Application and method of IL1R2 gene in regulation and control of pig placenta development or improvement of pig birth weight

Abstract

The invention discloses application of an IL1R2 gene in regulation and control of pig placenta development or improvement of pig birth weight. According to the present invention, transcriptome sequencing and whole genome methylation sequencing are performed on placenta samples of high birth weight (HBW) piglets and low birth weight (LBW) piglets in the same nest, such that the differential methylation differential expression gene IL1R2 is screened, and the methylation level of the gene in the HBW placenta is high, and the expression quantity is low; therefore, by regulating the methylation level or expression level of the IL1R2 gene, the development quality of the pig placenta can be improved, the birth weight and survival rate of piglets are further improved, and the breeding efficiency of sows and the market competitiveness of pig farms can be improved.

Description

Technical Field

[0001] The present invention relates to the field of gene regulation, and in particular to an application and method of an IL1R2 gene in regulating pig placenta development or increasing pig birth weight. Background Art

[0002] Piglet birth weight, along with the number of healthy piglets born, the number of live piglets born, and the total number of piglets born, are key indicators for evaluating sow reproductive performance. Studies have shown that piglet birth weight and uniformity are closely related to placental development and function during pregnancy. Poor placental development can lead to reduced piglet birth weight and uniformity, and can also affect piglet size.

[0003] The placenta relies on the expansion of the trophoblast in the uterine cavity to establish pregnancy and maintain the growth of the embryo and fetus. The rapid development of placental trophoblast cells promotes the increase of placental surface area and volume, which is conducive to the transport of nutrients to the fetus during pregnancy. Pigs are multiparous animals, and the development status of the placenta is closely related to the litter size and the rate of healthy piglets. Poor placental development can lead to a variety of developmental abnormalities such as fetal miscarriage, intrauterine growth retardation, stillbirth, and heart defects. Therefore, the proliferation and migration ability of porcine placental trophoblast cells (PTr2) (hereinafter referred to as "PTr2 cells") are important factors affecting the quality of pig placental development and further affecting piglet development (such as birth weight, etc.). Insufficient proliferation of PTr2 cells can lead to structural defects of the placenta, affect the nutrient supply of the fetus, and cause fetal growth restriction; poor migration ability affects angiogenesis and immune regulation, increasing the risk of miscarriage and weak piglets.

[0004] DNA methylation maintains precise and ordered gene expression and is crucial for mammalian embryonic attachment, implantation, and placental development. During early porcine embryonic development, DNA methylation patterns undergo global demethylation from fertilization to the blastocyst stage, followed by reestablishment through de novo methylation at approximately the 8- to 16-cell stage. DNA methylation influences porcine placental development by influencing vascularization and wrinkling, and also regulates placental energy and immune metabolism.

[0005] Interleukin 1 (IL1) is a key proinflammatory cytokine with broad inflammatory, metabolic, hematopoietic, and immunological properties. The IL1 system comprises two ligands, IL1a and IL1b, two cell surface receptors, IL1R1 and IL1R2, a receptor accessory protein, IL1RAP, and an inhibitor, designated as an IL1 receptor antagonist. IL1a and IL1b are encoded by different genes but possess identical biological activities. IL1R1 and IL1R2 share similar amino acid sequences and conserved exon and intron structures. However, unlike IL1R1, IL1R2 lacks the Toll / IL-1 receptor domain, which is responsible for IL1-triggered signal transduction. However, IL1R2, lacking a cytoplasmic signaling domain, acts as a decoy receptor, preventing IL1 from exerting its biological function. However, the role of IL1R2 in porcine placental development has not been reported. Summary of the Invention

[0006] The purpose of the present invention is to provide an application of an IL1R2 gene in regulating pig placenta development or increasing pig birth weight, so as to improve the development quality of sow placenta and further increase the birth weight of piglets.

[0007] According to a first aspect of the present invention, a method for regulating pig placental development or increasing pig birth weight is provided. By regulating the methylation or expression level of the IL1R2 gene, the development quality of the pig placenta can be improved, and the birth weight of piglets can be further increased, thereby enhancing the reproductive efficiency and market value of sows.

[0008] According to a second aspect of the present invention, the use of the IL1R2 gene in the preparation of a product for regulating pig placental development or increasing pig birth weight is provided. Thus, reagents that regulate the methylation or expression level of the IL1R2 gene are formulated into products that can be used to improve the development quality of the pig placenta, further increasing piglet birth weight, and enhancing the reproductive efficiency and market value of sows.

[0009] According to a third aspect of the present invention, the IL1R2 gene is used to promote the proliferation and / or migration of porcine placental trophoblast cells. By regulating the methylation level or expression level of the IL1R2 gene, the proliferation and / or migration of porcine placental trophoblast cells can be promoted, thereby improving the development quality of the sow's placenta, further increasing the birth weight of piglets, and enhancing the reproductive efficiency and market value of sows.

[0010] According to a fourth aspect of the present invention, a method for preparing a product for promoting the proliferation and / or migration of porcine placental trophoblast cells is provided. Thus, a reagent for regulating the methylation or expression level of the IL1R2 gene is formulated into a product that can be used to promote the proliferation and / or migration of porcine placental trophoblast cells, thereby improving the developmental quality of the sow's placenta, further increasing the birth weight of piglets, and enhancing the reproductive efficiency and market value of sows.

