SiRNA for targeted inhibition of chicken RUNX1 expression and application thereof
By targeting and inhibiting the chicken RUNX1 gene with siRNA, the technical gap in the research and treatment of broiler lignified muscle disease was solved, effective experimental tools and drug development pathways were provided, and in-depth research and treatment of broiler lignified muscle disease was achieved.
Patent Information
- Application Number
- CN202510902388.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-01
- Publication Date
- 2025-09-30
AI Technical Summary
There is no research in the prior art on using siRNA technology to interfere with the expression of the chicken RUNX1 gene, and the pathogenesis of broiler lignified myopathy is unclear, and there is a lack of effective prevention and treatment methods.
siRNAs targeting the inhibition of chicken RUNX1 gene expression, including RUNX1-566 siRNA, RUNX1-572 siRNA and RUNX1-577 siRNA, were designed and applied. By transfecting chicken embryo fibroblasts DF-1, the expression of RUNX1 gene was significantly inhibited and the expression of genes related to ECM synthesis was reduced.
It provides a simple, low-cost experimental tool for studying the pathogenesis of broiler lignified myopathy and the mechanism of action of the RUNX1 gene, and for developing drugs to prevent and treat broiler lignified myopathy, significantly inhibiting the progression of fibrosis.
Smart Images
Figure CN120718904A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and in particular relates to siRNA for targeted inhibition of chicken RUNX1 expression and application thereof. Background Art
[0002] Wooden breast (WB) is the most common heterogeneous meat in the broiler industry. Fibrosis, characterized by abnormal production and deposition of the extracellular matrix (ECM), is closely associated with the development of WB. Fibrosis is typically a process triggered by chronic tissue damage and inflammation, involving excessive and abnormal deposition of ECM, which impairs tissue function. Fibroblasts and myofibroblasts are the primary support cells that produce ECM components and are essential for the functional assembly of ECM fibers. Transforming growth factor-β (TGF-β) is the primary cytokine that induces the transformation of fibroblasts into myofibroblasts. Following tissue injury, local fibroblasts rapidly migrate to the site of injury and differentiate into contractile myofibroblasts, which synthesize and secrete ECM, thereby initiating muscle repair and remodeling. Once tissue repair is complete, myofibroblasts return to a quiescent state through apoptosis or reprogramming. However, under abnormal conditions such as chronic injury or pathological conditions, myofibroblasts will continue to be activated, causing excessive accumulation of ECM and disordered damage repair, which develops into a pathological fibrosis state. Summary of the Invention
[0003] To address the above technical issues, the present invention provides a siRNA that targets and inhibits chicken RUNX1 expression and its use. The siRNA provided by the present invention can effectively reduce the expression of the chicken RUNX1 gene, thereby inhibiting the differentiation of chicken embryonic fibroblasts DF-1 and reducing the expression of genes related to ECM synthesis. This siRNA can be used to study the pathogenesis of broiler chicken lignified myopathy and the mechanism of action of the RUNX1 gene in broiler chicken lignified myopathy, and can also be used to develop drugs to prevent and treat broiler chicken lignified myopathy.
[0004] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: The first aspect of the present invention provides an siRNA that targets and inhibits the expression of the chicken RUNX1 gene, wherein the siRNA is at least one of RUNX1-566 siRNA, RUNX1-572 siRNA and RUNX1-577 siRNA; the sense chain sequence of the RUNX1-566 siRNA is shown in SEQ ID NO. 1, and the antisense chain sequence is shown in SEQ ID NO. 2; the sense chain sequence of the RUNX1-572 siRNA is shown in SEQ ID NO. 3, and the antisense chain sequence is shown in SEQ ID NO. 4; the sense chain sequence of the RUNX1-577 siRNA is shown in SEQ ID NO. 5, and the antisense chain sequence is shown in SEQ ID NO. 6.
