New application of miR-363-5p

Abstract

The invention relates to the technical field of poultry breeding, and particularly provides a novel application of miR-363-5p. A hepatocyte model is established through overexpression and knock-down of the miR-363-5p so as to detect key indexes related to lipid synthesis, oxidative stress and inflammatory factors, and research results show that inflammatory response in chicken hepatocytes can be inhibited, oxidative stress can be reduced and lipid degradation can be prevented by reducing expression of the miR-363-5p. In addition, the invention also finds that the development of chicken liver cells is controlled by targeting the miR-363-5p to the CPEB2, ERK signal transduction can be activated by knocking down the miR-363-5p or overexpressing the CPEB2, so that the miR-363-5p-CPEB2-MAPK / ERK axis is determined to be a key regulatory factor in the pathogenesis of the chicken fatty liver. Therefore, the miR-363-5p and / or CPEB2 gene can be used as a detection target to develop a reagent or a kit for diagnosing the chicken fatty liver syndrome.

Description

Technical Field

[0001] The present invention relates to the technical field of poultry breeding, and in particular to a new application of miR-363-5p. Background Art

[0002] As the primary metabolic organ in chickens, the liver plays a key role in immune defense, coagulation regulation, and lipid synthesis, providing essential support for growth, development, and egg production. The liver is a central site of lipid metabolism, producing low-density lipoprotein (LDL), which provides essential nutrients for follicular growth and is crucial for maintaining metabolic homeostasis and reproductive efficiency. However, during the peak egg-laying period of laying hens, the demand for LDL increases dramatically, placing a greater metabolic burden on the liver. This leads to excessive lipid accumulation and, eventually, hepatic steatosis. This pathological change can further induce fatty liver syndrome (FLS), characterized by oxidative stress and inflammation, which severely impairs the production performance and economic benefits of poultry farming. Research has shown that the development of FLS may be caused by a variety of factors, including overnutrition, a high-fat diet, inappropriate feeding practices, and disease states. Furthermore, recent studies have revealed that abnormal miRNA expression is involved in regulating the pathophysiology of FLS.

[0003] miRNAs are an important class of small, non-coding RNA molecules, approximately 20-24 nucleotides in length. They primarily exist as single strands in eukaryotes and regulate gene expression by degrading mRNA or inhibiting translation. In animals, miRNAs are involved in a variety of biological processes, such as cell growth, proliferation, differentiation, and apoptosis. Therefore, it is crucial to identify miRNAs that may influence the regulatory functions of chicken livers, thereby advancing research on chicken FLS. Summary of the Invention

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

[0005] In order to achieve the above object, the technical solution of the present invention is: In a first aspect, the present invention provides the use of miR-363-5p and / or the target gene CPEB2 of miR-363-5p as a detection target in the preparation of a diagnostic reagent or a diagnostic kit for chicken fatty liver syndrome.

[0006] In a second aspect, the present invention provides use of a reagent for detecting miR-363-5p and / or the target gene CPEB2 of miR-363-5p in preparing a diagnostic reagent or a diagnostic kit for chicken fatty liver syndrome.

[0007] Preferably, the diagnostic reagent or diagnostic kit contains primers for detecting the miR-363-5p and / or the CPEB2 gene.

[0008] Further preferably, the primers for detecting 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.

[0009] In a third aspect, the present invention provides the use of a reagent for reducing or inhibiting the expression of miR-363-5p in the preparation of a product for preventing and treating chicken fatty liver syndrome, wherein the reagent has a sequence as shown in SEQ ID NO. 3.

[0010] In a fourth aspect, the present invention provides the use of the target gene CPEB2 of miR-363-5p in the preparation of a product for preventing and treating chicken fatty liver syndrome.

[0011] In a fifth aspect, the present invention provides the use of a reagent for enhancing or promoting the expression of the target gene CPEB2 of miR-363-5p or a reagent for reducing the adsorption of the target gene CPEB2 of miR-363-5p in the preparation of a product for preventing and treating chicken fatty liver syndrome.

