RNA methylation modified circRNA marker related to porcine adipocyte generation and application of RNA methylation modified circRNA marker

Abstract

The invention discloses an RNA (Ribonucleic Acid) methylation modified circRNA (Ribonucleic Acid) marker related to generation of porcine fat cells and an application of the RNA methylation modified circRNA marker. The invention finds that RNA methylation modification can regulate and control the expression of circRNA-circIGF1R of pigs for the first time, and has the function of regulating and controlling adipogenic differentiation of porcine adipocytes. The invention lays a theoretical foundation for understanding the effect of RNA methylation modification in regulating and controlling adipogenic differentiation of porcine cells through circRNA, and provides a new theoretical basis for improving pork quality by using RNA methylation modification.

Description

Technical Field

[0001] The present invention relates to the field of molecular genetic technology, and in particular to a circRNA marker of RNA methylation modification associated with pig adipocyte generation and an application thereof. Background Art

[0002] The most common epigenetic modification in eukaryotes is N6-methyladenosine (m 6 A) Methylation modification, whose role in adipogenesis is gradually revealed, but whether there is mRNA on noncoding RNA (ncRNAs) 6 A modification to regulate fat development has not yet been elucidated. Circular RNAs (circRNAs) have become an important tool for analyzing animal economic traits due to their good stability and RNase R resistance. Studies have shown that circRNAs play a regulatory role in cell proliferation, but there are few studies on pig fat development, and m 6 Whether A modification regulates adipogenic differentiation through circRNA remains to be studied. 6 A methylation modification regulates pig adipocyte production through circRNA, which can provide a theoretical basis for improving pork quality and healthy production of animal husbandry. Summary of the invention

[0003] The purpose of the present invention is to provide a circRNA marker of RNA methylation modification related to pig adipocyte generation and application thereof.

[0004] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides RNA m 6 A. Any of the following applications of methylation modification-related protein METTL3: (1) Used to regulate porcine adipocyte proliferation and adipogenic differentiation; Specifically, the regulation refers to positive regulation of porcine adipocyte proliferation and negative regulation of adipogenic differentiation; (2) mRNA for positive regulation of porcine circIGF1R 6 A modification level.

[0005] In a second aspect, the present invention provides RNA m 6 A. Any of the following uses of an inhibitor of a methylation modification-related protein METTL3: (1) Used to inhibit porcine adipocyte proliferation and promote adipogenic differentiation; (2) Used to reduce the m of porcine circIGF1R 6 A modification level.

[0006] The inhibitor may be selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, antibodies, and the like.

[0007] Preferably, the inhibitor is STM-2457, the chemical formula of which is C 25 H 28 N6O2 (CAS number is 2499663-01-1).

[0008] In a third aspect, the present invention provides RNA m 6 A. Any of the following applications of methylation modification-related protein FTO: (1) Used to regulate porcine adipocyte proliferation and adipogenic differentiation; Specifically, the regulation refers to positive regulation of pig adipocyte proliferation and adipogenic differentiation; (2) mRNA for negative regulation of porcine circIGF1R 6 A modification level.

[0009] In a fourth aspect, the present invention provides RNA m 6 A. The following applications of inhibitors of methylation modification-related protein FTO: (1) Used to inhibit the proliferation and adipogenic differentiation of porcine adipocytes; (2) Used to increase the m of porcine circIGF1R 6 A modification level.

[0010] The inhibitor may be selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, antibodies, and the like.

[0011] Preferably, the inhibitor is STM-2457, the molecular formula is C 18 H 15 Cl2N3O3 (CAS number is 2243736-45-8).

[0012] In the present invention, the reference sequence number of protein METTL3 in NCBI is NP_062695.2; the reference sequence number of protein FTO in NCBI is NP_001106162.1; the sequence of pig circIGF1R is shown in SEQ ID NO:1.

[0013] In a fifth aspect, the present invention provides a circRNA marker of RNA methylation modification associated with pig adipocyte formation, which is pig circIGF1R.

[0014] In a sixth aspect, the present invention provides the use of porcine circIGF1R in promoting adipogenic differentiation of porcine adipocytes.

[0015] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention provides a circRNA marker for RNA methylation modification related to pig adipocyte formation, and for the first time finds that the circRNA marker can be modified by changing RNA m 6 A modification level affects the proliferation and adipogenic differentiation of pig adipocytes. 6 The specific inhibitors of A methyltransferase METTL3, STM-2457 and m 6 After the induction of FB23-2, a specific inhibitor of A demethylase FTO, flow cytometry and EdU staining revealed that the number of cells in the cell proliferation stage decreased, and qRT-PCR results showed that the expression of proliferation-related genes was significantly reduced ( p <0.01), which showed that STM-2457 / FB23-2 could inhibit the proliferation of pig adipocytes. After adipogenesis induction, Oil Red O staining and qRT-PCR results showed that STM-2457 promoted cell adipogenesis, while FB23-2 had the opposite phenotype. In addition, circIGF1R was predicted to have multiple m 6 A modification site, and the results of qRT-PCR and MeRIP-qPCR showed that the addition of STM-2457 significantly reduced the m 6 A modification level ( P <0.01). When circIGF1R was interfered with in cells and adipogenic differentiation was induced, it was found that lipid droplets were significantly reduced and the expression of key adipogenic genes was extremely significantly reduced ( P <0.01); rescue experiments were designed to find that interfering with the expression of circIGF1R reduced the effect of STM-2457 on promoting adipogenic differentiation of cells. The present invention provides a new theoretical basis for the application of RNA methylation-modified circRNA markers in improving pork quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 In the preferred embodiment of the present invention, 6 A Expression analysis of methyltransferase METTL3. Among them, a: tissue expression profile of METTL3; b: expression of METTL3 at different stages of porcine preadipocyte differentiation; c: expression of METTL3 in adipose tissue of horse-skin pigs and Duroc pigs; d: expression of METTL3 in adipose tissue of obese model mice; ef: expression of METTL3 in adipose tissue of obese model mice 6 A: methylation modification level; g: interference efficiency of si-METTL3; hi: m after adding si-METTL3 6A: methylation modification level; j: expression level of METTL3 after adding STM-2457; kl: m 6 AMethylation modification level.

