Application of SETD1A protein

Abstract

The present invention belongs to the field of target drug technology, and in particular relates to an application of a SETD1A protein. The amino acid sequence of the SETD1A protein can be found in NCBI under the accession number NP_055527. The SETD1A protein is used as a drug target in the preparation of a drug for treating diseases caused by tobacco-induced DNA damage. By increasing SETD1A expression, for example, SETD1A can be overexpressed by plasmid transfection to repair DNA damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of target drugs, and in particular relates to an application of SETD1A protein. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease characterized by persistent airflow limitation, primarily caused by cigarette smoke exposure. COPD is currently the third leading cause of death in my country, resulting in a significant healthcare burden. In recent years, clinical interventions for COPD progression have primarily focused on medications such as glucocorticoids and bronchodilators, oxygen therapy, exercise rehabilitation, and surgery. While these interventions can alleviate symptoms, improve mobility, and enhance patients' quality of life to a certain extent, they are unlikely to effectively halt the disease's progression. DNA damage in bronchial and alveolar cells caused by the large amounts of reactive oxygen species (ROS) in cigarette smoke is a key factor in the development of COPD. Following DNA damage, the body initiates a series of DNA damage responses (DDRs) to activate repair pathways, restoring the structure or enabling cells to tolerate the damaged state and continue to survive. However, unrepaired DNA damage can lead to genetic instability and cellular senescence, inhibiting alveolar epithelial cell proliferation and enhancing inflammatory responses, thus promoting COPD progression. Therefore, identifying drugs that can repair cigarette smoke-induced DNA damage and effectively inhibit its accumulation would be of great value in inhibiting the development and progression of COPD. Summary of the Invention

[0003] The purpose of the present invention is to provide a SETD1A protein as a drug development target to solve the problem of how to repair tobacco-induced DNA damage and provide a target for the development of drugs for treating COPD.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] A use of a SETD1A protein, the amino acid sequence of which can be found in NCBI under accession number NP_055527. The use of the SETD1A protein as a drug target in the preparation of a drug for treating diseases caused by tobacco-induced DNA damage. The use of the SETD1A protein can repair DNA damage by increasing SETD1A expression, for example, by overexpressing SETD1A through plasmid transfection.

[0006] Furthermore, the DNA damage is DNA damage of bronchial epithelial cells induced by cigarette smoke.

[0007] Furthermore, the repairing of DNA damage refers to reducing the expression of γ-H2A.X (a marker of DNA double-strand breaks) protein.

[0008] Furthermore, the drug is a drug that promotes the expression of DNA damage repair proteins in bronchial epithelial cells, and the proteins include FANCD2 and BRCA1.

[0009] Furthermore, the disease is chronic obstructive pulmonary disease; the active ingredients of the drug include a carrier expressing SETD1A protein or its activity, and an agonist; the dosage form of the drug includes oral administration, injection, tablets, sustained-release preparations, and the like.

[0010] The advantages of the present invention are as follows: In this application, based on a DNA damage model constructed by BEAS-2B cells induced by cigarette smoke extract and a COPD mouse model induced by cigarette smoke, it was found that SETD1A protein was underexpressed in the DNA damage cell model and the COPD mouse model, indicating that reduced levels of SETD1A protein lead to or are accompanied by DNA damage; further, by establishing a SETD1A overexpression cell model, the present invention found that overexpression of SETD1A can activate the expression of DNA damage repair genes FANCD2 and BRCA1 and inhibit DNA damage in bronchial epithelial cells. By establishing a SETD1A knockout cell model, it was found that SETD1A knockout exacerbates DNA damage and inhibits the expression of damage repair genes FANCD2 and BRCA1; the present invention reveals the important role of SETD1A in repairing cigarette smoke-induced DNA damage. The further development of related DNA damage repair agents based on this target has very important technical theoretical and technical application significance for the prevention and treatment of COPD. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Figures 1 and 2 illustrate the cell model of DNA damage induced by cigarette smoke extract in Experiment 1 of the present invention. Figures A and B demonstrate the expression levels of the DNA damage marker γ-H2A.X protein after treatment with cigarette smoke extract at different concentrations and for different time periods. Figure C demonstrates the changes in 8-OHdG content after treatment with 1% cigarette smoke extract for different time periods. Figure D demonstrates the alkaline comet results induced by cigarette smoke extract and the olive distance of the comet tail analyzed using comet analysis software.

[0012] Figure 2 1 is an expression diagram of SETD1A and DNA repair indicators in Experiment 1 of the present invention. Graph A shows the SETD1A protein expression result; Graph B shows the DNA repair-related factor FANCD2 protein expression diagram.

