New use of miR-363-5p

Abstract

The application relates to the field of poultry breeding technology, and specifically provides a new application of miR-363-5p. A liver cell model is established by overexpressing and knocking down miR-363-5p to detect key indexes related to lipid synthesis, oxidative stress and inflammatory factors, and the research results show that the inflammatory reaction in chicken liver cells can be inhibited, oxidative stress can be reduced, and lipid degradation can be prevented by reducing the expression of miR-363-5p. In addition, it is found that miR-363-5p targets CPEB2 to control the development of chicken liver cells, and the ERK signal transduction can be activated by knocking down miR-363-5p or overexpressing CPEB2, and it is established that the miR-363-5p-CPEB2-MAPK / ERK axis is a key regulatory factor in the pathogenesis of chicken fatty liver. Therefore, the miR-363-5p and / or CPEB2 gene can be used as a detection target to develop a reagent or kit for diagnosing chicken fatty liver syndrome.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of poultry breeding technology, in particular to a new application of miR-363-5p. BACKGROUND

[0002] As the main metabolic organ of chicken, liver plays a key role in immune defense, coagulation regulation, and lipid synthesis, providing essential support for the growth, development, and egg production of chickens. Liver is the core site of lipid metabolism, which can produce low-density lipoprotein (LDL) to provide essential nutrients for follicle growth, which is crucial for maintaining metabolic balance and reproductive efficiency. However, during the peak egg-laying period of laying hens, the metabolic burden of the liver increases significantly due to the substantial increase in LDL demand, leading to excessive lipid accumulation and gradually causing liver steatosis. This pathological change can further induce fatty liver syndrome (FLS), which is characterized by oxidative stress and inflammation, severely damaging the production performance and economic benefits of poultry farming. Studies have shown that the occurrence of FLS can be caused by multiple factors, including excessive nutrition, high-fat diet, unreasonable feeding methods, and disease status. In addition, recent research has found that abnormal miRNA expression is also involved in the regulation of the pathophysiological mechanism of FLS.

[0003] MiRNAs are a class of important non-coding small RNA molecules, about 20-24 nucleotides in length, mainly existing in single-stranded form in eukaryotes, which regulate gene expression by degrading mRNA or inhibiting translation. In animals, miRNAs are involved in various biological processes such as cell growth, proliferation, differentiation, and apoptosis. Therefore, it is necessary to explore miRNAs that may affect the regulatory function of chicken liver, thereby promoting the progress of chicken FLS-related research. SUMMARY

[0004] Based on the series of miRNAs that may affect the metabolism and development of chicken liver in the applicant's previous research, miR-363-5p was preliminarily selected to elucidate the potential molecular mechanism and further explore the role of miR-363-5p in the development of chicken fatty liver disease, providing new ideas and methods for solving chicken FLS, thus proposing a new application of miR-363-5p.

[0005] To achieve the above purpose, the technical scheme of the present application is as follows:

[0006] In a first aspect, the present application provides the application of miR-363-5p and / or the target gene CPEB2 of miR-363-5p as a detection target in the preparation of a chicken fatty liver syndrome diagnostic reagent or diagnostic kit.

[0007] In a second aspect, the present application provides use of a reagent for detecting miR-363-5p and / or a target gene CPEB2 of miR-363-5p in preparation of a diagnostic reagent or a diagnostic kit for chicken fatty liver syndrome.

[0008] Preferably, the diagnostic reagent or the diagnostic kit contains primers for detecting the miR-363-5p and / or the CPEB2 gene.

[0009] Further preferably, the primers for detecting the miR-363-5p have sequences as shown in SEQ ID NO. 53 and SEQ ID NO. 54; and / or, the primers for detecting the CPEB2 gene have sequences as shown in SEQ ID NO. 35 and SEQ ID NO. 36.

[0010] In a third aspect, the present application provides use of a reagent for reducing or inhibiting expression of miR-363-5p in preparation of a product for preventing and treating chicken fatty liver syndrome, the reagent having a sequence as shown in SEQ ID NO. 3.

[0011] In a fourth aspect, the present application provides use of a target gene CPEB2 of miR-363-5p in preparation of a product for preventing and treating chicken fatty liver syndrome.

[0012] In a fifth aspect, the present application provides use of a reagent for promoting expression of a target gene CPEB2 of miR-363-5p or a reagent for reducing adsorption of a target gene CPEB2 of miR-363-5p in preparation of a product for preventing and treating chicken fatty liver syndrome.

[0013] In a sixth aspect, the present application provides a product for preventing and treating chicken fatty liver syndrome, comprising a sequence as shown in SEQ ID NO. 3.

