Application of MAGI3 gene in regulation and control of myoblast proliferation and differentiation
By regulating the MAGI3 gene and using plasmids and siRNA, the problem of unknown proliferation and differentiation of satellite cells in mouse C2C12 and sheep skeletal muscle was solved, and significant regulation of cell proliferation and differentiation was achieved, and livestock and poultry production was promoted. Improved meat traits.
Patent Information
- Application Number
- CN202411071417.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-06
- Publication Date
- 2024-12-13
AI Technical Summary
The prior art has not yet clarified the role of the MAGI3 gene in the proliferation and differentiation of mouse C2C12 cells and sheep skeletal muscle satellite cells, which has affected the growth and development of livestock and poultry meat products and meat production.
By overexpressing or inhibiting the MAGI3 gene, the proliferation and differentiation of mouse C2C12 and sheep skeletal muscle satellite cells are regulated by overexpressing or inhibiting the MAGI3 gene, and the proliferation and differentiation of plasmids and sheep skeletal muscle satellite cells are regulated by using plasmids, changing the promoter sequence, and using CRISPR and other technologies.
It significantly promotes or inhibits the proliferation and differentiation of cells, reveals the important regulatory role of MAGI3 in myoblasts, and lays the foundation for improving the meat production performance of livestock and poultry.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biological technology, in particular to the application of MAGI3 gene in regulating the proliferation and differentiation of myoblasts. Background Art
[0002] Skeletal muscle, also known as striated muscle, is composed of thousands of muscle cells with contractile ability, covered and connected by connective tissue. It accounts for about 40% of the body weight of mammals. It is the main metabolic activity site of the body and plays an important role in regulating the body's energy metabolism homeostasis. The formation of skeletal muscle mainly depends on complex and orderly processes such as cell differentiation and fusion. Skeletal muscle satellite cells are located between the sarcolemma and the basement membrane. Under normal circumstances, skeletal muscle satellite cells are in a dormant state. When muscle cells are damaged, stimulated or induced, satellite cells are activated. The mononuclear skeletal muscle satellite cells proliferate and differentiate to form multinuclear myotube cells, and then form new muscle fiber cells.
[0003] Myoblast proliferation and differentiation is one of the key factors affecting the growth, development and meat production of livestock and poultry. Analyzing the proliferation and differentiation of skeletal muscle satellite cells and their regulatory mechanisms is of great significance for revealing the regulatory mechanisms affecting muscle growth and development, improving the meat production performance of livestock and poultry, and genetic improvement.
[0004] MAGI3 (Membrane Associated Guanylate Kinase, WW and PDZ Domain Containing 3) is a protein-encoding gene belonging to the MAGUK family. It acts as a scaffold protein at intercellular junctions, thereby regulating various cellular processes and signal transduction pathways. Studies have shown that in gliomas, the expression level of MAGI3 is associated with tumor suppression. In addition, functional studies of the MAGI3 gene in colorectal cancer revealed that MAGI3 regulates the occurrence and development of colorectal cancer by degrading c-Myc. Previous studies have found that MAGI3 is differentially expressed in sheep skeletal muscle tissue at different developmental stages, and the role of MAGI3 in the proliferation and differentiation of mouse C2C12 cells and sheep skeletal muscle satellite cells is still unclear and needs further study.
[0005] Elucidating the role of MAGI3 in regulating myoblast proliferation and differentiation is of great significance for improving the meat production traits of meat-producing livestock and improving breeds. Therefore, it is necessary to further explore the role of MAGI3 in regulating myoblast proliferation and differentiation. Summary of the invention
[0006] The purpose of the present invention is to provide application of MAGI3 gene in regulating proliferation and differentiation of myoblasts.
[0007] To achieve the purpose of the present invention, in a first aspect, the present invention provides the use of the MAGI3 gene or a biological material containing the gene in regulating the proliferation and differentiation of myoblasts.
[0008] Among them, the Gene ID of the MAGI3 gene from mice in NCBI is 99470, and the Gene ID of the MAGI3 gene from sheep in NCBI is 101118910.
[0009] Furthermore, the regulation is positive regulation.
[0010] Specifically, the MAGI3 gene is overexpressed to promote the proliferation and differentiation of mouse C2C12 or sheep skeletal muscle satellite cells; the MAGI3 gene is inhibited or knocked down to inhibit the proliferation and differentiation of mouse C2C12 and sheep skeletal muscle satellite cells.
[0011] Furthermore, the overexpression method may be selected from the following 1) to 5), or an optional combination: 1) by introducing a plasmid carrying the gene; 2) by increasing the number of copies of the gene on the cell chromosomes; 3) by changing the promoter sequence of the gene on the cell chromosome; 4) by operably linking a strong promoter to the gene; 5) By introducing enhancers.
[0012] The plasmid is a eukaryotic expression vector, preferably pcDNA3.1(+).