[0012] In a sixth aspect, the present invention provides a product for preventing and treating chicken fatty liver syndrome, comprising the sequence shown in SEQ ID NO. 3.

[0013] In a seventh aspect, the present invention provides a method for promoting lipid metabolism balance in chicken liver and / or enhancing the antioxidant stress resistance of liver cells and / or weakening liver cell autophagy for non-therapeutic and non-diagnostic purposes, comprising: transfecting one of the sequences shown in SEQ ID NO. 3 into primary chicken liver cells.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This study establishes a hepatocyte model by overexpressing and knocking down miR-363-5p to examine key indicators related to lipid synthesis, oxidative stress, and inflammatory factors, further deepening our understanding of the mechanism of action of miR-363-5p in liver cell regulation. The results demonstrate that reducing miR-363-5p expression can inhibit inflammatory responses, reduce oxidative stress, and prevent lipid degradation in chicken hepatocytes. Furthermore, the study also discovered that miR-363-5p targets CPEB2 to control the development of chicken hepatocytes. Both knocking down miR-363-5p and overexpressing CPEB2 activate ERK signaling, establishing the miR-363-5p-CPEB2-MAPK / ERK axis as a key regulatory factor in the pathogenesis of fatty liver disease in chickens. Therefore, miR-363-5p and / or CPEB2 genes can also be used as detection targets to develop reagents or kits for diagnosing chicken fatty liver syndrome, providing new specific targets 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 THE DRAWINGS

[0015] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: 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.

[0016] 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.

[0017] Figure 3 shows the effect of miR-363-5p on regulating inflammation in chicken hepatocytes according to Example 1 of the present invention. (A and B) qPCR analysis of IL-1β, IL-6, IL-12, TNF-α, and INF-γ expression in hepatocytes overexpressing and knocking down miR-363-5p. (C and D) Western blotting assessment of IL-6 expression in hepatocytes after miR-363-5p knockdown and overexpression, and quantitative analysis of IL-6 in hepatocytes. (E, F, and G) ELISA analysis of IL-1β, IL-6, and TNF-α secreted in the supernatant of hepatocytes transfected with a miR-363-5p mimic, a mimic negative control, a miR-363-5p inhibitor, and an inhibitor negative control. Data are presented as mean ± standard error (SEM). *p < 0.05, **p < 0.01.

[0018] Figure 4 shows the results of a study of miR-363-5p's target genes and regulatory effects in Example 2 of the present invention. (A and B) qPCR analysis of the expression of target genes KPN4, CPEB2, PARD3B, CDC73, and NFATC2 following overexpression and knockdown of miR-363-5p. (C) Construction of wild-type and mutant CPEB2 dual-luciferase reporter genes. (D) Luciferase activity was measured in hepatocytes after co-transfection of wild-type or mutant dual-luciferase reporter genes with a miR-363-5p mimic or a mimic negative control. Data are presented as mean ± standard error (SEM). *p < 0.05, **p < 0.01.

[0019] Figure 5 shows the results of CPEB2 regulation of hepatocyte lipid synthesis in Example 2 of the present invention. (A) CPEB2 knockdown efficiency after transfection of chicken hepatocytes with three siRNAs. (B) Expression of lipid synthesis-related genes in hepatocytes was assessed by qPCR after CPEB2 knockdown. (C and D) Western blotting analysis of lipid synthesis-related gene expression in hepatocytes after CPEB2 knockdown. (D) Quantitative analysis of Western blotting. (E) Representative images of BODIPY 493 / 503 staining of hepatocytes after si-CPEB2 transfection. (F and G) TC and TG levels in hepatocytes were assessed using biochemical assay kits. Data are presented as mean ± standard error (SEM). *p < 0.05, **p < 0.01.

