A reagent for inhibiting CXCR4 gene expression and its application

Abstract

The present invention discloses a reagent and application for inhibiting CXCR4 gene expression, belonging to the field of biomedicine technology. The present invention provides an application of a reagent for inhibiting or knocking down CXCR4 gene expression in the preparation of a preparation for preventing and / or treating AFB1-induced cellular inflammation. The present invention uses a siRNA that can effectively inhibit CXCR4 gene expression, and regulates the damaging effect of AFB1 on mouse renal podocytes MPC-5 by affecting the function of the CXCR4 gene and the expression level of downstream signaling pathway genes, which helps to further clarify the molecular mechanism and regulatory mechanism of AFB1-induced glomerular podocyte inflammation, and is of great significance for the development of therapeutic drugs for AFB1-induced glomerular podocyte inflammation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a reagent for inhibiting CXCR4 gene expression and its application. Background Art

[0002] Aflatoxin B1 (AFB1), a chemically stable fungal metabolite, is the most common environmental carcinogen, posing a significant threat to human health and the economy. Given that some toxins can remain in the kidneys after metabolism, causing tissue damage and leading to renal dysfunction, it is imperative to quickly identify the specific molecular mechanisms underlying this damaging effect.

[0003] CXCR4 (CXC, chemokine receptor type 4) is an α-chemokine receptor that is highly distributed on the cell membrane of podocytes and plays a key role in directing leukocyte migration from the circulation to sites of inflammation. CXCR4 has been shown to play a crucial role in inflammatory diseases, pancreatic cancer invasion, gastrointestinal malignancies, and atherosclerosis. Furthermore, CXCR4 activation is associated with renal fibrosis caused by calcium oxalate crystal deposition in patients with kidney stones and in mice with hyperoxaluria. In spinal cord glial cells of mice induced by partial sciatic nerve ligation and in renal cells induced by oxidative stress induced by 2,2',4,4'-tetrabromodiphenyl ether, CXCR4 associates with thioredoxin-interacting protein (TXNIP) and NOD-like receptor pyrin domain-related protein 3 (NLRP3). Therefore, CXCR4 may trigger inflammatory responses by regulating the TXNIP / NLRP3 inflammasome axis. However, whether CXCR4-mediated TXNIP / NLRP3 activation is involved in AFB1-induced injury of mouse renal podocytes MPC-5 remains to be elucidated. Summary of the Invention

[0004] The purpose of the present invention is to provide a reagent and application for inhibiting CXCR4 gene expression, revealing that CXCR4 plays an important role in mediating AFB1-induced TXNIP and NLRP3 inflammasomes involved in podocyte inflammation, and that inhibiting CXCR4 gene expression can alleviate the damaging effect of AFB1 on cells.

[0005] The present invention provides use of a reagent for inhibiting or knocking down CXCR4 gene expression in preparing a preparation for preventing and / or treating AFB1-induced cellular inflammation.

[0006] The present invention provides an application of a reagent for inhibiting or knocking down CXCR4 gene expression in preparing a preparation for blocking the TXNIP / NLRP3 inflammatory pathway and the production of inflammatory factors.

[0007] Preferably, the method of inhibiting or knocking down CXCR4 gene expression comprises siRNA, shRNA, amiRNA or gene knockout.

[0008] Preferably, the AFB1-induced cellular inflammation includes AFB1-induced glomerular podocyte inflammation.

[0009] Preferably, the inflammatory factors include IL-1β, IL-6 and TNF-α.

[0010] The present invention also provides an siRNA for inhibiting the expression level of the CXCR4 gene. The siRNA comprises a positive chain of the nucleotide sequence shown in SEQ ID No. 1 and an antisense chain shown in SEQ ID No. 2.

[0011] The present invention also provides the use of the above siRNA in preparing a drug for preventing and / or treating cellular inflammation induced by AFB1.

[0012] Preferably, the prevention and / or treatment of AFB1-induced cellular inflammation is manifested in at least one of the following aspects:

[0013] (1) Blocking the TXNIP / NLRP3 inflammatory pathway;

[0014] (2) Inhibit inflammatory factors;

[0015] (3) Affecting CXCR4 gene function and downstream signaling pathway genes.