[0014] In a seventh aspect, the present application provides a method for promoting lipid metabolism balance in the liver of a chicken and / or enhancing the antioxidant stress resistance of liver cells and / or weakening autophagy of liver cells for non-treatment and non-diagnostic purposes, comprising: transfecting one of sequences as shown in SEQ ID NO. 3 in primary liver cells of a chicken.

[0015] Compared with the prior art, the present application has the following beneficial effects:

[0016] The application establishes a liver cell model by overexpression and knockdown of miR-363-5p to detect key indicators related to lipid synthesis, oxidative stress and inflammatory factors, and further deepen the understanding of the mechanism of miR-363-5p in the regulation of liver cells. The research results show that by reducing the expression of miR-363-5p, the inflammatory response in chicken liver cells can be inhibited, oxidative stress can be reduced, and lipid degradation can be prevented. In addition, the application also found that miR-363-5p targets CPEB2 to control the development of chicken liver cells, and the activation of ERK signaling can be achieved by knocking down miR-363-5p or overexpressing CPEB2, thereby establishing that the miR-363-5p-CPEB2-MAPK / ERK axis is a key regulator in the pathogenesis of chicken fatty liver. Therefore, the miR-363-5p and / or CPEB2 gene can also be used as a detection target to develop a reagent or kit for diagnosing chicken fatty liver syndrome, providing a new specific target for the early diagnosis and prevention of poultry FLS, and opening up new possibilities for the development of precise nutritional intervention measures for FLS. BRIEF DESCRIPTION OF DRAWINGS

[0017] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

[0018] Figure 1The results of miR-363-5p's effects in chicken hepatocytes in vitro in Example 1 of the present invention are shown. (A and B) qPCR was used to detect miR-363-5p expression in chicken hepatocytes after transfection with a miR-363-5p mimic or inhibitor. (C) qPCR was used to detect the expression of ACCa, SCD1, FASN, and PPARα in hepatocytes overexpressing miR-363-5p. (D) qPCR was used to assess the expression of lipid synthesis-related genes in hepatocytes knocked down by miR-363-5p. (E) Western blotting was used to analyze the protein expression of FASN and SCD1 in hepatocytes transfected with a miR-363-5p overexpression vector. (F) Statistical analysis of the relative protein expression levels of cellular lipid synthesis-related genes. (G) Western blotting was used to detect the protein expression of FASN and SCD1 in transfected hepatocytes. (H) Quantitative analysis of the Western blotting results. (I) Representative images of BODIPY 493 / 503 staining after treatment of hepatocytes with miR-363-5p mimics, mimic negative control (Mimic NC), miR-363-5p inhibitor, and inhibitor negative control (Inhibitor NC). Green in BODIPY represents hepatocyte lipid droplets, and blue in Hoechst represents the nuclei of all hepatocytes. The fusion image of the two is called an overlay. (J and K) Results of biochemical assays used to measure total cholesterol (TC) and triglyceride (TG) levels in hepatocytes. Data are expressed as mean ± standard error (SEM). *p < 0.05, **p < 0.01.

[0019] Figure 2 shows the results of miR-363-5p's effects on oxidative stress in hepatocytes in Example 1 of the present invention. (A and B) qPCR analysis of SOD, GPX7, TRX, and GST expression in hepatocytes overexpressing and knocking down miR-363-5p. (C and D) Western blotting analysis of SOD and GPX7 expression in hepatocytes after miR-363-5p overexpression and knockdown. (E and F) Statistical analysis of relative protein expression levels of genes related to lipid synthesis in cells. (G and H) Flow cytometry analysis of relative fluorescence values ​​of intracellular reactive oxygen species (ROS) after miR-363-5p knockdown and upregulation. (I) Results of a biochemical assay kit for measuring malondialdehyde (MDA) levels in hepatocytes. (J, K, and L) Results of biochemical assay kits for measuring SOD, total antioxidant capacity (T-AOC), and glutathione (GSH) in hepatocytes. The data are expressed as mean ± SEM, *p < 0.05, **p < 0.01.

[0020] Figure 3 is the effect of miR-363-5p on regulating inflammation in chicken hepatocytes in Example 1 of the present application, wherein (A and B) qPCR analysis of the expression of IL-1β, IL-6, IL-12, TNF-α and INF-γ in hepatocytes overexpressing and knocking down miR-363-5p. (C and D) Western blotting to evaluate the expression of IL-6 in hepatocytes after miR-363-5p knockdown and overexpression treatment, and quantitative analysis of the detection of IL-6 in hepatocytes. (E, F and G) ELISA was used to detect the contents of IL-1β, IL-6 and TNF-α secreted by hepatocytes in the supernatant after transfection of miR-363-5p mimics, negative control of mimics, miR-363-5p inhibitor and negative control of inhibitor. Data are expressed as mean ± standard error (Mean ± SEM), *p < 0.05, **p < 0.01.

