Use of mustn1 protein or its encoding gene in regulating adipocyte proliferation and differentiation

Abstract

The application discloses application of MUSTN1 protein or a coding gene thereof in regulation of adipocyte proliferation and differentiation. The application firstly finds that overexpression of the MUSTN1 gene can promote adipocyte proliferation and differentiation, and further proposes application of the MUSTN1 protein or the coding gene thereof or a biological material containing the coding gene thereof in regulation of adipocyte proliferation and differentiation and fat deposition. The application discloses that the MUSTN1 gene has the effect of promoting adipocyte proliferation and differentiation, and lays a foundation for further research on the mechanism of the MUSTN1 gene in cell proliferation and differentiation and application of the MUSTN1 gene in improvement of meat quality of domestic animals and treatment of human obesity diseases.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to the application of the MUSTN1 protein or its encoding gene in regulating adipocyte proliferation and differentiation. Background Technology

[0002] 3T3-L1 adipocytes are a cell line derived from mouse preadipocytes. After contact inhibition and exiting the growth cycle, they undergo a process of reversal from preadipocytes to adipocytes. 3T3-L1 cells can be used to study diabetes, obesity, and other conditions. They are an internationally recognized cell model for studying adipocyte differentiation, simulating the growth and development process of adipocytes under in vitro conditions to study the specific regulatory mechanisms of fat deposition.

[0003] Musculoskeletal (Embryonic Nuclear Protein 1) (MUSTN1) was discovered in a rat femoral fracture model and participates in the regulation of various biological processes, particularly playing a crucial role in the growth and development of bone fat in various animals such as chickens, ducks, and cattle. Furthermore, MUSTN1 may also play a role in ciliary function and tumor development, including tongue and lung cancer. Currently, there are no reported studies on the regulatory role of MUSTN1 in fat deposition, and its role in adipocyte proliferation and differentiation remains unclear.

[0004] For domesticated meat animals, a clear understanding of the role of the MUSTN1 gene in fat deposition is beneficial for accelerating animal breeding. Therefore, identifying the role of the MUSTN1 gene in adipocytes not only provides new insights into the expression regulation mechanisms of fat deposition, but also lays the foundation for further research on the regulatory role of the MUSTN1 gene in livestock meat quality traits and human obesity. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the MUSTN1 protein or its encoding gene in regulating adipocyte proliferation and differentiation.

[0006] In order to achieve the purpose of this invention, in a first aspect, this invention provides the application of MUSTN1 protein or its encoding gene, or biological materials containing its encoding gene, in regulating adipocyte proliferation and differentiation.

[0007] In this invention, the reference sequence number of the gene encoding the MUSTN1 protein in GenBank is GU345805.1.

[0008] The biological materials include, but are not limited to, recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0009] Secondly, the present invention provides the application of the MUSTN1 protein or its encoding gene, or biological materials containing its encoding gene, in the preparation of biological products for regulating lipid deposition.

[0010] Thirdly, the present invention provides the use of the MUSTN1 protein or its encoding gene, or biological materials containing its encoding gene, in the preparation of a medicament for treating obesity.

[0011] Fourthly, the present invention provides a method for promoting the proliferation and differentiation of adipocytes, the method comprising: overexpressing the gene encoding the MUSTN1 protein in adipocytes.

[0012] The CDS sequence of the gene encoding the MUSTN1 protein was amplified using porcine cDNA as a template. The CDS sequence (SEQ ID NO:5) was then constructed into a eukaryotic expression vector, and the recombinant expression vector was introduced into adipocytes.

[0013] Preferably, the primer sequences used to amplify the CDS sequence are as shown in SEQ ID NO:1 and 2.

[0014] Preferably, the eukaryotic expression vector is pCDH.

[0015] Preferably, the adipocytes are 3T3-L1 cells.

[0016] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0017] (I) This invention is the first to construct an expression vector pCDH-MUSTN1 that overexpresses the porcine MUSTN1 gene, which can significantly improve the expression of the MUSTN1 gene, filling the gap in the current lack of expression vectors for overexpressing the porcine MUSTN1 gene, and providing a research tool for exploring the biological function of the porcine MUSTN1 gene.

[0018] (II) This invention demonstrates that overexpression of MUSTN1 via the overexpression vector pCDH-MUSTN1 promotes the proliferation and differentiation of 3T3-L1 cells. It clarifies the important regulatory role of the porcine MUSTN1 gene in adipocyte proliferation and differentiation, providing new insights into the expression regulation mechanisms of fat deposition and laying the foundation for further research on the regulatory role of the MUSTN1 gene in livestock meat quality traits and human obesity. Attached Figure Description

[0019] Figure 1 This is a skeleton diagram of the pCDH carrier in this invention.

