SORBS1 inhibitor and product and application thereof

Abstract

The invention relates to the technical field of molecular biology, and discloses an SORBS1 inhibitor, which comprises at least one of SORBS1-siRNA1, SORBS1-siRNA2, and SORBS1-siRNA3, and one of the SORBS1 inhibitor, the SORBS1 inhibitor, the SORBS1 inhibitor, the SORBS1 inhibitor, the SORBS1 inhibitor and the SORBS1 inhibitor. The nucleic acid sequences of the SORBS1-siRNA1 are shown as SEQ ID NO. 1 and SEQ ID NO. 2; the nucleotide sequences of the SORBS1-siRNA2 are as shown in SEQ ID NO. 3 and SEQ ID NO. 4; the nucleotide sequences of the SORBS1-siRNA3 are as shown in SEQ ID NO. 5 and SEQ ID NO. 6. The second purpose of the invention is to provide a product which comprises the SORBS1 inhibitor. The third purpose of the invention is to provide the SORBS1 inhibitor, and the product is applied to any one of the following applications: B1) preparing a product for interfering SORBS1 expression; b2) preparing a product for inhibiting SORBS1 protein expression in the precursor fat cells; and B3) preparing a product for inhibiting the proliferation of the precursor fat cells. According to the invention, the breeding goal of lowering lipid without degrading quality is realized by accurately intervening the SORBS1 gene, and the method has theoretical innovation (revealing new functions of SORBS1), and provides a breakthrough solution for high-quality pig breeding and fat metabolism research.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology, and in particular to SORBS1 inhibitors, their products, and applications. Background Technology

[0002] Intramuscular fat (IMF) is a crucial factor affecting pork quality, directly influencing its flavor, tenderness, and juiciness. IMF deposition is influenced by the proliferation and adipogenic differentiation of intramuscular preadipocytes. In recent years, with the development of whole transcriptome sequencing technology, an increasing number of lipid metabolism-related genes have been discovered. The c-Cbl-related protein (CAP) encoded by the SORBS1 gene is an adaptor protein involved in cytoskeleton regulation and lipid metabolism. This invention aims to investigate the effects of interfering with SORBS1 gene expression on the proliferation and adipogenic differentiation of porcine intramuscular preadipocytes, providing a new target for molecular breeding of high-quality meat pigs. Summary of the Invention

[0003] To address the technical problems mentioned in the background art, the present invention provides SORBS1 inhibitors, their products, and applications.

[0004] The present invention is achieved by the following technical solution: One of the objectives of the present invention is to propose a SORBS1 inhibitor, comprising at least one of SORBS1-siRNA1, SORBS1-siRNA2, and SORBS1-siRNA3;

[0005] The nucleic acid sequences of SORBS1-siRNA1 are shown in SEQ ID NO.1 and SEQ ID NO.2: sense:GCCUCCUGGAUAUAUAUAUTT, antisense:AUAUAUAUAUCCAGGAGGCTT;

[0006] The nucleic acid sequence of SORBS1-siRNA2 is shown in SEQ ID NO.3 and SEQ ID NO.4: sense:CACAAGUCCUCUGCUAAAUTT, antisense:AUUUAGCAGAGGACUUGUGTT;

[0007] The nucleic acid sequence of SORBS1-siRNA3 is shown in SEQ ID NO.5 and SEQ ID NO.6: sense:CGACGCACCAUCAGUUUAUTT, antisense:AUAAACUGAUGGUGCGUCGTT.

[0008] The second objective of this invention is to provide a product comprising the aforementioned SORBS1 inhibitor.

[0009] Furthermore, the product also contains medically acceptable excipients.

[0010] A third objective of this invention is to provide the application of the SORBS1 inhibitor and the product in any of the following:

[0011] B1) Prepare products that interfere with SORBS1 expression;

[0012] B2) Prepare a product that inhibits the expression of SORBS1 protein in preadipocytes;

[0013] B3) Prepare products that inhibit the proliferation of proadipocytes;

[0014] B4) Prepare products that inhibit the expression of cell proliferation transcription factors in precursor adipocytes;

[0015] B5) Prepare products that inhibit adipogenic differentiation of precursor adipocytes;

[0016] B6) Prepare products that inhibit the expression of adipogenic differentiation genes in precursor adipocytes;

[0017] B7) Prepare products that inhibit lipid droplet formation or aggregation in precursor adipocytes;

[0018] B8) Prepare products that inhibit the accumulation of triglycerides in precursor adipocytes;

[0019] B9) Regulation of fat deposition traits in pig breeding.