[0013] Furthermore, the inhibition or knockdown method can be selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, antibodies, CRISPR, TALEN, and ZFN gene editing technologies.
[0014] For example, the substance for inhibiting the expression of MAGI3 gene is siRNA that interferes with the expression of MAGI3 gene.
[0015] Preferably, for the mouse MAGI3 gene, the nucleotide sequence encoding the siRNA is: siRNA-Mmu-1: 5′-GCAAGTCATCAGAGATAAT-3′ (SEQ ID NO: 1)
[0016] siRNA-Mmu-2: 5′-CCATCAGAATGTGCAGAAT-3′ (SEQ ID NO: 2)
[0017] Preferably, for the sheep MAGI3 gene, the sequence of the siRNA is: siRNA-oar-1: 5′-GGACUACAUCUGUCAGCAA-3′ (SEQ ID NO: 3)
[0018] 5′-UUGCUGACAGAUGUAGUCC-3′ (SEQ ID NO:4)
[0019] siRNA-oar-2: 5′-GCUGAUACUGGGUCUUCAA-3′ (SEQ ID NO: 5)
[0020] 5′-UUGAAGACCCAGUAUCAGC-3′ (SEQ ID NO:6)
[0021] In a second aspect, the present invention provides an agent for inhibiting the proliferation and differentiation of mouse C2C12 cells, wherein the active ingredient is siRNA, and the nucleotide sequence encoding the siRNA is: siRNA-Mmu-1: 5′-GCAAGTCATCAGAGATAAT-3′
[0022] siRNA-Mmu-2: 5′-CCATCAGAATGTGCAGAAT-3′
[0023] In a third aspect, the present invention provides an agent for inhibiting the proliferation and differentiation of sheep skeletal muscle satellite cells, wherein the active ingredient is siRNA, and the sequence of the siRNA is: siRNA-oar-1: 5′-GGACUACAUCUGUCAGCAA-3′
[0024] 5′-UUGCUGACAGAUGUAGUCC-3′
[0025] siRNA-oar-2: 5′-GCUGAUACUGGGUCUUCAA-3′
[0026] 5′-UUGAAGACCCAGUAUCAGC-3′
[0027] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention discovers for the first time that overexpression of MAGI3 can significantly promote the proliferation and differentiation of mouse C2C12 and sheep skeletal muscle satellite cells; knocking down the expression of MAGI3 can inhibit the proliferation and differentiation of mouse C2C12 and sheep skeletal muscle satellite cells. Further, the application of MAGI3 protein or its encoding gene or biological materials containing its encoding gene in regulating the proliferation and differentiation of mouse C2C12 cells and sheep skeletal muscle satellite cells is proposed. The present invention reveals that MAGI3 has an important regulatory effect on the proliferation and differentiation of mouse C2C12 cells and sheep skeletal muscle satellite cells, laying a foundation for further research on the mechanism of action of MAGI3 gene in myoblast proliferation and differentiation and the improvement of livestock meat production traits in the future. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 This is a detection diagram of the relative expression of mRNA of cell proliferation-related genes detected by RT-qPCR in Example 1 of the present invention after overexpression and interference of MAGI3 in C2C12 cells (Figure A shows the effect of overexpression of MAGI3 on the proliferation of C2C12 cells, and Figure B shows the effect of interference of MAGI3 on the proliferation of C2C12 cells); Figure 2 This is a WB detection diagram of protein expression of cell proliferation-related genes after overexpression and interference of MAGI3 in C2C12 cells in Example 1 of the present invention (Figure A shows the effect of overexpression of MAGI3 on the proliferation of C2C12 cells, and Figure B shows the effect of interference of MAGI3 on the proliferation of C2C12 cells); Figure 3 The effects of overexpression and interference of MAGI3 on the proliferation of C2C12 cells detected by CCK-8 in Example 1 of the present invention (Figure A shows the effect of overexpression of MAGI3 on the proliferation rate of C2C12 cells, and Figure B shows the effect of interference of MAGI3 on the proliferation rate of C2C12 cells); Figure 4 This is a detection diagram of the relative mRNA expression of myoblast differentiation-related genes detected by RT-qPCR in Example 2 of the present invention after overexpression and interference of MAGI3 in C2C12 cells (Figure A shows the effect of overexpression of MAGI3 on C2C12 cell differentiation, and Figure B shows the effect of interference of MAGI3 on C2C12 cell differentiation); Figure 5 This is a WB detection diagram of protein expression of myoblast differentiation-related genes after overexpression and interference of MAGI3 in C2C12 cells in Example 2 of the present invention (Figure A shows the effect of overexpression of MAGI3 on C2C12 cell differentiation, and Figure B shows the effect of interference of MAGI3 on C2C12 cell differentiation); Figure 6The immunofluorescence detection of the effects of overexpression and interference of MAGI3 on the differentiation of C2C12 cells in Example 2 of the present invention (Figure A shows the effects of overexpression of MAGI3 on the differentiation of C2C12 cells, and Figure B