[0020] Figure 2This is a comparison diagram of the sequencing results of the pCDH-MUSTN1 overexpression vector with the reference sequence in a preferred embodiment of the present invention.

[0021] Figure 3 This is a graph showing the efficiency detection of the overexpression vector in a preferred embodiment of the present invention. ***P<0.001.

[0022] Figure 4 This is a graph showing the mRNA expression detection of cell proliferation marker genes in a preferred embodiment of the present invention. *P<0.05, **P<0.01.

[0023] Figure 5 The image shown is an EdU staining detection of adipocyte proliferation in a preferred embodiment of the present invention. The two images on the far left show stained EdU-positive proliferating cells, the two images in the middle show DAPI-stained cell nuclei, and the two images on the far right (Merge) are an overlay of the two images.

[0024] Figure 6 This is a graph showing the mRNA expression detection of adipogenic differentiation marker genes in a preferred embodiment of the present invention. *P<0.05, **P<0.01.

[0025] Figure 7 This is a preferred embodiment of the present invention showing the detection of adipocyte differentiation by Oil Red O staining. The oil red O staining represents the differentiated fat droplets. The two leftmost images are images of fat droplets taken under a microscope, and the bar chart on the right is a quantitative analysis of the absorbance value of Oil Red O after isopropanol extraction, measured by an enzyme-linked immunosorbent assay (ELISA) reader.

[0026] Figure 8 This is a Bodipy staining detection of adipocyte differentiation in a preferred embodiment of the present invention. The top and bottom left images show the differentiated fat droplets stained with Bodipy, the top and bottom middle images show the cell nuclei stained with DAPI, and the top and bottom right images (Merge) are superimposed images of the two. Detailed Implementation

[0027] The present invention aims to provide an application of the MUSTN1 protein or its encoding gene in regulating adipocyte proliferation and differentiation.

[0028] Studies have found that the expression of the MUSTN1 gene differs significantly in the adipose tissue of pig breeds with different growth rates, indicating that MUSTN1 may be involved in the regulation of fat deposition. Further research was conducted based on this finding to obtain the solution of this invention.

[0029] The present invention adopts the following technical solution:

[0030] This invention provides the application of the MUSTN1 protein or its encoding gene, or biological materials containing its encoding gene, in regulating adipocyte proliferation and differentiation.

[0031] The present invention also provides the application of the MUSTN1 protein or its encoding gene, or biological materials containing its encoding gene, in the regulation of lipid deposition.

[0032] In this invention, the expression level of the MUSTN1 gene is increased to promote the proliferation and differentiation of adipocytes.

[0033] The present invention also provides the application of the MUSTN1 protein or its encoding gene, or biological materials containing its encoding gene, in animal breeding.

[0034] In this invention, the biological materials include, but are not limited to, expression cassettes, vectors, host cells, or recombinant bacteria.

[0035] The preferred biomaterial is the overexpression vector pCDH-MUSTN1.

[0036] When constructing the overexpression vector pCDH-MUSTN1, a pair of PCR primers were designed based on the porcine MUSTN1 gene sequence. The sequences of the PCR primers are shown in SEQ ID NO:1 and 2.

[0037] Specifically, the method for constructing the MUSTN1 gene overexpression vector pCDH-MUSTN1 is as follows:

[0038] S1. A pair of PCR primers were designed based on the porcine MUSTN1 gene sequence. BamHI and NotI restriction sites were introduced into the 5' ends of the two primers, respectively. The CDS region sequence of the MUSTN1 gene was amplified from porcine cDNA using this pair of primers. The length of the CDS region was 606 bp.

[0039] S2. The sequence obtained in step S1 and the pCDH vector are digested with BamHI and NotI double enzymes, and then ligated with ligase to insert the sequence into the BamHI and NotI restriction sites of the plasmid vector pCDH, thereby constructing the expression vector pCDH-MUSTN1 of the porcine MUSTN1 gene.

[0040] In this invention, the CDS sequence of the MUSTN1 gene is shown in SEQ ID NO:5.

[0041] This invention provides a method for regulating fat deposition in animals by using transgenic methods to regulate the expression of the MUSTN1 gene in animals.

[0042] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.

