Application of SPOP gene or protein in regulating fat production and thermogenic capacity of animals

Abstract

The application discloses application of SPOP gene or protein in regulating fat generation and heat production capacity of animals. The application discloses for the first time that SPOP plays a key role in fat cell generation and heat generation, especially in regulating gene expression related to fatty acid metabolism and energy consumption, which provides an important theoretical basis for developing a new method for regulating fat metabolism, and provides a new potential target for treatment of obesity and metabolic diseases.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically, it relates to the application of the SPOP gene or protein in regulating the fat production and thermogenesis in animals. Background Technology

[0002] Adipose tissue plays a crucial role in regulating energy balance and metabolic health. Adipocyte differentiation, or adipogenesis, is a complex process of transformation from precursor cells to adipocytes, involving the regulation of multiple genes and signaling pathways. Disorders of this process are closely associated with metabolic diseases such as obesity, diabetes, and metabolic syndrome. Therefore, a deeper understanding of the molecular mechanisms of adipocyte differentiation is essential for developing new strategies for treating metabolic diseases.

[0003] In adipogenesis, brown and beige adipose tissues play crucial roles in thermogenesis and energy expenditure. Brown adipocytes, through the action of uncoupling protein 1 (UCP1), can consume energy through thermogenesis, which is important for regulating body temperature and preventing obesity. Compared to white adipose tissue, brown and beige adipose tissues have a stronger energy metabolism capacity (Heeren, J et al., 2018). Therefore, activating the thermogenesis function of brown or beige adipocytes has become a potential approach for treating obesity and metabolic diseases.

[0004] SPOP (Speckle-type POZ protein) is an E3 ubiquitin ligase (Mani, R. S et al., 2014) that has been found to participate in the regulation of various biological processes, including cell development, aging, transcriptional regulation, and tumorigenesis. In recent years, the role of SPOP in tumors has been extensively studied, especially in prostate cancer and renal cell carcinoma.

[0005] Some recent studies have shown that SPOP plays an important role in regulating innate immune responses and hematopoiesis. However, the role of SPOP in adipocyte differentiation and thermogenesis remains unclear. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the SPOP gene or protein in regulating the fat production and thermogenesis in animals.

[0007] In order to achieve the purpose of this invention, in a first aspect, this invention provides the application of the SPOP gene or protein in regulating the fat production and thermogenesis in animals (including non-disease diagnosis and treatment purposes).

[0008] In this invention, the reference sequence number of the SPOP gene from mice on NCBI is NM_001359107.1.

[0009] Furthermore, inhibiting the expression or activity of the SPOP gene or its encoded protein at the transcriptional or translational level, or knocking out the SPOP gene from the animal genome, can reduce fat deposition in animals, decrease the adipogenic differentiation efficiency of animal preadipocytes, and promote the expression of thermogenic genes.

[0010] The thermogenic gene includes UCP1, and the reference sequence number of the mouse UCP1 gene on NCBI is NM_009463.3.

[0011] Secondly, the present invention provides the application of SPOP gene or protein as a target in the preparation of anti-obesity drugs.

[0012] Thirdly, this invention provides the application of SPOP gene or protein inhibitors in the preparation of formulations that enhance the cold resistance of animals.

[0013] The inhibitor may be selected from at least one of the following: small molecule inhibitors, oligonucleotides, shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, antibodies, fusion proteins, SPOP gene targeting vectors, etc.

[0014] The SPOP gene targeting vector can be constructed based on CRISPR, TALEN, or ZFN genome editing technologies.

[0015] Preferably, the inhibitor is an shRNA targeting the SPOP gene, with the following sequence:

[0016] 5′-UGCACCUCGGACUCCACAAAU-3′ (SEQ ID NO: 1).

[0017] Fourthly, the present invention provides any of the following applications of the SPOP gene:

[0018] 1) Used to construct animal models of fat development and / or thermogenicity;

[0019] 2) Animal models used to study obesity and related diseases;

[0020] 3) Used for breeding low-fat, high-lean-meat-ratio animals.

[0021] In this invention, the animals include, but are not limited to, mice.