shows the effects of interference of MAGI3 on the differentiation of C2C12 cells); Figure 7 This is a detection diagram of the relative expression of mRNA of cell proliferation-related genes detected by RT-qPCR in Example 3 of the present invention after overexpression of sheep skeletal muscle satellite cells and interference with MAGI3 (Figure A shows the effect of overexpression of MAGI3 on the proliferation of sheep skeletal muscle satellite cells, and Figure B shows the effect of interference with MAGI3 on the proliferation of sheep skeletal muscle satellite cells); Figure 8 This is a WB detection diagram of protein expression of cell proliferation-related genes after overexpression of sheep skeletal muscle satellite cells and interference with MAGI3 in Example 3 of the present invention (Figure A shows the effect of overexpression of MAGI3 on the proliferation of sheep skeletal muscle satellite cells, and Figure B shows the effect of interference with MAGI3 on the proliferation of sheep skeletal muscle satellite cells); Fig. 9 The effects of overexpression and interference of MAGI3 on the proliferation of sheep skeletal muscle satellite cells detected by CCK-8 in Example 3 of the present invention (Figure A shows the effect of overexpression of MAGI3 on the proliferation rate of sheep skeletal muscle satellite cells, and Figure B shows the effect of interference of MAGI3 on the proliferation rate of sheep skeletal muscle satellite cells); Fig.10 This is a detection diagram of the relative expression of mRNA of genes related to myoblast differentiation after overexpression of sheep skeletal muscle satellite cells and interference with MAGI3 detected by RT-qPCR in Example 4 of the present invention (Figure A shows the effect of overexpression of MAGI3 on the differentiation of sheep skeletal muscle satellite cells, and Figure B shows the effect of interference with MAGI3 on the differentiation of sheep skeletal muscle satellite cells); Fig.11 This is a WB detection of protein expression of myoblast differentiation-related genes after overexpression of sheep skeletal muscle satellite cells and interference with MAGI3 in Example 4 of the present invention (Figure A shows the effect of overexpression of MAGI3 on the differentiation of sheep skeletal muscle satellite cells, and Figure B shows the effect of interference with MAGI3 on the differentiation of sheep skeletal muscle satellite cells); Fig.12 This is the effect of overexpression and interference of MAGI3 on the differentiation of sheep skeletal muscle satellite cells detected by immunofluorescence in Example 4 of the present invention (Figure A shows the effect of overexpression of MAGI3 on the differentiation of sheep skeletal muscle satellite cells, and Figure B shows the effect of interference of MAGI3 on the differentiation of sheep skeletal muscle satellite cells).
[0029] DETAILED DESCRIPTION
[0030] The present invention aims to determine the effect of MAGI3 gene on the proliferation and differentiation of mouse C2C12 cells and sheep skeletal muscle satellite cells. The present invention firstly finds that MAGI3 regulates the proliferation and differentiation of mouse C2C12 cells and sheep skeletal muscle satellite cells.
[0031] The present invention adopts the following technical solution: To reveal the effect of MAGI3 on myoblast proliferation, the regulatory role of MAGI3 in the proliferation of mouse C2C12 cells and sheep skeletal muscle satellite cells was studied by constructing overexpression and interference fragments. It was found that overexpression of MAGI3 can promote the proliferation of mouse C2C12 cells and sheep skeletal muscle satellite cells, and interference with the expression of MAGI3 can inhibit the proliferation of mouse C2C12 cells and sheep skeletal muscle satellite cells.
[0032] Furthermore, the regulatory effect is that MAGI3 promotes the proliferation of mouse C2C12 cells and sheep skeletal muscle satellite cells.
[0033] To reveal the effect of MAGI3 on myoblast differentiation, the regulatory role of MAGI3 in the differentiation of mouse C2C12 cells and sheep skeletal muscle satellite cells was studied by constructing overexpression and interference fragments. It was found that overexpression of MAGI3 can promote the differentiation of mouse C2C12 cells and sheep skeletal muscle satellite cells, and interference with the expression of MAGI3 can inhibit the differentiation of mouse C2C12 cells and sheep skeletal muscle satellite cells.
[0034] Furthermore, the regulatory effect is that MAGI3 promotes the differentiation of mouse C2C12 cells and sheep skeletal muscle satellite cells.
[0035] The substance used to inhibit the expression of MAGI3 gene is siRNA that interferes with the expression of MAGI3 gene.
