Application of Rspo3 gene in regulation and control of skeletal muscle differentiation and muscle fiber type transformation

Abstract

The invention discloses application of an Rspo3 gene in regulation and control of skeletal muscle differentiation and muscle fiber type transformation. By regulating Rspo3 gene expression, differentiation and development of skeletal muscles can be effectively regulated, conversion of muscle fiber types of the skeletal muscles can also be regulated, a new scheme and direction are provided for improving meat quality, especially pig meat quality, and the Rspo3 gene has very important application value. Meanwhile, a new thought is provided for research on skeletal muscle development, and research and application of skeletal muscle development are promoted.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and particularly relates to the application of the Rspo3 gene in regulating skeletal muscle differentiation and muscle fiber type transformation. Background Art

[0002] In animal husbandry, the development of skeletal muscle is directly related to meat production, which is one of the most important economic traits. Skeletal muscle is a heterogeneous organ composed of muscle fibers, basement membranes, satellite cells, and nerves. Muscle fiber type is a key factor affecting pork quality. When the content of slow oxidative muscle fibers in muscle is high, the lactic acid content in postmortem pork is low, the pH value drops slowly, and the content of flavor substances such as myoglobin and intramuscular fat is high, resulting in good pork quality. Therefore, the exploration of skeletal muscle fiber type transformation plays an important role in improving the commercial performance of animals.

[0003] Skeletal muscle fibers first generate myogenic progenitor cells in the myotome region of the somites in the mesoderm. Myogenic progenitor cells further differentiate into mononuclear myoblasts, and myoblasts undergo a series of proliferation, differentiation, and fusion in vivo, and finally form multinucleated myotubes. The number of myofibrils in the myotubes continuously increases, and the nuclei continuously fuse to form multinucleated myotubes, which finally develop into mature muscle fibers. Mammalian skeletal muscle can be divided into type I, type IIa, type IIx, and type IIb according to four subtypes of myosin heavy chain MYHCⅠ, MYHCⅡa, MYHCⅡx, and MYHCⅡb, and different types of muscle fibers can follow the order of mutual transformation. According to the expression of the myosin heavy chain (MyHC) subtype of muscle fibers, it can be mainly divided into slow oxidative muscle fibers, fast glycolytic muscle fibers, and intermediate muscle fibers. Slow oxidative muscle fibers mainly express MyHC I, fast glycolytic muscle fibers mainly express MyHC IIb, and intermediate muscle fibers express MyHCⅡa and MyHCⅡx. Among them, slow oxidative muscle fibers are beneficial to oxidative metabolism and endurance, while fast glycolytic muscle fibers are mainly beneficial to glycolytic activities and contract faster.

[0004] RNA modification is a post-transcriptional regulation method widely existing in eukaryotes. At present, about 170 kinds of RNA modifications have been identified, which are distributed on messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and some non-coding RNAs, mainly including N7-methylguanine (m 7 G), N6-methyladenosine (m 6 A), and 5-methylcytosine (m 5 C), etc., which constitute an important part of "epitranscriptomics". N6-methyladenosine (m 6A) is the most abundant RNA modification in eukaryotic cells. m 6 A modifies approximately 0.1 - 0.4% of adenosines in eukaryotes. m 6 m6A RNA methylation occurs on the RRm6ACH consensus motif ([G / A / U][G / A]m6AC[U / A / C]). m6A modification also occurs in pre - RNAs (precursor mRNAs) and lncRNAs (long non - coding RNAs). 6 modification.

[0005] R - spondin (Rspo) proteins are a family composed of four secreted glycoproteins (Rsp1 - 4) and are pluripotent signaling ligands. The function of the Rspo family is to enhance the canonical Wnt / β - catenin signaling pathway. Rspo3 (R - spondin) is a secreted glycoprotein belonging to the Rspo subunit family. The Rspo3 gene is conserved in evolution and has one transcript in pigs. It has been found that the expression of the Rspo3 gene increases during embryonic heart development and is involved in the differentiation of cardiomyocytes and the formation of heart structure by activating the Wnt / β - catenin pathway. However, there is no report on the study of the Rspo3 gene on skeletal muscle differentiation and muscle fiber type transformation. Summary of the Invention

[0006] The object of the present invention is to provide an application of the Rspo3 gene in regulating skeletal muscle differentiation and muscle fiber type transformation, so as to provide a new method and application for regulating skeletal muscle differentiation and muscle fiber type transformation.

[0007] According to the first aspect of the present invention, there is provided an application of the Rspo3 gene in regulating skeletal muscle differentiation. Thus, by regulating the expression of the Rspo3 gene, the differentiation and development of skeletal muscle can be effectively regulated, providing a new application idea for further improving meat quality.