[0022] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0023] This invention reveals for the first time the crucial role of SPOP in adipocyte formation and thermogenesis, particularly in regulating gene expression related to fatty acid metabolism and energy expenditure. This provides an important theoretical basis for developing new methods to regulate lipid metabolism and also offers new potential targets for the treatment of obesity and metabolic diseases. Specific advantages are as follows:

[0024] (i) This invention provides the application of the SPOP gene or protein in regulating the ability of mice to produce fat and generate heat, thereby providing a new perspective for understanding fat metabolism and energy balance, and providing a theoretical basis for the study of obesity and related metabolic diseases.

[0025] (II) This invention provides the application of the SPOP gene as a target gene in anti-obesity treatment. This opens up new therapeutic avenues for anti-obesity therapy. By targeting SPOP, the generation and function of adipocytes can be regulated at the molecular level, helping to reduce fat accumulation and improve obesity-related symptoms.

[0026] (III) The SPOP inhibitor provided by this invention has significant application potential in improving cold resistance. By inhibiting the activity of SPOP, the adaptability of animals to cold environments can be enhanced, thereby improving their survival rate and health level. Attached Figure Description

[0027] Figures 1-2 This is the result of differential expression of UCP1 protein in SPOP conditional knockout mice in Example 1 of the present invention.

[0028] Figures 3-5 The results of the mouse feeding experiment in Example 2 of this invention are shown.

[0029] Figures 6-7 This is the result of the cold tolerance experiment of SPOP conditional knockout mice in Example 3 of the present invention.

[0030] Figures 8-9 The results of mouse adipocyte differentiation and Oil Red O staining are shown in Example 4 of this invention. Detailed Implementation

[0031] This invention aims to provide the application of the SPOP gene or protein in regulating the lipogenesis and thermogenesis in mice.

[0032] The present invention adopts the following technical solution:

[0033] This invention provides the application of the SPOP gene or protein in regulating the lipogenesis and thermogenesis in mice (including non-disease diagnosis and treatment purposes).

[0034] In this invention, the reference sequence number of the SPOP gene from mice on NCBI is NM_001359107.1.

[0035] Specifically, the number of fat droplets in the adipose tissue of SPOP-deficient mice was significantly reduced. Furthermore, UCP1 protein expression was significantly upregulated in the brown adipose tissue of SPOP-deficient mice, suggesting its crucial role in thermogenesis. Compared to wild-type mice, SPOP-deficient mice showed increased UCP1 expression, indicating that SPOP deficiency promotes the activation of thermogenesis-related genes.

[0036] This invention also provides the application of the SPOP gene as a target gene in anti-obesity treatment.

[0037] This invention also provides the application of SPOP gene or protein inhibitors in the preparation of formulations that enhance cold resistance.

[0038] The objective of this invention can be further achieved by the following technical measures.

[0039] The conditional SPOP knockout mice of this invention were prepared by Beijing VitaBio Technology Co., Ltd. using the classic Cre-LoxP recombination method. SPOP- / - mice were created by crossing SPOP- / 396 mice with Cre transgenic C57 BL / 6 mice. These mice were fed a high-fat diet (HFD) with 60% fat content purchased from Synergy Biotech for 15 weeks, and changes in body weight and lipid deposition were observed. Subsequently, the mice were subjected to a 9-hour cryogenic treatment at 4°C, and the cold tolerance of the knockout mice was significantly enhanced. Furthermore, proteomics analysis revealed that the expression of the heat generation-related gene UCP1 was significantly upregulated in the brown adipose tissue of the SPOP knockout mice, indicating accelerated energy metabolism. Further in vitro 3T3-L1 cell differentiation experiments showed that SPOP gene knockdown significantly inhibited lipid droplet formation. Finally, oxygen consumption and citrate synthase activity assays confirmed the active energy metabolism of the knockout mice. This invention reveals the important role of the SPOP gene in regulating adipogenesis, energy metabolism, and body temperature maintenance.