[0017] Figure 2 In the preferred embodiment of the present invention, 6 A Expression analysis of demethylase FTO. Among them, a: tissue expression profile of FTO; b: expression of FTO at different stages of porcine preadipocyte differentiation; c: expression of FTO in adipose tissue of horse-skin pigs and Duroc pigs; d: expression of FTO in adipose tissue of obese mice; ef: expression of FTO in adipose tissue of obese mice after adding FB23-2. 6 A methylation modification level

[0018] Figure 3 The effect of METTL3 / FTO on cell proliferation in the preferred embodiment of the present invention. Among them, a: the results of cell flow cytometry analysis after adding STM-2457; b: the effect of STM-2457 on the expression of cell proliferation-related genes; c: the results of EdU test after adding STM-2457 to cells; d: the results of cell flow cytometry analysis after adding FB23-2; e: the effect of FB23-2 on the expression of cell proliferation-related genes; f: the results of EdU test after adding FB23-2 to cells.

[0019] Figure 4 The effect of METTL3 / FTO on cell adipogenic differentiation in a preferred embodiment of the present invention. Among them, a: Oil Red O staining results of cells after adding STM-2457; b: changes in the expression level of adipogenic marker gene mRNA after adding STM-2457; c: Oil Red O staining results of cells after adding FB23-2; d: changes in the expression level of adipogenic marker gene mRNA after adding FB23-2.

[0020] Figure 5 In the preferred embodiment of the present invention, 6 A related genes affect circIGF1R. Among them, a: circIGF1Rm 6 A: Prediction of modification sites; bc: Effects of knocking down METTL3 on circIGF1R; de: Effects of knocking down FTO on circIGF1R; f: MeRIP-qPCR detection of the effect of adding STM-2457 on circIGF1R m 6 A. Effect of modification level.

[0021] Figure 6This is the preferred embodiment of the present invention that interferes with circIGF1R to inhibit the adipogenic differentiation of cells. Among them, a: si-circIGF1R interference efficiency detection; b: Oil Red O staining results; c: The expression changes of the mRNA level of adipogenic marker genes after adding si-circIGF1R.

[0022] Figure 7 This is a preferred embodiment of the present invention in which METTL3 affects cell adipogenic differentiation through circIGF1R. Wherein, a: Oil Red O staining results; b: Expression changes of adipogenic marker gene mRNA levels.

[0023] Figure 8 In the preferred embodiment of the present invention, circIGF1R can act as a sponge for miR-15a, miR-16 and miR-885-3p. Among them, ac: tissue expression profiles of miR-15a, miR-16 and miR-885-3p; df: expression of miR-15a, miR-16 and miR-885-3p at different stages of differentiation of porcine preadipocytes; gi: RNAhybird predicted the binding sites of circIG1R and miR-15a, miR-16 and miR-885-3p; jl: expression levels of miR-15a, miR-16 and miR-885-3p after interfering with circIGF1R; m: RIP test verified the binding of miR-15a, miR-16, miR-885-3p to circIGF1R.

[0024] Fig. 9 The effects of miR-15a, miR-16 and miR-885-3p on cell adipogenic differentiation in the preferred embodiment of the present invention. ac: miR-15a, miR-16 and miR-885-3p overexpression efficiency; d: Oil Red O staining results after adding miR-15a mimics, miR-16 mimics and miR-885-3p mimics; e: Expression changes of adipogenic marker gene mRNA levels after adding miR-15a mimics, miR-16 mimics and miR-885-3p mimics.

[0025] Fig.10NAT10 is a downstream target gene of miR-15a, miR-16 and miR-885-3p in a preferred embodiment of the present invention. Among them, a: tissue expression profile of NAT10; b: expression of NAT10 at different stages of differentiation of porcine preadipocytes; c: expression level of NAT10 in adipose tissue of horse-skinned pigs and Duroc pigs; df: RNAhybird predicted binding sites of NAT10 with miR-15a, miR-16 and miR-885-3p; g: expression level of NAT10 after overexpression of miR-15a, miR-16 and miR-885-3p; h: expression level of NAT10 after interference with circIG1R; i: RIP test verified the binding of miR-15a, miR-16, miR-885-3p to NAT10.

