1. The application of a reagent for detecting the expression quantity and/or the methylation degree of MEF2C in preparing a product for diagnosing whether a subject has asthma;

preferably, the asthma comprises bronchial asthma, allergic asthma, exercise-induced asthma, drug-induced asthma and dust-induced asthma;

preferably, the asthma is bronchial asthma.

2. The use of claim 1, wherein said MEF2C is highly expressed in a subject and said MEF2C is less methylated in a subject.

3. The use of claim 1, wherein the reagent for detecting the expression level of MEF2C comprises a reagent for detecting the expression level of MEF2C mRNA and/or the expression level of MEF2C protein.

4. The use of claim 3, wherein the reagent for detecting the expression level of MEF2C mRNA comprises a reagent used in any one or more of the following methods: PCR-based detection methods, Southern hybridization methods, Northern hybridization methods, dot hybridization methods, fluorescence in situ hybridization methods, DNA microarray methods, ASO methods, high throughput sequencing platform methods;

preferably, the PCR-based assay is an RT-qPCR assay.

5. The use of claim 3, wherein the reagent for detecting the expression level of MEF2C protein comprises a reagent used in any one or more of the following methods: western blotting, enzyme linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation, complement fixation assay, flow cytofluorimetry, and protein chip assay.

6. The use of claim 1, wherein the reagent for detecting the degree of methylation of MEF2C comprises a reagent used in any one or more of the following methods: MassARRAY methylation technology, pyrosequencing, bisulfite sequencing, methylation specific polymerase chain reaction, bisulfite specific polymerase chain reaction, methylation sensitive restriction enzyme-PCR/Southern method, bisulfite-bound restriction enzyme method, digital polymerase chain reaction, restriction landmark genome scanning, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, methylation profiling.

7. The use of claim 1, wherein the product further comprises reagents for collecting and/or processing the sample;

preferably, the sample includes, but is not limited to, peripheral blood, nasal epithelial cells, tissue, serum, plasma, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosol, ascites, pleural effusion, amniotic fluid, bladder irrigation fluid, and bronchoalveolar lavage fluid;

preferably, the sample is peripheral blood.

Use of an inhibitor of MEF2C, which can knock out MEF2C gene or increase the degree of methylation of MEF2C gene, in the manufacture of a product for treating a patient;

preferably, the patient is a patient diagnosed with an asthma disease;

preferably, the inhibitor comprises an agent used in siRNA interference, CRISPR/cas9 method, homologous recombination, gene knockout, gene replacement, gene silencing, site-directed mutagenesis, chemical drug method.

9. A kit for diagnosing whether a subject has asthma, wherein the kit comprises one or more of the reagent for detecting the expression level of MEF2C mRNA of claim 4, the reagent for detecting the expression level of MEF2C protein of claim 5 and the reagent for detecting the methylation degree of MEF2C of claim 6.

10. A pharmaceutical composition for treating an asthma patient comprising the inhibitor of MEF2C of claim 8.

Background

Asthma is a common respiratory disease, and its pathogenesis includes: allergy, airway chronic inflammation, airway hyperresponsiveness, airway neuromodulation disorder, genetic mechanism, respiratory virus infection, nerve signal transduction mechanism, airway remodeling and interaction thereof and the like, which are mainly manifested by symptoms such as paroxysmal wheeze, dyspnea, chest distress or cough, or the original symptoms are aggravated rapidly, and the breathing is difficult frequently, and the expiratory flow is reduced; are often induced by exposure to allergens, irritants or respiratory infections. The disease condition is aggravated by different degrees, and can appear within hours or days, and occasionally, the life can be threatened within minutes. Bronchial asthma (bronchial asthma) is a heterogeneous disease characterized by chronic inflammation of the airways involving various cells (e.g., eosinophils, mast cells, T lymphocytes, neutrophils, airway epithelial cells, etc.) and cellular components. It is usually attacked or aggravated at night and in the early morning, and most patients can relieve themselves or through treatment. Bronchial asthma, if not treated timely, can produce irreversible constriction of airway and airway remodeling along with the extension of disease course. In recent years, the incidence and mortality of bronchial asthma has been on the rise year by year worldwide.

Myocyte Enhancer Factor 2C (MEF 2C) is one of the members of MEF2 subfamily and is closely related to smooth muscle development, hematopoietic stem cell differentiation, and inflammatory pathway signaling.

Changes in methylation level alter gene expression by altering chromatin structure and stability, altering transcription factor binding activity, and the like. Generally, hypomethylation favors gene expression, and hypermethylation inhibits gene expression. Studies have now demonstrated that the state of DNA methylation is highly correlated with the occurrence of certain tumors. The occurrence of tumors and the therapeutic effect of therapeutic measures on tumors can be evaluated by DNA methylation.

There are a variety of methods in the art for detecting DNA methylation. The Methylation-Sensitive Restriction enzyme-PCR/Southern method (MSRE-PCR/Southern) is a method in which DNA is cleaved into fragments having different sizes by a Methylation-Sensitive Restriction enzyme and PCR/Southern analysis is performed. Also some Methylation Analysis methods based on Bisulfite treatment and derived from this, such as Bisulfite Sequencing (Bisulphite Sequencing), Methylation-Specific PCR (MS-PCR), Pyrosequencing (Pyrosequencing), Bisulfite-binding Restriction enzyme assay (COBRA), etc. In recent years, a Methylation analysis method based on a whole genome, also called Methylation Profiling (Methylation Profiling), has been emerging, which has a large detection sample size and can detect a plurality of sites.

Disclosure of Invention

The subject group found hypomethylation of MEF2C in peripheral blood and nasal epithelial cells of asthmatic patients through whole genome studies. The present study further provides the use of DNA expression levels and methylation levels in the diagnosis of airway inflammation in asthma, preferably bronchial asthma, by exploring the DNA methylation levels, expression level changes in MEF2C in patients with asthma; also provided is the use of an inhibitor of MEF2C in the treatment of asthma.

Use for diagnosing diseases

In one aspect, the invention provides the use of a reagent for detecting the expression level and/or methylation degree of MEF2C in the preparation of a product for diagnosing whether a subject has asthma.

Preferably, the asthma comprises bronchial asthma, allergic asthma, exercise-induced asthma, drug-induced asthma and dust-induced asthma.

Preferably, the asthma is bronchial asthma.

Preferably, the asthma comprises intermittent asthma or persistent asthma.

Preferably, the asthma comprises intrinsic asthma or extrinsic asthma.

Preferably, the subject is a human suspected of having asthma.

Preferably, said MEF2C is highly expressed in a subject.

Preferably, the MEF2C is less methylated in the subject.