[0011] According to a fifth aspect of the present invention, a method for regulating the inflammatory response of porcine placental trophoblast cells is provided. By regulating the methylation level or expression level of the IL1R2 gene, the inflammatory response of porcine placental trophoblast cells can be modulated. Furthermore, the inflammatory response in the placenta can be regulated to maintain placental function and development, thereby affecting the birth weight of piglets.

[0012] According to a sixth aspect of the present invention, a method for preparing a product for regulating the inflammatory response of porcine placental trophoblast cells is provided. Thus, a reagent for regulating the methylation or expression level of the IL1R2 gene is formulated into a product. This product can be used to regulate the inflammatory response of porcine placental trophoblast cells and further regulate the inflammatory response in the placenta to maintain placental function and development, thereby affecting the birth weight of piglets.

[0013] In certain embodiments, the use is achieved by increasing the methylation level of the IL1R2 gene or inhibiting the expression level of the IL1R2 gene.

[0014] According to a seventh aspect of the present invention, a method for regulating pig placental development and / or increasing piglet birth weight is provided. The method comprises adding an agent that increases IL1R2 gene methylation levels or inhibits IL1R2 gene expression to sow feed or drinking water; or directly injecting an agent that increases IL1R2 gene methylation levels or inhibits IL1R2 gene expression into the placenta. Thus, this method can regulate pig placental development and / or increase piglet birth weight, thereby improving the reproductive efficiency and market value of sows.

[0015] In certain embodiments, the agent capable of inhibiting IL1R2 gene expression is siRNA that interferes with IL1R2 gene expression, and the sequence of the siRNA is GCCCAUGGAGGAAGGUCAUTT, AUGACCUUCCUCCAUGGGCTT.

[0016] According to an eighth aspect of the present invention, a method for promoting the proliferation and / or migration of porcine placental trophoblast cells is provided. The method comprises adding a reagent that increases the methylation level of the IL1R2 gene to a porcine placental trophoblast cell culture medium, or injecting or transfecting a reagent that inhibits IL1R2 gene expression into the porcine placental trophoblast cells. The reagent that inhibits IL1R2 gene expression is an siRNA that interferes with IL1R2 gene expression, and the sequence of the siRNA is GCCCAUGGAGGAAGGUCAUTT, AUGACCUUCCUCCAUGGGCTT. Thus, this method can promote the proliferation and / or migration of porcine placental trophoblast cells, thereby improving the development quality of the sow placenta, further increasing the birth weight of piglets, and improving the reproductive efficiency and market economic value of sows.

[0017] According to a ninth aspect of the present invention, a product for promoting pig placental development and / or increasing piglet birth weight is provided. The product contains an agent capable of inhibiting IL1R2 gene expression, wherein the agent is an siRNA that interferes with IL1R2 gene expression, and the sequence of the siRNA is GCCCAUGGAGGAAGGUCAUTT, AUGACCUUCCUCCAUGGGCTT. Thus, this product can promote pig placental development and / or increase piglet birth weight, thereby improving the reproductive efficiency and market value of sows.

[0018] According to a tenth aspect of the present invention, a reagent that inhibits IL1R2 gene expression is used in the preparation of a product that promotes pig placental development and / or increases piglet birth weight. Thus, by formulating the reagent that inhibits IL1R2 gene expression into a product, pig placental development can be promoted and / or piglet birth weight can be increased, thereby improving the reproductive efficiency and market value of sows.

[0019] According to an eleventh aspect of the present invention, a reagent that inhibits IL1R2 gene expression is used in the preparation of a product for promoting the proliferation and / or migration of porcine placental trophoblast cells. Thus, by formulating the reagent that inhibits IL1R2 gene expression into a product, the proliferation and / or migration of porcine placental trophoblast cells can be promoted, thereby improving the development quality of the sow's placenta, further increasing the birth weight of piglets, and enhancing the reproductive efficiency and market value of sows.

[0020] According to a twelfth aspect of the present invention, a reagent that inhibits IL1R2 gene expression is used in the preparation of a product for regulating the inflammatory response of porcine placental trophoblast cells. Thus, by formulating the reagent that inhibits IL1R2 gene expression into a product, the inflammatory response of porcine placental trophoblast cells can be regulated, and further regulation of the inflammatory response in the placenta can maintain placental function and development, thereby affecting the birth weight of piglets.

[0021] In certain embodiments, the agent that inhibits IL1R2 gene expression is siRNA that interferes with IL1R2 gene expression, and the sequence of the siRNA is GCCCAUGGAGGAAGGUCAUTT, AUGACCUUCCUCCAUGGGCTT.

[0022] Beneficial effects of the present invention:

[0023] The present invention screened for differentially methylated and differentially expressed genes, IL1R2, by performing transcriptome sequencing and whole-genome methylation sequencing on placental samples from high birth weight (HBW) and low birth weight (LBW) piglets in the same litter. The gene had higher methylation levels and lower expression levels in HBW placentas. Then, by studying the methylation levels of the gene, it was found that the methylation levels of the IL1R2 gene in HBW placentas were significantly higher than those in LBW placentas, suggesting that the methylation status of the IL1R2 gene may be closely related to placental development and fetal growth regulation. The expression of IL1R2 in PTr2 cells was then regulated by DNA methylation. The results showed that the DNA methylation inhibitor 5-Aza could induce the expression of the IL1R2 gene in PTr2 cells, and its regulatory effect increased in a dose-dependent manner. It was further confirmed that during placental development, the expression of the IL1R2 gene was regulated by DNA methylation, and the methylation level of the IL1R2 promoter region was reduced in PTr2 cells treated with 5-Aza. Then, by inhibiting or overexpressing the IL1R2 gene in PTr2 cells, the results showed that inhibiting (interfering with) the IL1R2 gene can promote the proliferation and migration of PTr2 cells. PTr2 cells play an important role in placental development. By promoting the proliferation and migration of PTr2 cells, the development of the placenta can be effectively promoted, and the birth weight of piglets can be further increased. In addition, by inhibiting the expression of the IL1R2 gene in PTr2 cells, the mRNA expression of the inflammatory gene TNF-a can be significantly inhibited, and IL6 and IL8 can be downregulated, indicating that inhibiting the IL1R2 gene can also regulate the inflammatory response in the placenta to maintain the homeostasis and development of placental function, thereby affecting the birth weight of piglets. Based on this, the quality of pig placental development or the birth weight of piglets can be regulated by regulating the methylation level or expression level of the IL1R2 gene. The proliferation and / or migration of pig placental trophoblast cells can also be promoted by regulating the methylation level or expression level of the IL1R2 gene. The inflammatory response of pig placental trophoblast cells and the inflammatory response in the placenta can also be regulated by regulating the methylation level or expression level of the IL1R2 gene to maintain the homeostasis and development of placental function, thereby affecting the birth weight of piglets. Therefore, regulating the methylation level or expression level of the IL1R2 gene can be used to improve the quality of pig placental development, further increase the birth weight and survival rate of piglets, and improve the reproductive efficiency of sows and the market competitiveness of breeding pig farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the technical roadmap of the present invention;

[0025] Figure 2 A comparison of the two groups of sequenced piglet placentas;

[0026] Figure 3 The results of the comparative analysis of HBW and LBW placenta morphology are shown below: Figure 3 a represents HE staining analysis of placental morphology and structure, scale bar: 50 μm; Figure 3 b shows HE staining analysis of the number of placental red blood cells (green arrows indicate placental blood vessels), scale bars: 200 μm and 50 μm; Figure 3 c indicates histograms showing the placental vascular density of each group, *** indicates p < 0.001;

[0027] Figure 4 This is the result of the correlation analysis between placenta weight and piglet birth weight;

[0028] Figure 5 Screening of IL1R2 gene map for joint analysis: Figure 5 a is the GO enrichment analysis of DEGs in the joint analysis, 5b is the KEGG analysis of DEGs in the joint analysis, Figure 5 c is the methylation level and expression of IL1R2 gene (upper left corner), Figure 5 d is the IGV map of IL1R2 gene;

[0029] Figure 6 Figure 2 shows the results of RT-qPCR validation sequencing: * indicates P < 0.05, ** indicates P < 0.01;

[0030] Figure 7 This is the result of protein immunohistochemistry (IHC) staining analysis;

[0031] Figure 8 This is the result map of the IL1R2 gene promoter CpG island predicted by MethPrimer;

[0032] Figure 9 This is the result of detecting the methylation status of the promoter region of the IL1R2 gene in the placenta;

[0033] Figure 10 The results of RT-qPCR analysis of the relative expression level of IL1R2 gene mRNA in PTr2 cells after treatment with different concentrations of 5-Aza; ns indicates P>0.05, and * indicates P<0.05;

[0034] Figure 11 This is the result of MS-PCR verification of IL1R2 methylation level after 5-Aza treatment in PTr2 cells;

[0035] Figure 12 This is the result of BS-PCR verification of IL1R2 methylation level after 5-Aza treatment in PTr2 cells;

[0036] Figure 13The results of RT-qPCR analysis of the transfection efficiency of si-IL1R2 and pcDNA3.1-IL1R2 in PTr2 cells: *** indicates P < 0.001;

[0037] Figure 14 The figure shows the results of RT-qPCR detection of mRNA expression of proliferation and apoptosis marker genes after interference and overexpression of IL1R2: ns indicates P>0.05, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001;

[0038] Figure 15 The results of the test to interfere with the IL1R2 gene to promote the proliferation of PTr2 cells are shown in the figure: Figure 15 a is the result of CCK-8 detection of cell proliferation ability after interfering with IL1R2, Figure 15 b is the result of EdU detection of cell proliferation ability after interference with IL1R2, scale bar is 100 μm, * indicates P < 0.05, ** indicates P < 0.01;

[0039] Figure 16 Figure 1 shows the results of the detection of promoting the migration of PTr2 cells by interfering with the IL1R2 gene: the scale bar is 500 μm, and * indicates P < 0.05;

[0040] Figure 17 The results of the assay for overexpression of IL1R2 gene to inhibit the proliferation of PTr2 cells are shown in the figure: Figure 17 a is the result of CCK-8 detection of cell proliferation ability after overexpression of IL1R2, Figure 17 b is the result of EdU detection of cell proliferation ability after overexpression of IL1R2, scale bar is 100 μm, * indicates P < 0.05, ** indicates P < 0.01;

[0041] Figure 18 Figure 1 shows the results of the migration assay in which overexpression of IL1R2 gene inhibits PTr2 cells: Scale bar is 500 μm, ** indicates P < 0.01;

[0042] Figure 19 The results of RT-qPCR detection of the effect of IL1R2 on inflammation-related genes: Figure 19 a is the mRNA expression results of inflammatory marker genes after RT-qPCR detection of interference and overexpression of IL1R2. Figure 19 b shows the mRNA expression results of inflammatory factors IL6 and IL8 after interference with IL1R2 by RT-qPCR detection, ns indicates P>0.05, * indicates P<0.05, and ** indicates P<0.01. DETAILED DESCRIPTION

[0043] The technical roadmap of the present invention is as follows: Figure 1 shown.