[0040] This invention provides an application of the SPOP gene in regulating adipogenesis and thermogenesis in mice. Proteomics analysis revealed that UCP1 protein expression was significantly upregulated in the brown adipose tissue of SPOP-deficient mice, suggesting its crucial role in thermogenesis. Compared to wild-type mice, SPOP-deficient mice showed increased UCP1 expression, indicating that SPOP deficiency promotes the activation of thermogenesis-related genes. Using a second-generation lentivirus packaged with a shRNA plasmid targeting SPOP, the viral supernatant was collected, purified, concentrated, and then used to infect NIH3T3-L1 cells, obtaining a stable SPOP-interfering NIH3T3-L1 cell line. Quantitative real-time PCR results showed that SPOP expression was effectively inhibited in NIH3T3-L1 cells transfected with lentiviral shRNA targeting mouse SPOP. Oil Red O staining results showed that lipid droplet formation was significantly inhibited after SPOP downregulation.

[0041] Based on this, the present invention provides an application of the SPOP gene as a target gene in anti-obesity treatment.

[0042] Furthermore, this invention provides the application of SPOP gene or protein inhibitors in the preparation of formulations that enhance cold resistance.

[0043] The term "application" as used in this invention can refer to applications for therapeutic purposes or applications for non-therapeutic purposes, such as scientific research.

[0044] The "drug" or "inhibitor" described in this invention may be selected from small molecule inhibitors, oligonucleotides, antibodies, peptides, or fusion proteins. The oligonucleotide is preferably miRNA, siRNA, or shRNA, and more preferably shRNA targeting the SPOP gene.

[0045] 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 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.

[0046] The NIH3T3-L1 cells used in the following examples were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0047] Example 1: Differentially expressed protein analysis in SPOP conditional knockout mice

[0048] 1. Western blot

[0049] Protein lysates or immunoprecipitated samples were separated by electrophoresis on an SDS-PAGE gel and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was first blocked in TBST with 5% (w / v) skim milk powder, then incubated with the appropriate primary antibody (diluted in 5% skim milk powder TBST). After washing with TBST, the membrane was incubated with the appropriate secondary antibody (diluted in 5% skim milk powder TBST). Protein bands were visualized using Immobilon Western chemiluminescent HRP substrate (Millipore) according to the manufacturer's instructions.

[0050] 2. Proteomics analysis

[0051] Mouse kidney tissue samples were ground into powder using liquid nitrogen, lysed in RIPA buffer, and then sonicated three times on ice using a high-intensity sonication processor (Scientz). The supernatant was retained, and protein concentration was determined using a BCA kit according to the manufacturer's instructions. For mass spectrometry analysis, the protein samples were first reduced and then digested with trypsin. Trypsin peptides were dissolved in solvent A (0.1% formic acid, 2% acetonitrile / water) and directly loaded onto a self-made reversed-phase analytical column. The peptides were then separated in solvent B (0.4% copper divalent sulfate). The peptides were subsequently analyzed by a timsTOF Pro (Brooklyn Dalton) mass spectrometer via a capillary source.

[0052] 3. Results

[0053] See Figure 1 Western blot results showed that SPOP expression was effectively knocked down in mice.

[0054] See Figure 2 This study demonstrates that the protein UCP1 is significantly upregulated in SPOP knockout mice in proteomics analysis.

[0055] Example 2: SPOP knockout in mice leads to impaired weight gain

[0056] 1. Mouse grouping and feeding

[0057] SPOP conditional knockout mice and wild-type (WT) mice were randomly divided into two groups: the experimental group (SPOP gene knockout mice) and the control group (normal control mice). Both groups were fed the same standard diet and their body weight was monitored for ten weeks.

[0058] 2. Results

[0059] See Figure 3 The results showed that wild-type (WT) mice gained more than 50% more body weight than CKO mice during the feeding period (P<0.05). Figure 3 a), and significantly larger in size ( Figure 3b).

[0060] See Figure 4 Body composition analysis of mice showed that SPOP CKO mice had a slight decrease in lean body mass, while the reduction in fat mass was more significant.

[0061] See Figure 5 After measuring TG in ten-week-old mice, it was found that the TG and glucose levels in SPOP CKO mice were significantly lower than those in the WT control group.

