Function and application of kif5b gene in regulating physiological activity of myoblasts

Abstract

The application discloses functions and application of KIF5B gene in regulating physiological activity of myoblasts, and belongs to the technical field of animal cell culture. By investigating influences of KIF5B knockdown and overexpression on proliferation of myoblasts, it is found that KIF5B knockdown can significantly inhibit DNA synthesis speed of myoblasts, thereby inhibiting proliferation. In addition, overexpression of KIF5B can significantly inhibit expression of MYOD and MYOG, improve DNA synthesis speed of myoblasts, thereby significantly improving proliferation rate of myoblasts. The application provides an important key molecular target and technical support for efficient and rapid culture of myogenic cells.

Description

Technical Field

[0001] This invention relates to the function and application of the KIF5B gene in regulating the physiological activity of myoblasts, and belongs to the field of biotechnology. Background Technology

[0002] Myoblasts, also known as myosatellite cells, are precursor cells found in adult skeletal muscle tissue and play a crucial role in muscle tissue reconstruction after injury. These cells possess the ability to self-renew, proliferate, differentiate, fuse, and form myofibrils to maintain normal skeletal muscle function. In the medical field, myoblasts have broad application prospects. They are not only used as in vitro models in muscle biology research to study muscle development, metabolism, and differentiation, but also show great potential in gene therapy, neurological diseases, cardiovascular diseases, and orthopedics. In the food industry, myoblasts can be used to study the relationship between animal muscle development and meat quality, thereby improving the taste and nutritional value of meat products. Furthermore, cell-cultured meat products can be prepared by culturing and regulating the proliferation and differentiation of myoblasts in vitro. However, there are some challenges in the in vitro proliferation of myoblasts. Their in vitro proliferation activity is weak, their growth rate is relatively slow, and their growth cycle is long, which greatly limits related research and applications. Current technical strategies for addressing low in vitro proliferation activity mainly involve optimizing culture protocols, such as screening suitable serum and culture medium types, adding growth factors and proliferation-promoting compounds, and increasing serum concentration. However, these methods are costly, and different cells require different conditions, requiring repeated optimization of culture protocols, which is time-consuming, labor-intensive, and inefficient. Therefore, it is particularly important to fundamentally explore the key targets that promote cell proliferation, such as the stable upregulation of genes that promote proliferation and the downregulation of genes that inhibit proliferation.

[0003] The KIF5B (Kinesin Family Member 5B) gene encodes a kinesin, a member of the kinesin family. KIF5B's main functions within the cell include: microtubule-dependent motility: KIF5B is a microtubule-dependent motor protein responsible for transporting various intracellular cargoes (such as mitochondria and lysosomes) along microtubules. Simultaneously, KIF5B plays a crucial role in cell division, particularly during mitosis, helping to locate the centrosome and nucleus. In neurons, KIF5B has been reported to be responsible for forward transport within axons, ensuring normal neuronal function and signal transmission. KIF5B also plays a key role in the normal distribution and function of mitochondria. Mitochondria are the cell's energy factories, and their transport and localization are essential for energy metabolism in muscle cells. Mutations or abnormal expression of the KIF5B gene are associated with various diseases, such as Kearns-Sayre syndrome. Furthermore, variations in KIF5B may lead to skeletal diseases such as osteoogenesis imperfecta by affecting the mTOR signaling pathway. However, whether the KIF5B gene affects the proliferative activity of myoblasts is unknown, and its mechanism of action in myoblasts is also unclear. Therefore, the aim is to develop an effective strategy to enhance the survival and proliferative activity of myoblasts in vitro through precise regulation of the KIF5B gene. This is expected to provide new insights for research in muscle cell biology and open up new possibilities for the treatment of muscle-related diseases and regenerative medicine of muscle tissue. Summary of the Invention

[0004] To address the shortcomings of the existing technologies, this invention provides the function and application of the KIF5B gene in regulating the physiological activity of myoblasts, with the aim of effectively regulating the physiological activity of myoblasts by promoting or inhibiting the expression of the KIF5B gene.

[0005] The first technical solution provided by this invention is the application of the KIF5B gene in regulating myoblast proliferation.

[0006] In some embodiments, the nucleotide sequence of the KIF5B gene is shown in SEQ ID NO.1.

[0007] In some embodiments, the regulation is to inhibit myoblast proliferation by downregulating the expression of the KIF5B gene, or to promote myoblast proliferation by upregulating the expression of the KIF5B gene.