[0013] Figure 3 Figures 1 and 2 show DNA damage in COPD mice induced by cigarette smoke, as described in Experiment 1 of the present invention. Panel A shows the expression level of γ-H2A.X protein; Panels B and C show the expression levels of SETD1A protein and mRNA; and Panels D and E show the protein and mRNA expression results of the DNA repair proteins BRCA1 and FANCD2.

[0014] Figure 4 Figure 2 shows the repair of DNA damage in bronchial epithelial cells by overexpression of SETD1A in Experiment 2 of the present invention. Panel A shows the SETD1A protein expression level after transfection with the SETD1A overexpression plasmid; Panel B shows the γ-H2A.X protein expression level; and Panels C and D show the protein and mRNA expression results of the DNA repair proteins BRCA1 and FANCD2.

[0015] Figure 5 Figure 3 shows that SETD1A knockdown aggravates DNA damage in Experiment 3 of the present invention. Figure A shows the effect of transfection with siNC and siSETD1A on SETD1A protein expression; Figure B shows the expression level of γ-H2A.X protein; Figures C and D show the protein and mRNA expression results of DNA repair proteins BRCA1 and FANCD2. DETAILED DESCRIPTION

[0016] Experimental Materials:

[0017] Twenty SPF-grade female C57BL / 6 mice aged 6-8 weeks (20-25 g) were provided by Beijing Sibeifu Biotechnology Co., Ltd.; the BEAS-2B bronchial epithelial cell line was purchased from the Shanghai Life Science Cell Bank of the Chinese Academy of Sciences; Hongqiqu filter cigarettes (flue-cured tobacco type, tar content 10 mg, smoke nicotine content 1.0 mg, smoke carbon monoxide content 12 mg) were produced by Henan China Tobacco Industrial Co., Ltd.; the siRNA used for SETD1A gene silencing and the negative control siNC; pcDNA3.1 (Vector); and the pcDNA3.1-Flag-SETD1A plasmid were all constructed and synthesized by Hanbio Biotechnology Co., Ltd.

[0018] Experimental reagents:

[0019] DMEM / F-12 was provided by Pronose; Lipofectamine 3000 was purchased from Thermo Scientific; fetal bovine serum (FBS) and Opti-MEM medium were purchased from Gibco; antibodies used, including gamma H2A.X (phosphoS139), FANCD2, and BRCA1, were purchased from Cell Signaling; SETD1A was provided by Abcam; horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse IgG secondary antibodies were purchased from Wuhan Tri-Ting Biotechnology Co., Ltd.; 8-OHdG detection kit was purchased from Elabscience; comet assay kit was purchased from Yacoin; RT-qPCR-related reagents, including TRIzol, were purchased from TaKaRa; cDNA reverse transcription and SYBR Green kits were purchased from Novozymes; primers for SETD1A, FANCD2, FANCA, and BRCA1 were synthesized by Shanghai Sangon Biotechnology Co., Ltd.; RIPA lysis buffer, bovine serum albumin (BSA), Tween-20, skim milk powder, and ultrasensitive ECL kit were provided by Solebo.

[0020] Effect verification and application

[0021] Experimental Example 1: SETD1A protein is underexpressed in cigarette smoke-induced cell and mouse DNA damage models. I. Experimental Methods

[0022] 1. Construction of DNA Damage Cell Model

[0023] 1.1 Cell culture

[0024] Bronchial epithelial cell line BEAS-2B was cultured in DMEM-12 medium supplemented with 10% fetal bovine serum in an incubator at 37°C, 5% CO2, and 95% humidity. Fresh medium was replaced every two days, and cells were passaged when they reached 90% confluency.

[0025] 1.2 Cigarette smoke preparation

[0026] Cigarette smoke was prepared using a modified syringe-driven device as follows: smoke from one cigarette was slowly injected into 5 mL of serum-free culture medium. The optical density (OD) was measured at a wavelength of 320 nm. The OD was adjusted to 2.0 ± 0.1 with culture medium. The resulting suspension was defined as 100% cigarette smoke. The resulting solution was sterile-filtered through a 0.22 μm filter to remove bacteria and particulates and used for cell treatment within 30 minutes of preparation.

[0027] 1.3 Establishment of cigarette smoke-induced DNA damage model

[0028] BEAS-2B cells were seeded in 6-well plates. After 24 hours, the cells were treated with different concentrations of cigarette smoke (1%, 2%, and 4%). Total cell protein was extracted at 6, 12, 24, and 36 hours, respectively. The expression of gamma H2A.X (phospho S139) was detected by Western Blot, and the content of 8-ohdG was detected by ELISA kit to determine the experimental conditions for the stable induction of DNA damage model.