[0008] According to the second aspect of the present invention, there is provided an application of the Rspo3 gene in the preparation of a product capable of regulating skeletal muscle differentiation. Thus, by applying a product that can regulate the expression of the Rspo3 gene to the regulation of skeletal muscle differentiation, the differentiation and development of skeletal muscle can be effectively regulated, providing a new application idea for further improving meat quality.

[0009] According to the third aspect of the present invention, there is provided an application of a reagent for promoting or inhibiting the Rspo3 gene in regulating skeletal muscle differentiation. Thus, by promoting or inhibiting the expression of the Rspo3 gene, the differentiation and development of skeletal muscle can be effectively regulated, providing a new application idea for further improving meat quality.

[0010] According to the fourth aspect of the present invention, there is provided an application of a reagent for promoting or inhibiting the Rspo3 gene in the preparation of a product capable of regulating skeletal muscle differentiation. Thus, a reagent that can promote or inhibit the expression of the Rspo3 gene can be made into a product for regulating skeletal muscle differentiation, which can effectively regulate the differentiation and development of skeletal muscle and provide a new application idea for further improving meat quality.

[0011] According to the fifth aspect of the present invention, there is provided a method for regulating skeletal muscle differentiation, which realizes the regulation of skeletal muscle differentiation by regulating the expression of the Rspo3 gene. Thus, through this method, the regulation of skeletal muscle differentiation and development can be efficiently achieved, providing a new application idea for further improving meat quality.

[0012] In some embodiments, the method includes injecting or transfecting an Rspo3 overexpression vector to promote the expression of the Rspo3 gene, thereby promoting the differentiation of skeletal muscle.

[0013] In some embodiments, the method includes promoting the expression of the Rspo3 gene by increasing the m 6 A modification level, thereby promoting the differentiation of skeletal muscle.

[0014] In some embodiments, the method includes injecting or transfecting an Rspo3 inhibitor to inhibit the expression of the Rspo3 gene, thereby inhibiting skeletal muscle differentiation.

[0015] In some embodiments, the method includes inhibiting the expression of the Rspo3 gene by inhibiting the m 6 A modification level, thereby inhibiting skeletal muscle differentiation.

[0016] According to the sixth aspect of the present invention, there is provided an application of the Rspo3 gene in regulating the transformation of skeletal muscle fiber types. Thus, by regulating the expression of the Rspo3 gene, the transformation of skeletal muscle fiber types can be effectively regulated, the fiber types can be improved, and the meat quality, especially the meat quality of pigs, can be further improved, which has very important application value.

[0017] According to the seventh aspect of the present invention, there is provided an application of the Rspo3 gene in the preparation of a product capable of regulating the transformation of skeletal muscle fiber types. Thus, the product capable of regulating the expression of the Rspo3 gene can effectively regulate the transformation of skeletal muscle fiber types, improve the fiber types, and further improve the meat quality, especially the meat quality of pigs, which has very important application value.

[0018] According to the eighth aspect of the present invention, there is provided an application of a reagent for promoting or inhibiting the Rspo3 gene in regulating the transformation of skeletal muscle fiber types. Thus, by promoting or inhibiting the expression of the Rspo3 gene, the transformation of skeletal muscle fiber types can be effectively regulated, the fiber types can be improved, and the meat quality can be further improved, especially the meat quality of pigs, which has very important application value.

[0019] According to the ninth aspect of the present invention, there is provided an application of a reagent for promoting or inhibiting the Rspo3 gene in the preparation of a product capable of regulating the transformation of skeletal muscle fiber types. Thus, by making a reagent that can promote or inhibit the expression of the Rspo3 gene into a product, this product can effectively regulate the transformation of skeletal muscle fiber types, improve the fiber types, and further improve the meat quality, especially the meat quality of pigs, which has very important application value.

[0020] According to the tenth aspect of the present invention, there is provided a method for regulating the transformation of skeletal muscle fiber types, which realizes the regulation of the transformation of skeletal muscle fiber types by regulating the expression of the Rspo3 gene. Thus, through this method, the transformation of skeletal muscle fiber types can be efficiently regulated, the fiber types can be improved, and the meat quality can be further improved, especially the meat quality of pigs, which has very important application value.

[0021] In some embodiments, the method includes injecting or transfecting an Rspo3 overexpression vector to promote the expression of the Rspo3 gene, thereby inhibiting the formation of fast glycolytic muscle fibers, promoting the formation of slow oxidative muscle fibers or promoting the transformation of fast glycolytic muscle fibers into slow oxidative muscle fibers;

[0022] In some embodiments, the method includes promoting the expression of the Rspo3 gene by increasing the m 6 A modification level, thereby inhibiting the formation of fast glycolytic muscle fibers, promoting the formation of slow oxidative muscle fibers or promoting the transformation of fast glycolytic muscle fibers into slow oxidative muscle fibers.