[0026] Fig.11 This is a preferred embodiment of the present invention that interferes with NAT10 to inhibit the adipogenic differentiation of porcine preadipocytes. Among them, a: NAT10 interference efficiency detection; b: Oil Red O staining results after si-NAT10; c: Changes in the expression level of adipogenic marker gene mRNA after si-NAT10.

[0027] DETAILED DESCRIPTION

[0028] The present invention aims to provide a circRNA marker of RNA methylation modification related to pig adipocyte generation and application thereof.

[0029] The present invention adopts the following technical solution: The present invention provides RNA m 6 A. Effect of methylation modification-related protein METTL3 / FTO on porcine cell proliferation and adipogenic differentiation.

[0030] The present invention also provides RNA m 6 Application of A methylation modification-related protein METTL3 / FTO in regulating porcine circIGF1R.

[0031] The present invention also provides a circRNA marker of RNA methylation modification related to pig adipocyte generation and a method for using the same.

[0032] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0033] Example 1 RNA m 6 Effects of A methylation-related protein METTL3 / FTO on the proliferation and adipogenic differentiation of porcine adipocytes

[0034] 1. Materials and Methods

[0035] 1.1 Test materials

[0036] 1.1.1 Experimental animals, experimental cells and experimental reagents

[0037] In this study, three 6-month-old castrated boars were selected as biological replicates. After slaughter, the heart, liver, spleen, lung, kidney, abdominal subcutaneous fat, back subcutaneous fat, longissimus dorsi, psoas major and other tissues were collected, quickly put into liquid nitrogen, and then stored in a -80℃ refrigerator for a long time. Three 6-month-old Duchangda castrated boars were selected as biological replicates. After slaughter, the abdominal fat, subcutaneous fat, longissimus dorsi and other tissues were collected, quickly put into liquid nitrogen, and then stored in a -80℃ refrigerator for a long time.

[0038] Newborn piglets were used as experimental animals to isolate porcine preadipocytes.

[0039] STM-2457 and FB23-2 were purchased from MCE, USA; fetal bovine serum, collagenase type II and Opti-MEM were purchased from Gibco, USA; m 6 A Rabbit pAb (m 6 A antibody) was purchased from Wuhan ABclonal Company; trypsin, penicillin-streptomycin, high-glucose DMEM and insulin were purchased from Sigma Company, USA; PBS, dexamethasone, IBMX, 4% paraformaldehyde, dimethyl sulfoxide (DMSO), red blood cell lysis buffer and saturated oil red O stain were purchased from Solarbio Company, Beijing; indomethacin was purchased from Aladdin; Trizol Reagen and PARISTM Kit were purchased from Life Technologies Company, USA; m 6 A RNA methylation quantitative detection kit (colorimetric method) was purchased from Beijing Aide Technology Co., Ltd.; Prime Script RT reagent Kit with gDNA Eraser and SYBR Premix Ex Taq II were purchased from Beijing Quanshijin; Lipofectamine 3000 was purchased from Thermo Fisher; kFluor555 Click-iT EdU imaging detection kit was purchased from Ruibo Biotechnology Co., Ltd.; chloroform, isopropanol, anhydrous ethanol, and xylene were purchased from Zhiyuan Chemical Reagent Company.

[0040] 1.1.2 Preparation of test reagents

[0041] 15% complete culture medium: high glucose DMEM + 15% FBS + 1% penicillin-streptomycin double antibody.

[0042] Adipogenic induction medium: complete medium + 1 µmol / L DEX + 0.5 mmol / L IBMX + 10 µg / mL Ins + 100 µmol / L IND. Among them, DEX: Dexamethasone, IBMX: 3-Isobutyl-1-methylxanthine, Ins: Insulin, IND: Indomethacin.

[0043] Maintenance medium: complete medium + 10 µg / mL Ins.

[0044] 1.1.3 Test equipment

[0045] Real-time fluorescence quantitative PCR instrument (Bio-RAD, USA); ordinary PCR instrument (Bio-RAD, USA); WD-9403B UV transmission gel cutting station (Liuyi Biotechnology, Beijing); ultra-clean workbench (Purification, Suzhou); nucleic acid protein analyzer (Thermo, USA); electrophoresis instrument and transfer instrument (Bio-RAD, Beijing); constant temperature culture oscillator, electric constant temperature incubator (Zhicheng, Shanghai); high pressure steam sterilizer (PHCbi, Japan); ice machine (Panasonic, Japan); near-infrared dual-color laser and chemiluminescence dual-function imaging system (Odyssey FC, LI-COR, USA); PCR instrument (Applied Biosystems, USA) high-speed refrigerated centrifuge (Eppendorf, Germany); CO2 cell culture incubator (Thermo, USA); EVOS® FL Auto fully automatic cell imaging system (Life Technologies, USA).