[0008] In some implementations, downregulating the expression of the KIF5B gene inhibits DNA synthesis in myoblasts, thereby suppressing myoblast proliferation.

[0009] In some implementations, upregulating the expression of the KIF5B gene inhibits the expression of MYOD in myoblasts and / or promotes DNA synthesis in myoblasts, thereby promoting myoblast proliferation.

[0010] The second technical solution provided by this invention is the application of a KIF5B gene regulator in the preparation of products for regulating myoblast proliferation.

[0011] In some embodiments, the regulator of the KIF5B gene includes a KIF5B gene enhancer or a KIF5B gene inhibitor, wherein the KIF5B gene enhancer enhances the activity and / or expression of the KIF5B gene, and the KIF5B gene inhibitor inhibits the activity and / or expression of the KIF5B gene.

[0012] In some embodiments, the inhibitor of the KIF5B gene suppresses the proliferation of myoblasts, while the enhancer of the KIF5B gene promotes the proliferation of myoblasts.

[0013] In some embodiments, the inhibitor of the KIF5B gene is a lentiviral expression vector containing shRNA that targets and knocks down the expression of the KIF5B gene, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0014] In some embodiments, the KIF5B gene enhancer is a lentiviral expression vector containing a CDS sequence that targets the overexpression of the KIF5B gene, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0015] In some embodiments, the myoblasts include, but are not limited to, human, porcine, bovine, or mouse-derived myosatellite cells, myoblasts, C2C12 cells, etc.

[0016] The third technical solution provided by the present invention is a myoblast proliferation promoter, wherein the promoter is a lentiviral expression vector containing a CDS sequence that targets the overexpression of the KIF5B gene, and the nucleotide sequence of the CDS sequence is shown in SEQ ID NO.3.

[0017] The fourth technical solution provided by the present invention is an inhibitor of myoblast proliferation, wherein the inhibitor is a lentiviral expression vector containing shRNA that targets and knocks down the expression of the KIF5B gene, and the nucleotide sequence of the shRNA is shown in SEQ ID NO.2.

[0018] The fifth technical solution provided by the present invention is a method for inhibiting the proliferation of myoblasts in vitro, wherein the method involves knocking out or inhibiting the expression of the KIF5B gene in myoblasts.

[0019] In some embodiments, the expression of the KIF5B gene in myoblasts is knocked down by a lentiviral expression vector containing shRNA that targets and knocks down the expression of the KIF5B gene, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0020] The sixth technical solution provided by the present invention is a method for promoting the proliferation of myoblasts in vitro, wherein the method involves upregulating the expression of the KIF5B gene in myoblasts.

[0021] In some embodiments, the expression of the KIF5B gene in myocytes is upregulated by a lentiviral expression vector containing a CDS sequence that targets the overexpression of the KIF5B gene, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0022] The technical effects of this invention are as follows:

[0023] 1. This invention focuses on the role of the KIF5B gene in myoblast proliferation, which has a crucial impact on understanding myoblast activity and growth metabolism.

[0024] 2. This invention discovers that knocking down the KIF5B gene inhibits DNA synthesis in myoblasts and significantly suppresses their in vitro proliferation.

[0025] 3. This invention found that overexpression of the KIF5B gene inhibits the expression of MYOD in myoblasts, promotes DNA synthesis in myoblasts, and significantly enhances the in vitro proliferation activity of myoblasts. Attached Figure Description

[0026] Figure 1 This figure shows the validation effect of KIF5B gene knockdown in C2C12 cells transfected with the lentiviral expression vector pLKO.1-U6-KIF5B-shRNA-CMV-EGFP-hPGK-Puro.

[0027] Figure 2 The cell counts of control cells and KIF5B-KD cells were measured two days after proliferation.

[0028] Figure 3 The proliferation activity of control cells and KIF5B-KD was detected in the EdU assay.

[0029] Figure 4 The image shows the effect of KIF5B gene overexpression in C2C12 cells transfected with the lentiviral expression vector PHBLV-CMVd1-KIF5B-EF-1α-Zsgreen1-T2A-Puro.

[0030] Figure 5The values ​​represent the expression levels of MYOD and MYOG mRNA in control cells and KIF5B-OE cells.

[0031] Figure 6 The EdU assay was used to detect the proliferation activity of control cells and KIF5B-OE cells.