[0043] Example 1: Construction of a porcine MUSTN1 gene overexpression vector

[0044] 1. Obtaining the CDS region sequence of the porcine MUSTN1 gene

[0045] A pair of PCR primers was designed based on the porcine MUSTN1 gene sequence (GenBank: GU345805.1). A 15bp sequence near the BamHI and NotI restriction sites on the pCDH vector was introduced at the 5' end of each primer as the upper homologous arm (SEQ ID NO:3) and lower homologous arm (SEQ ID NO:4), respectively. The upstream and downstream primer sequences are shown in SEQ ID NO:1 and 2, respectively. Using this pair of primers and porcine cDNA as a template, the CDS region sequence of the MUSTN1 gene, MUSTN1-CDS (SEQ ID NO:5), was amplified. The sequence was 237bp in length, and the amplification product was recovered by gel electrophoresis. PCR amplification was performed using a 20 μL reaction system: 10 μL 2×Power Taq PCRMaster Mix, 8 μL ddH2O, 0.5 μL each of forward and reverse primers, and 1 μL cDNA template. PCR reaction conditions were: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 60 seconds, for 30 cycles; and a final extension at 72℃ for 5 minutes.

[0046] 2. Construction of pCDH-MUSTN1 overexpression vector

[0047] (1) pCDH vector backbone, such as Figure 1 As shown in Table 1 (purchased from Ubisoft Biotechnology), the pCDH circular plasmid was digested with BamHI and NotI. The digestion system was prepared according to Table 1, and after preparation, the system was placed in a 37°C water bath for 2 hours. After digestion, 1% agarose gel electrophoresis was performed, and the linearized vector after digestion was recovered and purified using a Tiangen gel recovery kit.

[0048] Table 1. Reagent preparation for the double enzyme digestion system

[0049]

[0050] (2) Connect the two recycled adhesive products, configure the connection system according to Table 2, and connect at 37°C for 15 minutes.

[0051] Table 2 Connection System

[0052]

[0053] (3) Transform the recombinant vector into DH5α competent Escherichia coli cells, mix well, incubate on ice for 30 minutes, then heat shock transformation at 42℃ for 90 seconds, followed by incubation on ice for 2 minutes, add 950 μL of sterile LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride) incubated at 37℃, shake culture at 180 rpm for 2 hours, take 100 μL of bacterial solution and spread it evenly on ampicillin-resistant solid LB medium (containing 100 μg / mL ampicillin), incubate overnight at 37℃ with inverted culture for 12-16 hours.

[0054] (4) Sequencing: Take a single colony and culture it in 1 mL of liquid LB medium containing 100 μg / mL ampicillin. Shake at 150 rpm until turbidity is reached. Send the bacterial culture to a sequencing company for sequencing verification. Figure 2 As shown, the sequencing results were completely consistent with the reference sequence, confirming the successful construction of the vector, which was named pCDH-MUSTN1.

[0055] 3. Overexpression efficiency detection

[0056] When 3T3-L1 cells in six-well plates reached 70%–90% confluence, the overexpression plasmid pCDH-MUSTN1 was transfected into the cells using the Lipo2000 transfection reagent. Cells transfected with the empty vector pCDH served as a control. The medium was changed 6 hours after transfection. Total RNA was extracted from adipocytes transfected for 48 hours using the Trizo1 method, and then reverse transcribed into cDNA using a Tiangen reverse transcription kit. Using cDNA as a template, quantitative PCR was performed using Bio-Rad's SYBR Green fluorescent dye and a Roche LightCycler 480 real-time PCR instrument, with β-actin as an internal control. The results were analyzed using 2... -ΔΔCt The relative mRNA expression level of each gene was calculated using a method with three replicates per group and three separate experiments. The reaction volume was 20 μL. The reaction conditions were: 95℃ pre-denaturation for 5 minutes, 95℃ denaturation for 15 seconds, 56℃ annealing for 15 seconds, and 72℃ for 15 seconds, repeated 40 times. The upstream and downstream primers used for quantitative real-time PCR detection of the MUSTN1 gene are shown in SEQ ID NO:6 and 7, respectively.

[0057] Overexpression vector efficiency detection, such as Figure 3 As shown, the expression level of MUSTN1 in adipocytes after transfection with the overexpression vector was significantly higher than that in the control group, indicating that the pCDH-MUSTN1 overexpression vector can increase the expression level of the target gene.

[0058] Example 2: The role of the MUSTN1 gene overexpression vector in mouse adipocyte proliferation

[0059] The overexpression vector pCDH-MUSTN1 was transfected into 3T3-L1 cells using the transfection reagent lipo2000, with cells transfected with the empty vector pCDH serving as a control. After 6 hours, the cells were replaced with DMEM complete medium for further culture. After 24-48 hours, RNA was extracted from a portion of the cells and reverse transcribed into cDNA. The expression changes of cell proliferation marker genes Ki67 (Gene ID: 17345), CDK4 (Gene ID: 12567), and Cyclin B (Gene ID: 22178) were detected using quantitative real-time PCR. EdU staining was performed on the other portion of the cells to assess cell proliferation.