[0046] 1.2 Test methods

[0047] 1.2.1 Isolation, culture and adipogenesis induction of porcine preadipocytes

[0048] Porcine preadipocytes are isolated from porcine adipose tissue. Select 0-day-old piglets and kill them by electroporation, and rinse the whole body with distilled water and 75% alcohol. Under sterile conditions, collect subcutaneous adipose tissue from the back and neck, put them into PBS with double antibodies, rinse the adipose tissue with PBS containing different concentrations of double antibodies, and trim the tissue blocks. Put the trimmed tissue blocks into a small beaker and cut them into pieces, then digest them with 2 mg / mL type II collagenase for 1 hour, and terminate the digestion with an equal amount of complete medium. After filtering the cell suspension through 70µm and 40µm filters in turn, resuspend the filtrate and inoculate it into a 100mm culture dish, and culture it in an incubator at 37°C, 5% CO2, and 95% O2. After the cells are full, replace the induction differentiation medium, replace the maintenance medium after 2 days of culture, collect the cells after 8 days of culture, and change the medium once every 2 days.

[0049] 1.2.2 Total RNA extraction, cDNA synthesis and qRT-PCR detection

[0050] Total RNA was extracted from tissues or cells according to the instructions of the Trizol kit and stored in a −80°C refrigerator. RNA was reverse transcribed into complementary DNA (cDNA) of mRNA according to the instructions of the PrimeScript RT kit. After the cDNA and primers were diluted to appropriate concentrations according to the instructions of the SYBR® PrimeScriptTM RT-PCR kit, qRT-PCR was performed on a real-time polymerase chain reaction system using cDNA as a template. 18S rRNA, β-actin or U6 was used as the internal reference gene, and each sample was repeated 3 times. The quantitative data was calculated according to 2 -ΔΔCt Calculation. Primer sequences are shown in Table 1.

[0051] Table 1 Primer sequences

[0052] 1.2.3 m 6 A methylation modification detection

[0053] Spot blotting: First, RNA was denatured. An equal amount of denaturing solution was added to the RNA sample and mixed. The mixture was reacted at 95°C for 5 min. The denatured sample was quickly spotted on the NC membrane and then irradiated under a 302 nm UV lamp, rotating every 30 s for a total of 6 min. The cross-linked NC membrane was blocked with milk and m 6 The same treatment was performed on another NC membrane, but after UV crosslinking, milk blocking and m 6 A antibody incubation, methylene blue staining and photography.

[0054] m 6A. Quantitative detection of RNA methylation (colorimetric method): Follow the instructions of the kit. First, add 200 ng of RNA of the sample to be tested to each well and add the binding solution. After incubation at 37°C for 90 min, remove the solution and wash each well. Then add the capture antibody solution, detection antibody solution and enhancer solution to each well (the capture antibody solution, detection antibody solution and enhancer solution are all reagents in the kit, and the kit was purchased from Beijing Aide Technology Co., Ltd., AP-9005), and wash. Finally, add the developer and incubate in the dark for 1 to 10 min and observe the color change. When the color of the positive control well turns to medium blue, add the stop solution and read the absorbance at 450 nm on the microplate reader. In this study, m 6 The relative quantification method of A was used for calculation, where S is the amount of input sample RNA in ng; P is the amount of input positive control in ng:

[0055] 1.2.4 Cell transfection

[0056] When the cell density reached about 60%, the cell culture medium was replaced with Opti-MEM basal medium, and the transfection reagent was prepared and transfected according to the instructions of Lipofectamine 3000. Three replicate wells were set up in each group, and the medium was changed 6-8 hours after transfection, and the cells were collected 48 hours later. The siRNA sequences are shown in Table 2.

[0057] Table 2 siRNA sequences

[0058] 1.2.5 EdU imaging to detect cell proliferation

[0059] Cells were seeded in 24-well plates, and EdU staining was performed when the cell density reached the logarithmic growth phase. The operation was performed according to the instructions of the kFluor 555 Click-iT EdU imaging detection kit. First, 2×EdU stock solution was diluted to 10 µM with complete culture medium, and 200 µL was added to each well. After incubation at 37°C for 2 hours, the culture medium was removed, and 200 µL of 4% paraformaldehyde was added to fix at room temperature for 30 minutes. Then, 200 µL of 2 mg / mL glycine solution, 400 µL of 0.5% Triton X-100 permeabilization solution, and Click-iT reaction mixture were added to each well in turn, and then washed. Finally, 200 μL of 5 μg / mL Hoechst33342 stock solution was added to each well, incubated at room temperature in the dark for 30 minutes, and then photographed, recorded, and analyzed using a fluorescence microscope after washing.

[0060] 1.2.6 Oil Red O staining

[0061] After 8 days of adipogenic differentiation, the cells were washed with PBS. After discarding the PBS, 4% paraformaldehyde was added, fixed for 30 min and then discarded. 60% isopropanol was added, and the cells were washed for 1 min and then discarded. The Oil Red O staining solution was prepared according to the instructions of the Oil Red O staining kit. After the cells were completely dry, the Oil Red O staining solution was added and stained for 10 min. After washing with PBS, the cells were observed and photographed under a microscope.