[0020] Furthermore, the preadipocytes are intramuscular preadipocytes, which are derived from mammals.

[0021] Furthermore, the mammal in question is a pig.

[0022] Furthermore,

[0023] In B4), the cell proliferation transcription factor is at least one of PCNA, CDK1, CDK2, and CCND1;

[0024] In B6), the adipogenic differentiation gene is at least one of PPARγ, C / EBPβ, FABP4, and FASN.

[0025] Furthermore, it is characterized by,

[0026] In B9), it is applied to molecular breeding of pork quality traits.

[0027] The fourth objective of this invention is to provide a method for inhibiting the expression of SORBS1 protein in preadipocytes, by transfecting preadipocytes with the aforementioned SORBS1 inhibitor.

[0028] Furthermore, the aforementioned SORBS1 inhibitor, Opti-MEM medium, and Lipofectamine 2000 were used to transfect the preadipocytes.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] This invention utilizes specific siRNA to target and interfere with the SORBS1 gene, significantly inhibiting the proliferation and adipogenic differentiation of porcine intramuscular preadipocytes: cell activity was reduced to below 60% of the control group, lipid droplets decreased by 70%, and triglyceride content decreased by more than 50%; proliferation marker genes (PCNA, CDK1 / 2, CCND1) and key adipogenic genes (PPARγ, C / EBPβ, FABP4, FASN) were all significantly downregulated. This provides the first confirmed evidence that SORBS1 is a novel functional target for regulating intramuscular fat deposition, which can be used for molecular breeding to rapidly cultivate low-fat, high-lean-ratio pig breeds with controllable meat flavor. The accompanying kit and validated cell model enable high-throughput screening, are easy to operate, and are compatible with existing breeding systems; they can also be used to develop flavor-enhancing biological agents or drugs. Compared to traditional breeding methods, the cycle is shortened by 50%, and the cost is reduced by 30%, possessing both academic value and industrial transformation potential. Attached Figure Description

[0031] Figure 1 This is the temporal expression profile of SORBS1 in the adipogenic differentiation process of porcine intramuscular preadipocytes, as proposed in this embodiment of the invention; wherein: all the above differences are compared with day 0.

[0032] Figure 2 The graph shows the detection results of the interference effect of SORBS1-siRNA proposed in the embodiments of the present invention; wherein: A is the siRNA interference efficiency determination; B is the SORBS1 protein detection; C is the SORBS1 protein quantification;

[0033] Figure 3 The figure shows the effect of SORBS1 interference on the proliferation of porcine intramuscular preadipocytes as proposed in the embodiments of the present invention; where: A is the cell proliferation measured by CCK8; B is the effect of SORBS1 interference on proliferation-related genes.

[0034] Figure 4 The figure shows the effect of interfering with SORBS1 on adipogenic differentiation of porcine intramuscular preadipocytes, as proposed in the embodiments of the present invention; where: A is Oil Red O staining; B is Oil Red O extracted with isopropanol; C is triglyceride content; and D is the effect of interfering with SORBS1 on adipogenic differentiation-related genes. Detailed Implementation

[0035] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0036] Example:

[0037] The present invention will be further explained below with reference to specific embodiments.

[0038] Primary culture of porcine intramuscular preadipocytes was conducted in a laboratory cell culture facility.

[0039] The main reagents used in the experiment are shown in Table 2-1:

[0040] Table 2-1: Main Reagents

[0041]

[0042]

[0043] In this embodiment, the main instruments used in the experiment are shown in Table 2-2.

[0044] Table 2-2: Main Instruments

[0045]

[0046]

[0047] In this embodiment, the primer design and synthesis steps are as follows:

[0048] The primers used in the experiment were designed and synthesized by Shanghai Sangon Biotech Co., Ltd., and the primer sequences are shown in Table 2-3. GAPDH was used as an endogenous control gene for mRNA.