[0032] Figure 7 The growth rates of control cells and KIF5B-OE cells are shown. Detailed Implementation

[0033] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0034] The detection methods involved in the embodiments are as follows:

[0035] I. Western blot method:

[0036] 1. Extraction and concentration determination of cellular proteins

[0037] Remove the culture medium and wash the cells 1-2 times with warm PBS. Add 100-150 μL of lysis buffer to each well and lyse on ice for 20-30 min. After lysis, collect the cells and centrifuge (14000 rcf, 6 min) to obtain the supernatant.

[0038] Lysis buffer: Lysis buffer + mixed protease inhibitor and phosphatase inhibitor (1x) and EDTA (1x)

[0039] 2. Protein quantification: Total protein was quantified using the Beyotime BCA protein concentration quantification kit.

[0040] 1) Take an appropriate amount of 25 mg / mL protein standard and dilute it with PBS to a final concentration of 0.5 mg / mL.

[0041] 2) Based on the sample quantity, prepare an appropriate mixture of BCA reagent A and BCA reagent B in a 50:1 ratio.

[0042] Take a volume of BCA working solution, mix thoroughly, and place on ice for later use.

[0043] 3) Add the standard to the 96-well plate at concentrations of 0, 1, 2, 4, 8, 12, 16, and 20 μL, then add PBS.

[0044] Bring the volume to 20 μL.

[0045] 4) Add 2 μL of the sample to be tested to the 96-well plate, and add 18 μL of PBS to make up to 20 μL.

[0046] 5) Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 min.

[0047] 6) Measure the absorbance of each well at a wavelength of 562 nm using an ELISA reader.

[0048] 7) Calculate the protein concentration of the test sample based on the standard curve and the volume of the test sample.

[0049] 3. Electrophoresis

[0050] 1) Add the quantified protein sample to 4x Loading buffer to a final concentration of 1x, and boil for 5-10 minutes.

[0051] Then let it cool to room temperature.

[0052] 2) Remove the electrophoresis gel, place it in the electrophoresis cartridge, and clamp it. Pour in the pre-prepared electrophoresis buffer.

[0053] 3) Shake the sample solution containing loading buffer, centrifuge briefly, load the sample, and turn on the power for electrophoresis.

[0054] 4) Electrophoresis conditions: constant voltage 100V, 120min, room temperature. If the bromophenol blue does not reach the end of the gel, the time can be increased appropriately.

[0055] 4. Transfer membrane

[0056] 1) Preparation of transfer buffer: Glycine (MW75.07): 15.11g; Tris (MW121.14): 3.03g, distilled water to 850mL, add 150mL methanol, dissolve completely, and pre-cool for 1h.

[0057] 2) Pry open the gel plate, cut out the corresponding gel containing the target protein band according to the protein marker, and cut the PVDF membrane according to its size. Activate the membrane with methanol for 15-30 seconds. Wash with water for 2 minutes, and then soak it together with the gel in transfer buffer for 10 minutes.

[0058] 3) Making the "sandwich" interlayer: black perforated plate - black sponge - white filter paper - gel - PVDF membrane - white filter paper

[0059] - Black sponge - Transparent perforated plate. Ensure the order is correct, remove air bubbles, and avoid touching the PVDF film with your hands.

[0060] 4) Transfer conditions: constant current 300mA, transfer at 4℃ for 180min.

[0061] 5. Antibody incubation

[0062] 1) Clean the PVDF membrane with TBST, add blocking solution, and seal on a shaker at room temperature for 0.5h-1h.

[0063] 2) Prepare the primary antibody. Antibody: Primary antibody dilution buffer = 1:1000 (dilution according to your optimized ratio). Mix the primary antibody,

[0064] The PVDF membrane was placed in an antibody incubation box and incubated overnight at 4°C.

[0065] 3) Prepare the secondary antibody: antibody: secondary antibody dilution solution = 1:2000.

[0066] 4) Recover the primary antibody, wash the PVDF membrane with TBST, gently shake on a shaker, wash 3 times, 5-10 minutes each time.

[0067] 5) Place the secondary antibody and PVDF membrane in an antibody incubation box and incubate on a shaker at room temperature for 2 hours.

[0068] 6) Recover the secondary antibody, wash the PVDF membrane with TBST, gently shake on a shaker, wash 3 times, 5 minutes each time.

[0069] 6. ECL development

[0070] 1) Add ECL working solution according to the kit, and use a gel imaging system to perform imaging. The gray values ​​of the bands can be analyzed on ImageJ software for semi-quantitative analysis.