[0005] RUNX1 (Runt-related transcription factor 1) is a key transcription factor involved in cell cycle regulation, apoptosis, differentiation, and cell migration, with a wide range of biological functions. RUNX1 activates genes involved in extracellular matrix (ECM) synthesis and mediates the development of various fibrotic diseases in mammals. However, research on RUNX1 in poultry is limited. There are no reports of using RUNX1 to treat lignified myopathy in broilers or other avian species, nor are there reports of using siRNA to interfere with RUNX1 gene expression in chickens.
[0006] The present invention has discovered through research that the RUNX1 gene can promote the differentiation of chicken embryo fibroblasts DF-1 and enhance the expression of genes involved in ECM synthesis. The siRNA provided by the present invention, when transferred into chicken embryo fibroblasts DF-1, can significantly inhibit the expression of the RUNX1 gene, inhibiting the differentiation of chicken embryo fibroblasts DF-1 and reducing the expression of genes involved in ECM synthesis. Therefore, the siRNA can be used to develop drugs for the prevention and treatment of broiler lignified myopathy, or to study the pathogenesis of broiler lignified myopathy and the role of the RUNX1 gene in broiler lignified myopathy through in vitro experiments.
[0007] Preferably, the siRNA is RUNX1-577 siRNA. Compared with RUNX1-566 siRNA and RUNX1-572 siRNA, RUNX1-577 siRNA has the best inhibitory effect on the RUNX1 gene, with an inhibition efficiency of more than 50%.
[0008] The second aspect of the present invention provides the use of the above siRNA in preparing a kit for inhibiting the expression of chicken RUNX1 gene.
[0009] Preferably, the kit comprises liquid A and liquid B, wherein the components of liquid A are opti-MEM culture medium and an aqueous solution of the siRNA with a concentration of 20 μM in a volume ratio of 25:1, and the components of liquid B are opti-MEM culture medium and Lipofectamine 3000 in a volume ratio of 125:4; the volume ratio of liquid A to liquid B is 130:129.
[0010] The third aspect of the present invention provides the use of the above-mentioned siRNA or kit in in vitro research on the pathogenesis of broiler lignified muscle disease.
[0011] A fourth aspect of the present invention provides the use of the above-mentioned siRNA or kit in in vitro research on the mechanism of action of the RUNX1 gene on lignified muscle disease in broiler chickens.
[0012] After the above-mentioned siRNA was transferred into chicken embryo fibroblasts DF-1, the expression of the chicken RUNX1 gene can be effectively reduced. Compared with gene knockout technology, it is simple to operate, has a short experimental cycle, and is low in price. It can provide a direct and effective experimental tool for in vitro research on the pathogenesis of broiler lignified myopathy and the mechanism of action of the RUNX1 gene on broiler lignified myopathy. The research results have certain guiding significance for further revealing the mechanism of action of the RUNX1 gene in the occurrence of fibrosis in broiler lignified myopathy.
[0013] A fifth aspect of the present invention provides the use of the above-mentioned siRNA in developing a drug for preventing and treating broiler lignified muscle disease, wherein the active ingredient of the drug includes the above-mentioned siRNA.
[0014] The above-mentioned siRNA can significantly inhibit the differentiation of chicken embryo fibroblasts DF-1, and at the same time inhibit the expression of genes related to ECM protein synthesis in the fibrosis progression of lignified myopathy, so it can be used to develop drugs for the prevention and treatment of broiler lignified myopathy.
[0015] Preferably, the drug is a drug that inhibits ECM protein synthesis.
[0016] Preferably, the drug further comprises a vector for transferring the siRNA into cells.
[0017] Further preferably, the vector is a transfection vector.
