Application of SETD1A protein

Abstract

The invention belongs to the technical field of target drugs, and particularly relates to application of SETD1A protein, the login number of an amino acid sequence of the SETD1A protein in NCBI (National Center of Biotechnology Information) is NP055527, the SETD1A protein can be inquired, the application of the SETD1A protein as a drug target in preparation of drugs for treating diseases caused by tobacco-induced DNA (Deoxyribose Nucleic Acid) damage is characterized in that the SETD1A expression is increased, for example, plasmid transfection overexpression of SETD1A can be utilized, and the DNA damage is repaired.

Description

Technical Field

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

[0002] Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease characterized by persistent airflow limitation, and smoke exposure is its main inducer. COPD has currently become the third leading cause of death due to diseases in China, causing a significant healthcare burden. In recent years, clinically, drugs such as glucocorticoids and bronchodilators, oxygen therapy, exercise rehabilitation, and surgical treatment have been mainly used to intervene in the course of COPD. These measures can relieve symptoms to a certain extent, improve activity ability, and improve the quality of life of patients, but it is difficult to effectively prevent the continuous development of the disease. DNA damage of pulmonary bronchial and alveolar cells caused by a large amount of reactive oxygen species (ROS) in cigarette smoke is one of the key factors leading to COPD. After DNA damage, the body will initiate a series of DNA damage responses (DDR) to activate the repair pathway to restore the structure to its original state or enable the cell to tolerate the DNA damage state and continue to survive, while the unrepaired DNA damage may lead to gene instability and cell senescence, thereby inhibiting the proliferation of alveolar epithelial cells and enhancing the inflammatory response to promote the development of the COPD process. Therefore, finding drugs that can repair smoke-induced DNA damage and effectively inhibit the accumulation of DNA damage has great application value for inhibiting the occurrence and development of COPD. Summary of the Invention

[0003] The purpose of the present invention is to provide a SETD1A protein as a drug research and 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] To achieve the above object, the technical solution adopted by the present invention is:

[0005] An application of a SETD1A protein, the amino acid sequence of the SETD1A protein can be queried with the accession number NP_055527 in NCBI. The application of the SETD1A protein as a drug target in the preparation of drugs for treating diseases caused by tobacco-induced DNA damage, by increasing the expression of SETD1A, for example, overexpressing SETD1A by plasmid transfection to repair DNA damage.

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

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

[0008] Further, 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] Further, the disease is chronic obstructive pulmonary disease; the active ingredients of the drug include a vector expressing SETD1A protein or its activity, an agonist; the dosage forms of the drug include oral preparations, injections, tablets, sustained-release agents, etc.

[0010] The advantages of the present invention are as follows: In this application, based on the DNA damage model constructed by cigarette smoke extract-induced BEAS-2B cells and the COPD mouse model induced by cigarette smoke, it is found that SETD1A protein is lowly expressed in the DNA damage cell model and the COPD mouse model, indicating that the decrease in the level of SETD1A protein leads to or is accompanied by DNA damage; further, by establishing a SETD1A overexpression cell model, it is 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 is found that knockout of SETD1A 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 DNA damage induced by cigarette smoke, and further developing related DNA damage repair agents based on this target has very important technical theory and technical application significance for the prevention and treatment of COPD. Description of the Drawings

[0011] Figure 1 It is a diagram of a DNA damage cell model induced by cigarette smoke extract in Test Example 1 of the present invention. Among them, Figures A and B show the expression levels of the DNA damage marker γ-H2A.X protein after treatment with different concentrations and different time periods of cigarette smoke extract; Figure C shows the change in 8-OHdG content after treatment with 1% concentration of cigarette smoke extract for different times; Figure D shows the alkaline comet results induced by cigarette smoke extract and the results of the olive tail moment of the comet analyzed by comet analysis software.

[0012] Figure 2 It is a diagram of the expression of SETD1A and DNA repair indexes in Test Example 1 of the present invention. Among them, Figure A is the expression result of SETD1A protein; Figure B is the expression diagram of the DNA repair-related factor FANCD2 protein.

[0013] Figure 3 It is a diagram of DNA damage in COPD mice induced by cigarette smoke in Test Example 1 of the present invention. Among them, Figure A is the expression level of γ-H2A.X protein; Figures B and C are the expression levels of SETD1A protein and mRNA; Figures D and E are the expression results of DNA repair proteins BRCA1 and FANCD2 proteins and mRNA.

[0014] Figure 4 It is the figure showing that overexpression of SETD1A repairs DNA damage in bronchial epithelial cells in Experimental Example 2 of the present invention. Among them, Figure A shows the expression level of SETD1A protein after transfection with the SETD1A overexpression plasmid; Figure B shows the expression level of γ-H2A.X protein; Figures C and D show the expression results of DNA repair proteins BRCA1 and FANCD2 proteins and mRNAs.