[0029] 1.4 Comet experiment

[0030] Add 100 μL Agarose to each well of the Comet Slide to completely cover it and create a basal layer. Then, mix the cell sample with Agarose and evenly spread it on the basal layer. Incubate at 4°C in the dark for 15 minutes, then lyse and unwind. Perform electrophoresis with alkaline electrophoresis buffer, stain with PI, and photograph under a fluorescence microscope. Analyze the results using OpenComet Comet Analysis Software.

[0031] 2. Preparation of COPD Mouse Model

[0032] 2.1 Mouse modeling

[0033] Twenty mice were divided into two groups of 10 mice each. The present invention established a COPD mouse model using continuous cigarette smoke stimulation. Mice in the model group were placed in a ventilated chamber filled with 4% cigarette smoke and exposed to three cigarettes three times daily for 16 consecutive weeks (five days per week). A peristaltic pump was used to deliver the smoke at a constant rate of 1 L / min. Mice in the control group were simultaneously placed in another ventilated chamber but exposed to fresh air.

[0034] 2.2 Lung tissue collection

[0035] Mouse lungs were washed with pre-chilled PBS and separated. The left lung was perfused with 10% paraformaldehyde for 15 minutes and then fixed for 72 hours, with the paraformaldehyde solution replaced every 24 hours, for immunohistochemistry and H&E staining. Right lung tissue blocks were aliquoted into cryovials, frozen in liquid nitrogen, and then stored at -80°C for gene and protein analysis.

[0036] 3.Indicator detection

[0037] Total RNA and total protein were extracted from cells and tissues. RT-qPCR and Western blot were used to detect the gene and protein expression levels of SETD1A, FANCD2, FANCI, and BRCA1.

[0038] 4. Data Processing

[0039] GraphPad Prism statistical software was used for analysis and processing, and all values ​​are expressed as mean ± standard deviation (mean ± SD). Statistics were performed using the unpaired Student's t test, and p < 0.05 was considered statistically significant.

[0040] 2. Experimental results

[0041] 1. Changes in various indicators in the DNA damage cell model

[0042] like Figure 1 As shown in Figure 2, the level of DNA damage marker γ-HA.X protein increased under treatment with different concentrations of cigarette smoke extract (CSE) ( Figure 1 Middle A), cells were treated with 1% CSE for different time periods, and it was found that the expression of γ-HA.X protein was the highest at 12 h, and the content of 8-ohdG was also the highest ( Figure 1 B, C); thus, it was determined that the condition for inducing cell DNA damage was 1% CSE for 12 hours. Comet assay showed that compared with the control group, the CSE group had a more obvious comet tail, and the olive moment and tail DNA percentage were higher ( Figure 1 Middle D) indicates that the cell DNA damage model was successfully established.

[0043] like Figure 2 As shown in Figure 2, compared with the control group, SETD1A expression levels were decreased after 6h and 12h of CSE induction ( Figure 2 Middle A), the expression level of FANCD2, a key protein in the DNA damage repair FA pathway, was reduced ( Figure 2 Middle B), indicating that CSE downregulates SETD1A expression and inhibits DNA repair.

[0044] 2. Changes in various indicators in the DNA damage mouse model

[0045] like Figure 3 As shown, compared with the control group, the level of DNA damage marker g-HA.X protein in the lung tissue of mice in the model group was increased ( Figure 3 In addition, the protein and mRNA expression levels of SETD1A and key factors in the DNA damage repair pathway (FANCD2 and BRCA1) were significantly decreased ( Figure 3 This application example demonstrates that SETD1A is underexpressed in DNA damage cell / animal models.

[0046] Experimental Example 2: SETD1A overexpression can repair CSE-induced DNA damage

[0047] 1. Experimental Methods

[0048] 1. Cell Culture and Grouping

[0049] BEAS-2B cell culture conditions were the same as those described in Experimental Method 1.1 of Experimental Example 1. The cells were divided into four groups: Vector group (transfected with empty vector plasmid pcDNA3.1), SETD1A-OE group (transfected with SETD1A full-length plasmid pcDNA3.1-Flag-SETD1A), Vector+CSE group (transfected with empty vector and treated with CSE), and SETD1A-OE+CSE group (transfected with SETD1A full-length plasmid and treated with CSE).