Preferably, the high expression refers to a MEF2C expression level that is greater than the expression level of MEF2C in a healthy control population, at least 1.1 fold, e.g., at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2.0 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3.0 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, or 3.5 fold or more, relative to the control expression level.

Preferably, the low degree of methylation means that MEF2C is less methylated than MEF2C in the healthy control population, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the methylation degree of control MEF 2C.

Preferably, the reagent for detecting the expression level of MEF2C comprises a reagent for detecting the expression level of MEF2C mRNA and/or the expression level of MEF2C protein.

Preferably, the reagent for detecting the expression level of MEF2C mRNA comprises the following reagents used in the following methods: PCR-based detection method, Southern hybridization method, Northern hybridization method, dot hybridization method, fluorescence in situ hybridization method, DNA microarray method, ASO method, high throughput sequencing platform method.

Preferably, the method is a quantitative or qualitative method.

Preferably, the PCR-based detection method comprises any one of: a step of extracting RNA, a step of reverse transcribing mRNA into cDNA, and a step of measuring the content of cDNA.

Preferably, the method of measuring the content of cDNA includes, but is not limited to, PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM.

Preferably, the PCR-based assay is an RT-qPCR (Real-time fluorescence quantitative PCR, RT-qPCR) assay.

Preferably, the reagent for detecting the expression level of MEF2C mRNA comprises a specific primer and/or a probe.

Preferably, the probes include hybridization-type probes and hydrolysis-type probes (Taqman probes).

Preferably, the two ends of the probe are connected with a quenching group and/or a fluorescent group.

Preferably, the reagent for detecting the expression level of MEF2C protein comprises the following reagents used in the following methods: western blotting (Western Blot), enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytofluorimetry, and protein chip methods.

Preferably, the reagent for detecting the expression level of MEF2C protein comprises a reagent used in Western blotting (Western Blot method) and/or ELISA method.

Preferably, the reagent for detecting the expression level of MEF2C protein comprises an antibody or fragment of MEF2C, and the antibody or fragment of MEF2C can be specifically combined with MEF2C protein.

Preferably, the reagent for detecting the expression level of MEF2C protein further comprises a secondary antibody, and the secondary antibody can be combined with the antibody or the fragment of MEF2C and develops color.

Preferably, the visualization is embodied by a visualization reagent including, but not limited to, a fluorescent dye (fluorescent molecule), a chemiluminescent label, an enzyme, a metal ion, biotin, a radioisotope, a molecule that absorbs in the UV spectrum, a molecule that absorbs in the near infrared radiation, or a molecule that absorbs in the far infrared radiation.

Preferably, the fluorescent dye includes, but is not limited to, rhodamine, p-methylaminophenol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, amino-p-methylaminophenol, carboxy-p-methylaminophenol, chloro-p-methylaminophenol, methyl-p-methylaminophenol, sulfop-methylaminophenol, aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, and sulforhodamine, cyanine, indocyanine, oxonol, thiacyanine, merocyanine, cyanine dye, oxadiazole derivative, pyridyloxazole, nitrobenzene oxadiazole, benzonitrobenzene, pyrene derivative, waterfall blue, oxazine derivative, nile red, nile blue, cresol purple, oxazine 170, azo derivative, proflavin, acridine orange, acridine yellow, arylmethine derivative, auramine, thioxanthene dye, Thioxanthene sulphonated dyes, alexas fluorescence (AlexaFluor), crystal violet, malachite green, tetrapyrrole derivatives, porphyrins, phthalocyanines, bilirubin, cy5.5, indocyanine green (ICG), DyLight750, or IRdye 800.

Preferably, the chemiluminescent label includes, but is not limited to, peroxidase, alkaline phosphatase, luciferase, aequorin, functionalized iron-porphyrin derivatives, luminol, isoluminol, acridinium esters, sulfonamides, and the like.

Preferably, the luciferase includes, but is not limited to, a pleiones magna (Gaussia) luciferase, a Renilla (Renilla) luciferase, a dinoflagellate luciferase, a firefly luciferase, a fungal luciferase, a bacterial luciferase, and a glowworm (vargula) luciferase.

Preferably, the antibody or fragment of MEF2C is also directly bound to a chromogenic agent, which is detected to indicate expression of MEF2C protein.

Preferably, the reagent for detecting the methylation degree of MEF2C is a reagent used in any one or more of the following methylation detection methods, wherein the methylation detection method comprises: pyrosequencing (Pyrosequencing), Bisulfite sequencing, Methylation-Specific polymerase chain reaction (MS-PCR), Bisulfite-Specific Polymerase Chain Reaction (PCR), Methylation-sensitive Restriction enzyme-PCR/Southern method, Bisulfite-bound Restriction enzyme method (COBRA), digital Polymerase Chain Reaction (PCR), Restriction landmark genomic scan, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension (SNUPE), Methylation Profiling (Methylation Profiling) method.

Preferably, the methylation detection method is methylation profiling.

Preferably, the methylation detection method is the MassARRAY technique, which belongs to methylation profiling.

Preferably, the product also comprises reagents for collecting and/or processing the sample.

Preferably, the sample comprises: peripheral blood, nasal epithelial cells, tissue, serum, plasma, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosol, ascites, pleural effusion, amniotic fluid, bladder irrigation fluid and bronchoalveolar lavage fluid.

Preferably, the sample is peripheral blood.

Use for treating patients

On the other hand, the invention provides application of an MEF2C gene inhibitor in preparation of a product for treating patients, wherein the MEF2C gene can be knocked out or the methylation degree of the MEF2C gene can be improved by the inhibitor, so that the effect of reducing the expression of the MEF2C gene is achieved.

Preferably, the patient is a patient diagnosed with an asthma disease.

Preferably, the inhibitor comprises an agent used in siRNA interference, CRISPR/cas9 method, homologous recombination, gene knockout, gene replacement, gene silencing, site-directed mutagenesis, chemical drug method.

Product(s)

In another aspect, the present invention provides a kit for diagnosing whether a subject suffers from asthma, the kit comprising the aforementioned reagents for detecting the expression level and/or methylation degree of MEF2C in the subject.

Preferably, the kit further comprises the aforementioned apparatus for detecting the expression level and/or methylation degree of MEF2C in a subject;

preferably, the kit further comprises reagents and/or instruments for detecting other disease markers.

Preferably, the other disease markers include IL-6, IL-8, TNF-alpha.

In another aspect the present invention provides a pharmaceutical composition for treating asthma in a patient, said pharmaceutical composition comprising an inhibitor of the aforesaid MEF2C gene.