[0015] Figure 5 It is the figure showing that knockdown of SETD1A aggravates DNA damage in Experimental Example 3 of the present invention. Among them, Figure A shows the effect of transfection with siNC and siSETD1A on the expression of SETD1A protein; Figure B shows the expression level of γ-H2A.X protein; Figures C and D show the expression results of DNA repair proteins BRCA1 and FANCD2 proteins and mRNAs. Specific implementation manners

[0016] Experimental materials:

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

[0018] Experimental reagents:

[0019] DMEM / F-12 was provided by Procell; 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-labeled goat anti-rabbit and goat anti-mouse IgG secondary antibodies were purchased from Wuhan Sanying 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 kit were purchased from Novoprotein; primers for SETD1A, FANCD2, FANCA, BRCA1, etc. were synthesized by Shanghai Sangon Biotech Co., Ltd.; RIPA lysis buffer, bovine serum albumin (BSA), Tween-20, skim milk powder, enhanced chemiluminescence (ECL) kit, etc. were provided by Solarbio.

[0020] Effect verification and application

[0021] Experimental example 1: SETD1A protein is lowly expressed 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] The bronchial epithelial cell line BEAS-2B was cultured in DMEM / F-12 medium supplemented with 10% fetal bovine serum in an incubator at 37 °C, 5% CO 2 , and 95% humidity. Fresh medium was changed every two days, and subculture was performed when the cell confluence reached 90%.

[0025] 1.2 Preparation of cigarette smoke

[0026] A modified syringe-driven device was used to prepare cigarette smoke as follows: The smoke of one cigarette was slowly injected into 5 mL of serum-free medium, and the optical density (OD) value at a wavelength of 320 nm was measured. The OD value was adjusted to 2.0 ± 0.1 with the medium, and the resulting suspension was defined as 100% cigarette smoke. The obtained solution was sterile filtered through a 0.22 μm filter membrane to remove bacteria and particles, and was used for cell treatment within 30 minutes after 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 cigarette smoke at different concentrations (1%, 2%, 4%). Total cell proteins were extracted at 6h, 12h, 24h, and 36h respectively. The expression level of gamma H2A.X (phospho S139) was detected by Western Blot, and the content of 8-ohdG was detected using an Elisa kit to determine the experimental conditions for a stable DNA damage induction model.

[0029] 1.4 Comet assay

[0030] 100 μL of agarose per well was added to the Comet Slide to completely cover and create a base layer. Subsequently, the cell samples were mixed with agarose and evenly spread on the base layer. After lysis and unwinding in the dark at 4°C for 15 minutes, electrophoresis was performed using an alkaline electrophoresis solution. After PI staining, photographs were taken under a fluorescence microscope, and the results were analyzed 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 each. In this invention, a method of continuous stimulation with cigarette smoke was used to construct a COPD mouse model. The mice in the model group were placed in a ventilated chamber filled with 4% cigarette smoke and exposed to 3 cigarettes three times a day for 16 weeks (5 consecutive days per week). A peristaltic pump was used to deliver gas at a constant rate of 1 L / min. The mice in the blank group were placed in another ventilated chamber but exposed to fresh air.

[0034] 2.2 Lung tissue sampling

[0035] The mouse lungs were washed with pre-cooled PBS, and the left and right lungs were 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 HE staining. The right lung tissue blocks were aliquoted into cryotubes, immersed in liquid nitrogen for freezing, and then transferred to an -80°C refrigerator for storage, for gene and protein detection.

[0036] 3. Index detection

[0037] Total RNA and total proteins in cells and tissues were extracted. The expression levels of SETD1A, FANCD2, FANCI, and BRCA1 genes and proteins were detected by RT-qPCR and Western Blot.

[0038] 4. Data processing

[0039] Analysis and processing were performed using GraphPad Prism statistical mapping software, and all numerical values were expressed as mean ± standard deviation (mean ± SD). Unpaired Student's t-test was used for statistics, and p < 0.05 was considered to have statistical significance.