[0062] 2. Experimental Results

[0063] 2.1 m 6 Expression analysis of A-related protein METTL3 / FTO

[0064] qRT-PCR detection revealed that METTL3 was expressed in a variety of tissues. The highest expression levels were in the lung and back adipose tissue, followed by the spleen and kidney ( Figure 1 a). In addition, METTL3 showed an overall upward trend during the adipogenic differentiation of porcine preadipocytes and was expressed most highly in the late differentiation stage ( Figure 1 b). In the subcutaneous fat tissue of the abdomen and back of pigs of different developmental breeds, the level of METTL3 in the fat tissue of horse-body pigs was significantly higher than that in DLY pigs ( Figure 1 c; P <0.01). To further explore the function of METTL3 in fat development, a C57BL / 6J obese mouse model was established using a high-fat diet, and it was found that the METTL3 level in obese model mice was significantly lower than that in normal mice ( Figure 1 d; P <0.01). By dot blot and m 6 A RNA methylation quantitative detection found that obesity model mice m 6 A methylation modification level was significantly lower than that of normal mice ( Figure 1 , ef). It is speculated that METTL3 may regulate the adipogenic differentiation of porcine preadipocytes. 6 A modification, using siRNA to interfere with METTL3, found that the addition of si-METTL3 significantly downregulated the expression of the METTL3 gene ( Figure 1 g; P <0.01), and si-METTL3-1 was used in subsequent experiments and was recorded as si-METTL3. 6 A RNA methylation quantitative detection found that RNA m 6 A methylation modification was significantly reduced ( Figure 1 , hi). In order to further explore the effect of METTL3 on m 6A specific inhibitor of METTL3, STM-2457, was selected. qRT-PCR detection showed that there was no significant difference in the expression of METTL3 after the addition of STM-2457 to the cells ( Figure 1 j), but RNA m 6 A methylation modification was significantly reduced ( Figure 1 , kl).

[0065] qRT-PCR detection revealed that FTO was expressed in a variety of tissues. The highest expression levels were found in the lungs and kidneys, followed by back fat and heart ( Figure 2 a). In addition, during the adipogenic differentiation of porcine preadipocytes, FTO showed an overall trend of first decreasing and then increasing, and its expression level was highest in the late stage of adipogenic differentiation ( Figure 2 -b). In the subcutaneous fat tissues of the abdomen and back of pigs of different developmental breeds, the expression level of FTO in the fat tissue of horse-body pigs was significantly higher than that in DLY pigs ( Figure 2 c; P <0.01); however, the expression level of FTO in obese model mice was significantly higher than that in normal mice ( Figure 2 d; P <0.01). In addition, we selected the FTO specific inhibitor FB23-2 and analyzed it by dot blot and m 6 A RNA methylation quantitative detection found that after adding FB23-2, RNA m 6 A methylation modification level increased significantly ( Figure 2 , ef).

[0066] 2.2 m 6 Effect of A-related protein METTL3 / FTO on cell proliferation

[0067] After adding STM-2457 to the cells, flow cytometry analysis of the cell cycle revealed that compared with the control group, the number of cells in the S phase in the STM-2457 group was significantly reduced, and the number of cells in the G2 / M phase was significantly increased ( Figure 3 a, P <0.05). The changes in the expression of cell proliferation-related genes were detected and it was found that the expression of PCNA was significantly reduced and the expression of Ki67 was significantly increased after the addition of STM-2457 ( Figure 3 b, P <0.05). The EdU results showed that the number of EdU positive cells in the STM-2457 group was significantly lower than that in the control group ( Figure 3 c). This indicates that the addition of STM2457 will inhibit cell proliferation. The reference sequence number of PCNA in NCBI is NM_011045.2, and that of Ki67 is NM_001081117.2.

[0068] After adding FB23-2 to the cells, flow cytometry analysis of the cell cycle revealed that compared with the control group, the number of cells in the S phase in the FB23-2 group was significantly reduced, and the number of cells in the G2 / M phase was significantly increased ( Figure 3 d, P <0.05). The changes in the expression of cell proliferation-related genes were detected and it was found that the expression of PCNA was significantly reduced and the expression of Ki67 was significantly increased after adding FB23-2 ( Figure 3 e, P <0.05). The EdU results showed that the number of EdU positive cells in the FB23-2 group was significantly lower than that in the control group ( Figure 3 f). This indicates that the addition of FB23-2 will inhibit cell proliferation.

[0069] 2.3 m 6 Effect of A-related protein METTL3 / FTO on cell adipogenic differentiation

[0070] STM-2457 was added to porcine primary preadipocytes and induced to differentiate into adipogenic cells for 8 days to detect the adipogenic status of cells. Compared with the control group, Oil Red O staining showed that the lipid droplets in the STM2457 group increased significantly ( Figure 4 a), qRT-PCR detection found that the expression levels of adipogenesis-related factors CEBPα, PPARγ and FABP4 were significantly increased ( Figure 4 b, P <0.01). The above results indicate that STM-2457 can promote adipogenic differentiation in primary porcine preadipocytes.

[0071] FB23-2 was added to primary porcine preadipocytes and induced to differentiate into adipogenic cells for 8 days to detect the adipogenic status of cells. Compared with the control group, Oil Red O staining showed that the lipid droplets in the group with FB23-2 addition were significantly reduced ( Figure 4 c), qRT-PCR detection found that the expression of adipogenesis-related factor mRNA was significantly reduced ( Figure 4 d, P <0.01). The above results indicate that FB23-2 can inhibit the adipogenic differentiation of primary porcine preadipocytes.

[0072] Example 2 RNA m 6 A methylation modification regulates porcine adipogenesis through circIGF1R

[0073] 1. Materials and Methods

[0074] 1.1 Test materials

[0075] 1.1.1 Test cells and test reagents

[0076] The test cells and test reagents are the same as those in Example 1.