[0036] Furthermore, the siRNA interfering with the expression of the MAGI3 gene includes two siRNAs, and the nucleotide sequences of the two siRNAs are as follows: For the mouse MAGI3 gene, the nucleotide sequence encoding the siRNA is: siRNA-Mmu-1: 5′-GCAAGTCATCAGAGATAAT-3′ (SEQ ID NO: 1)
[0037] siRNA-Mmu-2: 5′-CCATCAGAATGTGCAGAAT-3′ (SEQ ID NO: 2)
[0038] For the sheep MAGI3 gene, the sequence of the siRNA is: siRNA-oar-1: 5′-GGACUACAUCUGUCAGCAA-3′ (SEQ ID NO: 3)
[0039] 5′-UUGCUGACAGAUGUAGUCC-3′ (SEQ ID NO:4)
[0040] siRNA-oar-2: 5′-GCUGAUACUGGGUCUUCAA-3′ (SEQ ID NO: 5)
[0041] 5′-UUGAAGACCCAGUAUCAGC-3′ (SEQ ID NO:6)
[0042] si-NC control: 5′-UUCUCCGAACGUGUCACGU-3′
[0043] 5′-ACGUGACACGUUCGGAGAA-3′
[0044] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.
[0045] Example 1 Application of MAGI3 gene in C2C12 cell proliferation
[0046] 1. Cultivation and transfection of C2C12 cells
[0047] The MAGI3 RNA interference fragment and the pCDNA3.1 (+) overexpression vector overexpressing MAGI3 were transfected respectively. The specific implementation operations are as follows: (1) Cultivation of C2C12 cells
[0048] 1) Remove the cells from liquid nitrogen and shake them in a 37℃ constant temperature water bath to dissolve; transfer the cell suspension solution to a 15ml centrifuge tube, centrifuge at 1000r for 5min, and discard the supernatant; resuspend the cell pellet and transfer it to a 35mm culture flask, remove bubbles; and culture in a 37℃, 5% CO2 cell incubator.
[0049] 2) When the cells are about 80%-90% confluent, discard the culture medium and add PBS to gently wash 2-3 times.
[0050] 3) After discarding PBS, add trypsin to the cell culture flask and place it in a cell culture incubator at 37°C and 5% CO2 to digest the cells for 2-3 minutes; after observing the cells floating, add growth medium twice the amount of trypsin to terminate digestion; transfer the cell suspension to a centrifuge tube and centrifuge, then discard the supernatant.
[0051] 4) Add growth medium to the centrifuge tube to resuspend the cells, inoculate the cell suspension into a new culture flask or cell plate at a certain ratio, and grow at 37°C and 5% CO2.
[0052] 5) The proliferation medium formula is as follows: 89% DMEM-F12 + 10% FBS + 1% penicillin-streptomycin double antibody
[0053] (2) Cell transfection
[0054] 1) siRNA (si-NC and siRNA-Mmu-1, siRNA-Mmu-2) was synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., and the pcDNA3.1 (+) overexpression vector for overexpressing MAGI3 was synthesized by Qingke Biotechnology.
[0055] 2) C2C12 cells in good growth condition were seeded into six-well plates, and transfection experiments were performed when the cell density was about 40%-50%. The specific steps are as follows: a) Aspirate the culture medium, wash twice with PBS, and add new growth medium without double antibody; b) Preparation of reagents: siRNA mixed solution: Reagent A: 150ul Opti-MEM + 9ul Lipofectamine RNAiMAX Reagent
[0056] Reagent B: 150ul Opti-MEM + 3ul siRNA (10uM)
[0057] When preparing reagents A and B, be gentle and pipette slowly. Add A to B and mix, then let stand at room temperature for 5 minutes. Overexpression vector mixed solution: Reagent A: 125ul Opti-MEM+5ul P3000 Reagent+2.5ug plasmid pcDNA3.1 (+) or pcDNA3.1 (+) plasmid overexpressing MAGI3
[0058] Reagent B: 125ul Opti-MEM + 5ul Lipofectamine 3000 Reagent
[0059] Mix reagents A and B separately and let stand for 5 minutes, then add A to B and let stand at room temperature for 15 minutes; c) Add the mixed solution dropwise into the six-well plate containing culture medium, shake crosswise to mix, and place in a 37°C, 5% CO2 incubator for culture. Collect cell protein and RNA after 48 hours.
[0060] 2. Effect of MAGI3 on mRNA levels of myoblast proliferation marker genes
[0061] Total RNA was isolated from C2C12 cells using Trizol reagent. ® 1.5 μg of total RNA was reverse transcribed into cDNA using the III All-in-oneRT SuperMix Perfect for qPCR Kit. RT-qPCR analysis was performed using the Taq Pro Universal SYBR qPCR Master Mix Fluorescence Quantification Kit. −ΔΔCt The relative expression of the quantitative genes was normalized using ACTIN as the internal reference gene. The primer sequences, reaction system and procedures of the experiment are shown in Tables 1 to 3.