[0021] Figure 4 is the results of the target genes and regulatory effects of miR-363-5p in Example 2 of the present application, wherein (A and B) qPCR detection of the expression of target genes KPN4, CPEB2, PARD3B, CDC73 and NFATC2 after overexpression and knockdown of miR-363-5p. (C) Construction of wild-type and mutant dual luciferase reporter genes of CPEB2. (D) Detection of luciferase activity after co-transfection of wild-type or mutant dual luciferase reporter genes with miR-363-5p mimics or negative control of mimics in hepatocytes. Data are expressed as mean ± standard error (Mean ± SEM), *p < 0.05, **p < 0.01.

[0022] Figure 5 is the results of CPEB2 regulating lipid synthesis in hepatocytes in Example 2 of the present application, wherein (A) the knockdown efficiency of CPEB2 after transfection of chicken hepatocytes with three siRNAs. (B) Detection of the expression of lipid synthesis-related genes in hepatocytes after interference of CPEB2 by qPCR. (C and D) Western blotting analysis of the expression of lipid synthesis-related genes in hepatocytes after interference of CPEB2, and D is the quantitative analysis of Western blotting. (E) Representative images of BODIPY 493 / 503 staining of hepatocytes after si-CPEB2 transfection. (F and G) Detection of the contents of TC and TG in hepatocytes by biochemical detection reagent kit. Data are expressed as mean ± standard error (Mean ± SEM), *p < 0.05, **p < 0.01.

[0023] Figure 6 is the results of the study of CPEB2 inhibition of oxidative stress in hepatocytes in Example 2 of the present application, wherein (A) After interfering with CPEB2, the expression levels of antioxidant enzyme-related genes were evaluated by qPCR. (B) Flow cytometry analysis of the relative ROS fluorescence value in si-CPEB2-treated hepatocytes. (C) Western blot analysis of the expression of GPX7 and SOD in hepatocytes after transfection with si-CPEB2. (D) Statistical data of the relative protein expression of antioxidant enzyme-related genes. (E) Biochemical analysis kit detection results for evaluating the MDA content in hepatocytes. (F, G and H) Biochemical detection kit detection results for analyzing the levels of GSH, MDA, SOD and T-AOC in hepatocytes. Data are expressed as mean ± standard error (Mean ± SEM), *p < 0.05, **p < 0.01.

[0024] Figure 7 is (A) Quantitative analysis of the levels of inflammation-related genes after down-regulating CPEB2 using qPCR in Example 2 of the present application. (B) Western blot analysis of the expression of IL-6 in hepatocytes after transfection with si-CPEB2. (C, D and E) ELISA measurement of the levels of TNFα, IL-1β and IL-6 expressed by hepatocytes in the supernatant. Data are expressed as mean ± standard error (Mean ± SEM), *p < 0.05, **p < 0.01.

[0025] Figure 8 is the detection of the levels of extracellular signal-regulated kinase 1 / 2 (ERK1 / 2) and phosphorylated extracellular signal-regulated kinase 1 / 2 (P-ERK1 / 2) in transfected cells in Example 2 of the present application, wherein (A, B and C) Western blot analysis of the expression of ERK1 / 2 and P-ERK1 / 2 in hepatocytes after treatment with miR-363-5p mimic, miR-363-5p inhibitor and si-CPEB2. The right side is the statistical data of the relative protein expression levels of the cells. Data are expressed as mean ± standard error (Mean ± SEM), *p < 0.05, **p < 0.01.

[0026] Figure 9 is a model diagram of the regulation of hepatocyte lipid synthesis, oxidative stress and inflammation by miR-363-5p targeting CPEB2 through mediating the mitogen-activated protein kinase (MAPK) signaling pathway. DETAILED DESCRIPTION

[0027] In the description of the present application, it should be noted that, in the examples, if the specific conditions are not specified, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0028] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0029] Embodiment 1

[0030] This embodiment further explores the role of miR-363-5p in the development process of chicken fatty liver disease, as follows:

[0031] (1) Test animals

[0032] 30 300-day-old Tianfu broiler chickens were selected as experimental objects. Before treatment, the chickens were fasted for 3 hours. Subsequently, the chickens were anesthetized by intravenous injection of 4% chloral hydrate (dose: 50 mg / kg) and anticoagulated by injection of heparin sodium (dose: 1750 U / kg). All animal experiments were approved by the Animal Welfare Committee of Sichuan Agricultural University (Approval No.: 202300268).