[0016] Beneficial Effects: The present invention provides a reagent for inhibiting or knocking down CXCR4 gene expression for use in the preparation of a preparation for preventing and / or treating AFB1-induced cellular inflammation. This invention utilizes an siRNA that effectively inhibits CXCR4 gene expression, modulating the damaging effects of AFB1 on mouse renal podocytes (MPC-5) by affecting CXCR4 gene function and the expression levels of genes in the downstream signaling pathway. This helps to further elucidate the molecular and regulatory mechanisms of AFB1-induced glomerular podocyte inflammation and is of great significance for the development of therapeutic drugs for AFB1-induced glomerular podocyte inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of the mRNA and protein expression levels of the CXCR4 gene after siRNA interference are shown in the figure. “*” indicates p < 0.05 compared with the control siRNA group;

[0018] Figure 2Figure 3. Changes in the mRNA and protein expression levels of proinflammatory factors IL-1β, IL-6, and TNF-α in MPC-5 cells after siRNA transfection with AFB1. The relative expression levels of IL-1β, IL-6, and TNF-α mRNA were standardized with β-actin. "*" indicates p < 0.05 compared with the control group; "#" indicates p < 0.05 compared with the AFB1 and control siRNA groups.

[0019] Figure 3 The graph shows the changes in the CXCR4 / TXNIP / NLRP3 signaling axis and the protein expression levels of nephrin and podocin after siRNA transfection and AFB1 treatment of MPC-5; "*" indicates p < 0.05 compared with the control group; "#" indicates p < 0.05 compared with the AFB1 and control siRNA groups. DETAILED DESCRIPTION

[0020] The present invention provides use of a reagent for inhibiting or knocking down CXCR4 gene expression in preparing a preparation for preventing and / or treating AFB1-induced cellular inflammation.

[0021] The present invention is not particularly limited to the method of inhibiting or knocking down CXCR4 gene expression, and preferably includes siRNA, shRNA, amiRNA or gene knockout. In the embodiment, siRNA is used as an example for illustration, but it cannot be identified as the entire protection scope of the present invention. The cells of the present invention preferably include multiple types, such as multiple cells that induce inflammation by AFB1. In the embodiment, glomerular podocytes are used as an example for illustration, but it cannot be identified as the entire protection scope of the present invention. The inflammatory factors of cellular inflammation of the present invention preferably include IL-1β, IL-6 and TNF-α. The examples of the present invention demonstrate that knocking down CXCR4 gene expression can reduce AFB1-induced glomerular podocyte inflammation, such as reducing the expression of inflammatory factors, reducing the relative protein expression of nephrin and podocin induced by AFB1, and reducing the relative protein expression of CXCR4 / TXNIP / NLRP3 / ASC / Cleaved-caspase 1. This demonstrates that AFB1 can induce intracellular CXCR4 expression, thereby activating the TXNIP / NLRP3 inflammatory pathway and the production of inflammatory factors, leading to inflammation and damage of glomerular podocytes. Reducing CXCR4 expression blocks the activation of the TXNIP / NLRP3 inflammatory pathway, significantly reducing the production of inflammatory factors, and exhibiting a good therapeutic effect.

[0022] The present invention provides an application of a reagent for inhibiting or knocking down CXCR4 gene expression in preparing a preparation for blocking the TXNIP / NLRP3 inflammatory pathway and the production of inflammatory factors.

[0023] The application of the present invention is preferably the same as described above and will not be described again here.

[0024] The present invention also provides an siRNA for inhibiting the expression level of the CXCR4 gene. The siRNA comprises a positive chain of the nucleotide sequence shown in SEQ ID No. 1 and an antisense chain shown in SEQ ID No. 2.

[0025] The siRNA described in the present invention reveals that CXCR4 plays an important role in mediating AFB1-induced TXNIP and NLRP3 inflammasomes involved in podocyte inflammation; at the same time, siRNA can significantly attenuate the damage and inflammatory effects of AFB1 on glomerular podocytes, thus helping to further elucidate the molecular mechanism and regulatory mechanism of AFB1-induced glomerular podocyte inflammation, which is of great significance for the development of therapeutic drugs for AFB1-induced glomerular podocyte inflammation.

[0026] The present invention also provides the use of the above siRNA in preparing a drug for preventing and / or treating cellular inflammation induced by AFB1.