[0071] II. Assessment of Cell Proliferation Capacity

[0072] Cell proliferation was assessed using the EdU Imaging Kit (Cy5) (APExBIO Technology, USA). The procedure was briefly as follows: Cells were seeded in confocal culture dishes and allowed to adhere for 6 hours, then incubated with 10 μM EdU at 37°C for 4 hours to label newly synthesized DNA. Cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized with 0.5% Triton X-100 for 15 minutes. Click reaction solution was prepared according to instructions and added to the cells in the dark for 30 minutes. Finally, cells were counterstained with DAPI. Fluorescence images were captured using a confocal laser scanning microscope (N-SIM S, NIKON). Pearson correlation coefficients were calculated using NIS-Elements ER software to quantify the colocalization of EdU (CY5) and DAPI signals.

[0073] Raw materials used in the examples:

[0074] The in vitro expansion method for myogenic cells used below is consistent with the conventional in vitro culture method for myogenic cells.

[0075] The following examples involve culture media:

[0076] The myogenic growth medium (GM) consisted of 10 vol% fetal bovine serum, 79 vol% DMEM medium, and 1 vol% penicillin-streptomycin antibiotic solution, wherein the penicillin content was 10000 U / mL and the streptomycin content was 10 mg / mL.

[0077] The carriers involved in the following embodiments:

[0078] 1. The pLKO.1-U6-shRNA-CMV-EGFP-hPGK-Puro vector, i.e. the pLKO.1GFP shRNA plasmid, has been published in the following reference: Sancak Y, Peterson TR, Shaul YD, et al. The Rag GTPases bindraptor and mediate amino acid signaling to mTORC1[J]. Science, 2008, 320(5882):1496-1501. DOI:10.1126 / science.1157535, purchased from American Type Culture Collection.

[0079] 2. pMD2.G, purchased from Hanheng Biotechnology Co., Ltd.

[0080] 3. psPAX2, purchased from Hanheng Biotechnology Co., Ltd.

[0081] 4. PHBLV-CMVd1-EF-1α-Zsgreen1-T2A-Puro vector, purchased from Hanheng Biotechnology Co., Ltd.

[0082] The strains and cells involved in the following examples:

[0083] 1. HEK293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0084] 2. C2C12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0085] Example 1: Effects of KIF5B gene knockdown on myoblasts

[0086] I. Validation of KIF5B gene knockdown effect

[0087] (1) Culture of myoblasts

[0088] Mouse myoblasts C2C12, frozen in liquid nitrogen, were placed in a 25 cm³ container containing myogenic growth medium. 2 The cells were revived in small square bottles and cultured at 37°C with 5% CO2.

[0089] (2) Constructing the pLKO.1-U6-KIF5B-shRNA-CMV-EGFP-hPGK-Puro vector and lentivirus packaging

[0090] The mouse KIF5B-shRNA sequence (SEQ ID NO.2), 5'-CCTATGTTCCTTATCGAGATA-3', and the negative control scramble shRNA sequence: 5'-caacaagatgaagagcaccaa-3 were introduced into the pLKO.1-U6-shRNA-CMV-EGFP-hPGK-Puro vector to construct a lentiviral expression vector. The vector was then transformed into *E. coli* DH5α for large-scale plasmid amplification, followed by extraction of the endotoxin-free plasmid. HEK293T cells were transfected with the lentiviral vector and packaging vectors (pMD2.G and psPAX2) to generate lentiviral particles. In summary, HEK293T cells were seeded in T-75 culture flasks (Corning) and treated with 14 μg of lentiviral vector, 7 μg of pMD2.G, and 14 μg of psPAX2 and Lipo8000. TM HEK293T cells were transfected using Beyotime transfection reagent in Opti-MEM (Gibco). After 8 hours of incubation, the medium was replaced with fresh complete medium. Supernatant containing lentiviral particles was collected at 48 and 72 hours post-transfection, filtered through a 0.45 μm PVDF filter, and concentrated by ultracentrifugation at 82700 x g for 2 hours at 4°C. The viral pellet was resuspended in GM medium and stored at -80°C until use.

[0091] (3) Validation of lentiviral infection and knockdown effects

[0092] When C2C12 cells reached approximately 70% confluence, they were infected using 50% lentiviral particles supplemented with 10 μg / mL polybrene. This was repeated three times, followed by replacement with fresh complete culture medium. When cells reached approximately 70-80% confluence, positive cells were selected using 3 μg / mL puromycin. After culturing to approximately 500,000 cells, flow cytometry sorting and single-clone selection were performed, followed by culture in fresh complete culture medium. Finally, proteins were extracted from the control group (SCR) and knockdown group (KIF5B-KD) cells, and protein levels were validated using Western blot.