[0033] (2) Test method

[0034] (2-1) Isolation and culture of hepatocytes

[0035] After 5 minutes of anesthesia and anticoagulation treatment, the abdominal cavity of the chicken was opened by surgery, and the liver was completely removed. Then, the liver was perfused with 1 L of calcium-free HEPES buffer (pH 7.5) for 15 minutes, and then perfused with a buffer containing calcium chloride for 15 minutes. After perfusion, the liver was carefully washed and placed in a culture dish added with PBS on a sterile workbench, the membranes on the surface of the liver were removed using tweezers, and the appropriate amount of liver was cut off using scissors and placed in a new culture dish. A new pair of tweezers was used to remove the obvious blood vessels in the liver, and after removal, the liver was placed in a 50 ml beaker, and the appropriate amount of double antibody and PBS was added. A small pair of scissors was used to cut for 15 minutes, and then the supernatant was removed by centrifugation. Then, collagenase type II (Biofroxx, Einhoven, Germany) was used to perform enzyme digestion treatment in a 37°C water bath for 15 minutes, and the digestion was terminated after 15 minutes by adding an equal amount of medium. The cell suspension obtained after digestion was filtered using cell filters with pore sizes of 30 μm and 70 μm (Biologix, Jinan, Shandong, China). The supernatant was removed by centrifugation, and then 10 mL of red blood cell lysis buffer (Life-ilab, Shanghai, China) was added to the cell sediment and incubated at room temperature for 10 minutes to promote red blood cell lysis. When the liver cells were collected by centrifugation again, a mixture containing 10% fetal bovine serum (Gibco, Grand Island, New York, USA), 1% penicillin-streptomycin (Solarbio, Beijing, China), and M199 medium (Gibco, Grand Island, New York, USA) was used. The liver cells were cultured in a 37°C, 5% CO2 humidified environment, and the medium was replaced every 24 hours to maintain their optimal growth state.

[0036] (2-2) Plasmid construction and transfection

[0037] One hour before transfection, the growth medium (M199-DMEM, added with 10% FBS, 1% penicillin and 1% streptomycin) was replaced with double volume of penicillin / streptomycin-free medium (M199-DMEM, added with only 10% FBS). When the cell confluency reached 50%-60%, transfection was started. Subsequently, the miR-363-5p mimic, mimic negative control (mimic NC), miR-363-5p inhibitor, inhibitor negative control (inhibitor NC) synthesized by GenScript (Beijing, China) were transfected into cells using Opti-MEM (Gibco, Grand Island, NY, USA) and Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The miR-363-5p mimic, mimic negative control, miR-363-5p inhibitor, inhibitor negative control, si-CPEB2 and siRNA negative control were mixed with opti-MEM and Lipofectamine 3000 and incubated at room temperature for 15 minutes. The sequences of these constructs are listed in Table 1. After that, each construct was introduced into the cell culture plate, respectively. The hepatocytes were cultured in a 37°C, 5% CO2 environment, and the medium was replaced every 24 hours to maintain optimal growth conditions.

[0038] Table 1 Sequences of miR-363-5p related constructs

[0039]

[0040] Note: In the sequence listing file of the present application, the base 'u' in all sequences is represented by 'w'.

[0041] (2-3) RNA extraction, cDNA synthesis and quantitative real-time fluorescence quantitative PCR (qPCR)

[0042] After 24 hours of transfection, an appropriate amount of Trizol (Invitrogen, Carlsbad, CA, USA) was added to each cell culture plate to lyse the cells for RNA extraction from hepatocytes. Subsequently, the mRNA and miRNA were synthesized using the PrimeScript RT reagent kit and one-step miRNA synthesis kit from Bosheng Engineering (Beijing, China), respectively. Then, qPCR was performed using SYBR Green Master Mix (Bosheng Engineering, Beijing, China). β-actin was used as the internal control gene for mRNA, and U6 was used as the internal control gene for miRNA. The 2 -ΔΔCtMethod Relative expression of qPCR data was analyzed. Primer sequences are listed in Table 2.