[0050] 2. Plasmid Transfection

[0051] 24 hours before transfection, BEAS-2B cells in the logarithmic growth phase were harvested and trypsinized. The cells were then seeded into 6-well plates, ensuring even distribution of cells. When the cells reached approximately 80% confluence, a suspension of 2 μg of plasmid and the transfection reagent Lipfectamin 3000 was added. 48 hours after transfection, cells were treated with CSE and cultured for an additional 12 hours. Cells were then harvested for RNA and protein analysis.

[0052] 3. Index detection and data processing are the same as experimental methods 3 and 4 in Experimental Example 1.

[0053] 2. Experimental results

[0054] like Figure 4 As shown in Figure 3, compared with the Vector group, the SETD1A protein level in the SETD1A-OE group was significantly increased, indicating that we have successfully achieved SETD1A overexpression in BEAS-2B cells ( Figure 4 Compared with the Vector group, the expression of γ-H2A.X in the SETD1A-OE group was decreased, while that in the Vector+CSE group was increased ( Figure 4 Meanwhile, the expression levels of DNA damage repair-related protein genes FANCD2, BRCA1, and RAD51 increased in the SETD1A-OE group, while the expression of the above indicators decreased in the Vector+CSE group ( Figure 4 Compared with the Vector+CSE group, the expression level of γ-H2A.X in the SETD1A-OE+CSE group was significantly decreased, and the expression level of DNA damage repair-related protein genes was significantly increased ( Figure 4 These results indicate that overexpression of SETD1A can activate the DNA damage repair FA pathway, thereby repairing CSE-induced DNA damage. Experimental Example 3: SETD1A knockdown aggravates CSE-induced DNA damage

[0055] 1. Experimental Methods

[0056] 1. Cell Culture and Grouping

[0057] The BEAS-2B cell culture conditions were the same as those in Experimental Method 1.1 of Experimental Example 1. The cells were divided into four groups: siNC group (transfected with blank siRNA), siSETD1A group (transfected with SETD1A knockdown siRNA), siNC+CSE group (transfected with blank siRNA+CSE treatment), and siSETD1A+CSE group (transfected with SETD1A knockdown siRNA+CSE treatment).

[0058] 2. Gene Silencing

[0059] 24 hours before transfection, BEAS-2B cells in the logarithmic growth phase were harvested and trypsinized. The cells were then seeded into 6-well plates, ensuring even distribution of cells. When the cells reached approximately 80% confluence, a suspension of siRNA and transfection reagent Lipfectamin 3000 was added. CSE was treated 48 hours after transfection and cultured for an additional 12 hours. Cells were then harvested for RNA and protein analysis.

[0060] 3. Index detection and data processing are the same as experimental methods 3 and 4 in Experimental Example 1.

[0061] 2. Experimental results

[0062] like Figure 5 As shown in Figure 3, compared with the siNC group, the SETD1A protein level in the siSETD1A group was significantly decreased, indicating that we successfully knocked down SETD1A in BEAS-2B cells ( Figure 5 Compared with the siNC group, the expression of γ-H2A.X in the siSETD1A group and the siNC+CSE group was increased ( Figure 5 At the same time, the expression levels of DNA damage repair related protein genes FANCD2, BRCA1, RAD51, etc. were significantly reduced ( Figure 5 Compared with the siNC+CSE group, the expression level of γ-H2A.X in the siSETD1A+CSE group was significantly increased, and the expression level of DNA damage repair-related protein genes was significantly decreased ( Figure 5 These results indicate that SETD1A knockdown inhibits the DNA damage repair FA pathway, thereby aggravating CSE-induced DNA damage.

[0063] These experiments demonstrate for the first time that SETD1A protein is underexpressed in tobacco-induced DNA damage cell and animal models. Overexpression experiments have demonstrated that SETD1A overexpression activates the DNA damage repair pathway, thereby repairing CSE-induced DNA damage. Knockdown experiments have also demonstrated that inhibition of SETD1A protein exacerbates DNA damage. Therefore, SETD1A protein could be used as a target for screening and developing drugs that repair tobacco-induced DNA damage. SETD1A protein activators hold promise for development as therapeutics for COPD.

Claims

1. A use of SETD1A protein, characterized by: The amino acid sequence of the SETD1A protein is shown in SEQ ID No. 1, and the SETD1A protein is used in the preparation of a drug for treating tobacco-induced COPD.

2. The use according to claim 1, characterized in that: The drug can activate the expression of DNA damage repair genes FANCD2 and BRCA1, and inhibit DNA damage in bronchial epithelial cells.

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