[0062] Example 3: SPOP conditional knockout mice showed greater tolerance to cold.

[0063] 1. Cold tolerance test

[0064] Ten-week-old SPOP knockout mice and control mice underwent a cold tolerance test in a 4°C cold room for nine hours with free access to food and water. The mice's body temperature was measured hourly using a rectal probe connected to a digital thermometer.

[0065] 2. Oxygen Consumption Experiment

[0066] Oxygen consumption (VO2) was measured using the Integrated Laboratory Animal Monitoring System (CLAMS) (Colombus Instruments). Data were normalized to lean body mass as determined by EchoMRI. Mice were housed individually under a 12-hour light / 12-hour dark cycle.

[0067] 3. Results

[0068] See Figure 6 SPOP conditional knockout (CKO) mice were placed in an acute cold environment at 4°C for experiments. Compared with CKO mice, wild-type (WT) mice showed significantly greater cold tolerance, as their body temperature began to drop significantly after 3 hours of cold exposure.

[0069] See Figure 7 Metabolic responses in WT and SPOP CKO mice were analyzed using metabolic cages and the Integrated Laboratory Animal Monitoring System (CLAMS). Under normal conditions, carbon dioxide (VCO2) release in CKO mice was significantly enhanced, indicating that respiratory activity in SPOP CKO mice was higher than that in wild-type mice.

[0070] Example 4: Downregulation of SPOP inhibits adipocyte differentiation

[0071] 1. Adipocyte differentiation and Oil Red O staining

[0072] 3T3-L1 cells were cultured in DMEM / F12 medium supplemented with 10% FBS and 1% penicillin / streptomycin. Two days after cell confluence, 3T3-L1 cells were treated with medium containing 5 μg / mL insulin, 1 μmol / L dexamethasone, and 0.5 mmol / L 3-isobutyl-1-methylxanthine to induce differentiation. Two days later, the cells were transferred to medium containing 5 μg / mL insulin and 1 μmol / L rosiglitazone and cultured for 2 days, then maintained in the medium for 6 days.

[0073] For Oil Red O staining, cells were first washed twice with PBS, then fixed with 4% paraformaldehyde at room temperature. Cells were washed twice with distilled water and then rinsed with 60% isopropanol. They were then stained with freshly prepared Oil Red O for 10 minutes, washed with distilled water, and photographed under a microscope.

[0074] 2. Results

[0075] See Figure 8 SPOP expression was downregulated in NIH3T3-L1 cells. Quantitative real-time PCR results showed that SPOP expression was effectively suppressed in NIH3T3-L1 cells transfected with a genomic small interfering RNA (shRNA: 5′-UGCACCUCGGACUCCACAAAU-3′) lentivirus (purchased from Qingke Biotechnology's second-generation lentiviral vector; the lentiviral packaging system consists of a vector plasmid, helper plasmid psPAX2, and helper plasmid pMD2.G. The psPAX2 vector contains the HIV gag gene, encoding the major structural protein of the virus; the pol gene, encoding a virus-specific enzyme; and the rev gene, encoding a regulatory factor that regulates the expression of the gag and pol genes. The pMD2.G vector contains the VSV-G gene derived from herpes simplex virus, providing the envelope protein required for viral packaging).

[0076] 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. Application of SPOP gene knockout in regulating lipogenesis and thermogenesis in mice; in, The reference sequence number of the SPOP gene from mice on NCBI is NM_001359107.1; the regulation is to knock out the SPOP gene from the mouse genome to reduce fat deposition in animals, reduce the adipogenic differentiation efficiency of animal preadipocytes, and promote the expression of thermogenic genes. The thermogenic gene includes UCP1, and the reference sequence number of the mouse UCP1 gene on NCBI is NM_009463.3; The application is for purposes other than disease diagnosis and treatment.

2. Application of SPOP gene knockout in the construction of animal models for inhibiting adipocyte differentiation and / or increasing thermogenesis; in, The reference sequence number of the SPOP gene from mice on NCBI is NM_001359107.1; The animal in question is a mouse.

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