[0049] Table 2-3: Primer Sequences

[0050]

[0051]

[0052] The detailed steps for total RNA extraction and quality testing are as follows:

[0053] (1) Extraction of total RNA from cells

[0054] a. Discard the culture medium and wash the cells 1-2 times with pre-cooled PBS;

[0055] b. Per 10cm 2Add Trizol at a ratio of 1 mL Trizol to the bottom area of ​​the culture dish (e.g., 1 mL per well in a six-well plate). After pipetting 5-10 times, place the dish on ice and lyse for 5-10 minutes.

[0056] c. After mixing thoroughly by pipetting, transfer the solution to a 1.5 mL EP tube;

[0057] d. Add 200 μL of pre-cooled chloroform at a ratio of Trizol:chloroform = 5:1, shake vigorously for 15 seconds, let stand on ice for 10-15 minutes, and then centrifuge at 4°C and 12000 rpm for 15-20 minutes.

[0058] e. During the centrifugation process in step d, prepare 75% ethanol at a ratio of anhydrous ethanol: DEPC = 3:1, mix by inverting, and then pre-cool on ice;

[0059] f. Pipette 400 μL of the supernatant after centrifugation into a new EP tube, add 400 μL of pre-cooled isopropanol, invert the tube 10 times, let it stand on ice for 15-20 min, and then centrifuge at 4°C and 20,000 rpm for 15-20 min.

[0060] g. Discard the supernatant, add 1 mL of pre-cooled 75% ethanol to each EP tube, pipette and blow about 10 times, then centrifuge at 4°C and 20,000 rpm for 5-10 min.

[0061] h. Repeat step g 1-2 times;

[0062] i. After discarding the supernatant, open the cap and invert it onto ice to air dry the precipitate. Add pre-cooled DEPC water to dissolve the precipitate, and store the resulting RNA solution in a -80°C freezer.

[0063] (2) RNA concentration and quality detection

[0064] Take 1 μL of RNA solution and determine its concentration and OD. 260 / OD 280 OD 260 / OD 230 The value of OD 260 / OD 280 Within the range of 1.8-2.2 and OD 260 / OD 230 A value in the range of 2.0-2.5 is sufficient for subsequent experiments.

[0065] The detailed mRNA quantification procedure is as follows:

[0066] (1) Reverse transcription

[0067] According to HyperScript TMThe III RT SuperMix for qPCR with gDNA Remover (EnzyArtisan, China) kit provides the following steps for reverse transcription of mRNA. The reaction system and conditions are shown in Table 2-6.

[0068] Table 2-6: mRNA reverse transcription system and conditions

[0069]

[0070] (2) qPCR

[0071] Following the operating procedures provided with the 2×S6 Universal SYBR qPCR Mix (EnzyArtisan, China) kit, the BIO-RAD (CFX96) was used. TM qPCR was performed using a Real-Time System instrument. GAPDH was used as an endogenous control gene for mRNA. Three technical replicates were set up for each group. The reaction system and reaction conditions are shown in Table 2-7.

[0072] Table 2-7 qPCR system and conditions for mRNA

[0073]

[0074] Continued from Table 2-7: qPCR system and conditions for mRNA

[0075]

[0076] In detail, Westernblots operations are as follows:

[0077] (1) Discard the culture medium and wash the cells 2-3 times with pre-cooled PBS;

[0078] (2) Add 80 μL of LRIPA lysis buffer (containing 1% protease inhibitor and 1% phosphatase inhibitor) to each well of a six-well plate and lyse on ice for 10 min, shaking the plate repeatedly during the process to ensure that the lysis buffer fully contacts the bottom of the culture dish.

[0079] (3) Use a cell scraper to scrape cells, collect the liquid in a 1.5 mL EP tube with a pipette, and continue lysis on ice for 20 min, vortexing repeatedly during the process;

[0080] (4) Centrifuge at 4℃, 12000rpm for 10min, collect the supernatant into a new EP tube, add 5×SDS-PAGE protein loading buffer at a ratio of 4:1, and denature at 95℃ for 3min.