[0023] In some embodiments, the method includes injecting or transfecting an Rspo3 inhibitor to inhibit the expression of the Rspo3 gene, thereby inhibiting the formation of slow oxidative muscle fibers, promoting the formation of fast glycolytic muscle fibers or promoting the transformation of slow oxidative muscle fibers into fast glycolytic muscle fibers.

[0024] In some embodiments, the method includes inhibiting the expression of the Rspo3 gene by inhibiting the m 6 A modification level, thereby inhibiting the formation of slow oxidative muscle fibers, promoting the formation of fast glycolytic muscle fibers or promoting the transformation of slow oxidative muscle fibers into fast glycolytic muscle fibers.

[0025] Advantages of the present invention: By regulating the expression of the Rspo3 gene, the differentiation and development of skeletal muscle can be effectively regulated, and the transformation of skeletal muscle fiber types can also be regulated, providing a new solution and direction for improving meat quality, especially that of pigs, and having very important application value. At the same time, it also provides a new idea for the research on skeletal muscle development, which helps to promote the research and application of skeletal muscle development. Description of the Drawings

[0026] Figure 1 It is the interference efficiency result diagram of three pairs of si-Rspo3 gene interference fragments: among them, ns indicates P>0.05, * indicates P<0.05, ** indicates P<0.01;

[0027] Figure 2 It is the detection result diagram of the influence of interfering with the Rspo3 gene on myogenic differentiation marker genes: among them, si-Rspo3 represents the detection result after transfection with si-Rspo31-1, si-NC represents the detection result of the control group transfected with the nonsense fragment siRNA, ns indicates P>0.05, * indicates P<0.05, ** indicates P<0.01;

[0028] Figure 3 It is the detection result diagram of the influence of overexpressing the Rspo3 gene on myogenic differentiation marker genes: among them, pcDNA3.1-Rspo3 represents the detection result after transfection with the overexpression vector pcDNA3.1-Rspo3, pcDNA3.1 represents the detection result of the control group transfected with the empty vector pcDNA3.1, ns indicates P>0.05, * indicates P<0.05, ** indicates P<0.01;

[0029] Figure 4 It is the immunofluorescence experiment result diagram of interfering with the Rspo3 gene: among them, si-Rspo3 / si-RNA represents the detection result after transfection with si-Rspo31-1, si-NC represents the detection result of the control group transfected with the nonsense fragment siRNA, ns indicates P>0.05, ** indicates P<0.01, and the scale bar is 200μm;

[0030] Figure 5 It is the immunofluorescence experiment result diagram of overexpressing the Rspo3 gene: among them, pcDNA3.1-Rspo3 / overexpress represents the detection result after transfection with the overexpression vector pcDNA3.1-Rspo3, pcDNA3.1 represents the detection result of the control group transfected with the empty vector pcDNA3.1, ns indicates P>0.05, ** indicates P<0.01, and the scale bar is 200μm;

[0031] Figure 6Results of the effect of interfering with the Rspo3 gene on muscle fiber type marker genes: Among them, si-Rspo3 represents the detection result after transfection with si-Rspo31-1, si-NC represents the detection result of the control group transfected with the nonsense fragment siRNA, ns indicates P>0.05, and * indicates P<0.05;

[0032] Figure 7 Results of the effect of overexpressing the Rspo3 gene on muscle fiber marker genes: Among them, pcDNA3.1-Rspo3 represents the detection result after transfection with the overexpression vector pcDNA3.1-Rspo3, pcDNA3.1 represents the detection result of the control group transfected with the empty vector pcDNA3.1, ns indicates P>0.05, * indicates P<0.05, and ** indicates P<0.01;

[0033] Figure 8 Results of the effect of betaine and cyclo(leu)cine on the expression of the Rspo3 gene after treating cells: Among them, Figure 8 A shows the effect of betaine treatment on C2C12 on the expression level of the Rspo3 gene, Figure 8 B shows the effect of cyclo(leu)cine treatment on C2C12 on the expression level of the Rspo3 gene, * indicates P<0.05, and ** indicates P<0.01;

[0034] Figure 9 For m 6 Results of the change in the corresponding amount of genes related to m Figure 9 A shows the effect of betaine treatment on the expression levels of m 6 A-related proteins and enzymes, Figure 9 B shows the effect of cyclo(leu)cine treatment on the expression levels of m 6 A-related proteins and enzymes, ns indicates P>0.05, and * indicates P<0.05;

[0035] Figure 10 Results of HE staining and statistical chart of the gastrocnemius muscle in the thigh injected with lentivirus-packaged sh-NC and lentiviral vector sh-SiRNA of the interfering fragment: Among them, sh-SiRNA represents the detection result after lentivirus packaging with si-Rspo31-1, si-NC represents the detection result of the control group packaged with the nonsense fragment siRNA, and ** indicates P<0.01;

[0036] Figure 11 Results of immunofluorescence staining of the gastrocnemius muscle in the thigh injected with lentivirus-packaged sh-NC and lentiviral vector sh-SiRNA of the interfering fragment. Detailed implementation mode

[0037] The invention will be further described in detail below with reference to the accompanying drawings.