[0040] II. Experimental Results

[0041] 1. Changes in each index in the DNA damage cell model

[0042] As Figure 1 shown, under the treatment of cigarette smoke extract (CSE) at different concentrations, the protein level of the DNA damage marker γ-HA.X increased ( Figure 1 A in Figure 1 ). When cells were treated with 1% CSE for different times, it was found that the expression level 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 in

[0043] ). Thus, the condition for inducing cell DNA damage was determined to be 1% CSE induction for 12 h. The comet assay showed that compared with the control group, the CSE group had a more obvious comet tail, and the olive moment and the percentage of tail DNA were higher ( Figure 2 D in Figure 2 ), indicating that the cell DNA damage model was successfully established. Figure 2 As

[0044] shown, compared with the control group, the expression level of SETD1A decreased after 6 h and 12 h of CSE induction (

[0045] A in Figure 3 ), and the protein expression level of FANCD2, a key protein in the DNA damage repair FA pathway, decreased ( Figure 3 B in Figure 3 ), indicating that CSE down-regulates the expression of SETD1A and inhibits DNA repair.

[0046] Test Example 2: Overexpression of SETD1A can repair CSE-induced DNA damage

[0047] I. Experimental Methods

[0048] 1. Cell culture and grouping

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

[0050] 2. Plasmid transfection

[0051] Twenty-four hours before transfection, BEAS-2B cells in the logarithmic growth phase were taken, digested with trypsin, and then seeded into 6-well plates to ensure uniform distribution of the cells in the wells. When the cell confluence reached about 80%, a suspension of transfection reagent Lipfectamin 3000 and 2 μg plasmid was added. After 48 hours of transfection, CSE was added, and the cells were collected after continued culture for 12 hours. RNA and protein were extracted for analysis.

[0052] 3. Index detection and data processing were the same as those in Experimental Methods 3 and 4 of Test Example 1.

[0053] II. Experimental results

[0054] As Figure 4 shown, compared with the Vector group, the protein level of SETD1A in the SETD1A-OE group was significantly increased, indicating that we successfully achieved overexpression of SETD1A in BEAS-2B cells ( Figure 4 A in). Compared with the Vector group, the expression of γ-H2A.X in the SETD1A-OE group was decreased; the expression of γ-H2A.X in the Vector+CSE group was increased ( Figure 4 B in); meanwhile, the expression levels of DNA damage repair-related protein genes such as FANCD2, BRCA1, and RAD51 in the SETD1A-OE group were increased, and the expression of the above indicators in the Vector+CSE group was decreased ( Figure 4 C in). 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 levels of DNA damage repair-related protein genes were significantly increased ( Figure 4 B-D in). These results indicate that overexpression of SETD1A can activate the DNA damage repair FA pathway and thus repair CSE-induced DNA damage. Test Example 3: Knockdown of SETD1A exacerbates CSE-induced DNA damage

[0055] I. Experimental methods

[0056] 1. Cell culture and grouping

[0057] The culture conditions of BEAS-2B cells 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] Twenty-four hours before transfection, BEAS-2B cells in the logarithmic growth phase were taken. After trypsin digestion, the cells were seeded into 6-well plates to ensure uniform distribution of the cells in the wells. When the cell confluence reached about 80%, a suspension of transfection reagent Lipfectamin 3000 and siRNA was added. After 48 hours of transfection, CSE was treated, and the cells were collected after continued culture for 12 hours. RNA and protein were extracted for analysis.

[0060] 3. Index detection and data processing were the same as those in Experimental Methods 3 and 4 of Experimental Example 1.

[0061] II. Experimental results

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

[0063] The above experimental example first demonstrated that the protein of SETD1A was lowly expressed in the DNA damage cell / animal model induced by tobacco; through overexpression experiments, it was proved that overexpression of the protein of SETD1A could activate the FA pathway of DNA damage repair, thereby repairing CSE-induced DNA damage; through knockdown experiments, it was proved that inhibition of the protein of SETD1A aggravated DNA damage. Therefore, the protein of SETD1A can be used as a target for screening and preparing drugs for repairing tobacco-induced DNA damage, and the activator of the protein of SETD1A has the prospect of being developed into a drug for treating COPD.

Claims

1. An application of SETD1A protein, characterized in that: The amino acid sequence of the SETD1A protein is shown in SEQ ID No.

1. The SETD1A protein is used as a drug target in the preparation of drugs for treating diseases caused by tobacco-induced DNA damage, and the DNA damage is repaired by increasing the expression of SETD1A.

2. The use according to claim 1, characterized in that: The DNA damage is DNA damage of bronchial epithelial cells induced by cigarette smoke.

3. The use according to claim 1, characterized in that: The repairing of DNA damage refers to reducing the expression of γ-H2A.X protein.

4. The use according to claim 1, characterized in that: The drug is a drug for promoting the expression of DNA damage repair proteins in bronchial epithelial cells, and the proteins include FANCD2 and BRCA1.

5. The use according to claim 1, characterized in that: The disease is chronic obstructive pulmonary disease; the effective ingredients of the drug include a carrier expressing SETD1A protein or its activity and an agonist; the dosage form of the drug includes an oral agent, an injection, a tablet, and a sustained-release agent.

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