Preferably, the pharmaceutical composition may be a tablet (including a sugar-coated tablet, a film-coated tablet, a sublingual tablet, an orally disintegrating tablet, an oral tablet and the like), a pill, a powder, a granule, a capsule (including a soft capsule, a microcapsule), a lozenge, a syrup, a liquid, an emulsion, a suspension, a controlled release preparation (e.g., an instantaneous release preparation, a sustained release microcapsule), an aerosol, a film (e.g., an orally disintegrating film, an oral mucosa-adhesive film), an injection (e.g., subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), an intravenous drip, a transdermal absorption preparation, an ointment, a lotion, an adhesive preparation, a suppository (e.g., a rectal suppository, a vaginal suppository), a pellet, a nasal preparation, a pulmonary preparation (an inhalant), an eye drop and the like, an oral or parenteral preparation (e.g., intravenous, intramuscular, subcutaneous, intraorgan, intranasal, intradermal, instillation, intracerebral, intrarectal, etc. administration to the vicinity of tumors and directly to lesions).

Method

In another aspect, the invention provides a method of diagnosing whether a subject has asthma, the method comprising detecting in the subject the amount of expression and/or the degree of methylation of MEF 2C.

In another aspect the invention provides a method of treating a patient suffering from asthma, said method comprising reducing expression of the MEF2C gene and/or increasing the degree of methylation of the MEF2C gene in the patient.

Drawings

FIG. 1 is a graph showing the comparison between the relative expression levels of MEF2C mRNA in peripheral blood of a control group and that in asthma.

FIG. 2 is a graph showing the result of comparing the ratio of DNA methylation of MEF2C in the control group and the asthma group.

FIG. 3 is a graph showing the correlation between MEF2C mRNA and lung function, wherein A is a graph showing the correlation between MEF2C mRNA and the ratio of forced expiratory volume to forced vital volume in one second (FEV1/FVC), and B is a graph showing the correlation between MEF2C mRNA and the expected percentage of forced expiratory volume in 1 second (FEV 1% prep).

FIG. 4 is a graph showing the correlation between MEF2C mRNA and plasma IgE.

FIG. 5 ROC plot of MEF2C mRNA in the adjuvant diagnosis of asthma.

FIG. 6 is a graph showing the results of an analysis of the effect of HDM on BEAS-2B cell viability.

FIG. 7 is a graph showing the results of analysis of the effect of HDM on the expression levels of BEAS-2B cells L-6, IL-8 and TNF-. alpha.A, mRNA relative expression levels and B, protein expression levels.

FIG. 8 is a graph showing the results of 5-Aza-CdR effect on mRNA and protein levels of MEF2C, graph A is relative mRNA level and graph B is protein expression level.

FIG. 9 is a graph showing the results of percentage methylation of MEF2C DNA before and after 5-Aza-CdR treatment.

FIG. 10 is a graph showing the results of the effects of MEF2C demethylation on inflammation, where A is the mRNA level change, B is the protein expression change, C is the p65 protein expression change, D is the mRNA level of IL-6, E is the mRNA level of IL-8, and F is the mRNA level of TNF-. alpha..

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 correlation study of peripheral blood MEF2C Gene expression, DNA methylation level and asthma in asthmatic patients

In the early stage of the subject group, the genome-wide DNA methylation analysis of nasal epithelial cells and peripheral blood leukocytes of 6 bronchial asthma patients and 5 healthy volunteers shows that 13627 differential methylation sites of the gene promoter regions of the nasal mucosa epithelial cells and 3840 differential methylation sites of the gene promoter regions of the peripheral blood leukocytes are found, and the differential methylation sites are related to calcium signal channels, Rap1 signal channels, focal adhesion, epinephrine signal conduction and other channels through GO analysis and Pathway analysis. The level of DNA methylation of the MEF2C gene was altered in the nasal epithelium and peripheral blood leukocytes.

1. Study object

15 adult bronchial asthma patients and 15 healthy volunteers in a physical examination center are selected from the patients who are diagnosed in the department of respiratory medicine outpatient service of subsidiary hospitals of Jiangsu university from 9 months 2019 to 9 months 2020. And divided into an asthma group and a control group. In the asthmatic groups, 7 men and 8 women were diagnosed as bronchial asthma for the first time in the mean age (45. + -. 10.3) years. 8 men and 7 women in the control group, and the mean age (44. + -. 11.2) years. The two sets of baseline data were comparable (P > 0.05).

Inclusion and exclusion criteria

Inclusion criteria were: the method is used for diagnosing the bronchial asthma for the first time and accords with bronchial asthma diagnosis standards in bronchial asthma control guidelines (2016 edition); ② 18-65 years old; third, 2 or more doctors have the history of asthma diagnosis and obvious respiratory symptoms such as wheezing and chest distress appear when the doctor is put into the doctor or in the last 12 months; fourthly, the lung function test is performed in cooperation with the ability of autonomous behavior.

Exclusion criteria: other chronic respiratory diseases such as COPD, bronchiectasis, interstitial lung disease and the like; ② blood system diseases and rheumatism immune diseases; patients with diabetes, hypertension, coronary heart disease and tumor; fourthly, pregnant women and those who cannot cooperate.

2. Experimental methods

Collection of general data

All patients entering the group need to fill in a bronchial asthma epidemiological questionnaire and unify the survey information of all research objects, including (1) basic information of the patients, such as name, sex, age, height, weight, occupation and the like; (2) the past medical history data comprises chronic hereditary history such as hypertension and diabetes, familial hereditary history, smoking history and allergy history; (3) the onset of the disease, such as the first onset, the season of asthma onset, the cause of the onset, the frequency of the onset, the treatment, etc. All study populations received comprehensive medical assessments to ensure diagnostic accuracy. The study was approved by the ethical committee of the study at the affiliated hospital of Jiangsu university, all participants provided written informed consent.

Blood sample collection

Extracting peripheral venous blood 4ml of all patients with EDTA anticoagulation blood collection tube, mixing thoroughly, extracting peripheral blood leukocyte, or storing in refrigerator at-80 deg.C.

Isolation of peripheral blood leukocytes

(1) After venous blood is extracted by using an EDTA anticoagulant tube, 3500g of low-temperature ultracentrifuge is used for centrifuging for 5min, 2ml of blood plasma is collected and placed in a liquid nitrogen tank for 5min, and then the blood plasma is transferred into a refrigerator at the temperature of 70 ℃ below zero for storage.

(2) Sucking the middle white cell layer to an EP tube sterilized at high temperature and high pressure by using a liquid transfer device, adding 3 times of erythrocyte lysate, fully blowing, uniformly mixing, standing for 15min, centrifuging 3500g for 5min, discarding supernatant, and keeping the bottom white precipitate.