[0093] The results are as follows Figure 1 As shown, compared with the control group, the KIF5B protein level in the knockdown group was downregulated by about 85%, indicating that the lentiviral vector based on the SEQ ID NO.2 sequence has a significant effect on knocking down the KIF5B gene.

[0094] II. Effect of KIF5B knockdown on myoblast proliferation as measured by cell counting assay

[0095] SCR and KIF5B-KD cells were mixed at approximately 20 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well into 6-well plates containing complete culture medium and incubated at 37°C in a 5% CO2 incubator. Observation and photography were performed under a microscope during the incubation period. After 2 days of culture, cells were digested with trypsin and centrifuged (1200 rpm, 5 min) to collect the cells. The cells were resuspended in 3 mL of culture medium to obtain a homogeneous cell suspension, and counted using a hemocytometer.

[0096] The results are as follows Figure 2 As shown, compared with the control group, the number of cells in the KIF5B-KD group was significantly reduced, indicating that knocking down the KIF5B gene leads to a decrease in the physiological activity of myoblasts and a decrease in proliferation rate of about 50%.

[0097] III. EdU assay to detect the effect of KIF5B knockdown on cellular DNA synthesis

[0098] Cell proliferation capacity was assessed using the EdU Imaging Kit (Cy5) (APExBIO Technology, USA) according to the manufacturer's instructions.

[0099] The results are as follows Figure 3 As shown, compared with the control group, the Pearson correlation coefficient in KIF5B-KD cells was significantly lower than that in the SCR group, indicating that knockdown of KIF5B significantly inhibited DNA synthesis, thereby inhibiting the cell proliferation rate.

[0100] Example 2: Effects of KIF5B gene overexpression on myoblasts

[0101] I. Validation of KIF5B gene overexpression effect

[0102] (1) Construction of PHBLV-CMVd1-KIF5B-EF-1α-Zsgreen1-T2A-Puro vector and lentivirus packaging

[0103] The mouse KIF5B CDS sequence (SEQ ID NO.3) was introduced into the PHBLV-CMVd1-EF-1α-Zsgreen1-T2A-Puro vector to construct a lentiviral expression vector. The vector was then transformed in *E. coli* for large-scale plasmid amplification, followed by extraction of the endotoxin-free plasmid. HEK293T cells were transfected with the lentiviral vector and packaging vectors (pMD2.G and psPAX2) to generate lentiviral particles. In short, HEK293T cells were seeded in T-75 culture flasks (Corning) and treated with 14 μg of lentiviral vector, 7 μg of pMD2.G, and 14 μg of psPAX2 and Lipo8000. TM HEK293T cells were transfected using Beyotime transfection reagent in Opti-MEM (Gibco). After 8 hours of incubation, the medium was replaced with fresh complete medium. Supernatant containing lentiviral particles was collected at 48 and 72 hours post-transfection, filtered through a 0.45 μm PVDF filter, and concentrated by ultracentrifugation at 82700 × g for 2 hours at 4°C. The viral pellet was resuspended in GM medium and stored at -80°C until use.

[0104] (3) Validation of lentiviral infection and knockdown effects

[0105] When C2C12 cells reached approximately 70% confluence, they were infected using 50% lentiviral particles supplemented with 10 μg / mL Polybrene. This was repeated three times, followed by replacement with fresh complete culture medium. When cells reached approximately 70-80% confluence, positive cells were selected using 3 μg / mL Puromycin. After culturing to approximately 500,000 cells, flow cytometry sorting and single-clone selection were performed, followed by culture in fresh complete culture medium. Finally, proteins were extracted from the empty vector control group (Ctrl) and the knockdown group (KIF5B-OE), and protein levels were validated using Western blot.

[0106] The results are as follows Figure 4 As shown, compared with the control group, the KIF5B protein level in the overexpression group was upregulated by about 1.9 times, indicating that the effect of overexpressing the KIF5B gene is obvious.