[0018] The beneficial effects of the present invention are as follows: the present invention proves for the first time that the RUNX1 gene can promote the differentiation of chicken embryo fibroblasts DF-1 and enhance the expression of genes related to ECM synthesis. The siRNA provided by the present invention can effectively target and inhibit the expression of the chicken RUNX1 gene after being transferred into chicken embryo fibroblasts. Compared with gene knockout technology, it is simple to operate, has a short experimental cycle, and is inexpensive. It provides a direct and effective experimental tool for studying the mechanism of action of the RUNX1 gene in broiler lignified myopathy and other avian lignified myopathy. It can be applied to the study of the pathogenesis of broiler lignified myopathy and the study of the mechanism of action of the RUNX1 gene on broiler lignified myopathy. At the same time, the siRNA provided by the present invention can also inhibit the expression of genes related to ECM protein synthesis in the progression of fibrosis of lignified myopathy, and can be used to develop drugs for the prevention and treatment of broiler lignified myopathy. Therefore, the siRNA provided by the present invention has great economic value and scientific research value. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 The expression of RUNX1 in normal breast muscle and lignified breast muscle of white-feathered broiler chickens in Example 1 of the present invention (* indicates significant difference ( P <0.05)); Figure 2 The expression of RUNX1 in the chicken embryo fibroblast differentiation process in Example 1 of the present invention (different letters represent significant differences ( P <0.05)); Figure 3 The expression changes of RUNX1 gene after exogenous transfection of RUNX1 siRNA in Example 1 of the present invention (different letters represent significant differences ( P <0.05, the same letters indicate no significant differences ( P >0.05)); Figure 4 The expression changes of genes related to differentiation and ECM synthesis of chicken embryo fibroblasts after exogenous transfer of RUNX1-577 siRNA in Example 1 of the present invention (* indicates significant difference ( P <0.05)). DETAILED DESCRIPTION
[0020] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.
[0021] Lignified breast muscle is the most common type of heterogeneous meat in the current broiler industry. Fibrosis, characterized by abnormal production and deposition of ECM, is closely associated with the development of lignified breast muscle. Experimental studies have shown that the RUNX1 gene can promote the differentiation of chicken embryonic fibroblasts (DF-1) and enhance the expression of genes related to ECM synthesis. Targeting this gene, the present invention has designed siRNAs capable of targetedly inhibiting gene expression. The siRNAs are at least one of RUNX1-566 siRNA, RUNX1-572 siRNA, and RUNX1-577 siRNA. The sense strand sequence of the RUNX1-566 siRNA is shown in SEQ ID NO. 1, and the antisense strand sequence is shown in SEQ ID NO. 2. The sense strand sequence of the RUNX1-572 siRNA is shown in SEQ ID NO. 3, and the antisense strand sequence is shown in SEQ ID NO. 4. The sense strand sequence of the RUNX1-577 siRNA is shown in SEQ ID NO. 5, and the antisense strand sequence is shown in SEQ ID NO. 6. This siRNA can significantly inhibit the expression of RUNX1 gene when transferred into chicken embryo fibroblasts, inhibit the differentiation of chicken embryo fibroblasts DF-1, and reduce the expression of ECM synthesis-related genes.
[0022] The following is further described with reference to specific embodiments.
[0023] Unless otherwise specified, the raw materials, reagents, drugs, or instruments used in the following examples are all commercially available products. The methods used in the following examples are all conventional methods in the art unless otherwise specified.
[0024] Example 1 In this example, the expression of RUNX1 gene in lignified breast muscle tissue and fibroblasts of broiler chickens was detected by fluorescence quantitative PCR.
[0025] 1. Expression of RUNX1 in lignified pectoral muscle tissue RT-qPCR was used to detect the expression of RUNX1 in white-feathered broilers with normal breast muscle (CON) and white-feathered broilers with lignified breast muscle (WB). Figure 1 As shown in the results, there was a significant difference in the expression of RUNX1 in normal breast muscle and lignified breast muscle, which preliminarily indicated that RUNX1 may be involved in the occurrence of fibrosis in lignified breast muscle of broiler chickens.