(3) Adding 1ml erythrocyte lysate into an EP tube, blowing and uniformly mixing the erythrocyte lysate and the erythrocyte lysate again, standing the mixture for 15min at room temperature, centrifuging 3500g for 5min, and discarding the supernatant. This was repeated until no red color was evident in the precipitate.

(4) The extracted white blood cells are used for extracting RNA or DNA, or transferred into a refrigerator at the temperature of-70 ℃ for storage and standby.

Extraction of Total RNA from cells

(1) Taking out treated peripheral blood leukocyte, adding 1ml of RNAioso Plus into each 1.5ml EP tube, repeatedly pumping and mixing well by light shaking or pipette until no obvious precipitate is formed in the solution, and standing at room temperature for 5 min.

(2) Sucking 200 mul chloroform into a centrifugal tube according to the proportion of 1:5, violently shaking and uniformly mixing until the solution is milky white, and standing for 5min at room temperature. Placing into a precooled ultracentrifuge, and centrifuging for 15min at 12000g and 4 ℃.

(3) The centrifuged EP tube was carefully removed, whereupon the centrifugate was divided into three layers and the supernatant was colorless and clear and contains RNA. The supernatant is sucked into another new EP tube to avoid sucking the middle white layer and the lower pink liquid. Adding 500 μ l isopropanol into EP tube, turning upside down, mixing, standing in ice for 10min, then 12000g, centrifuging at 4 deg.C for 10 min.

(4) The centrifuge tube was removed, the supernatant was discarded, 1ml of 75% ethanol prepared from DEPC water and absolute ethanol was added to the EP tube, and the tube wall was washed by gently inverting the tube upside down. 7500g, 4 ℃, centrifuge for 5min and carefully discard the supernatant.

(5) The centrifuge tube cover is opened, and the mixture is dried for a plurality of minutes at room temperature until no obvious liquid adheres to the centrifuge tube. Simultaneously preheating the water bath to 65 ℃.

(6) Add 20. mu.l DEPC solution to the centrifuge tube to dissolve the precipitate, add to the preheated water bath for 10min to dissolve the RNA sufficiently.

(7) And detecting the concentration and purity of the RNA according to the operation instruction of an ultraviolet spectrophotometer, and measuring for three times to obtain an average value.

Preparation of cDNA (reverse transcription)

Based on the measured RNA concentration, the volume required to add 1000ng of total RNA per well was calculated.According to PrimeScript^TMThe RT Reagent Kit RT Reagent reverse transcription Kit protocol the 20. mu.l reaction system as shown in Table 1 below was prepared. The whole reaction solution preparation process is carried out on ice.

TABLE 1 reaction System for reverse transcription

After sample addition, the mixture was blown up and mixed by a pipette and centrifuged briefly. Placing 200 μ lEP tube into reverse transcription amplification instrument, setting reaction parameters at 37 deg.C, 15min (reverse transcription reaction), 85 deg.C, 5s (inactivation reaction of reverse transcriptase), 4 deg.C and infinity; after the reaction is finished, real-time fluorescence quantitative PCR reaction is carried out or the mixture is stored in a refrigerator at the temperature of minus 20 ℃ for later use.

Real-time fluorescent quantitative PCR

The PCR reaction solution was prepared using cDNA prepared by reverse transcription as a template according to the instructions of the SYBR Premix Ex Taq kit of Takara, and the reaction system is shown in Table 2 (the preparation process of the reaction solution was performed on ice).

TABLE 2 reaction System for real-time fluorescent quantitative PCR

After the sample is added, a pipettor is used for gently blowing, beating and uniformly mixing, and after the mixture is centrifuged for a short time, the mixture is placed into a PCR amplification instrument, and amplification parameters are set as follows: pre-denaturation: 3s at 95 ℃; and (3) PCR reaction: circulating for 40 times at 95 ℃ for 5 s; annealing of the primer: 60 ℃ for 34s, primer extension: 60 ℃ for 1 min.

And acquiring corresponding CT values, and calculating a relative quantitative result by adopting a 2-delta-CT method.

The primer sequence is as follows:

beta-Actin: forward 5'-CATCCGTAAAGACCTCTATGCCAAC-3' and Reverse 5'-ATGGAGCCACCGATCCACA-3'

MEF 2C: forward 5'-AGGATATCCATCAGCCATTTCAACA-3' and Reverse 5'-GCTGCTGCCAGCCAGTTACA-3'

Extraction of cellular DNA

(1) To the extracted cell pellet was added 500. mu.l of solution A, followed by thorough shaking and mixing.

(2) Mu.l RNase A was added to the suspension and left at 55 ℃ for 10 min.

(3) Adding 20 μ l proteinase K into the tube, mixing, digesting in 55 deg.C water bath for 30min, and mixing by reversing for several times. Centrifuge at 12000rpm for 10min and transfer the supernatant to a new centrifuge tube.

(4) Add 500. mu.l of solution B into the centrifuge tube, blow and mix well. If white precipitate appears, the mixture can be placed at 55 ℃ for 5min until the precipitate disappears.

(5) Adding 500 μ l anhydrous ethanol, mixing, transferring the solution and flocculent precipitate to adsorption column, centrifuging at 12000rpm for 2min, discarding waste liquid, and placing the adsorption column into collecting tube.

(6) Add 600. mu.l of rinsing solution (absolute ethanol was added before use) to the adsorption column, centrifuge at 12000rpm for 1min, discard the waste solution, place the adsorption column in the collection tube.

(7) Adding 600 μ l of rinsing solution into adsorption column, centrifuging at 12000rpm for 1min, discarding waste liquid, and placing adsorption column into collection tube.

(8) After centrifugation at 12000rpm for 2min, the column was left at room temperature for several minutes to remove the residual rinse from the column.

(9) Placing the adsorption column into a new centrifuge tube, adding the eluent preheated in 65 deg.C water bath, suspending and dripping 50-200 μ l into the center of the adsorption membrane, standing at room temperature for several minutes, and centrifuging at 12000rpm for 2 min.

(10) And slowly dripping the eluent obtained by centrifugation into an adsorption column, standing at room temperature for 2min, and centrifuging at 12000rpm for 2min to obtain the high-quality genome DNA.

(11) The DNA is dissolved in a proper amount of DEPC water, and the concentration of the DNA is detected for subsequent tests or stored in a refrigerator at the temperature of-70 ℃.

DNA methylation level detection by MassARRAY methylation technology

(1) Using EZ DNA Methylation-Gold^TMKit for performing genome DNA methylation conversion reaction

Adding 900 μ l of pure water, 300 μ l M-Dilution Buffer and 50 μ l M-dissolution Buffer into CT Conversion Reagent powder, and shaking rapidly for 10min at room temperature until the powder is completely dissolved.