[0038] Through the combined analysis of MeRIP-seq and RNA-seq sequencing data of the soleus muscle (SOL) with a high content of oxidative muscle fibers and the extensor digitorum longus (EDL) with a high content of glycolytic muscle fibers in pigs, it was found that the expression of the Rspo3 gene in SOL was significantly higher than that in EDL. It was inferred that this gene might promote the formation of slow muscle fibers, and further research was carried out on this gene.

[0039] Example 1. Construction of pcDNA3.1-Rspo3 overexpression vector

[0040] 1.1 Rspo3 gene sequence alignment

[0041] The nucleotide sequence of the CDS region of the porcine Rspo3 gene (Gene ID: 100155208; NC_010443.5) and the nucleotide sequence of the CDS region of the murine Rspo3 gene (ID: 72780; NC_000076.7) were downloaded from the NCBI database. Subsequently, the BLAST sequence alignment tool in the NCBI database was used to align the CDS regions of the sequences. The results showed that their homology was basically the same, the deviation was close to 0, and the confidence level was high. Moreover, the Rspo3 gene is a highly conserved gene with only one transcript and remains basically unchanged during the evolutionary process. Therefore, the constructed overexpression vector and interference fragment were used for functional verification and in vivo related verification in mouse myoblast C2C12.

[0042] 1.2 Construction of pcDNA3.1-Rspo3 overexpression vector

[0043] In this experiment, the transcript sequence of the porcine Rspo3 gene was downloaded from the NCBI database, specific primers for the CDS region were designed and synthesized, and restriction enzyme site sequences were added to the primers. The CDS fragment of the Rspo3 gene was amplified, and the amplified CDS fragment of the Rspo3 gene was ligated with the empty vector pcDNA3.1. The ligated product was detected by agarose gel electrophoresis and sequenced. The results were correct, indicating that the pcDNA3.1-Rspo3 overexpression vector was successfully constructed, and its nucleotide sequence is shown as SEQ ID No: 1.

[0044] Example 2. Synthesis and screening of Rspo3 interference fragment

[0045] Interference fragments were designed according to the porcine Rspo3 gene sequence and entrusted to Suzhou GenePharma Co., Ltd. for design and synthesis. The sequences are shown in Table 1:

[0046] Table 1 Interference fragment sequences

[0047]

[0048] The interfering fragments in Table 1, a pair of nonsense fragments si-NC, and the interfering fragment of the positive control GAPDH were transfected into C2C12 cells respectively, and myogenic differentiation was induced. The change in the mRNA expression level of the Rspo3 gene was detected by qPCR. The results were as Figure 1 shown in: The results showed that the expression level of Rspo3 in the treatment groups of the interfering fragments si-Rspo31-1 and si-Rspo31-2 was significantly down-regulated, indicating that these two pairs of interfering fragments could significantly inhibit the expression of the Rspo3 gene, and the inhibitory effect of si-Rspo31-1 was better. Therefore, the interfering fragment si-Rspo31-1 was selected for subsequent experiments.

[0049] Example 3. Effect of the Rspo3 gene on skeletal muscle myogenic differentiation

[0050] 3.1 Effect of interfering with the Rspo3 gene on the mRNA of myogenic differentiation marker genes

[0051] si-Rspo31-1 was transfected into C2C12 cells and induced to differentiate. Cell samples were collected to extract RNA, and the changes in the expression levels of the differentiation marker genes MyoD, MyoG, and MyHC were detected by qPCR experiments. The results were as Figure 2 shown in: The results showed that after interfering with the Rspo3 gene, the mRNA expression level of the MyoD gene decreased significantly (P<0.05), the mRNA expression level of the MyHC gene decreased extremely significantly (P<0.01), while the mRNA expression of MyOG decreased but the difference was not significant (P>0.05), indicating that interfering with the inhibition of Rspo3 gene expression could inhibit the myogenic differentiation of skeletal muscle.

[0052] 3.2 Effect of overexpressing the Rspo3 gene on the mRNA of myogenic differentiation marker genes

[0053] The overexpression vector pcDNA3.1-Rspo3 was transfected into C2C12 cells and induced to differentiate. Cell samples were collected to extract RNA, and the changes in the expression levels of the differentiation marker genes MyoD, MyoG, and MyHC were detected by qPCR experiments. The results were as Figure 3 shown in: The results showed that after overexpressing the Rspo3 gene, the mRNA expression level of the MyHC gene increased significantly (P<0.05), while the mRNA expressions of MyoG and MyoD increased but the differences were not significant (P>0.05), indicating that overexpressing the Rspo3 gene could promote the myogenic differentiation of skeletal muscle.