[0077] 1.1.2 Test equipment

[0078] The test apparatus is the same as that in Example 1.

[0079] 1.2 Test methods

[0080] 1.2.1 Cell transfection

[0081] The transfection method was the same as in Example 1, and the sequences are shown in Table 3.

[0082] Table 3 Transfection sequence

[0083] 1.2.2 MeRIP-qPCR

[0084] Total RNA was extracted using TRIzol reagent and the m 6 A RNA methylation fragment enrichment kit instructions were used for the experiment. First, the immune capture solution (purchased from Beijing Aide Technology Co., Ltd., AP-9018) was prepared and mixed with RNA samples, m 6 Add A antibody and magnetic beads to a 0.2 ml PCR tube, incubate at room temperature for 90 min, add 10 ul of NDE and 2 ul of CEM, incubate at room temperature for 4 minutes, and remove the supernatant on a magnetic rack. After washing, add 20 µl of Protein Digestion Solution, incubate at 55℃ for 15 minutes, and remove the supernatant on a magnetic rack. Then add 20 ul of RPS, 160 ul of 100% ethanol, and 2 µl of RNABinding Beads and incubate for 5 minutes. Remove the supernatant on the magnetic rack and wash with 90% ethanol. Finally, resuspend the beads in 13 µl Elution Buffer, incubate at room temperature for 5 min, and transfer the solution to a new 0.2 ml PCR tube. After reverse transcription using the kit, perform qRT-PCR to detect the expression of related genes.

[0085] 2. Experimental Results

[0086] 2.1 m 6 Effects of A modification on circIGF1R

[0087] SRAMP online website was used to predict that circIGF1R had multiple m 6 A modification site ( Figure 5 a). In order to detect m 6Effects of A-related genes on circIGF1R. si-METTL3 and STM2457 were added to cells, and qRT-PCR detection showed that the expression of circIGF1R increased significantly after knocking down METTL3 ( Figure 5 , bc; P <0.01). After adding si-FTO and FB23-2 to the cells, the expression of circIGF1R was significantly reduced ( Figure 5 ,de; P <0.01) In addition, MeRIP-qPCR results showed that the addition of STM-2457 to cells significantly reduced the m 6 A modification level ( Figure 5 f; P <0.01).

[0088] 2.2 circIGF1R promotes cell adipogenic differentiation

[0089] To reveal the role of circIGF1R in adipogenesis, si-circIGF1R was added to cells and its interference efficiency was detected ( Figure 6 a; P <0.01). After 8 days of adipogenic differentiation induction, Oil Red O staining revealed a significant decrease in lipid droplets ( Figure 6 b), and the mRNA levels of adipocyte markers were significantly decreased ( Figure 6 c; P <0.01). These data indicate that si-circIGF1R inhibits cell adipogenic differentiation.

[0090] 2.3 METTL3 regulates cell adipogenic differentiation through circIGF1R

[0091] In order to verify that METTL3 affects cell adipogenic differentiation by affecting the expression of circIGF1R, we designed a rescue experiment and divided it into three groups. Ctrl + si-NC, STM-2457 + si-NC, and STM2457 + si-circIGF1R were transfected into cells and induced cell adipogenic differentiation. It was found that interfering with the expression of circIGF1R reduced the effect of STM2457 on promoting cell adipogenic differentiation ( Figure 7 , ab; P<0.01). Among them, Ctrl is the control group without sequence, and the sequence of si-NC is shown in Table 2 (Negative Control).

[0092] Example 3 circIGF1R regulates cell adipogenic differentiation through miR-15a, miR-16 and miR-885-3p / NAT10 axis

[0093] circRNA can regulate cell proliferation and adipogenic differentiation through ceRNA. The ceRNA regulatory network is an important pathway for circRNA to exert its effects. miRNA can bind to the 3'UTR region of mRNA to promote its degradation, thereby leading to gene silencing. circRNA can competitively adsorb miRNA to avoid degradation of target genes.

[0094] From Example 2, it can be seen that m 6 A methyltransferase METTL3 6 In this study, the expression of circIGF1R was regulated in a manner dependent on miR-16 and miR-885-3p to affect the adipogenic differentiation of pig preadipocytes. In this study, the binding relationship between circIGF1R and miR-16 and miR-885-3p was verified by RIP to explore the downstream mechanism of circIGF1R in regulating cell adipogenic differentiation.

[0095] 1. Materials and Methods

[0096] 1.1 Test materials

[0097] 1.1.1 Test cells and test reagents

[0098] The experimental cells were the same as those in Example 1, and HEK293T cells were purchased from Shanghai Gaining Biotechnology.

[0099] Protein A / G Magnetic Beads were purchased from MCE, USA; Anti-AGO2 Antibody, 10% SDS, Proteinase K, IP Cell Lysis Buffer were purchased from Boster Biotechnology Co., Ltd.; Rabbit Control IgG was purchased from abclonal; Isoamyl alcohol, phenol, Tris-HCL were purchased from Beijing Solebao Company; m 6 A RNA methylation fragment enrichment kit (qPCR version) was purchased from Beijing Aide Technology Company.