② prepare 0.2ml PCR tubes, mark the tube caps, add 130. mu.l CT Conversion Reagent to each tube.

③ 20 mul of DNA is added into the PCR tube, and the mixture is repeatedly blown and evenly mixed and centrifuged for a short time.

Put the PCR tube into the PCR instrument and run the following program (activate the hot lid 105 ℃):

Step 1：98℃，10min；

Step 2：64℃，150min；

step 3: 4 ℃ and infinity. And taking out the PCR tube after the temperature of the PCR instrument is reduced to 4 ℃.

Taking Zymo-Spin^TMIC Column, the Column was placed in the collection tube provided in the kit and 600. mu. l M-Binding Buffer was added.

Sixthly, transferring all the products in the first PCR tube to a column containing M-Binding Buffer, turning upside down and mixing uniformly, centrifuging for 30s at 18000g, and sucking the effluent liquid in the collection tube by using a vacuum pump.

Seventhly, adding 100 mu l M-Wash Buffer into the column, and centrifuging for 30s at 18000 g.

(viii) adding 200. mu. l M-Desulphosphorylation Buffer to the column, incubating upright for 20min at room temperature, and centrifuging at 18000g for 30 s.

Ninthly, adding 200 mu l M-Wash Buffer to the column, and centrifuging for 30s at 18000 g. This step was repeated once.

The column was transferred to a 1.5ml centrifuge tube and 30. mu.l of Elution Buffer, 18000g, was added to the center of the column and centrifuged for 30 s.

The eluted CT-DNA may be immediately subjected to PCR or stored at-20 ℃.

(2) And (3) PCR reaction: the eluted DNA was removed, a PCR reaction system was prepared according to Table 3, and the reaction was carried out according to Table 4:

TABLE 3 PCR reaction System

TABLE 4 PCR amplification conditions

(3) SAP digestion reaction: the reaction plate after PCR was removed and SAP enzyme solution was prepared according to the following table 5:

TABLE 5 SAP enzyme solution composition

Name of reagent	Volume of
		ddH2O	1.53μl
SAP Buffer	0.17μl
		SAP Enzyme	0.30μl
Total volume	2.00μl

After the SAP reaction system is prepared, 2 mul is directly added into the mixed solution after the PCR reaction is finished. The digestion reaction was carried out as follows: 40min at 37 ℃, 5min at 85 ℃ and infinity at 4 ℃. After digestion, the reaction plate is removed.

(4) Mass CLEAVE reaction: a reaction system is prepared according to the following table 6, after a Mass CLEAVE reaction system is prepared, 5 mu l of the mixture is directly added into a mixed solution after SAP reaction is completed, and the reaction conditions are as follows: 3 hours at 37 ℃.

TABLE 6 Mass CLEAVE reaction System

Name of reagent	Volume of
		Ultrapure water	3.21μl
5x T7 Polymerase Buffer	0.89μl
		T Cleavage Mix	0.22μl
DTT	0.22μl
		T7 RNA&DNA Polymerase	0.4μl
Ribonuclease A	0.06μl
		Total volume	5.00μl

(5) Desalting, loading on a computer, making a score, and detecting an analysis result.

Pulmonary function detection

The lung function of the subject was examined using a Japanese HI-101 lung function examination apparatus.

Preparation before lung function detection: (1) and the parameters such as measuring range, precision and the like of the lung function instrument are subjected to quality control, so that the lung function instrument is ensured to be in a normal working state. And checking the switch connection and the switch, and connecting a power supply and a sensor to ensure that the equipment works normally. (2) Measuring vital signs of the patient before pulmonary function detection, confirming the past medical history of the patient, and eliminating the contraindication of pulmonary function detection. (3) The patient is rested for 15 minutes before examination, and after practicing deep inhalation, the patient can quickly exert force (explosive force) to blow for 6 seconds without interruption, and the patient needs to stop the corresponding bronchodilators such as theophylline, b2 receptor agonist, hormones, antiallergic agent, etc. according to the medical advice. (4) The indoor environment should be quiet, the examinee needs to have a rest for 15 minutes and then checks, and over-tight waistbands, chest straps, clothes and the like are avoided. Can be checked half an hour after meals to avoid the influence of satiety on respiration. (5) If necessary, emergency medicines and emergency equipment are prepared.

The lung function detection method comprises the following steps: (1) and (5) switching on the power supply, confirming that the power line, the sensor, the mouth sleeve and the respiratory filter are accurately connected, and performing zero calibration. Inputting the data of patient name, age, weight, race, room temperature and air pressure. (2) The patient takes the standing position, the nose clip clamps the nasal wings, and the oral lip wraps the oral piece, so that the nasal cavity and the oral cavity are ensured to be airtight. (3) After pressing the key (START), the patient breathes normally twice, breathes deeply at a higher speed to the greatest extent, breathes to the position where the patient cannot continue breathing, breathes deeply at a higher speed to the greatest extent, breathes deeply, breathes out continuously for six seconds in the process of breathing, and finally returns to normal breathing once. Throughout the respiratory process, interruptions, coughs or double breaths are avoided. (4) The determination is repeated for 3 times or more, the variation rate does not exceed 5 percent, and the optimal value is obtained by three times of inspection.

Hematology examination result collection

Blood routine and serum total IgE examination results were collected for all patients enrolled.

Statistical analysis

Statistical processing was performed using SPSS 23.0 statistical software. Normal distribution data is represented by mean plus or minus standard deviation (x plus or minus s), non-normal distribution measurement data is represented by median and quartile intervals (P50(P25-P75)), two-by-two comparison among groups adopts independent sample t test, and the comparison in groups adopts paired sample t test; correlation analysis was performed using Pearson analysis. P <0.05 is statistically significant for the differences.

3. Results of the experiment

Clinical characteristics of patients in group

15 subjects were collected from each of the asthmatic and normal controls, and there were no statistical differences in age, sex, BMI, etc. between the two groups (P > 0.05). Compared with the normal control group, the peripheral blood leukocyte count, the eosinophilic granulocyte count and the IgE of the bronchial asthma group are obviously increased (P <0.05), and the lung function of the bronchial asthma group patient is obviously higher than that of the normal control group (P < 0.05). Specific information is shown in the following table.

TABLE 7 clinical characteristics Collection Table for patients in group

MEF2C was expressed in peripheral blood leukocytes of patients with bronchial asthma increased compared to controls

The relative mRNA expression level of MEF2C in the asthma group and the control group was determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The results are shown in FIG. 1: the MEF2C mRNA level was higher in patients with bronchial asthma than in normal controls (P < 0.05).