[0020] Figure 6 shows the results of a study on the inhibition of oxidative stress in hepatocytes by CPEB2 in Example 2 of the present invention. (A) qPCR assessment of the expression levels of antioxidant enzyme-related genes after CPEB2 interference. (B) Flow cytometry analysis of relative ROS fluorescence in hepatocytes treated with si-CPEB2. (C) Western blotting analysis of GPX7 and SOD expression in hepatocytes transfected with si-CPEB2. (D) Statistical analysis of the relative protein expression levels of antioxidant enzyme-related genes. (E) Results of a biochemical assay kit for assessing MDA content in hepatocytes. (F, G, and H) Results of biochemical assay kits for analyzing GSH, MDA, SOD, and T-AOC levels in hepatocytes. Data are presented as mean ± standard error (SEM). *p < 0.05, **p < 0.01.

[0021] Figure 7 shows (A) quantitative analysis of inflammation-related gene levels by qPCR after CPEB2 downregulation in Example 2 of the present invention. (B) Western blotting analysis of IL-6 expression in hepatocytes after si-CPEB2 transfection. (C, D, and E) ELISA measurement of TNFα, IL-1β, and IL-6 levels in hepatocyte supernatants. Data are expressed as mean ± standard error (SEM). *p < 0.05, **p < 0.01.

[0022] Figure 8 shows the levels of extracellular signal-regulated kinases 1 / 2 (ERK1 / 2) and phosphorylated extracellular signal-regulated kinases 1 / 2 (P-ERK1 / 2) in transfected cells detected by Western blot analysis according to Example 2 of the present invention. (A, B, and C) Western blot analysis of ERK1 / 2 and P-ERK1 / 2 expression in hepatocytes treated with a miR-363-5p mimic, a miR-363-5p inhibitor, and si-CPEB2. Statistical data on relative protein expression levels are shown on the right. Data are presented as mean ± standard error (SEM). *p < 0.05, **p < 0.01.

[0023] FIG9 is a model diagram showing that miR-363-5p of the present invention targets CPEB2 to regulate hepatocyte lipid synthesis, oxidative stress and inflammation by mediating the mitogen-activated protein kinase (MAPK) signaling pathway. DETAILED DESCRIPTION

[0024] In the description of the present invention, it should be noted that if specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] Example 1:

[0027] This example further explores the role of miR-363-5p in the development of chicken fatty liver disease, as follows: (1) Experimental animals Thirty 300-day-old Tianfu broiler breeder chickens were selected for the experiment. Chickens were fasted for 3 hours before treatment. Subsequently, they were anesthetized with 4% chloral hydrate (50 mg / kg) injected via the wing vein and anticoagulated with sodium heparin (1750 U / kg). All animal experiments were approved by the Animal Welfare Committee of Sichuan Agricultural University (Approval Number: 202300268).

[0028] (2) Test method (2-1) Isolation and culture of hepatocytes After 5 minutes of anesthesia and anticoagulation, the chicken's peritoneal cavity was surgically opened and the liver removed intact. The liver was then perfused continuously with 1 L of calcium-free HEPES buffer (pH 7.5) for 15 minutes, followed by another 15-minute perfusion with calcium chloride buffer. Following perfusion, the liver was carefully rinsed and placed in a Petri dish containing PBS on a sterile workbench. Forceps were used to remove the membrane surrounding the liver. A suitable amount of liver was then cut with scissors and placed in a new Petri dish. Using fresh forceps, any visible blood vessels were removed. After this removal, the liver was placed in a 50 ml beaker, supplemented with the appropriate amount of double-stranded antibody and PBS, and minced with fine scissors for 15 minutes. The supernatant was then centrifuged and removed. The liver was then enzymatically digested with type II collagenase (Biofroxx, Einhausen, Germany) in a 37°C waterbath for 15 minutes. After 15 minutes, digestion was terminated with an equal volume of culture medium. The resulting cell suspension was filtered through 30μm and 70μm cell strainers (Biologix, Jinan, Shandong, China). The supernatant was removed by centrifugation, and 10 mL of erythrocyte lysis buffer (Life-ilab, Shanghai, China) was added to the cell pellet and incubated at room temperature for 10 minutes to promote erythrocyte lysis. Hepatocytes were collected by further centrifugation using a mixture containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 1% penicillin-streptomycin (Solarbio, Beijing, China), and M199 medium (Gibco, Grand Island, NY, USA). Hepatocytes were cultured at 37°C in a humidified atmosphere with 5% CO2, with the medium replaced every 24 hours to maintain optimal growth.