[0054] 3.3 Immunofluorescence staining to detect the effect of interfering with the Rspo3 gene on myoblast differentiation

[0055] To study the effect of the Rspo3 gene on the differentiation of C2C12 cells, the interference fragment si-Rspo31-1 was transfected into C2C12 cells. After 3 days of cell differentiation, the results of immunofluorescence detection were as Figure 4 shown: The results showed that after inhibiting the expression of the Rspo3 gene, the number of fused myotubes in C2C12 cells decreased extremely significantly (P<0.01), and the differentiated myotubes showed morphological characteristics of being shorter and narrower.

[0056] 3.4 Effect of immunofluorescence detection of overexpressed Rspo3 gene on myoblast differentiation

[0057] To study the effect of the Rspo3 gene on the differentiation of C2C12 cells, the overexpression vector pcDNA3.1-Rspo3 was transfected into C2C12 cells. The results of immunofluorescence detection were as Figure 5 shown: The results showed that compared with the control group, after 3 days of cell differentiation in the cells transfected with the overexpression vector, the number of fused myotubes in C2C12 cells increased extremely significantly (P<0.01), and the differentiated myotubes showed morphological characteristics of being slender.

[0058] The above results indicate that inhibiting the expression of the Rspo3 gene can inhibit myogenic differentiation of skeletal muscle, while promoting the expression of the Rspo3 gene can promote myogenic differentiation of skeletal muscle.

[0059] Example 4. Effect of the Rspo3 gene on muscle fiber type transformation

[0060] 4.1 Effect of interfering with the Rspo3 gene on the mRNA of muscle fiber type marker genes

[0061] To explore the regulatory effect of the Rspo3 gene on the transformation of skeletal muscle fiber types, si-Rspo31-1 was transfected into C2C12 cells and induced to differentiate. After obvious myotubes appeared in the cell differentiation, cell samples were collected respectively to extract total RNA, and the expression of the mRNA of muscle fiber type marker genes was detected by qPCR. The results were as Figure 6 shown: It was shown that after interfering with and inhibiting the expression of the Rspo3 gene, the expression level of the slow muscle marker gene MyHCⅠ decreased significantly (P<0.05), the fast muscle marker gene MyHCⅡb increased significantly (P<0.05), and the intermediate MyHCⅡa and MyHCⅡx had no significant changes (P>0.05).

[0062] It shows that interfering with or inhibiting the expression of the Rspo3 gene can inhibit the formation of slow oxidative muscle fibers, promote the formation of fast glycolytic muscle fibers, or promote the transformation of slow oxidative muscle fibers into fast glycolytic muscle fiber types.

[0063] 4.2 Effect of overexpressing the Rspo3 gene on the mRNA of muscle fiber marker genes

[0064] The overexpression vector pcDNA3.1-Rspo3 was transfected into C2C12 cells and induced to differentiate. When obvious myotubes appeared during cell differentiation, cell samples were collected and RNA was extracted. The expression levels of mRNAs related to marker genes of each muscle fiber type were detected by qPCR. The results were as Figure 7 shown: After overexpressing the Rspo3 gene, the expression level of MyHCⅠ increased significantly (P<0.05), while the expression level of MyHCⅡb decreased significantly (P<0.05), and the expression level of MyHCⅡa decreased extremely significantly (P<0.01). The results indicated that overexpressing the Rspo3 gene could promote the transformation of fast glycolytic muscle fiber type to slow oxidative muscle fiber type.

[0065] Example 5. Regulation of Rspo3 gene expression by m 6 Regulatory effect of A modification on Rspo3 gene expression

[0066] 5.1m 6 Expression regulation of Rspo3 gene by A modification

[0067] Methods for treating cells with betaine (Bet) and cycloleucine:

[0068] (1) Dissolve betaine and cycloleucine powders in sterile water to prepare a drug solution with a concentration of 1 mM, aliquot and store at -20 °C.

[0069] (2) Taking a six-well plate as an example, inoculate C2C12 cells with good growth status into a six-well cell culture plate and culture in a 37 °C cell incubator.

[0070] (3) When the cell density grows to 60%-70%, replace the fresh proliferation medium, add 40 μL of the drug solution (final concentration 20 μM) to the treatment group wells, and add an equal volume of sterile water to the control group wells, and continue to culture in a 37 °C cell incubator.