[0100] 1.1.2 Preparation of test reagents

[0101] RIP buffer (25 mL): Tris-HCL 0.5 mL, NaCl 219.15 mg, MgCl27.14 mg, glycerol 2.5 mL, NP40 1.25 mL, Triton-X100 1.25 mL.

[0102] 1.1.3 Test equipment

[0103] The test apparatus is the same as that in Example 1.

[0104] 1.2 Test methods

[0105] 1.2.1 RNA-binding protein immunoprecipitation (RIP)

[0106] (1) Collect cell samples: After the cells have been transfected and grown to 90%, add 1.1 mL of IP lysis buffer and lyse at 4°C for 2 h. Centrifuge at 12,000 r / min for 15 min and transfer the supernatant. Take 100 μL of cell lysate as the input group.

[0107] (2) Prepare magnetic beads: Mix the magnetic beads, take 30 μL of the magnetic bead suspension, add 500 μL of RIP buffer and mix well. Centrifuge at 3000 r / min for 1 min and remove the supernatant.

[0108] (3) RNA-binding protein immunoprecipitation: add 500 μL RIP buffer to resuspend the magnetic beads and add 5 μg of the corresponding antibody, and incubate overnight at 4°C with a tumbler; after completion, add 500 μL RIP buffer to mix, centrifuge at 3000 r / min for 1 min, and discard the supernatant; add 500 μL cell lysis buffer and incubate overnight at 4°C; after completion, centrifuge at 3000 r / min for 1 min at 4°C and discard the supernatant; add 1 mL RIP buffer to mix, centrifuge at 3000 r / min for 1 min, discard the supernatant, and repeat 5 times.

[0109] (4) RNA purification: Add 117 μL RIP buffer, 15 μL 10% SDS and 4.5 μL Proteinase K, incubate at 65°C for 45 min, centrifuge at 3000 r / min for 1 min, take the supernatant and make up to 200 μL with enzyme-free water, add 20 μL 3 M sodium acetate and 2 volumes of phenol: chloroform: isoamyl alcohol (25:24:1, volume ratio) solution, mix well, centrifuge at 12000 r / min for 2 min, transfer the supernatant, add 2 volumes of chloroform: isoamyl alcohol (24:1, volume ratio), mix well, let stand at room temperature for 5 min, centrifuge at 12000 r / min for 10 min; aspirate the upper aqueous phase and add 3 volumes of anhydrous ethanol and 2 μL glycogen, and place at -20°C for precipitation overnight. Centrifuge at 12000 r / min for 15 min, remove the supernatant; add 1 mL of pre-cooled 75% ethanol to wash the precipitate, centrifuge at 12000 r / min for 10 min, and remove the supernatant. Wait for the alcohol to evaporate and then add an appropriate amount of enzyme-free water.

[0110] 1.2.2 RNA extraction and qRT-PCR

[0111] The operation steps were the same as those in Example 1. The primer sequences are shown in Table 4. The miRNA primers were designed using the tailing method, and a tailing method reverse transcription kit was used. U6 was used as the internal reference, and 2 -ΔΔCt The relative expression was calculated by the method.

[0112] Table 4 Primer sequences

[0113] 2. Results and Analysis

[0114] 2.1 circIGF1R can act as a sponge for miR-15a, miR-16, and miR-885-3p (the reference sequence numbers of miR-15a, miR-16, and miR-885-3p in NCBI are NR_035364.1, NR_035366.1, NR_038543.1, AGO2, and NAT10, respectively)

[0115] Tissue expression analysis showed that miR-15a was most highly expressed in the lung and spleen, followed by the heart, liver, and kidney; miR-16 was most highly expressed in the lung, followed by the spleen; miR-885-3p was most highly expressed in the heart and kidney, followed by the lung. Overall, the expression levels of miR-16 and miR-885-3p in the back subcutaneous fat were higher than their expression levels in the longissimus dorsi muscle ( Figure 8 , ac). During the different stages of pig preadipocyte differentiation, the expressions of miR-15a, miR-16, and miR-885-3p showed a trend of first decreasing and then increasing. Their expression levels were lowest on the first day of differentiation and reached the highest on the seventh day ( Figure 8 , df).

[0116] RNAhybird software predicted that circIGF1R could target and bind to miR-15a, miR-16, and miR-885-3p ( Figure 8 , gi, and qRT-PCR experiments showed that after interfering with circIGF1R, the expression levels of miR-15a, miR-16, and miR-885-3p were significantly increased ( Figure 8 ,jl, P <0.01). The AGO2-RIP assay verified that circIGF1R binds to miR-15a, miR-16, and miR-885-3p. The results showed that the enrichment of circIGF1R in the AGO2 antibody group in the miR-15a, miR-16, and miR-885-3p mimics transfection groups was significantly higher than that in the si-NC group ( Figure 8 m).