[0081] (5) Load the sample after it has cooled and perform electrophoresis at a constant voltage of 150V for about 50 minutes.

[0082] (6) Immerse the PVDF membrane, which has been pre-activated with methanol, into the transfer solution, install the transfer device in sequence, and transfer the membrane at a constant current of 400mA for about 45 minutes.

[0083] (7) Transfer the PVDF membrane to a 5% skim milk powder solution (30mL 1×TBST solution + 1.5g skim milk powder) and seal it slowly on a shaker for 1-2 hours.

[0084] (8) The SORBS1 antibody is a rabbit antibody. Dilute the antibody with 1×TBST solution. Dilute the SORBS1 antibody at 1:500 and incubate overnight at 4°C using the inverted method.

[0085] (9) Place the PVDF membrane in 1×TBST solution and wash it on a shaker for 10 min. Repeat 4 times.

[0086] (10) The secondary antibody is goat anti-rabbit antibody. The antibody is diluted with 1×TBST solution (1:20000) to prepare 20mL of secondary antibody dilution solution. The PVDF membrane is placed in it and incubated on a shaker for 1.5h.

[0087] (11) Place the PVDF membrane in 1×TBST solution and wash it on a shaker for 10 min. Repeat 4 times.

[0088] (12) In a light-protected environment, prepare the luminescent solution by mixing ECL chemiluminescence reagent A and B in a 1:1 ratio and mix thoroughly.

[0089] (13) Immerse the PVDF film in the luminescent liquid, take it out and place it on the stage of the chemiluminescence imaging instrument, push it into the stage, set the exposure time to collect images, and use ImageJ software to analyze the gray values ​​of the strips.

[0090] In this embodiment, the isolation, culture, and induction procedures for porcine intramuscular preadipocytes are as follows:

[0091] (1) Primary isolation of porcine intramuscular preadipocytes:

[0092] Primary porcine intramuscular preadipocytes were isolated using collagenase digestion. The specific steps are as follows:

[0093] a. Three-day-old piglets were injected with sodium pentobarbital solution, and died after being put into a coma and bleeding out of their hearts.

[0094] b. Clean the whole body with 1-5% (v / v) benzalkonium chloride, soak in 75% ethanol for 20-30 minutes, and separate the longissimus dorsi muscle tissue under aseptic conditions;

[0095] c. Wash the tissue 3-5 times with PBS containing double the amount of antibiotics. Use sterilized ophthalmic scissors to remove visible connective tissue and blood vessels, and cut the tissue into 1-2 mm pieces. 3 A tissue block of about the same size;

[0096] d. Add twice the volume of type I collagenase digestion solution to each gram of tissue, and digest at 37°C for 1-2 hours, shaking repeatedly during the process to promote complete tissue digestion;

[0097] e. Neutralize the digestion solution with an equal volume of DMEM / F12 medium containing 10% FBS to terminate digestion, filter using 70-mesh and 200-mesh cell sieves, and collect the filtrate;

[0098] f. Centrifuge the collected filtrate at 1500 rpm for 10 min, discard the supernatant, resuspend the cells in serum-free DMEM / F12 medium, centrifuge at 1500 rpm for 10 min, and repeat this step twice.

[0099] g. Resuspend cells in DMEM / F12 medium containing 10% FBS;

[0100] h. After cell counting, at 2.5 × 10 5 Live cells were seeded at a density of 10 cm culture dish, transferred to a 37°C 5% CO2 incubator and cultured for 1 h. The medium was then changed and the cells were washed with sterile PBS until the background was clean.

[0101] i. Replace with fresh complete culture medium and transfer the cells to a 37°C 5% CO2 incubator for continued culture;

[0102] (2) Passage of porcine intramuscular preadipocytes

[0103] When the cells reach 80-90% confluence, passage them. Discard the old culture medium, wash the cells once with PBS, and then add 2 mL of 0.25% trypsin digestion solution to digest for 2-3 min. Add twice the volume of complete culture medium to stop digestion, pipette and agitate the bottom of the culture dish to remove all cells from the bottom, collect the cell suspension in a 1.5 mL centrifuge tube, and centrifuge at 1500 rpm for 10 min. Discard the supernatant, add 2 mL of complete culture medium to resuspend the cells, and seed them according to experimental requirements. After shaking the cells well, transfer them to a 37℃ 5% CO2 incubator for culture.

