Application of SOX2 protein or its encoding gene in regulating skeletal muscle satellite cell proliferation and / or myogenic differentiation

Abstract

The present invention relates to the field of biotechnology, and specifically discloses the use of SOX2 protein or its encoding gene in regulating the proliferation and / or myogenic differentiation of skeletal muscle satellite cells. The present invention has found that after overexpression of SOX2, the proliferation ability and myogenic differentiation ability of sheep skeletal muscle satellite cells significantly increased; after knocking down SOX2, the proliferation ability and myogenic differentiation ability of sheep skeletal muscle satellite cells significantly decreased, and then proposed the use of SOX2 protein or its encoding gene, or a biomaterial containing its encoding gene in regulating the proliferation and / or myogenic differentiation of skeletal muscle satellite cells. The present invention provides a new way to regulate the proliferation and myogenic differentiation ability of skeletal muscle satellite cells, which can be used to improve the meat production performance of animals.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to the application of SOX2 protein or its encoding gene in regulating the proliferation and / or myogenic differentiation of skeletal muscle satellite cells. Background Art

[0002] Mutton is an extremely important meat product with high protein content and moderate fat content. It is one of the most important meat foods for human health. In animal husbandry, mutton production is one of the important economic traits of livestock and poultry. Increasing mutton production performance and improving mutton quality are one of the main goals in animal breeding. Therefore, analyzing the regulatory mechanism of sheep skeletal muscle development is of great significance for improving meat product yield, quality and biological breeding.

[0003] Skeletal muscle satellite cells are located between the skeletal muscle basement membrane and the plasma membrane. They are a type of mononuclear myoblasts with the potential for proliferation and differentiation. Normally, skeletal muscle satellite cells are in a relatively static state. When muscle tissue is damaged, skeletal muscle satellite cells will be activated and re-enter the cell cycle, thus having the potential to differentiate to replace the injured muscle tissue. In this process, skeletal muscle satellite cells are activated, proliferated, migrated, and differentiated into myoblasts. Myoblasts undergo terminal differentiation, myotube fusion, and myonuclear rearrangement to form multinuclear myotube cells, and finally myotube cells form new skeletal muscle fibers. Therefore, the proliferation and differentiation of skeletal muscle satellite cells determine the number of muscle fibers, which further affects muscle production.

[0004] SOX2 (Sex Determining Region Y-Box Protein 2) is an important transcription factor that can maintain the multidirectional differentiation and self-renewal of various stem cells, and is also important in regulating cell proliferation and differentiation. Studies have reported that SOX2 is a key regulator of nervous system development. In humans, SOX2 mutations can lead to eye development defects, hearing development defects, and central nervous system abnormalities. Currently, SOX2 research mainly focuses on regulating embryonic development and the occurrence and development of diseases, and its role in regulating muscle development and its specific regulatory mechanism are still unclear. Summary of the invention

[0005] One of the purposes of the present invention is to provide a new method for regulating the proliferation and differentiation of skeletal muscle satellite cells.

[0006] The present invention provides the use of SOX2 protein (NCBI accession number is NM_001318074) or its encoding gene, or a biological material containing the encoding gene, in regulating the proliferation and / or myogenic differentiation of skeletal muscle satellite cells.

[0007] In the application of the present invention, when the expression of SOX2 gene is reduced, the proliferation and / or myogenic differentiation of skeletal muscle satellite cells is inhibited; when the expression of SOX2 gene is increased, the proliferation and / or myogenic differentiation of skeletal muscle satellite cells is promoted.

[0008] The present invention found that the proliferation and differentiation of sheep skeletal muscle satellite cells were inhibited after transfecting siRNA interference agent of SOX2 into skeletal muscle satellite cells; after transfecting overexpression plasmid of SOX2, the proliferation and differentiation of sheep skeletal muscle satellite cells were promoted, thus proving that SOX2 can effectively promote the proliferation and differentiation of sheep skeletal muscle satellite cells. The present invention not only provides a new scientific basis and method for regulating the proliferation and differentiation of sheep skeletal muscle satellite cells, but also provides a new target for improving the meat production performance of mutton sheep and genome breeding.

[0009] The present invention provides an application of SOX2 protein or its encoding gene, or a biological material containing the encoding gene thereof, in regulating the expression of one or more of CDK1 (NCBI accession number is NM_001142508), PCNA (NCBI accession number is XM_004014340), MyoG (NCBI accession number is NM_001174109), and MyHC (NCBI accession number is XM_004012706) proteins in skeletal muscle satellite cells.