[0043] Table 2 mRNA sequences

[0044]

[0045]

[0046] (2-4) Protein extraction and Western blot (WB)

[0047] After transfection for 48 hours, total protein was extracted from hepatocytes using a tissue or cell total protein extraction kit (Solarbio, Beijing, China). Protein concentration was determined using a bicinchoninic acid (BCA) protein quantification kit (BestBio, Shanghai, China). In the experiment, β-tubulin was used as a control. Proteins were separated using 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride membrane (PVDF membrane, Millipore, Massachusetts, USA). The PVDF membrane was then subjected to related experiments. After treatment with the primary antibody for 12 hours at 4°C, the secondary antibody was incubated for 1 hour at 4°C. Table 3 shows the concentration of the related antibodies. After adding the ECL luminescence solution (Beyotime, Shanghai, China), the protein bands were analyzed using Image J (National Institutes of Health, Bethesda, Maryland, USA).

[0048] Table 3 Antibody dilution concentration

[0049]

[0050] (2-5) Detection of inflammatory cytokines

[0051] After transfection for 48 hours, the treated cell culture medium was collected, and the concentrations of tumor necrosis factor-α (TNFα), interleukin 1β (IL-1β), and interleukin 6 (IL-6) in the medium were determined using an ELISA kit (Newgeorge, Shanghai, China) according to the manufacturer's instructions.

[0052] (2-6) Flow cytometry analysis

[0053] Intracellular reactive oxygen species (ROS) levels were measured by flow cytometry. After 48 hours of transfection, hepatocytes were harvested and ROS probes were introduced into the cells using a ROS detection kit (Beyotime, Shanghai, China) to detect ROS. The fluorescence values were then measured using a CytExpert flow cytometer. The measurement of these fluorescence values in hepatocytes was performed by Sichuan Chengdu Auragene Bio-tech Co., Ltd. Data processing and evaluation were performed using Kaluza 2.1 software.

[0054] (2-7) BODIPY 493 / 503 staining

[0055] After 48 hours of transfection, hepatocytes were fixed with 4% paraformaldehyde (Beyotime) for 30 minutes at room temperature. After labeling the lipid droplets of hepatocytes with BODIPY 493 / 503 (Mogao Biotech, Shanghai, China) for 30 minutes, the cell nuclei were stained with Hoechst 33342 (RiboBio, Guangzhou, China) for 15 minutes. Three fields of view were randomly selected for observation using an IX53 biological microscope (Olympus, Tokyo, Japan).

[0056] (2-8) Evaluation of lipid and oxidative stress indicators

[0057] After 48 hours of transfection, the levels of triglycerides (TG) and total cholesterol (TC) in hepatocytes were measured using biochemical detection kits (Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the manufacturer's instructions.

[0058] In addition, the levels of glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) in hepatocytes were measured using biochemical detection kits (Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the instructions in the kits.

[0059] (2-9) Dual-luciferase reporter gene assay

[0060] Dual-luciferase reporter assay was performed using chicken fibroblast cell line (DF-1 cells). We obtained the 3' untranslated region (3'UTR) sequence of CPEB2 from Ensembl (https: / / www.ensembl.org / index.html?redirect=no) and constructed wild type (WT) and mutant (MT) dual-luciferase reporter genes, and cloned the WT and MT into pmirGLO dual-luciferase reporter vector. DF-1 cells were cultured in 48-well plates and co-transfected with miR-363-5p mimic or mimic negative control, and CPEB2-WT (wild type) or CPEB2-MT (mutant). After 48 hours of culture, luciferase luminescence activity was detected according to the instructions of the dual-luciferase reporter assay reagent kit (Promega, Madison, WI, USA).

[0061] (2-10) Statistical analysis

[0062] Statistical analysis of all data in this experiment was performed using SPSS 17.0 software (SPSS, Chicago, IL, USA). Each experiment contained at least three biological replicates, and the results were expressed as the least square mean ± standard error of the mean (SEM). For comparison between two groups, unpaired t test was used; for statistical significance evaluation among multiple groups, one-way ANOVA was used. Significant difference was indicated when P value was less than 0.05 (* p < 0.05; ** p < 0.01).