[0029] (2-2) Plasmid construction and transfection One hour before transfection, replace the growth medium (M199-DMEM supplemented with 10% FBS, 1% penicillin, and 1% streptomycin) with double the volume of penicillin / streptomycin-free medium (M199-DMEM supplemented with 10% FBS only). Begin transfection when cells reach 50%-60% confluency. Subsequently, miR-363-5p mimic, mimic negative control (mimic NC), miR-363-5p inhibitor, and inhibitor negative control (inhibitor NC) synthesized by Qingke Biotechnology (Beijing, China) were transfected into the 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. Each construct was then introduced into cell culture plates separately. Hepatocytes were cultured at 37°C with 5% CO2, and the medium was changed every 24 hours to maintain optimal growth conditions.

[0030] Table 1 Sequences of miR-363-5p related constructs

[0031] Note: In the sequence listing of the present invention, the base 'u' in all sequences is represented by 'w'.

[0032] (2-3) RNA extraction, cDNA synthesis, and quantitative real-time fluorescence quantitative PCR (qPCR) 24 hours after transfection, an appropriate amount of Trizol (Invitrogen, Carlsbad, CA, USA) was added to each cell culture plate to lyse the cells and extract RNA from the hepatocytes. Subsequently, mRNA and miRNA were synthesized using the PrimeScript RT Reagent Kit and the One-Step miRNA Synthesis Kit from Takara Biotech (Beijing, China), respectively. qPCR was then performed using SYBR Green Master Mix (Takara Biotech, Beijing, China). β-actin was used as an internal reference gene for mRNA, and U6 was used as an internal reference gene for miRNA. 2 -ΔΔCt Methods: Relative expression levels of qPCR data were analyzed. Primer sequences are listed in Table 2.

[0033] Table 2 mRNA sequences

[0034]

[0035] (2-4) Protein extraction and Western blotting (WB) 48 hours after transfection, 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). β-tubulin was used as a control. Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (PVDF membranes, Millipore, MA, USA). The PVDF membranes were then used for the relevant experiments. After treatment with primary antibodies at 4°C for 12 hours, secondary antibodies were incubated for 1 hour at 4°C. Table 3 shows the concentrations of the relevant antibodies. Protein bands were analyzed using Image J (National Institutes of Health, Bethesda, MD, USA) after adding ECL luminescence solution (Beyotime, Shanghai, China).

[0036] Table 3 Antibody dilution concentration

[0037] (2-5) Detection of inflammatory cytokines The treated cell culture medium was collected 48 h after transfection, and the concentrations of tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in the culture medium were determined using ELISA kits (Newgeorge, Shanghai, China) according to the manufacturer's instructions.

[0038] (2-6) Flow cytometry analysis Intracellular reactive oxygen species (ROS) levels were measured by flow cytometry. Hepatocytes were harvested 48 hours after transfection, and a ROS probe was introduced into the cells using a ROS detection kit (Beyotime, Shanghai, China) for ROS detection. Fluorescence was then measured using a CytExpert flow cytometer. Fluorescence measurements in hepatocytes were performed by Sichuan Chengdu AoChuang Biotechnology Co., Ltd. Data were processed and evaluated using Kaluza 2.1 software.

[0039] (2-7) BODIPY 493 / 503 staining Forty-eight hours after transfection, hepatocytes were fixed with 4% paraformaldehyde (Beyotime) for 30 minutes at room temperature. Hepatocyte lipid droplets were labeled with BODIPY 493 / 503 (Maokang Biotechnology, Shanghai, China) for 30 minutes, followed by nuclei staining with Hoechst 33342 (Reibo Biotechnology, Guangzhou, China) for 15 minutes. Three randomly selected fields of view were observed using an IX53 biological microscope (Olympus, Tokyo, Japan).