[0010] In the application of the present invention, when the expression of the SOX2 gene is reduced, the expression of one or more of the CDK1, PCNA, MyoG, and MyHC proteins in skeletal muscle satellite cells is inhibited; when the expression of the SOX2 gene is increased, the expression of one or more of the CDK1, PCNA, MyoG, and MyHC proteins in skeletal muscle satellite cells is promoted.

[0011] The present invention also found that the expression levels of skeletal muscle satellite cell proliferation-related markers CDK1 and PCNA, and skeletal muscle satellite cell myogenic differentiation-related markers MyoG and MyHC will also change with the expression of SOX2 gene, and then proposed a method for changing the expression of the above proteins.

[0012] The present invention provides the use of SOX2 protein or its coding gene, or biological materials containing its coding gene in regulating the meat production performance of animals.

[0013] In the application of the present invention, when the expression of the SOX2 gene is reduced, the meat production performance of the animal decreases; when the expression of the SOX2 gene is increased, the meat production performance of the animal is increased.

[0014] The proliferation and differentiation of skeletal muscle satellite cells determine the number of muscle fibers, which further affects muscle production. Therefore, the meat production (muscle production) performance of animals can be regulated by changing the expression of the SOX2 gene.

[0015] In the application of the present invention, the biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.

[0016] In the application of the present invention, the skeletal muscle satellite cells are derived from sheep.

[0017] The present invention provides a method for promoting the proliferation and differentiation of skeletal muscle satellite cells, and the skeletal muscle satellite cells are made to over-express the SOX2 gene through a transgenic method.

[0018] In the method of the present invention, the transgenic method comprises the step of transfecting skeletal muscle satellite cells with a vector carrying the SOX2 gene.

[0019] The beneficial effects of the present invention are at least:

[0020] The present invention discovered the role of SOX2 in the proliferation and differentiation of skeletal muscle satellite cells, and revealed the function and molecular mechanism of SOX2 in regulating skeletal muscle development, which will help to further understand the molecular mechanism of muscle development, and is expected to accelerate the improvement of meat-producing animals, improve the meat production performance of animals, and provide a new method for the breeding of new varieties of high-quality and high-yield animals and the improvement of meat quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are the results of detecting CCK-8 in sheep skeletal muscle satellite cells after interfering with and overexpressing SOX2, where A is the proliferation of sheep skeletal muscle satellite cells after interfering with SOX2, and B is the proliferation of sheep skeletal muscle satellite cells after overexpressing SOX2.

[0022] Figure 2 The results of CDK1 and PCNA protein expression in sheep skeletal muscle satellite cells after SOX2 interference and SOX2 overexpression, where A is the protein expression of CDK1 and PCNA after SOX2 interference, and B is the protein expression of CDK1 and PCNA after SOX2 overexpression.

[0023] Figure 3 These are the results of MyoG and MyHC protein expression in sheep skeletal muscle satellite cells after SOX2 interference and SOX2 overexpression, where A is the MyoG and MyHC protein expression after SOX2 interference, and B is the MyoG and MyHC protein expression after SOX2 overexpression.

[0024] In each figure (if any), represent P <0.05, represent P <0.01, representP <0.001. DETAILED DESCRIPTION

[0025] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0026] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0027] Example 1 Isolation and culture of skeletal muscle satellite cells

[0028] (1) Wash the sheep longissimus dorsi muscle tissue with PBS containing 2% streptomycin / penicillin and place it in a 10 cm sterile culture dish. Use sterile ophthalmic forceps and ophthalmic scissors to peel off the visible nerves, blood vessels, connective tissues, etc.

[0029] (2) Use sterile ophthalmic scissors to cut the longissimus muscle tissue into 1 mm 3 Small tissue pieces of different sizes were transferred into a sterile 1.5 ml centrifuge tube and minced using sterile ophthalmic scissors.

[0030] (3) Centrifuge the 1.5 ml centrifuge tube at 1000 g for 5 minutes, discard the supernatant, add 0.1% type I collagenase to resuspend the precipitate, and place in a 37°C water bath to digest the tissue for 1 hour, inverting it every 15 minutes, centrifuge at 1000 g for 5 minutes, discard the supernatant; then add 0.25% trypsin to digest for 30 minutes, inverting it every 10 minutes.

[0031] (4) Add an equal volume of complete medium (DMEM medium containing 20% ​​FBS and 1% streptomycin / penicillin) to terminate digestion, then pass the digestion solution through 100-mesh and 200-mesh cell sieves in turn, centrifuge the filtrate at 1000g for 10 min, discard the supernatant, resuspend the cells in complete medium, and inoculate them in a 35 cm culture flask at 37°C and 5% CO. 2 Sheep skeletal muscle satellite cells were successfully isolated by culturing in an incubator.