[0026] 2. Expression of RUNX1 in fibroblasts RT-qPCR was used to detect the expression of RUNX1 before and after fibroblast differentiation. Figure 2As shown in the results, the expression of RUNX1 was significantly increased after differentiation of the chicken embryo fibroblast cell line DF-1, and the expression of differentiation marker genes and ECM synthesis-related genes was significantly increased, indicating that RUNX1 may play a role in promoting chicken fibroblast differentiation and excessive ECM deposition.
[0027] Example 2 This example provides siRNA that targets and inhibits the expression of the chicken RUNX1 gene and its inhibitory effect on the RUNX1 gene, and examines the changes in the expression levels of genes related to cell differentiation and ECM synthesis after RUNX1 gene knockdown.
[0028] 1. RUNX1 siRNA design The chicken RUNX1 gene mRNA sequence was obtained from the NCBI online database (accession number: NM_001396968.1). Three pairs of siRNAs targeting the CDS region of the chicken RUNX1 gene were designed. Each siRNA sequence was suffixed with a TT overhang. The designed siRNAs were synthesized by Shanghai Sangon Biotechnology Co., Ltd. After synthesis, 125 μL of DEPC water was added to each OD of synthesized siRNA to a final concentration of 20 μM / μL and stored at -20°C until use. The sequences of the three designed siRNA pairs are shown in Table 1.
[0029] Table 1 Three pairs of siRNA sequences
[0030] 2. siRNA transfection into chicken embryo fibroblasts DF-1 After recovery, chicken embryo fibroblasts DF-1 were resuspended in DMEM medium, then seeded into 6-well culture plates and placed in a 37°C incubator for normoxia culture. When the growth density reached 70%, the original medium in the culture plate was aspirated and replaced with fresh medium. Three pairs of siRNAs targeting the inhibition of the chicken RUNX1 gene and a negative control siRNA (NC) were transfected into chicken embryo fibroblasts DF-1 using the transfection reagent Lipofectamine 3000, with three replicate wells set up for each group.
[0031] The specific transfection system is as follows: Experimental group: Solution A: 125 μL opti-MEM medium + 5 μL 20 μM siRNA solution in DEPC water; Solution B: 125 μL opti-MEM medium + 4 μL Lipofectamine 3000; Control group: Solution A: 125 μL opti-MEM medium + 5 μL 20 μM negative sequence solution in DEPC water; Solution B: 125 μL opti-MEM medium + 4 μL Lipofectamine 3000. After preparing Solution A and Solution B, gently pipette to mix them separately and let them stand for 5 minutes. Then, add all of Solution A to Solution B, gently pipette to mix them, and let them stand for 20 minutes. Then, add 259 μL of the A and B mixture to a 6-well plate at a rate of 2 mL / well. Complete medium was then added to bring the volume up to 2 mL.
[0032] 3. RNA Extraction and cDNA Preparation 24 hours after transfection, each well was washed three times with PBS, and then 1 mL of Trizol was added to each well to collect the cells. Total RNA was extracted from cells transfected with RUNX1 siRNA and from the control group using the RNA isolater Total RNA Extraction Reagent kit (Nanjing Novozymes Biotechnology Co., Ltd.). RNA concentration was determined using a Nanodrop ND-1000 nucleic acid quantifier. Reverse transcription was performed according to the instructions for the ABScript Neo RT Master Mix for gPCR with gDNA Remover kit (Wuhan Abotek Biotechnology Co., Ltd.).
[0033] 4. Real-time fluorescence quantitative PCR detection of siRNA interference efficiency Real-time quantitative PCR (qPCR) was performed using SYBR Green I using 2× Universal SYBR Green Fast gPCR Mix reagent (available from Wuhan Abotek Biotechnology Co., Ltd.) to detect the efficiency of siRNA-specific interference with chicken RUNX1 gene expression. Three replicates were performed for each sample. The RT-qPCR protocol was as follows: 95°C for 30 seconds, followed by 40 cycles of (95°C for 10 seconds, 60°C for 30 seconds, 95°C for 15 seconds, and 60°C for 1 minute); and 95°C for 15 seconds.
