRNA1 and sgRNA2 described in D7).

6. The application according to claim 5, wherein The recombinant microorganism is a recombinant virus.

7. The ENO1 inhibitor described in claim 5.

8. A method for promoting myoblast differentiation, characterized in that, The method includes reducing the content and / or activity of ENO1 protein in myoblasts.

9. The method according to claim 8, wherein The reduction of the content and / or activity of ENO1 protein in myoblasts is achieved by reducing the expression level of the coding gene of ENO1 protein in myoblasts.

10. A product for promoting animal muscle development or skeletal muscle development, characterized in that, The product contains the ENO1 inhibitor described in any one of claims 2-6.

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