[0060] Figure 4 To detect changes in the mRNA expression levels of cell proliferation marker genes using quantitative real-time PCR, it was found that the gene expression level in the overexpression group was significantly higher than that in the control group, indicating that pCDH-MUSTN1 promotes adipocyte proliferation by overexpressing the MUSTN1 gene.

[0061] Figure 5 To detect adipocyte proliferation using EdU staining, cells transfected with pCDH-MUSTN1 showed more EdU-positive proliferating cells than cells transfected with the control pCDH empty vector, indicating that the MUSTN1 gene promotes adipocyte proliferation.

[0062] Example 3: The role of the MUSTN1 gene overexpression vector in mouse adipocyte differentiation

[0063] The overexpression vector pCDH-MUSTN1 was transfected into 3T3-L1 cells using the transfection reagent lipo2000. Cells transfected with the empty vector pCDH served as a control. After 6 hours, the medium was replaced with adipogenic differentiation induction medium I (DMEM complete medium containing 0.5 mM IBMX, 1 μM DEX, and 5 μg / ml insulin) to induce cell differentiation. After 4 days of differentiation, the medium was replaced with adipogenic differentiation induction medium II (DMEM complete medium containing 5 μg / ml insulin), and thereafter the medium was changed every two days. RNA was extracted from a portion of the cells and reverse transcribed into cDNA. Quantitative real-time PCR was used to detect changes in the expression of adipogenic differentiation marker genes Cebpβ (Gene ID: 12608), Fabp4 (Gene ID: 11770), and PPARγ (Gene ID: 19016). The remaining cells were stained with Oil Red O and Bodipy staining to detect the formation of lipid droplets during differentiation.

[0064] Figure 6 To detect changes in the mRNA expression levels of adipogenic differentiation marker genes in cells using quantitative real-time PCR, the overexpression group showed higher expression levels of adipogenic differentiation marker genes than the control group, indicating that pCDH-MUSTN1 promotes adipocyte differentiation by overexpressing the MUSTN1 gene.

[0065] Figure 7 and Figure 8 Oil Red O and Bodipy staining were used to measure the adipogenic differentiation of adipocytes. Compared with the control group, the overexpressing group differentiated more lipid droplets, indicating that overexpression of the MUSTN1 gene can promote adipocyte differentiation.

[0066] In summary, this invention is the first to construct an expression vector pCDH-MUSTN1 that overexpresses the porcine MUSTN1 gene, which can significantly improve the expression of the MUSTN1 gene, filling the current gap in the lack of expression vectors for overexpressing the porcine MUSTN1 gene, and providing a research tool for exploring the biological function of the porcine MUSTN1 gene.

[0067] This invention demonstrates that overexpression of MUSTN1 via the overexpression vector pCDH-MUSTN1 promotes the proliferation and differentiation of mouse adipocytes. It clarifies the important regulatory role of the porcine MUSTN1 gene in adipocyte proliferation and differentiation, providing new insights into the expression regulation mechanisms of fat deposition and laying the foundation for further research on the regulatory role of the MUSTN1 gene in livestock meat quality traits and human obesity.

[0068] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of MUSTN1 protein or its encoding gene, or biological materials containing its encoding gene, in promoting adipocyte proliferation and differentiation; in, The gene encoding the MUSTN1 protein has a reference sequence number of GU345805.1 in GenBank.

2. The application of the MUSTN1 protein or its encoding gene, or biological materials containing its encoding gene, in the preparation of biological products for promoting lipid deposition; in, The gene encoding the MUSTN1 protein has a reference sequence number of GU345805.1 in GenBank.

3. The application according to claim 1 or 2, characterized in that, The biological materials are recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

4. A method for promoting adipocyte proliferation and differentiation, characterized in that, The method includes: overexpressing the gene encoding the MUSTN1 protein in adipocytes; The gene encoding the MUSTN1 protein has a reference sequence number of GU345805.1 in GenBank.

5. The method according to claim 4, characterized in that, The CDS sequence of the gene encoding the MUSTN1 protein was amplified using porcine cDNA as a template, the CDS sequence was constructed into a eukaryotic expression vector, and the recombinant expression vector was introduced into adipocytes. The primer sequences used to amplify the CDS sequence are shown in SEQ ID NO:1 and 2.

6. The method according to claim 5, characterized in that, The eukaryotic expression vector is pCDH.

7. The method according to claim 6, characterized in that, The CDS sequence is shown in SEQ ID NO:

5.

8. The method according to any one of claims 4-7, characterized in that, The adipocytes were 3T3-L1 cells.

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