[0062] Table 1 RT-qPCR primer sequences
[0063] Table 2 RT-qPCR reaction system
[0064] Table 3 RT-qPCR reaction procedure
[0065] The results are as follows Figure 1 As shown in the figure, the expression of CDK1 mRNA was significantly upregulated after overexpression of MAGI3, and the expression of CDK1 mRNA was significantly downregulated after interference with MAGI3 expression. This indicates that overexpression of MAGI3 can promote the proliferation of C2C12 cells, and interference with MAGI3 can inhibit the proliferation of C2C12 cells.
[0066] 3. Effect of MAGI3 on the protein levels of myoblast proliferation marker genes
[0067] C2C12 cells overexpressing or interfering with MAGI3 were lysed at low temperature for 20 minutes in RIPA supplemented with PMSF, collected in a 1.5 ml centrifuge tube, and centrifuged at 4°C and 13000g for 5 minutes. 80ul of supernatant was taken, 20ul of protein loading buffer was added, mixed and centrifuged instantly, and the protein sample was denatured at 98°C for 10 minutes and placed on ice. Gel electrophoresis was performed using 10% SDS-PAGE to separate proteins according to molecular weight, and then the separated proteins were transferred to a PVDF membrane. The PVDF membrane with protein blot was blocked with a rapid blocking solution for 40 minutes and incubated with the corresponding primary antibody overnight at 4°C. After washing with 1x TBST containing Tween-20, the PVDF membrane was incubated with the corresponding secondary antibody at room temperature for 40 minutes. The immunoblotting results were detected using an ECL kit, and the grayscale value of the bands was analyzed using ImageJ software. The WB (Western blot) detection results are shown in Figure 2. Figure 2 As shown in the figure, the protein expression levels of myoblast proliferation-related genes PCNA and CDK1 were significantly upregulated after overexpression of MAGI3; the protein expression levels of myoblast proliferation-related genes Pax7 and CDK1 were significantly downregulated after interference with MAGI3 expression. This indicates that overexpression of MAGI3 can promote the proliferation of C2C12 cells, and conversely, interference with MAGI3 inhibits the proliferation of C2C12 cells.
[0068] 4. CCK-8 assay to detect the effect of MAGI3 on C2C12 cell proliferation
[0069] C2C12 cells were seeded in 96-well plates, and MAGI3 was interfered with and overexpressed, respectively. At 12, 24, 36, and 48 hours after transfection, 10ul of CCK-8 reagent and 90ul of complete medium were added to each well, incubated for 2h30min, and the absorbance at 450nm was detected.
[0070] CCK8 test results Figure 3 As shown, compared with the control group, the proliferation ability of C2C12 cells was significantly upregulated after overexpression of MAGI3, while the opposite result occurred after interference with MAGI3.
[0071] Example 2 Application of MAGI3 in C2C12 cell differentiation
[0072] 1. Effect of MAGI3 on mRNA levels of myoblast differentiation marker genes
[0073] Total RNA was isolated from C2C12 cells differentiated for 5 days using Trizol reagent. ®IIIAll-in-one RT SuperMix Perfect for qPCR Kit was used to reverse transcribe 1.5 μg of total RNA into cDNA. RT-qPCR analysis was performed using TaqPro Universal SYBR qPCR Master Mix Fluorescence Quantification Kit. −ΔΔCt The relative expression of the quantified genes was normalized with ACTIN as the internal reference gene. The primer sequences of the experiment are shown in Table 4.
[0074] Table 4 RT-qPCR primer sequences
[0075] The results are as follows Figure 4 As shown in the figure, the expression of MyoG mRNA was significantly upregulated after overexpression of MAGI3, and the expression of MyoG mRNA was significantly downregulated after interference with MAGI3 expression. This indicates that overexpression of MAGI3 can promote the differentiation of C2C12 cells, and interference with MAGI3 can inhibit the differentiation of C2C12 cells.
[0076] 2. Effect of MAGI3 on the protein levels of myoblast differentiation marker genes
[0077] C2C12 cells that were differentiated for 5 days after overexpression or interference with MAGI3 were lysed at low temperature in RIPA supplemented with PMSF for 20 minutes, collected in a 1.5 ml centrifuge tube, and centrifuged at 4°C and 13000g for 5 minutes. 80ul of supernatant was taken, 20ul of protein loading buffer was added, mixed and centrifuged instantly, and the protein sample was denatured at 98°C for 10 minutes and placed on ice. Gel electrophoresis was performed using 10% SDS-PAGE to separate proteins according to molecular weight, and then the separated proteins were transferred to a PVDF membrane. The PVDF membrane with protein blot was blocked with a rapid blocking solution for 40 minutes and incubated with the corresponding primary antibody overnight at 4°C. After washing with 1x TBST containing Tween-20, the PVDF membrane was incubated with the corresponding secondary antibody at room temperature for 40 minutes. The immunoblotting results were detected using an ECL kit, and the grayscale value of the bands was analyzed using ImageJ software. The WB (Western blot) detection results are shown in Figure 2. Figure 5 As shown in the figure, the protein expression levels of myoblast differentiation-related genes MyHC and MyoG were significantly upregulated after overexpression of MAGI3; the protein expression levels of myoblast differentiation-related genes MyHC and MyoG were significantly downregulated after interference with MAGI3 expression. This indicates that overexpression of MAGI3 can promote C2C12 cell differentiation, and conversely, interference with MAGI3 inhibits C2C12 cell differentiation.