[0063] (3) Results and analysis

[0064] (3-1) miR-363-5p promotes lipid synthesis in hepatocytes

[0065] To investigate the role of miR-363-5p in chicken hepatocytes in vitro, miR-363-5p overexpression and knockdown vectors were constructed. The effectiveness of the overexpression and knockdown strategies was confirmed by qPCR (Figure 1, A and B). qPCR analysis showed that miR-363-5p overexpression significantly upregulated the expression of genes such as acetyl-CoA carboxylase α (ACCa), stearoyl-CoA desaturase-1 (SCD1), fatty acid synthase (FASN), and peroxisome proliferator-activated receptor α (PPARα) (Figure 1, C). Western blotting analysis revealed that miR-363-5p overexpression also significantly increased the expression of FASN and SCD1 proteins (Figure 1, E and F). In contrast, the expression of genes and proteins related to lipid synthesis was reduced in the miR-363-5p inhibitor-treated group (Figure 1, D, G, and H). BODIPY 493 / 503 staining results showed that compared with the control group, the miR-363-5p mimics treatment group successfully increased the production of lipid droplets in hepatocytes, while knockdown of miR-363-5p effectively reduced the accumulation of lipid droplets in hepatocytes ( Figure 1 In addition, we used biochemical assays to measure TC and TG levels in hepatocytes following transfection with a miR-363-5p mimic, a mimic negative control, a miR-363a-5p inhibitor, and an inhibitor negative control. The results showed that overexpression of miR-363-5p significantly increased TC and TG levels in hepatocytes, while knockdown of miR-363-5p had the opposite effect (Figure 1, J and K). These results collectively indicate that miR-363-5p plays a role in lipid synthesis in hepatocytes.

[0066] (3-2) miR-363-5p induces oxidative stress in hepatocytes

[0067] To investigate whether miR-363-5p affects oxidative stress in hepatocytes, qPCR analysis was performed on antioxidant enzyme genes (superoxide dismutase (SOD), glutathione peroxidase (GPX), thioredoxin (TRX), and glutathione S-transferase (GST)). The results showed that overexpression of miR-363-5p significantly downregulated the expression of these antioxidant stress-related genes ( Figure 2The expression of these antioxidant-related genes was significantly increased after miR-363-5p interference (Fig. 2B). Similar results were also verified by Western Blot analysis of SOD and GPX7 protein content (Fig. 2C-F). Flow cytometry analysis showed that overexpression of miR-363-5p significantly increased the intracellular ROS level, while its knockdown decreased the ROS fluorescence intensity in hepatocytes (Fig. 2G and H). Biochemical kit analysis showed that knockdown of miR-363-5p significantly decreased the expression of MDA in hepatocytes, while overexpression significantly increased the expression of MDA (Fig. 2I). Figure 2 In addition, knockdown of miR-363-5p significantly increased the levels of key antioxidant enzymes (T-AOC, GSH and SOD), while overexpression did the opposite, decreasing the levels of these antioxidant markers (Fig. 2J, K and L). These findings highlight the important role of miR-363-5p in the oxidative stress process in hepatocytes. Figure 2

[0068] (3-3) miR-363-5p exacerbates inflammation in chicken hepatocytes

[0069] Lipid overaccumulation in the liver is associated with oxidative stress and inflammatory response. qPCR analysis showed that overexpression of miR-363-5p significantly upregulated the expression of pro-inflammatory cytokines, including interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interleukin 12 beta (IL-12β), tumor necrosis factor - alpha (TNF-α) and interferon - gamma (IFN-γ) (Fig. 3A). In contrast, knockdown of miR-363-5p significantly inhibited the expression of these inflammatory mediators (Fig. 3B). Western Blot analysis also showed that overexpression of miR-363-5p significantly enhanced the protein expression of IL-6 in hepatocytes (Fig. 3C), while knockdown of miR-363-5p effectively attenuated IL-6 production (Fig. 3D). ELISA analysis of inflammatory cytokines showed that overexpression of miR-363-5p significantly enhanced the secretion of IL-1β, IL-6 and TNF-α compared to the mimic negative control (mimic NC). In contrast, knockdown of miR-363-5p significantly attenuated the release of these pro-inflammatory cytokines (Fig. 3E, F and G). These findings collectively highlight the key role of miR-363-5p in regulating inflammation in chicken hepatocytes. Figure 3 Figure 3 Figure 3 Figure 3 Figure 3

[0070] ​​​​​​Therefore, by monitoring the expression of miR-363-5p in chicken liver cells, it can be used as one of the criteria for judging chicken FLS. Further, miR-363-5p can be developed as a detection target to develop related reagents and kits.

[0071] Example 2:

[0072] This example investigates the target gene of miR-363-5p and its role in the development of chicken fatty liver disease, as follows:

[0073] (1) miR-363-5p targets CPEB2 gene

[0074] In this example, miRDB (https: / / mirdb.org / ) was used to predict 5 target genes of miR-363-5p, namely KPNA4, CPEB2, PARD3B, CDC73 and NFATC2, which are known to have higher expression levels in normal liver than in fatty liver. qPCR analysis showed (the primer sequences used are shown in Table 4) that, referring to the plasmid construction and transfection in (2-2) and the RNA extraction, cDNA synthesis and quantitative real-time fluorescence quantitative PCR (qPCR) in (2-3) of Example 1, knocking down miR-363-5p significantly up-regulated the mRNA level of CPEB2, while overexpressing miR-363-5p significantly reduced its expression (A and B in Figure 4). To confirm the direct regulatory interaction between miR-363-5p and CPEB2, a dual luciferase reporter gene experiment was performed using wild-type (CPEB2-WT) and mutant (CPEB2-MT) constructs of CPEB2 (C in Figure 4). Luciferase activity detection showed that transfection of miR-363-5p mimics significantly inhibited the luciferase activity in DF-1 cells transfected with CPEB2-WT, but had no effect on cells transfected with CPEB2-MT (D in Figure 4). These findings collectively confirm that CPEB2 is a direct functional target gene of miR-363-5p in chicken liver cells. Therefore, CPEB2 can also be used as a detection target to construct a chicken fatty liver syndrome diagnostic reagent or diagnostic kit, wherein the diagnostic reagent can use the primer pair shown as SEQ ID NO. 35 and SEQ ID NO. 36, or the diagnostic kit contains the primer pair shown as SEQ ID NO. 35 and SEQ ID NO. 36. By monitoring the expression of CPEB2 in the liver, it can be used as one of the criteria for judging chicken FLS.

[0075] Table 4 Primer sequences

[0076]

[0077] (2) CPEB2 inhibits lipid synthesis in hepatocytes

[0078] To investigate the functional role of CPEB2 in lipid metabolism, three siRNA constructs targeting CPEB2 (s1-CPEB2, s2-CPEB2 and s3-CPEB2, sequences shown in Table 5) were designed and transfected into hepatocytes as described in (2-2) of Example 1, followed by RNA extraction, cDNA synthesis and quantitative real-time PCR (qPCR) as described in (2-3) of Example 1. Although all constructs showed significant knockdown efficiency in hepatocytes (Fig. 2A), s3-CPEB2 was chosen for further experiments due to its better silencing effect (Fig. 2B). Figure 5 Figure 5 Figure 6 Figure 7 Figure 8 Figure 5 Figure 5 Figure 5 Figure 5

[0079] Table 5 siRNA sequences of CPEB2

[0080]

[0081] (3) CPEB2 inhibits oxidative stress in hepatocytes

[0082] ​​​​​​​​​This example also investigated whether CPEB2 induces oxidative stress in hepatocytes, referring to protein extraction and western blotting (WB) (2-4) and flow cytometry analysis (2-6) in Example 1. qPCR analysis revealed a significant decrease in the expression of antioxidant genes after hepatocyte transfection with si-CPEB2 (Figure 6A). Flow cytometry analysis demonstrated a dramatic increase in reactive oxygen species (ROS) levels in hepatocytes with CPEB2 silencing (Figure 6B). Western blotting further revealed a significant decrease in the protein expression of SOD and GPX7 (Figure 6C and D). Biochemical assays confirmed these observations, demonstrating that CPEB2 deficiency resulted in increased malondialdehyde (MDA) levels and decreased total antioxidant capacity (T-AOC), SOD, and glutathione (GSH) levels (Figure 6EH). Conversely, enrichment or overexpression of CPEB2 upregulated the expression of antioxidant genes, reduced ROS levels, and increased hepatocyte T-AOC, SOD, and GSH levels. These data strongly suggest that CPEB2 plays a key role in maintaining oxidative homeostasis in hepatocytes.

[0083] (3-7) CPEB2 inhibits chicken hepatocyte inflammation

[0084] To investigate the role of CPEB2 in chicken hepatocyte inflammation, this example first measured inflammation-related genes and proteins in hepatocytes using qPCR and Western blotting. The results showed that knockdown of CPEB2 significantly upregulated the expression of inflammation-related genes. Furthermore, IL-6 protein expression also increased significantly ( Figure 7 Quantitative ELISA analysis (referring to the inflammatory cytokine assays in Example 1 (2-5)) further demonstrated increased secretion of inflammatory cytokines, including IL-1β, IL-6, and TNF-α, in CPEB2-deficient hepatocytes (Figure 7, C, D, and E). Conversely, enrichment or overexpression of CPEB2 significantly downregulated the expression of inflammation-related genes and reduced the secretion of inflammatory cytokines, including IL-1β, IL-6, and TNF-α. These findings establish CPEB2 as a key regulator of inflammatory signaling in chicken hepatocytes.