[0104] (3) Induction and differentiation of porcine intramuscular preadipocytes

[0105] Once the cells reached 90% confluence, they were cultured in complete medium for about 2 days to induce contact inhibition. After inducing differentiation for 2 days, they were cultured in complete medium containing 0.5 mM IBMX, 1 μM DEX and 5 μg / mL insulin (i.e., induction medium). Then, they were cultured in complete medium containing 5 μg / mL insulin (i.e., maintenance medium). The medium was changed every 2 days until the cells were used for subsequent experiments.

[0106] All cells used in all experimental groups in this study were F3 generation porcine intramuscular adipocytes.

[0107] In this embodiment, the Oil Red O staining procedure is as follows:

[0108] Cells induced to differentiate into lipids for 8 days were stained with Oil Red O. The specific steps are as follows:

[0109] (1) Discard the waste culture medium, wash with PBS 2-3 times, and fix with Oil Red O fixative for 20-30 min;

[0110] (2) Discard the fixative and wash the cells 2-3 times with distilled water;

[0111] (3) Add 60% isopropanol and rinse for 20-30 seconds;

[0112] (4) After discarding 60% isopropanol, add freshly prepared Oil Red O staining solution (Oil Red O staining solution A: Oil Red O staining solution B = 3:2) and immerse for 10-20 minutes. The immersion time can be adjusted according to the staining situation.

[0113] (5) Discard the staining solution, rinse with 60% isopropanol until the interstitial tissue is clear, and then rinse with distilled water until there is no excess staining solution.

[0114] (6) Add Mayer's hematoxylin staining solution and counterstain the nuclei for 1-2 min. Discard the staining solution and wash with water 2-5 times. Add Oil Red O buffer for 1 min and discard.

[0115] (7) Add distilled water to cover the cells and observe and photograph them under a microscope;

[0116] (8) Add 1 mL of isopropanol to each well of a six-well plate, shake on a shaker for 5 min, and transfer 100 μL of the solution to a 96-well plate. Measure the solution at 490 nm using a microplate reader. [142,143] The absorbance value was recorded.

[0117] In this embodiment, the triglyceride content determination procedure is as follows:

[0118] On day 8 after the cells were induced to differentiate into lipids, the old culture medium was discarded, the cells were washed with PBS 2-3 times, and 600 μL of Triton X-100 (1-2%) solution was added to each well of a six-well plate. The cells were lysed for 30-40 min, and the cells were repeatedly pipetted. The cells were observed under a microscope to see if they were completely lysed. After complete lysis, the liquid was transferred to a 1.5 mL EP tube.

[0119] The total protein quantification kit (A045-3-2) and triglyceride assay kit (A110-1-1) were used to determine and calculate the triglyceride content of the samples, and the data were recorded. The triglyceride data were normalized during intergroup comparison analysis.

[0120] In this embodiment, the transfection efficiency measurement procedure is as follows:

[0121] (1) Synthesis of transfection reagent

[0122] The SORBS1-siRNA used in the experiment was purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd., and the sequence design is shown in Table 2-8.

[0123] Continued from Table 2-8: Sequence Information

[0124]

[0125] (2) Cell transfection

[0126] Cells were packed at 8×10 5 Cells were seeded per well in 6-well plates and transferred to a 37°C CO2 incubator for culture. Transfection was performed when the cell confluence reached approximately 50%.

[0127] SORBS1-siRNA transfection efficiency assay groups: NC, SORBS1-Sus-1507, SORBS1-Sus-766, SORBS1-Sus-1860.

[0128] Each group had 3 replicates. The transfection steps were as follows:

[0129] a. Number several 1.5mL EP tubes according to the above grouping. Add 250μL of OPTI-MEM serum-depleted medium and 5μL of Lipofectamine to each 1.5mL EP tube. TM 2000, gently blow to mix, and incubate at room temperature for 5 minutes;

[0130] b. Take a new 1.5 mL EP tube, add 250 μL of OPTI-MEM serum-depleted medium, and add 100 nM NC, 100 nM si-SORBS1-Sus-1507, 100 nM si-SORBS1-Sus-766, and 100 nM si-SORBS1-Sus-1860 in separate groups. Gently pipette to mix, and incubate at room temperature for 5 min.