[0078] 3. Immunofluorescence detection of the effect of MAGI3 on C2C12 cell differentiation
[0079] C2C12 cells differentiated for 5 days were fixed in 4% paraformaldehyde at 4°C overnight, permeabilized with 0.25% Triton X-100 for 30 minutes, and blocked with 3% goat serum for 1 hour. The cells were then incubated with the corresponding primary antibody at 37°C for 1 hour. After washing with phosphate-buffered saline (PBS), the cells were incubated with fluorescent secondary antibodies at room temperature for 1 hour in the dark. Finally, 4',6'-diamidino-2-phenylindole (DAPI) was used to stain in the dark for 15 minutes, and immunofluorescence staining was observed under a confocal microscope. The immunofluorescence results are shown in Figure 6 As shown, compared with the control group, the number of C2C12 cell myotubes increased and the differentiation ability was significantly improved after overexpression of MAGI3, while the opposite result occurred after interference with MAGI3.
[0080] Example 3 Application of MAGI3 in the proliferation of sheep skeletal muscle satellite cells
[0081] 1. Culture and transfection of sheep skeletal muscle satellite cells
[0082] The MAGI3 RNA interference fragment and the pcDNA3.1 (+) overexpression vector overexpressing MAGI3 were transfected respectively. The specific implementation operations are as follows: (1) Culture of sheep skeletal muscle satellite cells
[0083] 1) Remove the cells from liquid nitrogen and shake them in a 37℃ constant temperature water bath to dissolve; transfer the cell suspension solution to a 15ml centrifuge tube, centrifuge at 1000r for 5min, and discard the supernatant; resuspend the cell pellet and transfer it to a 35mm culture flask, remove bubbles; and culture in a 37℃, 5% CO2 cell incubator.
[0084] 2) When the cells are about 80%-90% confluent, discard the culture medium and add PBS to gently wash 2-3 times.
[0085] 3) After discarding PBS, add trypsin to the cell culture flask and place it in a cell culture incubator at 37°C and 5% CO2 to digest the cells for 2-3 minutes; after observing the cells floating, add growth medium twice the amount of trypsin to terminate digestion; transfer the cell suspension to a centrifuge tube and centrifuge, then discard the supernatant.
[0086] 4) Add growth medium to the centrifuge tube to resuspend the cells, inoculate the cell suspension into a new culture flask or cell plate at a certain ratio, and grow at 37°C and 5% CO2.
[0087] 5) The proliferation medium formula is as follows: 79% DMEM-F12 + 20% FBS + 1% penicillin-streptomycin double antibody
[0088] (2) Cell transfection
[0089] 1) siRNA (si-NC and siRNA-oar-1, siRNA-oar-2) and pCDNA3.1 (+) overexpression vector for overexpressing MAGI3 were synthesized by Qingke Biotechnology.
[0090] 2) Inoculate sheep skeletal muscle satellite cells in good growth condition into a six-well plate, and perform transfection experiments when the cell density is about 40%-50%. The specific steps are as follows: a) Aspirate the culture medium, wash twice with PBS, and add new growth medium without double antibody; b) Preparation of reagents: siRNA mixed solution: Reagent A: 150ul Opti-MEM + 9ul Lipofectamine RNAiMAX Reagent
[0091] Reagent B: 150ul Opti-MEM + 3ul siRNA (10uM)
[0092] When preparing reagents A and B, be gentle and pipette slowly. Add A to B and mix, then let stand at room temperature for 5 minutes.
[0093] Overexpression vector mixed solution: Reagent A. 125ul Opti-MEM + 5ul P3000 Reagent + 2.5ug plasmid pcDNA3.1 (+) or pcDNA3.1 (+) plasmid overexpressing MAGI3
[0094] Reagent B.125ul Opti-MEM+5ul Lipofectamine 3000 Reagent
[0095] Mix reagents A and B separately and let stand for 5 minutes, then add A to B and let stand at room temperature for 15 minutes; c) Add the mixed solution dropwise into the six-well plate containing culture medium, shake crosswise to mix, and place in a 37°C, 5% CO2 incubator for culture. Collect cell protein and RNA after 48 hours.
[0096] 2. Effect of MAGI3 gene on mRNA levels of myoblast proliferation marker genes
[0097] Total RNA was isolated from sheep skeletal muscle satellite cells using Trizol reagent. ®IIIAll-in-one RT SuperMix Perfect for qPCR Kit was used to reverse transcribe 1.5 μg of total RNA into cDNA. RT-qPCR analysis was performed using TaqPro Universal SYBR qPCR Master Mix Fluorescence Quantification Kit. −ΔΔCt The relative expression of the quantified genes was normalized with ACTIN as the internal reference gene. The primer sequences of the experiment are shown in Table 5.