[0027] In the present invention, the prevention and / or treatment of AFB1-induced cellular inflammation is preferably manifested in at least one of the following aspects:

[0028] (1) Blocking the TXNIP / NLRP3 inflammatory pathway;

[0029] (2) Inhibit inflammatory factors;

[0030] (3) Affecting CXCR4 gene function and downstream signaling pathway genes.

[0031] To further illustrate the present invention, the reagent for inhibiting CXCR4 gene expression and its application provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Unless otherwise specified, the methods used in this example are conventional methods known to those skilled in the art, and the reagents and other materials used are commercially available products unless otherwise specified.

[0033] Example 1

[0034] 1. siRNA sequence design

[0035] The CXCR4 gene sequence (GeneID: 12767) was obtained from GeneBank using bioinformatics methods. Based on the siRNA sequence design principles, several pairs of siRNA interference sequences were designed. The blast function of the GeneBank database was used to compare the sequences with the human genome to ensure that there was no homology. Finally, an siRNA was screened out, whose nucleic acid sequence is:

[0036] Sense strand (SEQ ID No. 1): 5′GGUUACCAGAAGAAGCUAATT 3′;

[0037] Antisense strand (SEQ ID No. 2): 5'UUAGCUUCUUCUGGUAACCTT 3'.

[0038] 2. Detection of CXCR4 gene mRNA and protein expression after siRNA interference

[0039] 2.1 Detection of CXCR4 gene mRNA expression after siRNA interference

[0040] MPC-5 cells were seeded in 12-well plates and transfected when the cell density reached 75-80%. Lipofectamine 2000 was used for transfection. The specific procedures were performed according to the product instructions, ensuring a final concentration of 100 nM for siRNA and control siRNA. Twenty-four hours after transfection, the cells were harvested, washed with PBS, and the supernatant removed by centrifugation. Total cellular RNA was extracted using Trizol and used for cDNA synthesis. After reverse transcription, qRT-PCR was performed to measure CXCR4 mRNA expression.

[0041] Table 1 qPCR primer sequences

[0042]

[0043] 2.2 Detection of CXCR4 gene protein expression after siRNA interference

[0044] MPC-5 cells were seeded in 6-well plates and transfected when the cell density reached 75-80%. The transfection method was lipofectamine 2000 transfection. The specific operation was carried out according to the product instructions to ensure that the final concentration of siRNA and control siRNA was 100nM. 24 hours after transfection with siRNA and control siRNA, the cells were harvested and washed with PBS. Protein samples were prepared by SDS-PAGE electrophoresis and transferred to PVDF membranes. After blocking with 5% skim milk for 2 hours, the membranes were washed with PBST for 5 minutes, repeated three times, and incubated with primary antibodies (β-actin antibody (Abcam) and CXCR4 antibody (Abcam)) overnight at 4°C. The membranes were washed with TBST for 5 minutes, repeated three times, and the secondary antibody (goat anti-rabbit IgG H&L (HRP) (CST)) was added. The membranes were incubated at room temperature for 1 hour, washed with TBST for 5 minutes, repeated three times, and exposed for 3-5 minutes. After development, the relative protein expression of the CXCR4 gene was observed.

[0045] The results of the determination of CXCR4 gene mRNA and protein expression after siRNA interference are as follows Figure 1 As shown, the mRNA and protein expression levels of the CXCR4 gene in MPC-5 transfected with siRNA were significantly decreased compared with those in MPC-5 not transfected with siRNA (p<0.05). Therefore, this siRNA can be used as an interference sequence to detect the function of the CXCR4 gene and downstream genes.

[0046] 3. Detection of IL-1β, IL-6, and TNF-α mRNA and protein expression after siRNA interference with AFB1 treatment of MPC-5

[0047] 3.1 Detection of IL-1β, IL-6, and TNF-α mRNA expression after siRNA interference with AFB1 treatment of MPC-5

[0048] MPC-5 was seeded in a 12-well plate and transfected when the cell density reached 75-80%. The transfection method was lipofectamine 2000 transfection. The specific operation was carried out according to the product instructions to ensure that the final concentration of siRNA and control siRNA was 100nM. 24 hours after transfection of siRNA and control siRNA, both were replaced with complete culture medium containing 20μM AFB1, and MPC-5 was cultured for another 24 hours. The cells in the wells of the challenge group and the control group were collected, washed with PBS, and the supernatant was removed by centrifugation. The total cell RNA was extracted using the Trizol method for cDNA synthesis. After reverse transcription, qRT-PCR reaction was performed to detect the mRNA expression levels of IL-1β, IL-6, and TNF-α.