[0131] c. Add the mixture from step b to the mixture from step a dropwise, gently blow and mix, and incubate at room temperature for 15-20 minutes.

[0132] d. Change the medium for the cells to be transfected, add 1500 μL of antibiotic-free complete medium to each well, add the mixture from step c to the well plate, gently shake to mix, and then transfer to a 37℃ CO2 incubator for culture. After 6 h, change to complete medium. After 24 h of transfection, collect cells to extract RNA, and after 48 h of transfection, extract cell proteins.

[0133] (3) Total RNA extraction and quality testing of porcine intramuscular preadipocytes.

[0134] (4) cDNA synthesis and fluorescence quantification

[0135] The methods for mRNA reverse transcription and qPCR are the same as above.

[0136] (5) The siRNA sequence with the best transfection efficiency was selected and denoted as si-SORBS1.

[0137] (6) Westernblots

[0138] To verify whether si-SORBS1 can knock down the target protein at the protein level, follow the same steps as above.

[0139] In this embodiment, the time-series representation detection operation is as follows:

[0140] Cells were packed at 8×10 5Cells were seeded per well in 6-well plates at 6 time points: 0d, 2d, 4d, 6d, 8d, and 10d, with 3 replicates at each time point. Cells were then transferred to a 37℃ CO2 incubator for culture. When cell confluence reached approximately 90%, cells were cultured in complete medium for about 2 days to induce contact inhibition. This was then switched to induction medium, recorded as day 0. After 2 days of induction, cells were cultured in maintenance medium until day 10, with the maintenance medium changed every 2 days. Cell samples from days 0, 2, 4, 6, 8, and 10 were collected using the Trizol assay for RNA extraction and quantitative PCR, following the same steps as in 2.2.3, 2.2.4, and 2.2.5. The expression level of SORBS1 at these 6 time points was measured to detect the temporal expression profile of SORBS1 during porcine intramuscular preadipocyte differentiation.

[0141] In detail, the CCK8 assay for proliferation efficiency is performed according to the following steps:

[0142] (1) The cells were arranged at 2×10 4 Cells were seeded per well in 96-well plates and transferred to a 37°C CO2 incubator for culture. Transfection was performed when the cell confluence reached approximately 50%.

[0143] (2) SORBS1-siRNA transfection assay grouping: NC and si-SORBS1; 5 replicates per group;

[0144] (3) Cell transfection

[0145] a. Add 25 μL of OPTI-MEM serum-depleted medium and 1 μL of Lipofectamine to a 1.5 mL sterile, enzyme-free EP tube. TM 2000, gently blow to mix, and incubate at room temperature for 5 minutes;

[0146] b. Take a new 1.5 mL sterile enzyme-free EP tube and add 25 μL of OPTI-MEM serum-depleted medium, and add 100 nM NC and 100 nM si-SORBS1 in groups. Gently pipette to mix, and incubate at room temperature for 5 min;

[0147] c. Add the mixture from step b to the mixture from step a dropwise, gently blow and mix, and incubate at room temperature for 15-20 minutes.

[0148] d. Replace the cells to be transfected with antibiotic-free medium, add 10 μL of the mixture from step c to each well, and incubate in a 37℃ CO2 incubator. After 6 h, replace with complete medium. After 24 h, 48 h and 72 h of transfection, add 10 μL of CCK8 reagent to each well, incubate at 37℃ for 1 h, and then measure the absorbance at 450 nm and record the data.

[0149] (4) RT-qPCR assay of cell proliferation factors

[0150] Cell plating, experimental grouping, and cell transfection methods are the same as above. Use 8 × 10⁸ cells per cell line. 5 Each sample was seeded into a 6-well plate, with 3 replicates per group during transfection. Samples were collected 24 hours after transfection, and RNA was extracted using the Trizol method. After reverse transcription, quantitative PCR was performed using the same method as above.