[0085] (3-8) miR-363-5p mediates the MAPK / ERK signaling pathway by targeting CPEB2

[0086] To elucidate the molecular mechanism of miR-363-5p regulating chicken fatty liver, the present embodiment predicts the signaling pathway of CPEB2 by Kyoto Encyclopedia of Genes and Genomes (KEGG, https: / / www.kegg.jp / kegg / pathway.html) and finds that it can play a role through the classic MAPK signaling pathway. The levels of extracellular signal-regulated kinase 1 / 2 (ERK1 / 2) and phosphorylated extracellular signal-regulated kinase 1 / 2 (P-ERK1 / 2) in cells after knocking down CPEB2 are detected by Western blotting (for protein extraction and Western blotting, refer to (2-4) in Embodiment 1). It is found that the expression levels of P-ERK1 / 2 / ERK1 / 2 are significantly decreased after overexpression of miR-363-5p (A in FIG. 8), while the expression levels of P-ERK1 / 2 / ERK1 / 2 are opposite after knocking down miR-363-5p (B in FIG. 8). Similarly, the expression levels of P-ERK1 / 2 / ERK1 / 2 also significantly decrease after knocking down CPEB2 (C in FIG. 8).

[0087] The present application shows that miR-363-5p induces inflammatory response, exacerbates oxidative stress and promotes lipid degradation in chicken hepatocytes, as shown in Figure 9 To explore the potential mechanism, the present application predicts and confirms the function of the downstream target gene CPEB2. The research results show that miR-363-5p targets CPEB2 to mediate the MAPK / ERK signaling pathway, thereby exacerbating the development of chicken FLS. The present application establishes that the miR-363-5p-CPEB2-MAPK / ERK axis is a key regulator in the pathogenesis of chicken fatty liver. This pathway provides a specific target for the treatment of poultry FLS and opens up new possibilities for the development of precise nutritional intervention measures for FLS.

[0088] Embodiment 3:

[0089] The present embodiment investigates the application of miR-363-5p and its target gene CPEB2 as a target for diagnosing chicken FLS, as follows:

[0090] (1) Construction of chicken FLS model

[0091] 160-day-old Tianfu green-shell laying hens were selected and randomly divided into two groups: a control group (80) and a test group (80). The control group was fed with a diet having a metabolic energy of 11.51 MJ / kg and a crude protein content of 15.58%, while the test group was fed with a diet having a metabolic energy of 14.47 MJ / kg and a crude protein content of 12.55%. After 25 days of feeding, the body weight of the chickens in the control group and the test group and the levels of triglyceride (TG) and total cholesterol (TC) in the serum were detected. The results showed that the body weight, serum TG and TC levels of the chickens in the test group were significantly higher than those in the control group (shown in Table 6), indicating that the FLHS model of the test group was successfully constructed.

[0092] Table 6 Test group and control group

[0093]

[0094] (2) qPCR analysis

[0095] RNA in the serum of the chickens in the control group and the test group was extracted by using RNAeasy™ blood RNA extraction kit (centrifugal column type) (Shanghai Biyun Tian Biotechnology Co., Ltd.), and qPCR analysis was performed on miR-363-5p and its target gene CPEB2. The qPCR reaction system is shown in Table 7, the primer sequence of miR-363-5p is shown in Table 8, the primers of CPEB2 are shown in SEQ ID NO. 35 and SEQ ID NO. 36, and the qPCR results are shown in Table 9.

[0096] Table 7 qPCR reaction system

[0097]

[0098] Table 8 qPCR primers of miR-363a-5p

[0099]

[0100] The qPCR reaction conditions are as follows: 98°C pre-denaturation for 2 min; 98°C denaturation for 2 s, 65°C annealing for 15 s, 72°C extension for 10 s, a total of 40 cycles; and the melting curve generation stage temperature increases from 65°C to 95°C every second, with an increase of 0.5°C every second.

[0101] Table 9 qPCR results

[0102]

[0103] According to the data in Table 9, in all samples of the control group and the test group, the expression amount of miR-363-5p in the test group was significantly increased, while the expression amount of CPEB2 was significantly decreased. The specific judgment criteria were: the Ct value of miR-363-5p in the test group was less than 34, and / or the Ct value of CPEB2 was higher than 35, which was determined as FLS positive.

[0104] Although the embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents. The above-described embodiments only express several implementation manners of the present application, which are described in a more specific and detailed manner, but should not be understood as a limitation on the patent scope of the present application. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, which are all within the protection scope of the present application. Therefore, the patent protection scope of the present application should be subject to the appended claims.

Claims

1. Application of CPEB2 as a detection target in the preparation of a diagnostic kit for chicken fatty liver syndrome, characterized in that: The kit is used to detect the expression level of CPEB2, wherein the primers for detecting the expression level of CPEB2 have the sequences shown as SEQ ID NO.35 and SEQ ID NO.36.

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