[0117] 2.2 miR-15a, miR-16, and miR-885-3p can inhibit cell adipogenic differentiation

[0118] The miR-15a, miR-16 and miR-885-3p mimics were transfected into cells and their overexpression efficiency was detected. The results showed that compared with the si-NC group, the expression levels of miR-15a, miR-16 and miR-885-3p mimics were significantly increased ( Fig. 9 ,ac, P <0.01), and subsequent experiments can be carried out. MiR-15a mimics, miR-16 mimics and miR-885-3p mimics were transfected into porcine preadipocytes, and the cells were induced to differentiate into adipocytes for 8 days to detect the adipogenesis of the cells. Compared with the control group, Oil Red O staining showed that the lipid droplets of cells in the transfected miR-15a mimics, miR-16 mimics and miR-885-3p mimics groups were significantly reduced ( Fig. 9 d), qRT-PCR detection found that the expression of adipogenesis-related factor mRNA was significantly reduced ( Fig. 9 e, P <0.01). The above results indicate that miR-15a, miR-16 and miR-885-3p can inhibit the adipogenic differentiation of porcine preadipocytes.

[0119] 2.3 NAT10 It is a downstream target gene of miR-15a, miR-16 and miR-885-3p

[0120] The expression of NAT10 in the lung was significantly higher than that in other tissues, and the expression in the back subcutaneous fat was higher than that in the longissimus dorsi muscle ( Fig.10 a); The expression level was different at different stages of pig preadipocyte differentiation induction, and the lowest expression level was on the 3rd day ( Fig.10 b). There are differences in the expression of NAT10 in the abdominal and back fat tissues of pigs of different developmental breeds. Overall, the level of NAT10 in the fat tissue of horse-body pigs is significantly higher than that of DLY pigs ( Fig.10 c; P <0.01).

[0121] RNAhybird software predicted that miR-15a, miR-16 and miR-885-3p had binding sites with NAT10 ( Fig.10 , df, and qRT-PCR experiments showed that the expression of NAT10 was significantly reduced after overexpression of miR-15a, miR-16 and miR-885-3p ( Fig.10 g, P <0.01), after interfering with circIGF1R, the expression of NAT10 was significantly increased ( Fig.10 h, P<0.01). The results of AGO2-RIP showed that in the mimics groups transfected with miR-15a, miR-16 and miR-885-3p, the enriched NAT10 in the AGO2 antibody group was significantly higher than that in the si-NC group ( Fig.10 i).

[0122] 2.4 NAT10 regulates cell adipogenic differentiation

[0123] After transfection of si-NAT10 in porcine preadipocytes ( Table 3 ) and detection of interference efficiency, si-NAT10-2 (denoted as si-NAT10) was selected for subsequent experiments ( Fig.11 a, P <0.01). At the same time, the cells were induced to differentiate into adipocytes for 8 days to detect the adipogenesis of the cells. Compared with the control group, Oil Red O staining showed that the lipid droplets in the si-NAT10 transfected group were significantly reduced ( Fig.11 b), qRT-PCR detection found that the expression of adipogenesis-related factor mRNA was significantly reduced ( Fig.11 c, P <0.01). The above results indicate that si-NAT10 can inhibit the adipogenic differentiation of porcine preadipocytes.

[0124] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. RNA m 6 A. Any of the following applications of methylation modification-related protein METTL3: (1) Used to regulate porcine adipocyte proliferation and adipogenic differentiation; Specifically, the regulation refers to positive regulation of porcine adipocyte proliferation and negative regulation of adipogenic differentiation; (2) mRNA for positive regulation of porcine circIGF1R 6 A modification level; in, The reference sequence number of protein METTL3 in NCBI is NP_062695.2; the sequence of pig circIGF1R is shown in SEQ ID NO:

1.

2. RNA m 6 A. Any of the following uses of an inhibitor of a methylation modification-related protein METTL3: (1) Used to inhibit porcine adipocyte proliferation and promote adipogenic differentiation; (2) Used to reduce the m of porcine circIGF1R 6 A modification level; in, Protein METTL3 and porcine circIGF1R are the same as those described in claim 1.

3. The inhibitor according to claim 2, characterized in that The inhibitor is selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, and antibodies; Preferably, the inhibitor is STM-2457, the chemical formula of which is C 25 H 28 N6O2.

4. RNA m 6 A. Any of the following applications of methylation modification-related protein FTO: (1) Used to regulate porcine adipocyte proliferation and adipogenic differentiation; Specifically, the regulation refers to positive regulation of pig adipocyte proliferation and adipogenic differentiation; (2) mRNA for negative regulation of porcine circIGF1R 6 A modification level; in, The reference sequence number of protein FTO in NCBI is NP_001106162.1; pig circIGF1R is the same as described in claim 1.

5. RNA m 6 A. The following applications of inhibitors of methylation modification-related protein FTO: (1) Used to inhibit the proliferation and adipogenic differentiation of porcine adipocytes; (2) Used to increase the m of porcine circIGF1R 6 A modification level; in, The protein FTO is the same as that described in claim 4, and the porcine circIGF1R is the same as that described in claim 1.

6. The inhibitor according to claim 5, characterized in that The inhibitor is selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, and antibodies; Preferably, the inhibitor is FB23-2, with a molecular formula of C 18 H 15 Cl2N3O3.

7. A circRNA marker of RNA methylation modification associated with pig adipocyte formation, which is pig circIGF1R; in, Porcine circIGF1R is the same as that described in claim 1.

8. Application of porcine circIGF1R in promoting adipogenic differentiation of porcine adipocytes; in, Porcine circIGF1R is the same as that described in claim 1.

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