[0098] Table 5 RT-qPCR primer sequences
[0099] The results are as follows Figure 7 As shown in the figure, after overexpression of MAGI3, CDK1 mRNA expression was upregulated; after interference with MAGI3 expression, CDK1 and PCNA mRNA expression was downregulated. This indicates that overexpression of MAGI3 can promote the proliferation of sheep skeletal muscle satellite cells, and interference with MAGI3 can inhibit the proliferation of sheep skeletal muscle satellite cells.
[0100] 3. Effect of MAGI3 on the protein levels of myoblast proliferation marker genes
[0101] Sheep skeletal muscle satellite cells after overexpression or interference of MAGI3 were lysed at low temperature in RIPA supplemented with PMSF for 20 minutes, collected in a 1.5 ml centrifuge tube, and centrifuged at 4°C and 13000g for 5 minutes. 80ul of supernatant was taken, 20ul of protein loading buffer was added, mixed and centrifuged instantly, and the protein sample was denatured at 98°C for 10 minutes and placed on ice. Gel electrophoresis was performed using 10% SDS-PAGE to separate proteins according to molecular weight, and then the separated proteins were transferred to a PVDF membrane. The PVDF membrane with protein blot was blocked with a rapid blocking solution for 40 minutes and incubated with the corresponding primary antibody overnight at 4°C. After washing with 1x TBST containing Tween-20, the PVDF membrane was incubated with the corresponding secondary antibody at room temperature for 40 minutes. The immunoblotting results were detected using an ECL kit, and the grayscale value of the bands was analyzed using ImageJ software. The WB (Western blot) detection results are shown in Figure 2. Figure 8 As shown in the figure, overexpression of MAGI3 upregulated the protein expression levels of myoblast proliferation-related genes PCNA and Pax7; interference with MAGI3 expression downregulated the protein expression level of myoblast proliferation-related gene CDK1. This indicates that overexpression of MAGI3 can promote the proliferation of sheep skeletal muscle satellite cells, while interference with MAGI3 can inhibit the proliferation of sheep skeletal muscle satellite cells.
[0102] 4. CCK-8 assay to detect the effect of MAGI3 on the proliferation of sheep skeletal muscle satellite cells
[0103] Sheep skeletal muscle satellite cells were inoculated in 96-well plates, and MAGI3 was interfered with and overexpressed, respectively. At 12, 24, 36 and 48 hours after transfection, 10ul CCK-8 reagent and 90ul complete culture medium were added to each well, incubated for 4h, and the absorbance at 450nm was detected.
[0104] CCK8 test results Fig. 9 As shown, compared with the control group, the proliferation ability of sheep skeletal muscle satellite cells was significantly increased after overexpression of MAGI3, while the opposite result occurred after interference with MAGI3.
[0105] Example 4 Application of MAGI3 in differentiation of sheep skeletal muscle satellite cells
[0106] 1. Effect of MAGI3 on mRNA levels of myoblast differentiation marker genes
[0107] Total RNA was isolated from sheep skeletal muscle satellite cells using Trizol reagent. ® IIIAll-in-one RT SuperMix Perfect for qPCR Kit was used to reverse transcribe 1.5 μg of total RNA into cDNA. RT-qPCR analysis was performed using TaqPro Universal SYBR qPCR Master Mix Fluorescence Quantification Kit. −ΔΔCt The relative expression of the quantified genes was normalized using ACTIN as the internal reference gene. The primer sequences of the experiment are shown in Table 6.
[0108] Table 6 RT-qPCR primer sequences
[0109] The results are as follows Fig.10 As shown in the figure, after overexpression of MAGI3, the expression of MyHC and MyoG mRNA was significantly upregulated; after interference with MAGI3 expression, the expression of MyHC and MyoG mRNA was significantly downregulated. This indicates that overexpression of MAGI3 can promote the differentiation of sheep skeletal muscle satellite cells, and interference with MAGI3 can inhibit the differentiation of sheep skeletal muscle satellite cells.