[0044] Example 1 Screening of differentially methylated and differentially expressed genes from HBW and LBW pig placentas 1.1 Sample collection and data analysis

[0045] Placenta and umbilical cord samples were collected from high birth weight (HBW) and low birth weight (LBW) piglets in the same litter, and the placental characteristics and tissue morphology were compared. Finally, placenta samples of piglets from two sows were selected during delivery, and the birth weight (also referred to as "birth weight") and placental weight of the corresponding piglets were recorded (Table 1). The sampling results showed that the placenta weight of piglets in the HBW group was generally higher than that in the LBW group. In addition, morphological observations found that the umbilical cords of HBW piglets were slender, smooth in appearance, and contained a small amount of blood, while the umbilical cords of LBW piglets were enlarged and accompanied by local thrombosis or occlusive thrombosis ( Figure 2 ).

[0046] Table 1 Sequencing piglet and farrowing sow information

[0047]

[0048] The morphological characteristics of pig placenta tissue were compared under a microscope, and paraffin sections were made from the placenta and HE staining was performed on the sections. The HE staining results showed that the number of chorionic villi in the HBW piglet placenta was sufficient, the structure of the chorionic epithelial cells was plump, and there was no obvious edema and necrosis of the chorionic epithelial cells. The overall structure of the placenta tissue of the LBW piglet was slightly abnormal, the arrangement of the chorionic epithelial cells was irregular, and some chorionic epithelial cells were obviously desquamated, pyknotic and necrotic ( Figure 3 a). HBW placentas have higher numbers of red blood cells in the blood vessels and intervillous spaces compared to LBW placentas ( Figure 3 bc).

[0049] In order to explore the relationship between placenta weight and piglet birth weight, this study conducted a correlation analysis between placenta weight and piglet birth weight. Figure 4 As shown: The data point distribution shows that as the piglet birth weight increases, the placenta weight also shows an upward trend. The placenta weight is significantly positively correlated with the piglet birth weight (R = 0.89), indicating that the increase in placenta weight may have a positive effect on the piglet birth weight. The linear regression equation is y = -210 + 0.51x, R 2 =0.83, indicating that placental weight can explain 83% of the variation in piglet birth weight.

[0050] 1.2 Transcriptome sequencing and whole-genome methylation sequencing analysis of HBW and LBW piglet placenta samples

[0051] Transcriptome sequencing (RNA-seq) and whole genome methylation sequencing (WGBS) were performed on HBW and LBW piglet placenta samples, and the data quality was assessed. The data are summarized in Table 2:

[0052] (1) RNA-seq data: The number of clean reads for all samples exceeded 39 million, and the unique alignment rate ranged from 89.19% to 93.66%, indicating that the alignment efficiency of RNA-seq data was high and could meet the requirements of subsequent gene expression analysis. The alignment rates of HBW1 and HBW2 were slightly higher than those of LBW1 and LBW2, indicating that the sequencing data quality of the HBW group samples was slightly better.

[0053] (2) WGBS data: The number of clean reads for all samples exceeded 290 million, and the sulfite conversion rate was higher than 99.6%, indicating that the sulfite treatment was highly efficient and could effectively distinguish methylated and unmethylated cytosines. The unique alignment rate ranged from 80.79% to 81.81%, meeting the standards for whole-genome methylation sequencing and supporting subsequent DNA methylation analysis.

[0054] The quality assessment of the sequencing data confirmed that the RNA-seq and WGBS data of the HBW and LBW piglet placenta samples met the analysis requirements, indicating that the sequencing data quality of all samples was reliable and suitable for subsequent analysis.

[0055] Table 2. Statistics of raw sequencing data quality

[0056]

[0057] Combining the results of genome-wide methylation and transcriptome sequencing data analysis, 330 DEGs regulated by DNA methylation in promoter regions were identified, of which 104 belonged to promoter-negative genes (PNGs). GO and KEGG analyses showed that these PNGs were enriched in placental development-related pathways, including "chemokine signaling pathway," "extracellular matrix organization," "integrin-mediated signaling," "oxidative phosphorylation," and "Rap1 signaling pathway." Figure 5 Finally, the differentially methylated and differentially expressed gene IL1R2 was screened and functionally verified. The IGV map showed that the methylation level of IL1R2 was higher and the expression level was lower in HBW placenta ( Figure 5 cd).

[0058] Example 2 IL1R2 gene and DNA methylation in placenta

[0059] 2.1 Detection of placental DNMTs and IL1R2 expression by RT-qPCR

[0060] The expression of DNA methyltransferases DNMT3A, DNMT3L, and DNA methylation maintenance enzyme DNMT1 in HBW and LBW placentas was detected by RT-qPCR. Figure 6 As shown in the results, the expression levels of DNMT3A, DNMT3L and DNMT1 in the HBW placenta group were significantly or extremely significantly higher than those in the LBW placenta group (P < 0.05 or P < 0.01), but the expression level of IL1R2 in the HBW placenta group was significantly lower than that in the LBW placenta group (P < 0.05).