[0032] Example 2 Method for knocking down the SOX2 gene in sheep skeletal muscle satellite cells

[0033] Well-growing sheep skeletal muscle satellite cells were inoculated into six-well cell culture plates, and cells were transfected after the cell density reached 70%-80%. The transfection system was: 150μl opti-MEM diluted 4μl LipofectamineRNAiMAX as solution A, 150μl opti-MEM diluted 2μl 20μM SOX2 interference fragment as solution B; solution B was mixed with solution A and allowed to stand for 5 minutes before slowly adding to sheep skeletal muscle satellite cells for transfection for 48 hours. The SOX2 interference sequence was provided by Ruibo Bio. The SOX2 interference target sequence was: CTTCACATGTCCCAGCACT (SEQ ID No.1).

[0034] Example 3 Method for overexpressing SOX2 gene in sheep skeletal muscle satellite cells

[0035] Well-growing sheep skeletal muscle satellite cells were digested with 0.25% trypsin, then inoculated into a six-well cell culture plate and cultured with DMEM medium containing 20% ​​FBS. Cell transfection was performed when the cell density reached 70%-80%. The transfection system was: 125μl opti-MEM diluted 7.5μl Lipofectamine 3000 as solution A, 125μl opti-MEM diluted 5μl P3000 and 5μg SOX2 overexpression plasmid as solution B; after the A solution and B solution were allowed to stand for 5 minutes, the A solution was added to the B solution and then allowed to stand for 20 minutes. Finally, the mixed solution was added to the six-well cell culture plate containing DMEM medium containing 20% ​​FBS and transfected for 48 hours. The SOX2 overexpression plasmid was provided by Qingke Bio.

[0036] Example 4 SOX2 Regulatory effect on proliferation of sheep skeletal muscle satellite cells

[0037] (1) Well-growing sheep skeletal muscle satellite cells were digested with 0.25% trypsin, then inoculated into a 96-well cell culture plate and cultured with DMEM medium containing 20% ​​FBS. When the cell density reached 70%-80%, cell transfection was performed. 10 μl of the SOX2 interference fragment transfection system prepared in Example 2 was added to each well of the 96-well cell culture plate; 10 μl of the SOX2 overexpression transfection system prepared in Example 3 was added to each well of the 96-well cell culture plate, and transfected for 48 hours respectively. 10 μl of CCK-8 solution was diluted with 90 μl of DMEM medium containing 20% ​​FBS and then added to the 96-well cell culture plate, and placed at 37 °C and 5% CO 2Incubate in an incubator for 4 hours, and then measure the OD value at 450nm with a microplate reader. SiNC was used as the control group for the interference experiment, and pcDNA3.1 was used as the control group for the overexpression experiment. Three biological replicates were set for each group of experiments. The results showed that interference with SOX2 inhibited the proliferation of sheep skeletal muscle satellite cells ( Figure 1 Overexpression of SOX2 promoted the proliferation of sheep skeletal muscle satellite cells ( Figure 1 B in the figure).

[0038] (2) 300 μl of the SOX2 interference fragment transfection system prepared in Example 2 was added to each well of a six-well cell culture plate, and 250 μl of the SOX2 overexpression plasmid transfection system prepared in Example 3 was added to each well of a six-well cell culture plate; 48 hours after transfection, the cells were washed twice with pre-cooled PBS and the cell proteins were collected.

[0039] (3) Detection of expression levels of proteins related to proliferation of sheep skeletal muscle satellite cells:

[0040] The cell proteins collected in the above step (2) were used to detect the proteins CDK1 and PCNA related to the proliferation of sheep skeletal muscle satellite cells by western blotting, and grayscale quantitative analysis was performed.

[0041] The specific operations are as follows:

[0042] 1) Taking a six-well cell culture plate as an example, aspirate the culture medium in the wells and wash twice with pre-cooled PBS.

[0043] 2) Add 300 μl of cell lysis buffer containing 1% protease inhibitor to each well, scrape the transfected sheep skeletal muscle satellite cells with a cell scraper, transfer them to a 1.5 ml centrifuge tube with a pipette, and incubate on ice for 20 minutes, pipetting several times during the process.

[0044] 3) After incubation, centrifuge at 4°C and 13,000 rpm for 10 min.

[0045] 4) Pipette the protein supernatant and add protein loading buffer at a ratio of 5:1 and place in a 100°C water bath for denaturation for 10 minutes.

[0046] 5) Use precast gel to add 10 μl of denatured protein solution to each well, add 5 μl of protein maker to the outermost wells on both sides, place it at a constant voltage of 150 V, and perform electrophoresis for 55 minutes.