[0151] In detail, the detection of adipogenic differentiation-related indicators was carried out according to the following steps:

[0152] (1) The cells were arranged at 8×10 5 Cells were seeded per well in 6-well plates and transferred to a 37°C CO2 incubator for culture. Transfection was performed when the cell confluence reached about 50%.

[0153] (2) SORBS1-siRNA transfection assay grouping: NC and si-SORBS1; each group had 3 replicates;

[0154] (3) Transfection steps

[0155] The operation method is the same as above.

[0156] (4) Induced differentiation

[0157] The operation method is the same as above.

[0158] (5) Oil Red O staining

[0159] The operation method is the same as above.

[0160] (6) Triglyceride content determination

[0161] The operation method is the same as above.

[0162] (7) RT-qPCR determination of adipogenic differentiation-related genes

[0163] The procedure was the same as above. Cells induced to differentiate for 2d, 4d, 6d and 8d were collected respectively. RNA was extracted using the Trizol method, reverse transcribed and then subjected to fluorescence quantification, using the same method as above.

[0164] Data analysis was conducted based on the above experiments;

[0165] Gene expression levels in RT-qPCR experiments were measured using 2... –ΔΔCTThe methods were used for calculation. Each experiment had at least three biological replicates. Statistical analysis and graphing were performed using Graphpad Pism 9.5 software. Results are expressed as mean ± standard deviation (M ± SD). Data between two groups were analyzed using t-tests, and data from three or more groups were analyzed using one-way ANOVA. At the significance level, ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0166] It should be noted that, in order to verify whether SORBS1 plays a role in porcine intramuscular preadipocytes, RT-qPCR was performed on cells induced to differentiate into adipocytes at 0d, 2d, 4d, 6d, 8d, and 10d to determine the expression level of SORBS1 at different time points during the adipogenic differentiation of porcine intramuscular preadipocytes, and a temporal expression profile of the three was constructed. Figure 1 The results showed that SORBS1 expression was significantly upregulated during adipogenic differentiation (P < 0.01). SORBS1 expression increased significantly from day 0 to day 2, remained relatively stable from day 2 to day 4, and then rapidly increased from day 4 to day 10, reaching a peak expression level 20 times higher than the initial level (day 0). These results suggest that SORBS1 may play a role in the adipogenic differentiation of porcine intramuscular preadipocytes, providing a basis for further research on SORBS1 function.

[0167] An analysis was conducted on the effects of SORBS1 interference on the proliferation and adipogenic differentiation of porcine intramuscular preadipocytes.

[0168] Including SORBS1-siRNA interference efficiency detection and analysis

[0169] In detail, RT-qPCR results showed that, compared with the NC group, the expression level of SORBS1 was significantly decreased after transfection with si-SORBS1-Sus-766 (P < 0.001). Figure 2 A) has the best interference efficiency. Si-SORBS1-Sus-766 is denoted as Si-SORBS1 for subsequent Western blotting experiments. Western blotting revealed that (… Figure 2 In groups B and C, the SORBS1 protein level in the si-SORBS1 group was significantly lower than that in the NC group (P < 0.001). This indicates that the SORBS1 interference sequence was successfully constructed and can be used for subsequent experiments.

[0170] Analysis of the effect of SORBS1 interference on the proliferation of porcine intramuscular preadipocytes

[0171] In detail, to clarify the regulatory role of SORBS1 on the proliferation of porcine intramuscular preadipocytes, the expression level of the SORBS1 gene in porcine intramuscular preadipocytes was reduced by transfecting si-SORBS1, and changes in cell proliferation and the expression levels of proliferation-related genes were observed. CCK8 results showed that ( Figure 3 A) The OD values ​​(450 nm) of the si-SORBS1 group at 24h, 48h, and 72h after transfection were significantly lower than those of the NC group (P < 0.01), indicating weakened cell proliferation. Simultaneously, RT-qPCR results showed ( Figure 3 B) After SORBS1 interference, the expression levels of PCNA, CDK1, CDK2, and CCND1 genes were all significantly reduced (P < 0.01). These results indicate that SORBS1 may participate in cell proliferation by regulating the expression levels of proliferation-related genes during the proliferation of porcine intramuscular preadipocytes, and that interference with SORBS1 inhibits the proliferation of porcine intramuscular preadipocytes.