[0061] The primers for RT-qPCR detection are shown in Table 3:

[0062] Table 3 qPCR primer sequences

[0063]

[0064] 2.2 Protein immunohistochemistry (IHC) staining analysis

[0065] The HBW and LBW placentas were subjected to protein immunohistochemistry (IHC) staining analysis. Figure 7 The results showed that DNMT3A was more expressed in HBW placenta than in LBW placenta ( Figure 7 a), and the IL1R2 protein content in HBW placenta was lower than that in LBW placenta ( Figure 7 b)

[0066] 2.3 Sequence analysis of the IL1R2 gene promoter region

[0067] The methylation status of the porcine IL1R2 gene promoter region was analyzed using MethPrimer software, with the criteria set as G+C content > 50% and CpG observed / expected ratio > 0.6. Figure 8 As shown in Table 4, a single CpG island is predicted to be present in the porcine IL1R2 promoter region. Studies have shown that even promoter regions with low CpG island density may still exhibit epigenetic characteristics similar to genes with high CpG island density. To further investigate the methylation status of the IL1R2 gene during placental development, MethPrimer software was used to design methylated and unmethylated primers targeting CpG sites in the promoter region (see Table 4).

[0068] Table 4 Methylation and non-methylation specific primers

[0069]

[0070]

[0071] 2.4 Methylation status of the IL1R2 gene promoter region

[0072] Genomic DNA was extracted from HBW and LBW placentas. The DNA concentration was suitable and the purity was good (OD260 / 280 ratio was between 1.8 and 2.0), which met the requirements of subsequent experiments. Subsequently, the placental tissue DNA was treated with bisulfite and the converted DNA was used as a template for MS-PCR amplification (primers are shown in Table 4) to detect the methylation status of the IL1R2 gene promoter region. The results are as follows: Figure 9 The results showed that the methylation level of IL1R2 gene in HBW placenta was significantly higher than that in LBW placenta, suggesting that the methylation status of IL1R2 gene may be closely related to placental development and fetal growth regulation.

[0073] MS-PCR detection method:

[0074] (1) Using MethPrimer software, DNA-specific primers for methylated (Methylated-Specific) and unmethylated (Unmethylated-Specific) CpG sites in the IL1R2 promoter were designed (see Table 4);

[0075] (2) Using bisulfite-treated DNA as a template, MS-PCR amplification experiments were performed using 2× Taq PCR Star Mix. The reaction system was as shown in Table 5, and the reaction procedure was as shown in Table 6.

[0076] Table 5 MS-PCR reaction system

[0077]

[0078] Table 6 qPCR reaction procedure

[0079]

[0080] (3) Detect the degree of methylation of the amplified product by agarose gel electrophoresis. After the PCR amplification reaction is completed, the product is subjected to agarose gel electrophoresis. After the electrophoresis is completed, the gel is placed in an imaging system to observe the bands.

[0081] (4) Judgment criteria: Unmethylated: only unmethylated bands appear in the electrophoresis; Completely methylated: only methylated bands appear in the electrophoresis; Partially methylated: both methylated and unmethylated bands appear in the electrophoresis. The degree of methylation is calculated using the grayscale value: Methylation degree = grayscale value of methylated band / (grayscale value of methylated band + grayscale value of unmethylated band) × 100%.

[0082] Example 3 DNA methylation regulates the expression of IL1R2 in PTr2 cells

[0083] 3.1 Effect of 5-Aza Treatment on IL1R2 Expression in PTr2 Cells

[0084] In order to explore the regulatory effect of DNA methylation on IL1R2 gene expression in PTr2 cells, this study used the DNA methylation inhibitor 5-Aza to treat PTr2 cells. First, the DNA methylation inhibitor 5-Aza powder was dissolved in DMSO to prepare a 1mM solution, which was then divided and stored at -20°C. To determine the optimal treatment concentration, four gradient concentrations of 0μmol / L (DMSO control group), 5μmol / L, 10μmol / L and 20μmol / L were set. The expression level of the IL1R2 gene was detected by RT-qPCR, and the results are as follows Figure 10 RT-qPCR results showed that 5-Aza treatment significantly increased IL1R2 mRNA expression in PTr2 cells, and the relative mRNA expression of IL1R2 gradually increased with increasing 5-Aza concentration. When the 5-Aza treatment concentration reached 20 μmol / L, IL1R2 mRNA expression was significantly higher than that in the blank control group. These results indicate that the DNA methylation inhibitor 5-Aza can induce IL1R2 expression in PTr2 cells, and its regulatory effect increases in a dose-dependent manner. This further confirms that IL1R2 expression is regulated by DNA methylation during placental development.

[0085] 3.2 MS-PCR detection of IL1R2 methylation levels in PTr2 cells after 5-Aza treatment

[0086] The methylation level of IL1R2 in PTr2 cells after 5-Aza treatment was detected by MS-PCR. Figure 11 As shown in: MS-PCR experiments verified that in PTr2 cells treated with DMSO and 5-Aza, the methylation level of the IL1R2 promoter region in the 5-Aza group was reduced.

[0087] 3.3 BS-PCR detection of IL1R2 methylation levels in PTr2 cells after 5-Aza treatment

[0088] BS-PCR was used to detect the methylation level of IL1R2 in PTr2 cells after 5-Aza treatment. Figure 12 Shown: BS-PCR experiments further verified that in PTr2 cells treated with DMSO and 5-Aza, the methylation level of the IL1R2 promoter region in the 5-Aza group was reduced.