[0071] (4) When the cells are about to grow confluent, replace with fresh differentiation medium. Add differentiation medium containing 10 μM cycloleucine or 10 μM betaine to the treatment group wells respectively, add 40 μL of the drug solution (final concentration 20 μM) to the treatment group wells, and add an equal volume of sterile water to the control group wells, and continue to culture in a 37 °C cell incubator.

[0072] (5) After inducing differentiation for 2 days, replace with a new differentiation medium with the same drug concentration and continue to culture in a 37 °C cell incubator for 1 day.

[0073] Culture C2C12 cells with medium containing betaine or cycloleucine respectively to increase or decrease the overall m of cells 6After the methylation modification level, the qPCR technology was used to detect the mRNA expression level of the Rspo3 gene in C2C12. The results are as Figure 8 shown: when the overall m 6 A modification level in C2C12 cells increased, the expression level of the Rspo3 gene increased extremely significantly accordingly (P<0.01)( Figure 8 A); when the overall m 6 A modification level in C2C12 cells decreased, the expression level of the Rspo3 gene decreased significantly accordingly (P<0.05)( Figure 8 B). These results indicate that m 6 A modification can promote the expression of the Rspo3 gene and play a positive regulatory role on it.

[0074] Cycloleucine is a competitive inhibitor of methionine adenosyltransferase and can inhibit methylation by reducing the concentration of S-adenosylmethionine (SAM). Betaine is a methyl donor and can increase the m 6 A methylation level. In this invention, C2C12 cells were treated with betaine and cycloleucine respectively to explore the effect on the expression of the Rspo3 gene after increasing or inhibiting the m 6 A level, indicating that m 6 A modification has a promoting effect on the expression of the Rspo3 gene.

[0075] 5.2 Effects of m 6 A modification-related proteins

[0076] The methyltransferase METTL3 is the main catalytic enzyme that catalyzes the m 6 A modification of mRNA. FTO, also known as the demethylase, mainly functions to remove the m 6 A modification. IGF2BP3 is a common recognition protein for m 6 A modification. After treating the cycloleucine and betaine treatment groups and the control group, qPCR was used to verify three common m 6 A modification enzymes and proteins selected to prove that the corresponding changes occurred in the m 6 A modification level in the cells. The results are as Figure 9 shown: in the betaine group, the number of the recognition protein IGF2BP3 increased significantly (P<0.05), and the methyltransferase METTL3 and the demethylase FTO had an upward trend but not significantly (P>0.05)( Figure 9 A); while in the cycloleucine group, the methyltransferase METTL3 and the demethylase FTO decreased significantly (P<0.05), and the recognition protein IGF2BP3 had a downward trend but not significantly (P>0.05)( Figure 9 B). It shows that the expression of m 6 A modification-related factors was affected by the drug, changing the m in the cells.6 The modification level of A.

[0077] m 6 m6A methylation is one of the most common mRNA modifications in eukaryotes, affecting multiple aspects such as RNA stability, translation efficiency, and splicing, and thus regulating gene expression. METTL3 (methyltransferase-like protein 3) is the key catalytic enzyme for m 6 6A (N6-methyladenosine) methylation. As the core component of the m 6 6A methylation complex, it acts together with proteins such as METTL14 and WTAP to be responsible for adding methyl groups to specific adenosine residues on RNA molecules. FTO (Fat mass and obesity-associated protein) is the first enzyme found to have m 6 6A demethylation activity and plays an important role in the dynamic regulation of m 6 6A RNA methylation modification. The IGF2BP family (insulin-like growth factor 2 binding protein family), including three main members IGF2BP1, IGF2BP2, and IGF2BP3, is a group of RNA-binding proteins. They can recognize and bind to specific sequences of mRNA, regulate its stability and translation. The members of the IGF2BP family do not directly recognize m 6 6A modification, but they indirectly participate in the m 6 6A-related regulatory network by binding to m 6 6A-modified RNA or affecting the metabolic process of m 6 6A-modified RNA. IGF2BP3 is overexpressed in various cancers and is closely related to tumor progression and prognosis, promoting tumor growth and metastasis by regulating the mRNA stability and translation of tumor-related genes. Therefore, in order to verify that the intracellular m 6 6A modification level has changed after adding cyclo-leucine and betaine, three enzymes and proteins closely related to m 6 6A, namely METTL3, FTO, and IGF2BP3, were selected for qPCR verification. The results showed that the intracellular m 6 6A modification level has indeed changed.