[0047] 6) Before transfer, activate the PVDF membrane with an activation reagent, gently close the transfer clip in the order of the black side of the transfer clip, sponge, filter paper, precast gel, PVDF membrane, filter paper, sponge, and white side of the transfer clip, transfer the transfer clip to the transfer tank, and transfer for 45 minutes at a constant current of 500mA under ice bath conditions.

[0048] 7) After transfer, wash with 1× TBST for 5 minutes, add 10 ml of rapid blocking solution, and block at room temperature for 1 hour.

[0049] 8) Incubate with primary antibody at 4°C overnight.

[0050] 9) After incubation, recover the antibody and wash with 1× TBST for 10 min. Repeat this step 3 times.

[0051] 10) Incubate the secondary antibody for 40 min at room temperature.

[0052] 11) After incubation, recover the antibody and wash with 1× TBST for 10 min. Repeat this step 3 times.

[0053] 12) Prepare ECL chemiluminescent solution in a 1:1 ratio according to the instructions and keep it away from light. Finally, place it in a chemiluminescent imager for imaging.

[0054] 13) Use ImageJ software to quantify protein bands and calculate statistics.

[0055] The results showed that interference with SOX2 inhibited the expression of CDK1 and PCNA proteins in sheep skeletal muscle satellite cells ( Figure 2 Overexpression of SOX2 promoted the expression of CDK1 and PCNA proteins in sheep skeletal muscle satellite cells ( Figure 2 B in the figure).

[0056] Example 5 SOX2 Regulation of sheep skeletal muscle satellite cell differentiation

[0057] The experimental operation of the protein immunoblotting method in this example is the same as that in Example 4.

[0058] Well-growing sheep skeletal muscle satellite cells were digested with 0.25% trypsin, then inoculated into a 96-well cell culture plate and cultured in DMEM medium containing 20% ​​FBS. When the cell density reached 70%-80%, the following operations were performed:

[0059] 300 μl of the SOX2 interference fragment transfection system configured in Example 2 was added to each well of a six-well cell culture plate, and after 8 hours of transfection, the culture medium was replaced with a differentiation medium containing 2% horse serum DMEM culture medium for myogenic induction differentiation for 3 days; 250 μl of the SOX2 overexpression transfection system configured in Example 3 was added to each well of a six-well cell culture plate, and after 8 hours of transfection, the culture medium was replaced with a differentiation medium containing 2% horse serum DMEM culture medium for myogenic induction differentiation for 3 days. The above two groups of cells were collected to extract protein samples, and the sheep skeletal muscle satellite cell differentiation-related proteins MyoG and MyHC were detected by protein immunoblotting. The results showed that interference with SOX2 inhibited the expression of MyoG and MyHC proteins in sheep skeletal muscle satellite cells ( Figure 3 Overexpression of SOX2 promoted the expression of MyoG and MyHC proteins in sheep skeletal muscle satellite cells ( Figure 3 B in the figure).

[0060] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Use of SOX2 protein or its encoding gene, or biological materials containing its encoding gene, in regulating skeletal muscle satellite cell proliferation and / or myogenic differentiation for purposes other than disease diagnosis or treatment.

2. The use according to claim 1, characterized in that: When the expression of SOX2 gene is reduced, the proliferation and / or myogenic differentiation of skeletal muscle satellite cells is inhibited; when the expression of SOX2 gene is increased, the proliferation and / or myogenic differentiation of skeletal muscle satellite cells is promoted.

3. Use of SOX2 protein or its encoding gene, or biological materials containing its encoding gene, in regulating the expression of one or more of CDK1, PCNA, MyoG, and MyHC proteins in skeletal muscle satellite cells for purposes other than disease diagnosis or treatment.

4. The use according to claim 3, characterized in that: When the expression of SOX2 gene is reduced, the expression of one or more of CDK1, PCNA, MyoG, and MyHC proteins in skeletal muscle satellite cells is inhibited; when the expression of SOX2 gene is increased, the expression of one or more of CDK1, PCNA, MyoG, and MyHC proteins in skeletal muscle satellite cells is promoted.

5. The use according to any one of claims 1 to 4, characterized in that: The biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.

6. The use according to any one of claims 1 to 4, characterized in that: The skeletal muscle satellite cells are derived from sheep.

7. A method for promoting the proliferation and differentiation of skeletal muscle satellite cells for non-disease diagnosis or treatment purposes, characterized in that: Through transgenic methods, skeletal muscle satellite cells were made to overexpress the SOX2 gene.

8. The method according to claim 7, characterized in that The transgenic method comprises the step of transfecting skeletal muscle satellite cells with a vector carrying the SOX2 gene.

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