[0089] BS-PCR detection method

[0090] (1) Forward and reverse DNA-specific primers for the IL1R2 promoter CpG site were designed using MethPrimer software (see Table 7). Subsequent steps were performed according to the MS-PCR protocol.

[0091] Table 7 Forward and reverse specific primers

[0092]

[0093] (2) After agarose gel electrophoresis, the PCR product was recovered according to the instructions of the Gel Extraction Kit and the purified product was stored at -20°C.

[0094] (3) Thaw the Trans10 competent cells on ice, add 1 μL of the ligation product, mix gently, and place on ice for 30 minutes. Heat shock the cells in a 42°C water bath for 45 seconds, then immediately place on ice for 2-3 minutes. Add 500 μL of LB medium and incubate on a constant temperature air shaker at 37°C and 220 rpm / min for 1 hour.

[0095] (4) Spread 20 μL of the transformation mixture evenly on LA solid medium and incubate at 37°C for 12-16 h. Pick a single colony and inoculate it into 1 mL of LA medium. Incubate on a shaker at 37°C, 220 rpm / min for 2-4 h. Take 1 μL of the bacterial solution for PCR identification.

[0096] (5) The bacterial solution identified as positive was sent to the company for sequencing, ensuring that at least 10 valid sequencing results were obtained for each cell sample. The sequencing results were analyzed using BiQ Analyzer software, and a methylation bead map was generated.

[0097] Example 4 Effect of IL1R2 on PTr2 Cell Function

[0098] Synthesis of IL1R2 overexpression vector pcDNA3.1-IL1R2

[0099] The steps for constructing the IL1R2 overexpression vector pcDNA3.1-IL1R2 are as follows: First, the porcine IL1R2 gene (NCBI Gene: 100628112), design specific primers and amplify the target fragment; then, the amplified product is cloned into the multiple cloning site of the eukaryotic expression vector pcDNA3.1 to construct the IL1R2 overexpression vector pcDNA3.1-IL1R2. The nucleotide sequence of the IL1R2 overexpression vector pcDNA3.1-IL1R2 is shown in SEQ ID No: 15. After the IL1R2 overexpression vector pcDNA3.1-IL1R2 is constructed, the correctness and integrity of the inserted fragment are verified by sequencing to ensure that the IL1R2 gene can be efficiently expressed. The IL1R2 overexpression vector pcDNA3.1-IL1R2 can be used in subsequent cell experiments to study the function of the IL1R2 gene in placental development and its regulatory mechanism.

[0100] Synthesis of IL1R2 interference fragments

[0101] The IL1R2 interference fragment was designed and synthesized by Suzhou Genema Gene Co., Ltd. The sequence is shown in Table 8.

[0102] Table 8 IL1R2 interference fragment sequence

[0103]

[0104] To explore the role of IL1R2 in PTr2 cells, si-IL1R2 and si-NC (sequences shown in Table 8) were first transfected into PTr2 cells. The expression level of IL1R2 after transfection was detected by RT-qPCR. Compared with the si-NC group, the expression level of IL1R2 in the si-IL1R2 group was significantly decreased, indicating that the interference efficiency of si-IL1R2 was better ( Figure 13 a). In contrast, after transfection of the IL1R2 overexpression vector pcDNA3.1-IL1R2 into PTr2 cells, the expression level of pcDNA3.1-IL1R2 was significantly increased compared with the empty vector pcDNA3.1 group ( Figure 13 b) shows a high transfection efficiency, indicating that the synthesized si-IL1R2, si-NC, pcDNA3.1, and pcDNA3.1-IL1R2 can be used for subsequent experimental studies.

[0105] 4.1 Effects of IL1R2 on proliferation- and apoptosis-related genes in PTr2 cells

[0106] PTr2 cells were transfected with si-IL1R2 or si-NC; or the IL1R2 overexpression vector pcDNA3.1-IL1R2 or the empty vector pcDNA3.1 was transfected into PTr2 cells. Then, RT-qPCR technology was used to explore the effect of IL1R2 on cell proliferation and apoptosis-related genes in PTr2 cells. The experimental results are as follows Figure 14As shown: After interfering with IL1R2, the mRNA expression level of the proliferation-related gene Ki67 was significantly upregulated, while the mRNA expression levels of the apoptosis-related genes BAX, CASP3 and CASP9 were significantly downregulated ( Figure 14 a). In contrast, overexpression of IL1R2 significantly inhibited the mRNA expression of the proliferation-related gene PCNA, while promoting the mRNA expression of the apoptosis-related gene BAX ( Figure 14 b).

[0107] The primers for RT-qPCR detection are shown in Table 9:

[0108] Table 9oPCR primer sequences

[0109]

[0110] 4.2 Interference with IL1R2 gene promotes PTr2 cell proliferation

[0111] PTr2 cells were transfected with si-IL1R2 or si-NC, and then CCK-8 cell proliferation assay and EdU cell proliferation assay were performed.