[0078] Example 6. In vivo verification experiment

[0079] The Rspo3 interfering small RNA (si-Rspo31-1) and the control NC were sent to Suzhou GenePharma Co., Ltd. for lentivirus packaging. The packaged viruses were respectively denoted as sh-SiRNA and sh-NC. Then the packaged viruses were injected into the hind leg muscles of 6-week-old Kunming mice. sh-NC was injected into the left leg and sh-SiRNA was injected into the right leg. The concentration of the packaged virus was 2×10 8TU / mL. The virus was diluted with 1×PBS to a final concentration of 2×10 7 TU / mL. 100 μL was injected into both the left and right legs once a week for 4 consecutive times, after which the mice were sacrificed and the gastrocnemius muscles were collected. Before sacrificing the mice and collecting the gastrocnemius muscles, the left and right legs were weighed respectively, and the results are shown in Table 2 below:

[0080] Table 2 Statistical results of the thigh weights of mice

[0081]

[0082]

[0083] The results in Table 2 show that the thigh weights in the lentivirus - injected sh - SiRNA group were significantly reduced, suggesting that interfering with the expression of the Rspo3 gene can inhibit cell proliferation.

[0084] The collected gastrocnemius muscles of the mice were subjected to HE staining experiments, and the results were as Figure 10 shown: In the lentivirus - injected sh - SiRNA group (i.e., after interfering with the expression of the Rspo3 gene), the number of fused myotubes in the gastrocnemius muscle was significantly lower than that in the control group sh - NC (P < 0.05), and the myofiber area became smaller.

[0085] The collected gastrocnemius muscles of the mice were used to detect the changes in the number of gastrocnemius muscle fibers by immunofluorescence staining technology, and the results were as Figure 11 shown: In the lentivirus - injected sh - SiRNA group (i.e., after interfering with the expression of the Rspo3 gene), the number of fused myotubes in the gastrocnemius muscle was significantly lower than that in the control group sh - NC, and the myofiber area became smaller.

[0086] Through in - vivo experiments, one month after injecting lentivirus sh - SiRNA and the control group sh - NC, the mice were sacrificed and the gastrocnemius muscles were extracted. From the HE staining results, it can be concluded that compared with the control group, the number of myotubes in the gastrocnemius muscle of the interference group was significantly reduced, and the differentiated myotubes showed morphological characteristics of being shorter and narrower. It can be concluded that interfering with the expression of the Rspo3 gene can inhibit the myogenic differentiation of skeletal muscle. By weighing the mice in the interference group and the control group, it can be obtained that the thigh weights of the mice in the interference group were significantly reduced, suggesting that interfering with the expression of the Rspo3 gene can inhibit cell proliferation. It was also found by immunofluorescence that the number of differentiated myotubes in the gastrocnemius muscle of the interference group was reduced, which was basically consistent with the results of the HE - stained sections, further indicating that interfering with the expression of the Rspo3 gene can inhibit the myogenic differentiation of skeletal muscle.

[0087] In summary, skeletal muscle is composed of different types of muscle fibers. Different types of muscle fibers can not only determine the function of skeletal muscle, but also determine the quality of meat products. Therefore, exploring the key genes that regulate skeletal muscle differentiation, development, and muscle fiber type transformation is of great significance for improving meat quality and enhancing the economic benefits of livestock and poultry products. The above research shows that:

[0088] (1) qPCR analysis was performed on the differentially expressed gene Rspo3 screened from fast and slow muscles in the early stage in the myofibers of induced-differentiated porcine skeletal muscle satellite cells. The results showed that the Rspo3 gene regulated the expression of the slow muscle fiber marker gene MYH7 (encoding MYHCI protein) in the induced-differentiated skeletal muscle myotubes (P<0.05), indicating that the Rspo3 gene can promote the formation of slow oxidative muscle fibers.

[0089] (2) Since the encoded proteins of the Rspo3 gene are highly conserved in pigs and mice, in order to further study the effect of the Rspo3 gene on myoblast differentiation, the classical muscle cell model C2C12 cells were used to study the effect of the Rspo3 gene on myoblast proliferation, differentiation, and muscle fiber type transformation. After interfering with the Rspo3 gene in C2C12 cells, it was detected by qPCR experiment that interfering with the Rspo3 gene could inhibit the expression of MYHC at the RNA level (P<0.05), inhibiting the myogenic differentiation of skeletal muscle. In addition, it was detected by immunofluorescence staining technology that the positive cell rate of MYHC in the cells with the Rspo3 gene interfered also decreased significantly, while the results obtained by overexpressing the Rspo3 gene were opposite to those of interference. It was proved that the Rspo3 gene has a promoting effect on the myogenic differentiation of skeletal muscle.

[0090] (3) In order to study the effect of the Rspo3 gene on muscle fiber type transformation, the Rspo3 gene was interfered and overexpressed respectively in the C2C12 cell line. It was found that the Rspo3 gene positively regulated and promoted the expression of the slow oxidative muscle fiber marker gene MYHCⅠ and inhibited the expression of the fast glycolytic muscle fiber marker gene MYHCⅡb at the RNA level (P<0.05), indicating that promoting the expression of the Rspo3 gene can positively regulate and promote the transformation of muscle fiber type into slow oxidative muscle fibers, and vice versa, inhibiting the expression of the Rspo3 gene promotes the transformation of muscle fiber type into fast glycolytic muscle fibers.