MEF2C gene DNA methylation level is reduced compared with control group

The MassARRAY methylation technology is used for simultaneously detecting and analyzing 94 percent of CpG islands in a detection section in one reaction, and the MassARRAY methylation technology is used for carrying out semi-quantitative methylation analysis on DNA methylation of peripheral blood leukocyte MEF2C genes of a healthy control group and an asthma group. The results are shown in FIG. 2: the MEF2C gene has 4 methylated fragments, and compared with a control group, the methylation level of the asthma group is remarkably reduced (P < 0.01).

Correlation analysis of relative levels of MEF2C mRNA with Lung function

The correlation between the relative expression level of peripheral blood MEF2CmRNA in asthmatics and the ratio of forced expiratory volume per forced vital volume in one second (FEV1/FVC) to the expected percentage of forced expiratory volume in 1 second (FEV 1% prep) was compared in a Spearman correlation analysis. As shown in fig. 3, MEF2C mRNA levels were positively correlated with FEV1/FVC and FEV 1% prep, with a correlation coefficient rs of 0.27240 with FEV1/FVC, p of 0.046; the correlation coefficient rs is 0.2160 and p is 0.0409 with FEV 1% prep.

Correlation analysis of peripheral blood leukocyte genes and plasma IgE levels

The correlation between the relative expression level of MEF2CmRNA in peripheral blood of asthmatic patients and the plasma IgE level was compared in a Spearman correlation assay. Results figure 4 shows: the methylation rate is positively correlated with plasma IgE, the correlation coefficient rs is 0.3464, and p is 0.021.

Effect verification of peripheral blood leukocyte gene diagnosis asthma

An ROC curve was drawn based on the expression levels of MEF2CmRNA in peripheral blood of 15 subjects in each of the asthmatic group and the normal control group, and the results are shown in FIG. 5, in which the AUC of MEF2C (standard error: 0.085, 95% CI: 0.619-0.926) was 0.804, sensitivity was 73.33%, and specificity was 80.00% when MEF2C was used for assisting asthma diagnosis.

The research shows that compared with a normal control group, the peripheral blood MEF2C expression of the bronchial asthma patient is increased, and the methylation level detection by using a DNA methylation detection technology shows that the peripheral blood DNA methylation level of the bronchial asthma patient is reduced, and the peripheral blood DNA methylation level has correlation with the lung function and the plasma IgE level. According to the ROC curve, MEF2C has good specificity and sensitivity when used for diagnosing asthma, and can be used for clinical diagnosis.

Example 2 correlation of DNA methylation of MEF2C Gene with HDM-induced BEAS-2B cell inflammation

1. Test object

Human bronchial epithelial cell line BEAS-2B, Shanghai cell Bank of Chinese academy of sciences, China

2. Experimental methods

Experiment grouping

The method mainly comprises the following steps:

(1) effect of HDM on cell viability: 0U/ml, 200U/ml, 400U/ml, 600U/ml, 800U/ml.

(2) Effect of HDM on airway epithelial cells: control and HDM groups were divided.

(3) Effect of 5-Aza-CdR on Gene expression: the control group and 5-Aza-CdR group were divided.

(4) Role of DNA methylation in the inflammatory response of asthma: HDM + DMSO group, 5-Aza-CdR + HDM group.

Cell recovery:

(1) the frozen BEAS-2B cells were taken out from the freezer at-80 ℃ and rapidly shaken in a water bath at 37 ℃ to be thawed and thawed within 1 to 2 minutes.

(2) The vial was wiped with alcohol, the cell suspension was transferred in a clean bench to a 15ml centrifuge tube, 5ml of medium was added and mixed well. Centrifuging at 1000rpm for 5min, discarding supernatant, adding 1ml culture medium, and gently pumping.

(3) All cell suspensions were added to a 10cm petri dish, approximately 6ml of medium was added and mixed gently. The petri dish was placed in an incubator (37 ℃ C., containing 5% CO)₂) Overnight, the next day the fluid was changed and cell density and morphology were examined.

Cell passage

(1) The culture dish was removed from the incubator, the original culture medium was discarded, and rinsed 2 times with autoclaved PBS free of calcium and magnesium ions.

(2) Adding 1ml of Trypsin digestive juice (0.25Trypsin-0.53mM EDTA) into a culture dish, placing the culture dish in an incubator at 37 ℃ for digesting for 2 minutes, observing the cell digestion condition under a microscope, if most of cells retract to become round and the cell gap increases, quickly taking back to a super clean bench, adding 5ml of culture medium, slightly blowing and beating a cell layer, and blowing and scattering all cells as far as possible.

(3) Transferring all cell suspension into a 15ml centrifugal tube, centrifuging for 5min at 1000rpm, discarding supernatant, supplementing 1ml culture medium, and blowing uniformly.

(4) The cell suspension was divided into new dishes containing 6ml of medium at a ratio of 1:2, placed in an incubator and observed for cell morphology after 24 hours.

Cell cryopreservation

(1) When the cell growth state is good and the cell density reaches 95%, the cells can be frozen. After discarding the medium, 2ml PBS was added for washing 2 times, 1ml trypsin was added and digested in the incubator for 2 min. After the cells became round and fell off, the cells were collected by centrifugation and centrifuged at 1000RPM for 5 min.

(2) After centrifugation, the supernatant was removed, 1ml of the cryopreservation solution was aspirated to resuspend the cell pellet, and the cell suspension was transferred into a cryopreservation tube. And (4) sterilizing the cryopreserved pipe, sealing the pipe orifice by using a sealing film, and filling information such as cell names, cryopreserved dates and operators on a pipe wall label.

(3) And finally, carrying out programmed cooling at 4 ℃ for 20 minutes, at-20 ℃ for 1 hour, at-80 ℃ for overnight, and then placing the mixture in a liquid nitrogen tank for storage.

Construction of house dust mite induced bronchial epithelial cell inflammation model

And (3) taking BEAS-2B cells which have good growth state and are paved on a cell culture dish by more than 80%, digesting, centrifuging, discarding supernatant, adding 1ml of culture medium without double antibody for heavy suspension, and counting by using a cell counting plate. According to 5 x 10 per hole⁵Cells were seeded into 6-well plates, 5 x 10 per well⁴Inoculating each cell in a 96-well plate, shaking gently, mixing uniformly to distribute the cells uniformly, placing the culture dish flat, and culturing in a 5% CO2 incubator at 37 ℃. When the cells in the culture plate are cultured to 80% confluence, the original culture medium is discarded, and HDM (0U/ml, 200U/ml, 400U/ml, 600U/ml and 800U/ml) culture media with different concentrations are respectively added for continuous culture for 12 hours.