[0112] CCK-8 cell proliferation assay:

[0113] (1) Cell culture and treatment: Cell proliferation activity was measured at 12, 24, 48, and 72 h after transfection;

[0114] (2) CCK-8 assay: Add 10 μL of CCK-8 solution to each well of a 96-well plate and incubate in an incubator for 2 h;

[0115] (3) OD value determination: Use a microplate reader to measure the optical density (OD) of each well at 450 nm. EdU cell proliferation detection method:

[0116] (1) EdU labeling: 48 h after transfection, EdU solution was added to the cell culture medium and incubated at 37°C and 5% CO2 for 2 h.

[0117] (2) Cell fixation and permeabilization: Remove the culture medium and wash the cells twice with DPBS for 3 minutes each time. Fix the cells with 4% paraformaldehyde at room temperature for 15 minutes and wash them twice with DPBS. Add cell permeabilization solution for 10 minutes and wash them twice with DPBS.

[0118] (3) Click reaction and staining: Add 70 μL of Click reaction solution to each well, incubate at room temperature for 30 min in the dark, discard the reaction solution, and wash twice with DPBS. Add DAPI staining solution, incubate at room temperature for 8 min, discard the staining solution, and wash twice with DPBS.

[0119] (4) Image acquisition and analysis: Images were captured using a fluorescence microscope, and four fields of view were randomly selected from each well for observation.

[0120] The results are as follows Figure 15 The results showed that transfection of si-IL1R2 could promote the proliferation of PTr2 cells. The CCK-8 assay showed that interference with IL1R2 expression could significantly promote the proliferation of PTr2 cells (P < 0.01). Figure 15 a); EdU experiments also confirmed that downregulation of IL1R2 could significantly promote the proliferation of PTr2 cells (P<0.01) ( Figure 15 b).

[0121] 4.3 Interference with IL1R2 gene promotes PTr2 cell migration

[0122] PTr2 cells were transfected with si-IL1R2 or si-NC, and then cell migration was detected by wound healing assay.

[0123] Scratch assay to detect cell migration:

[0124] (1) Cell seeding and scratching: Cells were seeded in a 6-well plate. After 36 h of transfection, the cells were allowed to fuse into a monolayer and scratched with a 200 μL pipette tip. The plates were washed with DPBS to remove detached cells and serum-free medium was added to continue culturing for 12 h.

[0125] (2) Image acquisition and analysis: Observe and photograph the wound closure status under a microscope. Randomly select three fields of view and use Image J software to calculate the wound area at 0 h and 12 h. Calculate the migration rate according to the formula:

[0126] Migration rate = (0 h wound area - 12 h wound area) / 0 h wound area × 100%.

[0127] Test results such as Figure 16 As shown in the figure, the migration speed and distance of PTr2 cells in the si-IL1R2 group were significantly accelerated within the same period of time, indicating that downregulation of the IL1R2 gene significantly promoted the migration ability of PTr2 cells (P < 0.05).

[0128] 4.4 Overexpression of IL1R2 gene inhibits the proliferation of PTr2 cells

[0129] To further investigate the changes in PTr2 cell function after upregulation of IL1R2, the synthetic IL1R2 overexpression vector pcDNA3.1-IL1R2 and the empty vector pcDNA3.1 were transfected into PTr2 cells, and then CCK-8 cell proliferation assay and EdU cell proliferation assay were performed. Figure 17As shown in a: The results show that upregulating IL1R2 can significantly inhibit the proliferation of PTr2 cells (P < 0.01). Figure 17 As shown in b: The results showed that after overexpression of IL1R2, the percentage of proliferating cells was significantly reduced (P < 0.05), further indicating that overexpression of IL1R2 inhibits the proliferation of PTr2 cells.

[0130] 4.5 Overexpression of IL1R2 gene inhibits the migration of PTr2 cells

[0131] In order to study the effect of overexpression of IL1R2 gene on the migration of PTr2 cells, IL1R2 overexpression vector pcDNA3.1-IL1R2 and empty vector pcDNA3.1 were transfected into PTr2 cells, and then the scratch test was performed. The experimental results are shown in Figure 2. Figure 18 The results showed that the migration speed of PTr2 cells overexpressing IL1R2 was significantly slowed down (P < 0.01) and the migration distance was significantly reduced in the same period of time, indicating that overexpression of the IL1R2 gene significantly inhibited the migration ability of PTr2 cells.

[0132] Example 5 Effect of IL1R2 on Inflammation-Related Genes in PTr2 Cells

[0133] PTr2 cells were transfected with si-IL1R2 or si-NC; or the IL1R2 overexpression vector pcDNA3.1-IL1R2 or the empty vector pcDNA3.1 was transfected into PTr2 cells. Then, the effect of IL1R2 on inflammation-related genes in PTr2 cells was investigated by RT-qPCR. The experimental results are shown in Figure 2. Figure 19 Results showed that IL1R2 interference significantly inhibited the mRNA expression of the inflammatory gene TNF-α (P < 0.05), while overexpression of IL1R2 significantly increased TNF-α mRNA expression (P < 0.01). Furthermore, when IL1R2 was interfered with, the downstream inflammatory factors of TNF-α, IL6 and IL8, showed a downward trend.