[0107] II. Effects of KIF5B gene overexpression on MYOD and MYOG expression

[0108] Ctrl and KIF5B-OE cells were placed at 12 × 10⁻⁶ cells per well. 4 -15×10 4Cells were seeded at a density of 1000 cells / well in 12-well plates containing complete culture medium and incubated at 37°C in a 5% CO2 incubator. When the cell density reached approximately 90%-95%, total RNA was extracted from cells in the Ctrl and KIF5B-OE treatment groups using the Total RNA Kit I (Omega Bio-tek, catalog number R6834). Sample preparation: Cell or tissue samples were homogenized. The samples were first added to lysis buffer and thoroughly mixed to lyse the cells. The lysed samples were then added to... In the RNA Mini Column, RNA binds to the column. Three rapid washes using a waslbuffer are performed to remove cell debris and other contaminants. Finally, the purified RNA is eluted from the column using elution buffer. RNA concentration and purity are assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). 1 μg of total RNA is reverse transcribed into cDNA using a HiScript IIQ Select RT SuperMix (Vazyme). RT-qPCR is performed using a ChamQ Universal SYBR qPCR Master Mix (Vazyme) on a CFX96 real-time PCR detection system (Thermo Fisher Scientific). The following cycling conditions are used: initial denaturation at 95°C for 3 minutes, followed by 40 cycles of 10 seconds at 95°C followed by 30 seconds at 60°C. Relative gene expression is calculated using the 2^-ΔΔCt method, with GAPDH as an internal control. Results are expressed as fold changes relative to the KIF5B-OE or D0 group.

[0109] Table 1 Primer List

[0110]

[0111] The results are as follows Figure 5 As shown, compared with the control group, the expression level of MYOD mRNA in the overexpression group was almost completely inhibited, and the expression level of MYOG mRNA was downregulated to 50% of that in the control group, indicating that the overexpression of KIF5B significantly inhibited the expression of MYOD and MYOG.

[0112] III. Effects of KIF5B gene overexpression on myoblast DNA synthesis as detected by EDU

[0113] Cell proliferation capacity was assessed using the EdU Imaging Kit (Cy5) (APExBIO Technology, USA) according to the manufacturer's instructions.

[0114] The results are as follows Figure 6As shown, compared with the control group, the Pearson correlation coefficient in the overexpression group was significantly higher than that in the Ctrl group, increasing by about 1.5 times, indicating that overexpression of KIF5B significantly promoted DNA synthesis and further promoted cell proliferation.

[0115] IV. Effect of KIF5B gene overexpression on myoblast proliferation as determined by cell counting assay

[0116] Ctrl and KIF5B-OE cells were spaced at approximately 10 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well into 12-well plates containing complete culture medium and incubated at 37°C in a 5% CO2 incubator. Observation and photography were performed under a microscope midway through the incubation. After one day, the cells were trypsinized, diluted, and counted using a hemocytometer. Then, 10 × 10⁶ cells were collected. 5 Cells were seeded into 12-well plates containing complete culture medium and cultured at 37°C in a 5% CO2 incubator. Cell counts were performed after 1 day, and the culture was repeated for 6 consecutive days. The cell counts were then statistically analyzed.

[0117] The results are as follows Figure 7 As shown, compared with the control group, the cell proliferation rate in the KIF5B-OE group was significantly higher than that in the control group, indicating that overexpression of KIF5B can enhance the physiological activity of myoblasts and significantly promote cell proliferation.

[0118] This invention claims protection for the application of the KIF5B gene in regulating the physiological activity of myoblasts. This invention provides important technical support and theoretical indicators for in vitro cell culture, and can screen key compounds with KIF5B as a key target.

[0119] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The application of a KIF5B gene regulator in the preparation of products for regulating myoblast proliferation, characterized in that, The regulator of the KIF5B gene is either a KIF5B gene enhancer or a KIF5B gene inhibitor. The KIF5B gene inhibitor inhibits the proliferation of myoblasts, while the KIF5B gene enhancer promotes the proliferation of myoblasts. The KIF5B gene inhibitor is a lentiviral expression vector containing shRNA that targets and knocks down KIF5B gene expression, the nucleotide sequence of which is shown in SEQ ID NO.

2. The KIF5B gene enhancer is a lentiviral expression vector containing a CDS sequence that targets and overexpresses KIF5B gene expression, the nucleotide sequence of which is shown in SEQ ID NO.

3.

2. The application according to claim 1, characterized in that, The myoblasts are C2C12 cells.

3. A method for inhibiting myoblast proliferation in vitro, characterized in that, The method involves knocking down the expression of the KIF5B gene in myoblasts using a lentiviral expression vector containing shRNA that targets and knocks down the KIF5B gene. The nucleotide sequence of the shRNA is shown in SEQ ID NO.

2.

4. A method for promoting myoblast proliferation in vitro, characterized in that, The method involves upregulating the expression of the KIF5B gene in muscle cells using a lentiviral expression vector containing a CDS sequence that targets the overexpression of the KIF5B gene. The nucleotide sequence of the CDS sequence is shown in SEQ ID NO.3.

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