[0040] (2-8) Assessment of lipid and oxidative stress indicators Forty-eight hours after transfection, triglyceride (TG) and total cholesterol (TC) levels in hepatocytes were measured using biochemical detection kits (Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the manufacturer's instructions.

[0041] 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 (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) according to the instructions in the kits.

[0042] (2-9) Dual luciferase reporter gene assay Dual-luciferase reporter gene studies were performed using a 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. Both WT and MT were cloned into the pmirGLO dual-luciferase reporter vector. DF-1 cells were cultured in 48-well plates and co-transfected with miR-363-5p mimics or a mimic negative control, along with CPEB2-WT (wild-type) or CPEB2-MT (mutant). After 48 hours of culture, luciferase activity was measured using the Dual-Luciferase Reporter Gene Assay Kit (Promega, Madison, WI, USA).

[0043] (2-10) Statistical analysis All statistical analyses of data in this experiment were performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Each experiment included at least three biological replicates, and results are presented as least-squares means ± standard error of the mean (SEM). Comparisons between two groups were performed using an unpaired t-test, while statistical significance among multiple groups was assessed using one-way analysis of variance. P values less than 0.05 were considered significant (* indicates p < 0.05; ** indicates p < 0.01).

[0044] (3) Results and analysis (3-1) miR-363-5p promotes lipid synthesis in hepatocytes 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.

[0045] (3-2) miR-363-5p induces oxidative stress in hepatocytes 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 2 Figure 2A). After interfering with miR-363-5p, the expression of these antioxidant-related genes increased significantly (Figure 2B). Similar results were also verified by protein immunoblotting analysis of SOD and GPX7 protein levels (Figure 2CF). Flow cytometry analysis showed that miR-363-5p overexpression significantly increased the level of intracellular ROS, while its knockdown reduced the fluorescence intensity of ROS in hepatocytes (Figure 2G and H). Biochemical kit analysis showed that knockdown of miR-363-5p significantly reduced the expression of MDA in hepatocytes, while overexpression significantly increased the expression of MDA ( Figure 2 Furthermore, knockdown of miR-363-5p significantly increased the levels of key antioxidant enzymes (T-AOC, GSH, and SOD), whereas overexpression of miR-363-5p reduced the levels of these antioxidant markers ( Figure 2 These findings highlight the important role of miR-363-5p in oxidative stress in hepatocytes.

[0046] (3-3) miR-363-5p exacerbates chicken hepatocyte inflammation Excessive lipid accumulation in the liver is associated with oxidative stress and inflammatory responses. qPCR analysis showed that miR-363-5p overexpression significantly upregulated the expression of proinflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-12β (IL-12β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) compared with the control group ( Figure 3 In contrast, knockdown of miR-363-5p significantly inhibited the expression of these inflammatory mediators ( Figure 3 Western blot analysis also showed that miR-363-5p overexpression significantly enhanced the protein expression of IL-6 in hepatocytes ( Figure 3 C), while knockdown of miR-363-5p effectively attenuated the production of IL-6 ( Figure 3ELISA analysis of inflammatory cytokines showed that miR-363-5p overexpression significantly enhanced the secretion of IL-1β, IL-6, and TNF-α compared with the mimic negative control (mimic NC). Conversely, knockdown of miR-363-5p significantly attenuated the release of these pro-inflammatory cytokines ( Figure 3 These findings together highlight the critical role of miR-363-5p in regulating inflammation in chicken hepatocytes.

[0047] Therefore, monitoring the expression of miR-363-5p in chicken hepatocytes can be used as one of the criteria for determining chicken FLS. Furthermore, miR-363-5p can be used as a detection target to develop related reagents and kits.