[0134] The primers for RT-qPCR detection are shown in Table 10:

[0135] Table 10 qPCR primer sequences

[0136]

[0137] In summary, the present invention collects placenta and umbilical cord samples from high birth weight (HBW) and low birth weight (LBW) piglets in the same litter, compares placental traits and tissue morphology, and then performs transcriptome sequencing and whole-genome methylation sequencing on the placenta samples of HBW and LBW piglets to screen out the differentially methylated and differentially expressed gene IL1R2. This gene has a higher methylation level and lower expression in the HBW placenta. Then, by studying the methylation level of this gene, it was found that the methylation level of the IL1R2 gene in the HBW placenta was significantly higher than that in the LBW placenta, suggesting that the methylation status of the IL1R2 gene may be closely related to placental development and fetal growth regulation. Then, the expression of IL1R2 in PTr2 cells was regulated by DNA methylation. The results showed that the DNA methylation inhibitor 5-Aza can induce the expression of the IL1R2 gene in PTr2 cells, and its regulatory effect increases in a dose-dependent manner. It was further confirmed that during placental development, the expression of the IL1R2 gene is regulated by DNA methylation, and the methylation level of the IL1R2 promoter region in PTr2 cells treated with 5-Aza is reduced. Then, by inhibiting or overexpressing the IL1R2 gene in PTr2 cells, the results showed that inhibiting (interfering with) the IL1R2 gene can promote the proliferation and migration of PTr2 cells. PTr2 cells play an important role in placental development. By promoting the proliferation and migration of PTr2 cells, the development of the placenta can be effectively promoted, and the birth weight of piglets can be further increased. Moreover, by inhibiting the expression of the IL1R2 gene in PTr2 cells, the mRNA expression of the inflammatory gene TNF-α can be significantly inhibited, and IL6 and IL8 can be downregulated, indicating that inhibiting the IL1R2 gene can also regulate the inflammatory response in the placenta to maintain the homeostasis and development of placental function, thereby affecting the birth weight of piglets. Based on this, the quality of pig placental development or the birth weight of pigs can be regulated by regulating the methylation level or expression level of the IL1R2 gene; the proliferation and / or migration of pig placental trophoblast cells can also be promoted by regulating the methylation level or expression level of the IL1R2 gene; the inflammatory response of pig placental trophoblast cells and the inflammatory response in the placenta can also be regulated by regulating the methylation level or expression level of the IL1R2 gene to maintain the homeostasis and development of placental function, thereby affecting the birth weight of piglets. Therefore, regulating the methylation level or expression level of the IL1R2 gene can be used to improve the quality of pig placental development, further improve the birth weight and survival rate of piglets, and improve the reproductive efficiency of sows and the market competitiveness of breeding pig farms. IL1R2 overexpression vector pcDNA3.1-IL1R2 nucleotide sequence (SEQ ID No: 15):

[0138]

[0139]

Claims

1. Application of the IL1R2 gene in regulating pig placental development or increasing pig birth weight; or application of the gene in preparing a product for regulating pig placental development or increasing pig birth weight.

2. Use of the IL1R2 gene in promoting the proliferation and / or migration of porcine placental trophoblast cells; or use of the gene in preparing a product that promotes the proliferation and / or migration of porcine placental trophoblast cells.

3. Application of the IL1R2 gene in regulating the inflammatory response of porcine placental trophoblast cells; or application in preparing products for regulating the inflammatory response of porcine placental trophoblast cells.

4. The use according to any one of claims 1 to 3, wherein The application is achieved by increasing the methylation level of the IL1R2 gene or inhibiting the expression level of the IL1R2 gene.

5. A method for regulating pig placental development and / or increasing piglet birth weight, wherein: The method comprises adding a reagent capable of increasing the methylation level of the IL1R2 gene or a reagent capable of inhibiting the expression of the IL1R2 gene to sow feed or drinking water; or directly injecting the reagent capable of increasing the methylation level of the IL1R2 gene or a reagent capable of inhibiting the expression of the IL1R2 gene into the placenta.

6. The method according to claim 5, wherein: The reagent capable of inhibiting IL1R2 gene expression is siRNA that interferes with IL1R2 gene expression, and the sequence of the siRNA is GCCCAUGGAGGAAGGUCAUTT.AUGACCUUCCUCCAUGGGCTT.

7. A method for promoting the proliferation and / or migration of pig placental trophoblast cells, wherein: The method comprises adding a reagent capable of increasing the methylation level of the IL1R2 gene to a culture medium of pig placental trophoblast cells or injecting or transfecting a reagent for inhibiting the expression of the IL1R2 gene into the pig placental trophoblast cells. The reagent for inhibiting the expression of the IL1R2 gene is an siRNA that interferes with the expression of the IL1R2 gene, and the sequence of the siRNA is GCCCAUGGAGGAAGGUCAUTT, AUGACCUUCCUCCAUGGGCTT.

8. A product that can promote pig placenta development and / or increase piglet birth weight, wherein: The product contains a reagent capable of inhibiting IL1R2 gene expression, wherein the reagent is siRNA that interferes with IL1R2 gene expression, and the sequence of the siRNA is GCCCAUGGAGGAAGGUCAUTT, AUGACCUUCCUCCAUGGGCTT.

9. Use of an agent that inhibits IL1R2 gene expression in the preparation of a product that promotes pig placental development and / or increases piglet birth weight; or in the preparation of a product that promotes the proliferation and / or migration of pig placental trophoblast cells; or in the preparation of a product that regulates the inflammatory response of pig placental trophoblast cells.

10. The use according to claim 9, wherein: The reagent for inhibiting IL1R2 gene expression is siRNA that interferes with IL1R2 gene expression, and the sequence of the siRNA is GCCCAUGGAGGAAGGUCAUTT.AUGACCUUCCUCCAUGGGCTT.

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