[0110] 2. Effect of MAGI3 on the protein levels of myoblast differentiation marker genes
[0111] Sheep skeletal muscle satellite cells that were differentiated for 3 days after overexpression or interference with MAGI3 were lysed at low temperature in RIPA supplemented with PMSF for 20 minutes, collected in a 1.5 ml centrifuge tube, and centrifuged at 4°C and 13000g for 5 minutes. 80ul of supernatant was taken, 20ul of protein loading buffer was added, mixed and centrifuged instantly, and the protein sample was denatured at 98°C for 10 minutes and placed on ice. Gel electrophoresis was performed using 10% SDS-PAGE to separate proteins according to molecular weight, and then the separated proteins were transferred to a PVDF membrane. The PVDF membrane with protein blot was blocked with a fast blocking solution for 40 minutes and incubated with the corresponding primary antibody overnight at 4°C. After washing with 1x TBST containing Tween-20, the PVDF membrane was incubated with the corresponding secondary antibody at room temperature for 40 minutes. The immunoblotting results were detected using an ECL kit, and the grayscale value of the bands was analyzed using ImageJ software. The WB (Western blot) detection results are shown in Figure 2. Fig.11 As shown in the figure, the protein expression levels of MyHC and MyoG genes related to myoblast differentiation were significantly upregulated after overexpression of MAGI3; the protein expression levels of MyHC and MyoG genes related to myoblast differentiation were significantly downregulated after interference with MAGI3 expression. This indicates that overexpression of MAGI3 can promote the differentiation of sheep skeletal muscle satellite cells, and conversely, interference with MAGI3 can inhibit the differentiation of sheep skeletal muscle satellite cells.
[0112] 3. Immunofluorescence detection of the effect of MAGI3 on the differentiation of sheep skeletal muscle satellite cells
[0113] Sheep skeletal muscle satellite cells differentiated for 3 days were fixed in 4% paraformaldehyde at 4°C overnight, permeabilized with 0.25% Triton X-100 for 30 minutes, and blocked with 3% goat serum for 1 hour. The cells were then incubated with the corresponding primary antibody at 37°C for 1 hour. After washing with phosphate-buffered saline (PBS), the cells were incubated with fluorescent secondary antibodies at room temperature for 1 hour in the dark. Finally, 4',6'-diamidino-2-phenylindole (DAPI) was used for staining in the dark for 15 minutes, and immunofluorescence staining was observed under a confocal microscope. The immunofluorescence results are shown in Fig.12 As shown, compared with the control group, the number of sheep skeletal muscle satellite cell myotubes increased and the differentiation ability was significantly improved after overexpression of MAGI3, while the opposite result occurred after interference with MAGI3.
[0114] 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 can be made on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.
Claims
1. Application of MAGI3 gene in regulating myoblast proliferation and differentiation; in, The Gene ID of the mouse MAGI3 gene in NCBI is 99470, and the Gene ID of the sheep MAGI3 gene in NCBI is 101118910.
2. The use according to claim 1, characterized in that: The regulation is positive regulation.
3. The use according to claim 1, characterized in that: Overexpression of the MAGI3 gene promotes the proliferation and differentiation of mouse C2C12 or sheep skeletal muscle satellite cells; inhibition or knockdown of the MAGI3 gene inhibits the proliferation and differentiation of mouse C2C12 and sheep skeletal muscle satellite cells.
4. The use according to claim 3, characterized in that: The overexpression method is selected from the following 1) to 5), or an optional combination: 1) by introducing a plasmid carrying the gene; 2) by increasing the number of copies of the gene on the cell chromosomes; 3) by changing the promoter sequence of the gene on the cell chromosome; 4) by operably linking a strong promoter to the gene; 5) By introducing enhancers.
5. The use according to claim 4, characterized in that: The plasmid is a eukaryotic expression vector, preferably pcDNA3.1(+).
6. The use according to claim 3, characterized in that: The inhibition or knockdown method is selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, antibodies, CRISPR, TALEN, and ZFN gene editing technologies.
7. The use according to claim 6, characterized in that: For the mouse MAGI3 gene, the nucleotide sequence encoding the siRNA is: siRNA-Mmu-1: 5′-GCAAGTCATCAGAGATAAT-3′ siRNA-Mmu-2: 5′-CCATCAGAATGTGCAGAAT-3′ For the sheep MAGI3 gene, the sequence of the siRNA is: siRNA-oar-1: 5′-GGACUACAUCUGUCAGCAA-3′ 5′-UUGCUGACAGAUGUAGUCC-3′ siRNA-oar-2: 5′-GCUGAUACUGGGUCUUCAA-3′ 5′-UUGAAGACCCAGUAUCAGC-3′.
8. An agent for inhibiting the proliferation and differentiation of mouse C2C12 cells, characterized in that: The active ingredient is siRNA, and the nucleotide sequence encoding the siRNA is: siRNA-Mmu-1: 5′-GCAAGTCATCAGAGATAAT-3′ siRNA-Mmu-2: 5′-CCATCAGAATGTGCAGAAT-3′.
9. An agent for inhibiting the proliferation and differentiation of sheep skeletal muscle satellite cells, characterized in that: The active ingredient is siRNA, and the sequence of the siRNA is: siRNA-oar-1: 5′-GGACUACAUCUGUCAGCAA-3′ 5′-UUGCUGACAGAUGUAGUCC-3′ siRNA-oar-2: 5′-GCUGAUACUGGGUCUUCAA-3′ 5′-UUGAAGACCCAGUAUCAGC-3′.