[0049] Table 2 qPCR primer sequences

[0050]

[0051] 3.2 Detection of protein expression of IL-1β, IL-6, and TNF-α after siRNA interference with AFB1 treatment of MPC-5

[0052] MPC-5 cells were seeded in 6-well plates and transfected when the cell density reached 75-80%. The transfection method was lipofectamine 2000 transfection. The specific operation was carried out according to the product instructions to ensure that the final concentration of siRNA and control siRNA was 100nM. 24 hours after transfection of siRNA and control siRNA, both were replaced with complete culture medium containing 20μM AFB1, and MPC-5 was cultured for another 24 hours. The supernatant in the wells of the challenge group and the control group was aspirated, and the protein levels of IL-1β, IL-6, and TNF-α in MPC-5 were detected using ELISA kits (R&D Systems Inc.).

[0053] The results of the determination of IL-1β, IL-6 and TNF-α mRNA and protein expression levels after siRNA interference AFB1 treatment of MPC-5 are shown in Figure 2. Figure 2 As shown in the results, CXCR4 was interfered with in MPC-5 cells by siRNA, and after challenge treatment, the downregulation of CXCR4 significantly reduced the mRNA and protein expression of AFB1-induced proinflammatory factors IL-1β, IL-6 and TNF-α compared with the control siRNA (p<0.05).

[0054] 4. Detection of CXCR4 / TXNIP / NLRP3 signaling axis and protein expression of nephrin and podocin after siRNA interference with AFB1 treatment of MPC-5

[0055] MPC-5 cells were seeded in 6-well plates and divided into four groups: a control group, a virus-only challenge group, and a virus-challenged group plus siRNA or control siRNA group. Transfection was performed when the cell density reached 75-80%. Lipofectamine 2000 was used for transfection according to the product instructions, ensuring a final concentration of 100 nM for the siRNA and control siRNA. Twenty-four hours after transfection with siRNA and control siRNA or without transfection reagent, the three groups were replaced with complete culture medium containing 20 μM AFB1 and cultured for an additional 24 hours. The cells in the challenge group and the control group were collected and washed with PBS. Protein samples were prepared and electrophoresed by SDS-PAGE. The samples were transferred to PVDF membranes and blocked with 5% skim milk for 2 h. The membranes were washed with PBST for 5 min, repeated 3 times, and incubated with primary antibodies (β-actin antibody (Abcam), nephrin antibody (Abcam), podocin antibody (Abcam), CXCR4 antibody (Abcam), ASC antibody (Abcam), NLRP3 antibody (Abcam), Pro-caspase1 antibody (CST), Cleaved-caspase 1 antibody (CST), TXNIP antibody (Abcam)) at 4 ° C overnight. The membranes were washed with TBST for 5 min, repeated 3 times, and the secondary antibodies (goat anti-rabbit IgG) were added. The cells were incubated at room temperature for 1 hour, washed with TBST for 5 minutes, and repeated three times. The cells were exposed for 3-5 minutes. After development, the relative protein expression levels of nephrin and podocin and the CXCR4 / TXNIP / NLRP3 signaling axis, including the relative protein expression levels of CXCR4 / TXNIP / NLRP3 / ASC / Cleaved-caspase 1, were observed.

[0056] The results of the determination of CXCR4 / TXNIP / NLRP3 signaling axis and nephrin and podocin protein expression levels after siRNA interference AFB1 treatment of MPC-5 are shown in Figure 2. Figure 3 As shown in the results, siRNA was used to interfere with CXCR4 in MPC-5 cells. After challenge treatment, the downregulation of CXCR4 significantly reduced the relative protein expression of nephrin and podocin induced by AFB1 and the relative protein expression of CXCR4 / TXNIP / NLRP3 / ASC / Cleaved-caspase 1 compared with the control siRNA (p<0.05).

[0057] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Use of siRNA in the preparation of a drug for preventing and / or treating AFB1-induced glomerular podocyte inflammation, characterized in that: The siRNA comprises a positive strand as shown in SEQ ID No. 1 and an antisense strand as shown in SEQ ID No. 2.

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