[0048] Example 2:

[0049] This example investigates the target genes of miR-363-5p and its role in the development of chicken fatty liver disease, as follows: (1) miR-363-5p targets the CPEB2 gene In this example, using miRDB (https: / / mirdb.org / ), we predicted five target genes for miR-363-5p: KPNA4, CPEB2, PARD3B, CDC73, and NFATC2. These genes are known to be expressed at higher levels in normal liver than in fatty liver. qPCR analysis (primer sequences are shown in Table 4) showed that, following the same procedures as in Example 1 (2-2) for plasmid construction and transfection, and (2-3) for RNA extraction, cDNA synthesis, and quantitative real-time PCR (qPCR), knockdown of miR-363-5p significantly upregulated CPEB2 mRNA levels, while overexpression of miR-363-5p significantly decreased its expression (Figure 4, A and B). To confirm the direct regulatory interaction between miR-363-5p and CPEB2, dual-luciferase reporter assays were performed using wild-type (CPEB2-WT) and mutant (CPEB2-MT) CPEB2 constructs (Figure 4, C). Luciferase activity assays showed that transfection with a miR-363-5p mimic significantly inhibited luciferase activity in DF-1 cells transfected with CPEB2-WT, but had no effect in cells transfected with CPEB2-MT (Figure 4D). These findings confirm that CPEB2 is a direct functional target gene of miR-363-5p in chicken hepatocytes. Therefore, CPEB2 can also be used as a detection target to construct a diagnostic reagent or kit for chicken fatty liver syndrome. Such a diagnostic reagent can utilize the primer pair set forth in SEQ ID NOs. 35 and 36, or the diagnostic kit can include the primer pair set forth in SEQ ID NOs. 35 and 36. Monitoring CPEB2 expression in the liver can be used as a basis for determining the presence of FLS in chickens.

[0050] Table 4 Primer sequences

[0051] (2) CPEB2 inhibits hepatocyte lipid synthesis To investigate the functional role of CPEB2 in lipid metabolism, three small interfering RNA (siRNA) constructs targeting CPEB2 (s1-CPEB2, s2-CPEB2, and s3-CPEB2, sequences shown in Table 5) were designed. Transfection and RNA extraction, cDNA synthesis, and quantitative real-time fluorescence quantitative PCR (qPCR) were performed according to (2-2) of Example 1. Although all constructs showed significant knockdown efficiency in hepatocytes ( Figure 5 However, s3-CPEB2 was selected for subsequent experiments because of its better silencing effect ( Figure 5 Medium BG, Figure 6 、 Figure 7 、 Figure 8 qPCR analysis showed that knockdown of CPEB2 significantly upregulated the mRNA levels of key lipogenic genes compared with the control group ( Figure 5 Consistent with the transcriptional changes, western blot analysis confirmed that the expression of marker proteins related to adipogenesis was increased in si-CPEB2-treated cells ( Figure 5 BODIPY 493 / 503 staining further demonstrated that intracellular lipid accumulation increased after CPEB2 silencing ( Figure 5 These findings were confirmed by biochemical analysis, which showed that intracellular triglyceride (TG) and total cholesterol (TC) levels were significantly increased in CPEB2-deficient hepatocytes ( Figure 5 Conversely, enrichment or overexpression of CPEB2 downregulated the mRNA levels of key lipogenic genes and the expression of lipogenesis-related marker proteins, and significantly reduced triglyceride and tc levels in hepatocytes. These results collectively indicate that CPEB2 is a key regulator of hepatic lipogenesis.

[0052] Table 5 siRNA sequences for CPEB2

[0053] (3) CPEB2 inhibits hepatocyte oxidative stress 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.

[0054] (3-7) CPEB2 inhibits chicken hepatocyte inflammation 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.

[0055] (3-8) miR-363-5p mediates the MAPK / ERK signaling pathway by targeting CPEB2 To elucidate the molecular mechanism by which miR-363-5p regulates fatty liver in chickens, this example used the Kyoto Encyclopedia of Genes and Genomes (KEGG, https: / / www.kegg.jp / kegg / pathway.html) to predict the CPEB2 signaling pathway and found that it may act through the canonical MAPK signaling pathway. Western blotting (see protein extraction and Western blotting in (2-4) of Example 1) was used to examine 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 CPEB2 knockdown. Overexpression of miR-363-5p significantly decreased the expression levels of P-ERK1 / 2 / ERK1 / 2 (Figure 8A), while knockdown of miR-363-5p exhibited the opposite effect (Figure 8B). Similarly, the expression levels of P-ERK1 / 2 / ERK1 / 2 were significantly decreased after knockdown of CPEB2 (Figure 8C).