CCK-8 detection of cell viability

Groups were made according to different concentrations of HDM, with 3-5 replicate wells per group. The cells were seeded at 5X 104 cells per well in 96-well plates and 100. mu.l of medium with different concentrations of HDM was added after the cells had adhered. Culturing at 37 deg.C in 5% CO2 incubator for 12h, taking out the culture plate, adding 10 μ l/hole CCK-8 reagent in a super clean bench under dark condition, mixing well, and placing in the incubator for 1-2 hr.

And taking out the culture plate with 96 holes, setting parameters of an enzyme-labeling instrument, detecting the absorbance at the wavelength of 450nm at 37 ℃, and recording the detection result. Cell viability ═ 100% x [ (experimental-blank)/(control-blank).

Extraction of Total RNA from cells

(1) The treated 6-well plate was removed from the incubator, the original medium was aspirated, and gently washed 2 times with 1 × PBS buffer.

(2) Add 1ml of RNAioso Plus to each well and mix by gentle shaking to distribute the lysate evenly on the cell surface. Reference "Chapter 2.3.4 extraction of Total cellular RNA"

Real-time fluorescent quantitative PCR (in the same front)

Extraction of Total cellular protein

(1) The PIPA lysate was dissolved and mixed well, and phenylmethylsulfonyl fluoride (PMSF) was added to the lysate to a final concentration of 1mM, within a few minutes before use.

(2) The treated 6-well plate was removed, the medium was removed, and washed 2 times with PBS buffer, 200. mu.l of lysis buffer was added to each well. Gently blowing and beating for several times by using a pipette gun to make the lysate and the cells fully contact, and standing for 5min on ice.

(3) Cells were scraped off with a cell scraper and the cell suspension was pipetted into a 1.5ml EP tube and labeled. Centrifuge at 12000g 4 ℃ for 5 min. The supernatant was pipetted into a new EP tube, labeled and the protein content recorded.

Extraction of nuclear and cytoplasmic proteins

The operation is carried out according to the instructions of the Biyunshi cell nucleus protein and cytoplasm protein extraction kit.

(1) The three reagents were dissolved at room temperature and stored on ice, and PMSF was added to the cell nuclear protein extraction reagent and the cytoplasmic protein extraction reagent A, respectively, several minutes before use, so that the final concentration of PMSF was 1 mM.

(2) The treated 6-well plate was removed, the medium was removed, washed 2 times with PBS buffer, the cells were scraped off with a cell scraper, and transferred to an EP tube with a pipette. Centrifuge at 12000g 4 ℃ for 5 min. The supernatant was aspirated off and the cell pellet was retained for use.

(3) Adding 50 μ l of the cell plasma protein extraction reagent A added with PMSF into the cell sediment, violently shaking and uniformly mixing for 5 seconds to completely suspend and disperse the cell sediment, and carrying out ice bath for 15 min.

(4) Add 10 μ l of cytoplasmic protein extraction reagent B into the tube, mix by vigorous shaking, ice-wash for 1 min.

(5) Mix by vigorous shaking for 5s, 12000g, 4 ℃, and centrifuge for 5 min.

(6) The supernatant was pipetted into a new EP tube (without touching the pellet), which was the cytosolic protein.

(7) After the supernatant was removed, 50. mu.l of the nuclear protein extraction reagent containing PMSF was added, followed by shaking vigorously and mixing for 30 seconds, and ice-cooling for 30 min. Shaking vigorously every 1-2min for 15s during ice bath.

(8)12000g, 4 ℃, centrifugation for 10 min. The supernatant was aspirated into a new EP tube, which was the nuclear protein.

Protein concentration determination

The procedure was performed according to the instruction of BCA protein concentration assay kit (Biyunna).

(1) Preparation of protein standard: adding 0.8ml of the protein standard preparation solution into 20mg of protein standard (BSA), fully shaking and uniformly mixing to prepare 25mg/ml of protein standard solution. An appropriate amount of 25mg/ml protein standard solution was diluted to 0.5mg/ml with 0.9% NaCl solution.

(2) Preparing a BCA working solution: the BCA working solution was prepared in a volume ratio of 50:1 between the BCA reagent A and the reagent B, and was mixed well.

(3) Adding 0, 1, 2, 4, 8, 12, 16 and 20 mul of protein standard substance into a 96-well plate respectively, and adding the standard substance diluent to 20 mul to ensure that the concentrations of the standard substance are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5mg/ml in sequence.

(4) And (3) taking a proper volume of the protein sample to a 96-well plate, supplementing the protein sample to 20 mu l by using a standard substance diluent, and recording the volume of the added protein sample.

(5) Add 200. mu.l BCA working solution to each of the empty and sample wells of the standards and leave them at 37 ℃ for 30 min.

(6) The microplate reader parameters were set and the absorbance at the wavelength of a562nm was measured.

(7) And (5) making a standard curve and calculating the concentration of the protein sample.

Western blot assay

(1) Preparation work: the prepared proteins were removed and added to a suitable volume of 5 SDS-PAGE at a ratio of 4:1Loading buffer solution, blowing, beating, mixing, and boiling at 100 deg.C for 5min to denature protein. Cleaning the glass plate and the comb, vertically drying at room temperature, oppositely arranging the glass plate on a glue filling frame, adding ddH₂And O, checking whether liquid leakage exists or not.

(2) Preparing glue and pouring the glue: a10% separation gel and a 4% concentration gel were prepared according to the molecular weight of the protein, and the system is shown in Table 8.

TABLE 8 composition System for separation gums and concentrating gums

Glue pouring: slowly adding the prepared 10% separation glue to 2/3 parts in the glass plate along the wall of the glass plate, then adding double distilled water, standing at room temperature for 30min until the glue at the lower layer is solidified, inclining to the place where the double distilled water is evaporated and sucking the glue with absorbent paper. Then 4% concentrated glue is added, a comb is inserted to avoid generating bubbles, and the mixture is kept stand for 30min at room temperature until the upper layer glue is solidified.

(3) Loading: and (3) putting the prepared rubber plate into an electrophoresis tank, adding a proper volume of electrophoresis liquid, slightly pulling out a comb, and adding 10-20 mu l of protein sample and 2.5 mu l of protein marker into each hole according to experimental requirements.

(4) Electrophoresis: adding electrophoresis liquid, connecting a power supply, setting the electrophoresis of the upper layer gel at 80V for 20min, adjusting the electrophoresis parameters to 110V for 90min when the protein strips run to the lower layer gel until the marker strips with the required protein molecular weight are obviously separated.