[0034] Upstream primer: 5′-CCTCAGCCTCAGAGTCAGATTCAG-3′; Downstream primer: 5′-AGGTACTGGTAGGACTGGTCGTAG-3′.
[0035] Detection of chicken internal reference 18s RNA The primer pairs for gene expression are as follows: Upstream primer: 5′-TAGAGGGACAAGTGGCGTTC-3′; Downstream primer: 5′-GGCGTAGGGTAGACACAACC-3′.
[0036] The efficiency of 3 pairs of siRNA in specifically inhibiting chicken RUNX1 gene expression is as follows Figure 3 As shown. Figure 3 It can be seen that compared with the control group, the three pairs of siRNAs can significantly inhibit the expression of RUNX1 gene in chicken embryo fibroblasts DF-1 ( P <0.05), RUNX1-577 siRNA had the best interference efficiency, with an inhibition efficiency of more than 50%. In subsequent experiments, RUNX1-577 was selected to explore the mechanism of RUNX1 gene in broiler chicken lignified myopathy fibrosis.
[0037] 5. Effects of RUNX1 gene inhibition on the expression of genes related to cell differentiation and ECM synthesis 24 hours after RUNX1-577 siRNA transfection, Western blot and RT-qPCR were used to detect the expression changes of differentiation markers and ECM synthesis-related genes in chicken embryo fibroblasts DF-1. Figure 4 It can be seen that transfection with RUNX1-577 siRNA inhibited the differentiation of chicken embryo fibroblasts DF-1, and compared with the control group, the expression of genes related to ECM synthesis was significantly reduced. The experiment proves that the RUNX1 gene can promote the differentiation of chicken embryo fibroblasts DF-1 and enhance the expression of genes related to ECM synthesis.
[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A siRNA targeting and inhibiting the expression of chicken RUNX1 gene, characterized in that: The siRNA is at least one of RUNX1-566 siRNA, RUNX1-572 siRNA and RUNX1-577 siRNA; the sense chain sequence of the RUNX1-566 siRNA is shown as SEQ ID NO.1, and the antisense chain sequence is shown as SEQ ID NO.2; the sense chain sequence of the RUNX1-572 siRNA is shown as SEQ ID NO.3, and the antisense chain sequence is shown as SEQ ID NO.4; the sense chain sequence of the RUNX1-577 siRNA is shown as SEQ ID NO.5, and the antisense chain sequence is shown as SEQ ID NO.
6.
2. The use according to claim 1, characterized in that The siRNA is RUNX1-577 siRNA.
3. Use of the siRNA according to claim 1 in preparing a kit for inhibiting chicken RUNX1 gene expression.
4. The use according to claim 3, characterized in that The kit includes liquid A and liquid B, wherein the components of liquid A are opti-MEM culture medium and an aqueous solution of the siRNA with a concentration of 20 μM in a volume ratio of 25:1, and the components of liquid B are opti-MEM culture medium and Lipofectamine 3000 in a volume ratio of 125:4; the volume ratio of liquid A to liquid B is 130:
129.
5. Use of the siRNA according to claim 1 or the kit according to claim 3 or 4 in in vitro studies on the pathogenesis of lignified myopathy in broiler chickens.
6. Use of the siRNA according to claim 1 or the kit according to claim 3 or 4 in in vitro studies on the mechanism of action of the RUNX1 gene on lignified myopathy in broiler chickens.
7. Use of the siRNA according to claim 1 in developing a drug for preventing and treating broiler chicken lignified muscle disease, characterized in that: The active ingredient of the medicine is the siRNA.
8. The use according to claim 7, characterized in that The drug is a drug that inhibits the synthesis of ECM proteins.
9. The use according to claim 7, characterized in that The drug also includes a vector for transferring the siRNA into cells.
10. The use according to claim 9, characterized in that The vector is a transfection vector.