[0056] The present invention shows that miR-363-5p induces inflammatory response, aggravates oxidative stress and promotes lipid degradation in chicken hepatocytes, e.g. Figure 9To explore the underlying mechanism, the present invention predicted and confirmed the function of its downstream target gene CPEB2. The results showed that miR-363-5p targets CPEB2 to mediate the MAPK / ERK signaling pathway, thereby exacerbating the development of FLS in chickens. This invention establishes the miR-363-5p-CPEB2-MAPK / ERK axis as a key regulatory factor in the pathogenesis of fatty liver disease in chickens. This pathway provides a specific target for the treatment of FLS in poultry and opens up new possibilities for the development of precise nutritional interventions for FLS.

[0057] Example 3:

[0058] This example investigates the application of miR-363-5p and its target gene CPEB2 as targets for diagnosing chicken FLS, as follows: (1) Construction of chicken FLS model A total of 160 Tianfu green-shell laying hens, aged 160 days, were randomly divided into two groups: a control group (80 birds) and an experimental group (80 birds). The control group was fed a diet with a metabolizable energy of 11.51 MJ / kg and a crude protein content of 15.58%. The experimental group, on the other hand, was fed a diet with a diet that increased its metabolizable energy to 14.47 MJ / kg and reduced its crude protein content to 12.55%. After 25 days of feeding, the body weights, serum triglyceride (TG), and total cholesterol (TC) levels of the birds in the control and experimental groups were measured. The results showed that the body weights, serum TG, and TC levels of the birds in the experimental group were significantly higher than those in the control group (Table 6), indicating that the FLHS model was successfully established in the experimental group.

[0059] Table 6 Experimental group and control group

[0060] (2) qPCR analysis RNA was extracted from the serum of chickens in the control and experimental groups using the RNAeasy™ Blood RNA Extraction Kit (Spin Column) (Shanghai Biotech Co., Ltd.). qPCR analysis of miR-363-5p and its target gene CPEB2 was performed. The qPCR reaction system is shown in Table 7, the primer sequences used for miR-363-5p are shown in Table 8, and the primers used for CPEB2 are shown in SEQ ID NOs. 35 and 36. The qPCR results are shown in Table 9.

[0061] Table 7 qPCR reaction system

[0062] Table 8 miR-363a-5p qPCR primers

[0063] The qPCR reaction conditions were as follows: pre-denaturation at 98°C for 2 min; 40 cycles of denaturation at 98°C for 2 s, annealing at 65°C for 15 s, and extension at 72°C for 10 s; during the melting curve generation phase, the temperature increased from 65°C to 95°C at an increment of 0.5°C per second.

[0064] Table 9 qPCR results

[0065] According to the data in Table 9, among all samples in the control and experimental groups, miR-363-5p expression was significantly increased in the experimental group, while CPEB2 expression was significantly decreased. The specific criteria for FLS were: a Ct value of miR-363-5p below 34 and / or a Ct value of CPEB2 above 35 in the experimental group.

[0066] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents. The embodiments described above merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent of the present invention. It should be pointed out that, without departing from the concept of the present invention, a number of variations and improvements may be made by those skilled in the art, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the appended claims.

Claims

1. Use of miR-363-5p and / or the target gene CPEB2 of miR-363-5p 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 miR-363-5p and / or the target gene CPEB2 of miR-363-5p. Among them, the primers for detecting the expression of miR-363-5p have the sequences shown in SEQ ID NO.53 and SEQ ID NO.54; And / or, the primers for detecting the expression level of the target gene CPEB2 of miR-363-5p have the sequences shown as SEQ ID NO.35 and SEQ ID NO.36.

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