(5) Film transfer: and sequentially putting the film transferring clamp, the spongy cushion, the two layers of thick filter paper and the one layer of thin filter paper into the film transferring liquid for soaking. Cutting a PVDF membrane with proper size, putting the PVDF membrane into methanol for soaking for 3s for activation, and then putting the PVDF membrane into a membrane transferring solution for soaking. And removing the rubber plate after electrophoresis, cutting off the rubber block, paving the PVDF film, marking, clamping the film transferring clamp, and placing in a film transferring groove. And setting the film transfer parameter to 300mA for 90 min. After the transfer of the membrane, the membrane was washed 1 × TBST 3 times for 15min each.

(6) And (3) sealing: and (3) putting the washed PVDF membrane into the prepared BSA blocking solution, blocking for 2 hours at room temperature with the front side upward and the back side downward. After the end of blocking, the cells were washed 1 × TBST 3 times for 15min each.

(7) Primary antibody incubation: the primary antibody was diluted as recommended by the antibody instructions and the PVDF membrane was placed in the primary antibody and incubated overnight at 4 ℃ with shaking. After incubation of primary antibody, primary antibody was recovered and washed 3 times with 1 × TBST, 15min each time.

(8) And (3) secondary antibody incubation: the secondary antibody was diluted as recommended by the antibody instructions and the PVDF membrane was placed in the secondary antibody and incubated for 2h at room temperature. After the incubation of the secondary anticaking agent, the secondary antibody was recovered and washed 3 times with 1 × TBST, 15min each time.

(9) Exposure and development: preparing exposure liquid and storing in the dark, dropping the exposure liquid on the membrane to avoid generating bubbles, scanning the PVDF membrane by a FluorChem FC3 software system, analyzing the relative molecular content of a target strip by a gel image processing system, and comparing the relative expression quantity of each histone.

Enzyme-linked immunosorbent assay (ELISA)

(1) Sample treatment: the cell culture dish supernatant was collected in a sterile 1.5ml EP tube, centrifuged at 2000rpm for 20 minutes, and the supernatant was collected.

(2) Loading a standard substance: and arranging a standard product hole and a sample hole, and sequentially adding 50ul of standard products with different concentrations into the standard flat hole.

(3) Sample adding: setting a blank control hole and a sample hole to be detected, adding 40ul of sample diluent into the sample hole to be detected on the enzyme label plate, and then adding 10ul of sample to be detected, so that the sample is finally diluted by 5 times. When in sample adding, the sample is added to the bottom of the hole of the ELISA plate, and the hole wall is not touched as much as possible. Gently shaking and mixing.

(4) Adding an enzyme: 100ul of enzyme-labeled reagent was added to each well, except for blank wells.

(5) And (3) incubation: after sealing the plate with a sealing plate, the microplate was incubated in an incubator at 37 ℃ for 60 minutes.

(6) Preparing liquid: the 20 × concentrated washings were diluted 20 times with distilled water.

(7) Washing: carefully tearing off the sealing plate film, sucking off liquid, drying by spin, filling washing liquid into each hole, standing for 30 seconds, discarding, repeatedly washing for 5 times, and patting on absorbent paper to dry.

(8) Color development: 50ul of color developing agent A and 50ul of color developing agent B are added into each hole, the mixture is gently shaken and uniformly mixed, and the mixture is subjected to dark color development for 15 minutes at the temperature of 37 ℃.

(9) And (3) stopping color development: 50ul of stop solution was added to each well to terminate the color reaction.

(10) And (3) determination: within 15 minutes after the addition of the stop solution, the absorbance of each well was measured by zeroing the blank well.

3. Results

Effect of different concentrations of HDM on BEAS-2B cell proliferation

The BEAS-2B cells were cultured for 12h by diluting HDM to various concentrations (0, 200, 400, 600, 800U/ml) with cell culture medium containing 10% FBS and no diabodies. The CCK-8 method measures the effect of different concentrations of HDM on cell proliferation. The results are shown in FIG. 6: BEAS-2B cell viability was significantly increased at 600U/ml HDM compared to the control group (P < 0.01).

Effect of HDM on BEAS-2B cell inflammatory factor expression

According to the experimental results, the HDM concentration of 600U/ml can obviously promote the proliferation of airway epithelial cell BEAS-2B. The airway epithelial cells are cultured for 12 hours by using an HDM culture medium containing 600U/ml, the relative expression quantity of mRNA of IL-6, IL-8 and TNF-alpha in the cells is detected by a real-time fluorescent quantitative PCR method, and the content of each cytokine in cell supernatant is detected by an ELISA method. The results are shown in FIG. 7: compared with the control group, after the BEAS-2B cells are stimulated by the HDM, the relative expression quantity of the mRNA of IL-6, IL-8 and TNF-alpha and the content of the supernatant fluid are obviously increased (P is less than 0.01).

5-Aza-CdR increases mRNA and protein levels of MEF2C

Culturing airway epithelial cells for 72h by using a DMEM medium containing 20 mu mol/L5-Aza-CdR, extracting total RNA, and detecting the mRNA level of MEF2C by real-time fluorescence quantitative PCR; extracting total protein, and detecting MEF2C protein expression by western blotting method. The results are shown in FIG. 8: 5-Aza-CdR treated BEAS-2B cells for 72h, MEF2C mRNA increased by about 3-fold, and protein expression increased compared to the control group (P < 0.05).

5-Aza-CdR reduces the percent methylation of MEF2C CpG islands

Methylation inhibitor 5-Aza-CdR increased mRNA and protein expression of MEF2C, and was probably related to DNA methylation level of MEF2C gene. Airway epithelial cells were cultured in DMEM medium containing 20 μmol/L5-Aza-CdR for 72h, and after 12h of HDM stimulation, the cells were harvested, cell DNA was extracted, and methylation levels were measured using MassARRAY methylation technique, and the results are shown in fig. 9: compared with the control group, the MEF2C DNA methylation ratio of the 5-Aza-CdR treatment group is reduced.

Role of MEF2C hypomethylation in HDM-constructed models of cellular inflammation

To investigate the role of MEF2C hypomethylation in the model of cellular inflammation constructed by HDM, we continued culturing for 12h after BEAS-2B cell attachment in the HDM +5-Aza-CdR group with DMEM medium containing 5-Aza-CdR for 3 days and then with 600U/ml of HDM, and continued culturing for 12h in the HDM group with DMEM medium containing no 5-Aza-CdR for 3 days and then with 600U/ml of DMEM medium. MEF2C methylation levels, mRNA and protein expression levels were measured.

The results are shown in FIG. 10: MEF2C methylation decreased, mRNA levels decreased (a), and protein expression decreased (B). Meanwhile, in the MEF 2C/NF-kB signal channel, cytoplasmic NF-k B p65 protein is reduced, NF-kB in a cell nucleus is increased (C), and mRNA levels of IL-6, IL-8 and TNF-alpha are increased (D, E, F), which indicates that MEF2C demethylation aggravates airway inflammatory response to a certain extent.