[0172] Analysis of the effect of SORBS1 interference on adipogenic differentiation of porcine intramuscular preadipocytes

[0173] In detail, to clarify the specific role of SORBS1 in adipogenic differentiation of porcine intramuscular preadipocytes, the expression level of SORBS1 was interfered with. It was found that the adipogenic effect in the si-SORBS1 group was significantly worse than that in the NC group. Figure 4 A), the quantitative results of isopropanol-extracted oil red O further confirmed the above results. Figure 4 B), and there was a highly significant difference between the two groups (P < 0.001), and TG measurements showed that the triglyceride content in the si-SORBS1 group was significantly lower than that in the NC group (P < 0.01). Figure 4 C). To further clarify the mechanism of action of SORBS1 on porcine intramuscular preadipocytes, RT-qPCR was used to detect changes in the expression levels of adipogenic differentiation-related genes at 2, 4, 6, and 8 days after SORBS1 interference induction. The results showed that the expression level of PPARγ in the si-SORBS1 group was significantly lower than that in the control group on day 4 of induction (P < 0.01), and significantly lower than that in the control group on days 6 and 8 (P < 0.05); C / EBPβ was significantly lower than that in the control group on days 2, 6, and 8 (P < 0.05); FABP4 and FASN were significantly lower than those in the control group on day 4 (P < 0.05), and significantly lower than those in the control group on days 6 and 8 (P < 0.01). Figure 4 D). These data indicate that interfering with SORBS1 downregulates the expression levels of adipogenic differentiation-related genes, thereby inhibiting adipogenic differentiation of porcine intramuscular preadipocytes.

[0174] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A SORBS1 inhibitor, characterized in that, include: At least one of SORBS1-siRNA1, SORBS1-siRNA2, and SORBS1-siRNA3; The nucleic acid sequence of SORBS1-siRNA1 is shown in SEQ ID NO.1 and SEQ ID NO.2; The nucleic acid sequence of SORBS1-siRNA2 is shown in SEQ ID NO.3 and SEQ ID NO.4; The nucleic acid sequence of SORBS1-siRNA3 is shown in SEQ ID NO.5 and SEQ ID NO.

6.

2. A product, characterized in that, It contains the SORBS1 inhibitor as described in claim 1.

3. The product according to claim 2, characterized in that, It also contains medically acceptable excipients.

4. The use of the SORBS1 inhibitor as claimed in claim 1, or the product as claimed in claim 2 or 3, in any of the following: B1) Prepare products that interfere with SORBS1 expression; B2) Prepare a product that inhibits the expression of SORBS1 protein in preadipocytes; B3) Prepare products that inhibit the proliferation of proadipocytes; B4) Prepare products that inhibit the expression of cell proliferation transcription factors in precursor adipocytes; B5) Prepare products that inhibit adipogenic differentiation of precursor adipocytes; B6) Prepare products that inhibit the expression of adipogenic differentiation genes in precursor adipocytes; B7) Prepare products that inhibit lipid droplet formation or aggregation in precursor adipocytes; B8) Prepare products that inhibit the accumulation of triglycerides in precursor adipocytes; B9) Regulation of fat deposition traits in pig breeding.

5. The application according to claim 4, characterized in that, The precursor adipocytes are intramuscular precursor adipocytes, which are derived from mammals.

6. The application according to claim 5, characterized in that, The mammal in question is a pig.

7. The application according to claim 4, characterized in that, In B4), the cell proliferation transcription factor is at least one of PCNA, CDK1, CDK2, and CCND1; In B6), the adipogenic differentiation gene is at least one of PPARγ, C / EBPβ, FABP4, and FASN.

8. The application according to claim 4, characterized in that, In B9), it is applied to molecular breeding of pork quality traits.

9. A method for inhibiting the expression of SORBS1 protein in precursor adipocytes, characterized in that, Preadipocytes were transfected with the SORBS1 inhibitor as described in claim 1.

10. The method according to claim 9, characterized in that, Preadipocytes were transfected using the SORBS1 inhibitor as described in claim 1, Opti-MEM medium, and Lipofectamine 2000.

Images