[0091] (4) In C2C12 cells, the levels of m 6 A RNA methylation were up-regulated and down-regulated respectively by drug treatment, and it was found that the expression level of the Rspo3 gene increased with the overall increase in the level of m 6 A RNA methylation modification, indicating that m 6ARNA methylation modification can positively regulate and promote the expression of the Rspo3 gene, thereby promoting the transformation of muscle fiber types into slow oxidative muscle fibers; conversely, inhibition of m 6 ARNA methylation modification can inhibit the expression of the Rspo3 gene, thereby promoting the transformation of muscle fiber types into fast glycolytic muscle fibers.

[0092] (6) A mouse lentiviral interference model was constructed. sh-SiRNA and sh-NC packaged in lentivirus were injected into the left and right leg muscles of mice respectively for 4 consecutive weeks. Through HE staining and other techniques, it was detected that the muscle fiber area in the gastrocnemius muscle of mice decreased and the number of muscle fibers was significantly reduced compared with the control group (P<0.05). Further in vivo experiments proved that interfering with the inhibition of Rspo3 gene expression could inhibit the myogenic differentiation of skeletal muscle.

[0093] The above results indicate that regulating the expression of the Rspo3 gene can regulate the differentiation (myogenic differentiation) of skeletal muscle, and regulating the expression of the Rspo3 gene can also regulate the transformation of skeletal muscle fiber types. This has very important application value for regulating the differentiation and development of porcine skeletal muscle, regulating the transformation of skeletal muscle fiber types, and further improving the meat quality of pigs.

[0094]

Claims

1. Use of the Rspo3 gene in regulating skeletal muscle differentiation or in the preparation of a product capable of regulating skeletal muscle differentiation.

2. Use of a reagent that promotes or inhibits the Rspo3 gene in regulating skeletal muscle differentiation or in the preparation of a product capable of regulating skeletal muscle differentiation.

3. A method for regulating skeletal muscle differentiation, wherein, The method realizes the regulation of skeletal muscle differentiation by regulating the expression of the Rspo3 gene.

4. According to the method described in claim 3, wherein, The method includes injecting or transfecting an Rspo3 overexpression vector to promote the expression of the Rspo3 gene, thereby promoting the differentiation of skeletal muscle; or promoting the expression of the Rspo3 gene by increasing the m 6 A modification level, thereby promoting the differentiation of skeletal muscle.

5. The method according to claim 3, wherein The method includes injecting or transfecting an Rspo3 inhibitor to inhibit the expression of the Rspo3 gene, thereby inhibiting skeletal muscle differentiation; or inhibiting the expression of the Rspo3 gene by inhibiting the level of m 6 A modification, thereby inhibiting skeletal muscle differentiation.

6. Use of the Rspo3 gene in regulating the transformation of skeletal muscle fiber types or in the preparation of a product capable of regulating the transformation of skeletal muscle fiber types.

7. Use of a reagent that promotes or inhibits the Rspo3 gene in regulating the transformation of skeletal muscle fiber types or in the preparation of a product capable of regulating the transformation of skeletal muscle fiber types.

8. A method for regulating the transformation of skeletal muscle fiber types, wherein, The method realizes the regulation of the transformation of skeletal muscle fiber types by regulating the expression of the Rspo3 gene.

9. According to the method described in claim 8, wherein The method includes injecting or transfecting an Rspo3 overexpression vector to promote the expression of the Rspo3 gene, thereby inhibiting the formation of fast glycolytic muscle fibers, promoting the formation of slow oxidative muscle fibers, or promoting the transformation of fast glycolytic muscle fibers into slow oxidative muscle fibers; or promoting the expression of the Rspo3 gene by increasing the m 6 A modification level, thereby inhibiting the formation of fast glycolytic muscle fibers, promoting the formation of slow oxidative muscle fibers, or promoting the transformation of fast glycolytic muscle fibers into slow oxidative muscle fibers.

10. According to the method described in claim 8, wherein, The method includes injecting or transfecting an Rspo3 inhibitor to inhibit the expression of the Rspo3 gene, thereby inhibiting the formation of slow oxidative muscle fibers, promoting the formation of fast glycolytic muscle fibers or promoting the transformation of slow oxidative muscle fibers into fast glycolytic muscle fibers; or inhibiting the expression of the Rspo3 gene by inhibiting the level of m 6 A modification, thereby inhibiting the formation of slow oxidative muscle fibers, promoting the formation of fast glycolytic muscle fibers or promoting the transformation of slow oxidative muscle fibers into fast glycolytic muscle fibers.

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