阅读说明：本技术 一种copd早期诊断标志物及其应用 (COPD early diagnosis marker and application thereof ) 是由 刘持 秦岭 袁琳 杜茜子 秦晓群 向阳 瞿湘萍 刘惠君 于 2019-09-30 设计创作，主要内容包括：本发明涉及医药技术领域,具体涉及用于COPD的生物标志物及其应用。本发明提供了一种COPD早期诊断药物标志物及包含此标志物的诊断药物试剂盒,所述标志物为8个在COPD中差异表达的衰老相关基因：FOXO3,TP53,TGFβ1,HDAC1,NUF2,ATG3,AREG,E2F1中的一个或任意几个的组合,或在COPD中差异表达的衰老相关基因的13个甲基化位点的一个或任意几个的组合。本发明标志物在对照组与COPD组的甲基化程度有统计学意义(P<0.05),可作为生物标志物应用于临床COPD的诊断药物。(The invention relates to the technical field of medicines, in particular to a biomarker for COPD and application thereof. The invention provides a COPD early diagnosis drug marker and a diagnosis drug kit comprising the marker, wherein the marker comprises 8 senescence-associated genes which are differentially expressed in COPD: FOXO3, TP53, TGF β 1, HDAC1, NUF2, ATG3, AREG, E2F1, or one or any combination of 13 methylation sites of senescence-associated genes differentially expressed in COPD. The marker has statistical significance (P <0.05) in the methylation degree of a control group and a COPD group, and can be used as a biomarker applied to clinical COPD diagnosis medicines.)

1. An early diagnosis marker of COPD, wherein the marker is one or a combination of any two of 8 senescence-associated genes differentially expressed in COPD, and the 8 senescence-associated genes differentially expressed in COPD are respectively: FOXO3, TP53, TGF β 1, HDAC1, NUF2, ATG3, AREG, E2F 1.

2. The marker according to claim 1, wherein the marker is one or a combination of any of 13 methylation sites of 8 senescence-associated genes differentially expressed in COPD, the 13 methylation sites being: chr 6: 108879506, chr 20: 32274142, chr 3: 112281632, chr 20: 32274387, chr 3: 112280747, chr 6: 108879098, chr 1: 32757856, chr 19: 41858066, chr 6: 108880486, chr 6: 108879086, chr 3: 112280605, chr 1: 163292017, chr 19: 41858034.

3. use of the marker of claim 1 in a medicament for the early diagnosis of COPD.

4. Use of the marker of claim 2 in a medicament for the early diagnosis of COPD.

5. Use of a marker according to claim 4 in a medicament for the early diagnosis of COPD comprising the steps of:

(1) collecting peripheral blood from COPD patients and healthy control testers;

(2) assessing the accuracy of the methylation sites for a COPD diagnostic drug;

(3) assessing the optimal diagnostic drug threshold for the methylation site;

(4) the rate of variation was evaluated for the changes in 13 methylation sites in each sample.

6. The use of the marker in the medicine for the early diagnosis of COPD according to claim 5, wherein the variation rate of 0-23.08% in the step (4) indicates negative COPD, the variation rate of 23.08% -61.54% indicates high risk of COPD, and the variation rate of 61.54-100% indicates positive COPD.

7. A kit for early diagnosis of COPD, comprising 13 methylation PCR primer pairs for amplifying the marker of claim 2.

8. The kit of claim 7, wherein the kit comprises a Taq enzyme polymerase chain reaction system and the following methylation PCR primer pairs:

9. use of a kit according to claim 7 or 8 in the early diagnosis of COPD.

Technical Field

The invention relates to the technical field of medicines, in particular to a biomarker for identifying chronic obstructive pulmonary disease and detection application thereof.

Background

Chronic Obstructive Pulmonary Disease (COPD) is a disease state characterized by airflow limitation that is not fully reversible, usually progressive, with the pathological features of persistent progressive airflow obstruction and persistent exacerbation of airway inflammation. COPD is one of the diseases with high morbidity and mortality in the world at present, and the incidence rate of the COPD is in a gradually rising trend. COPD is predicted to be the third leading cause of death in the world by 2020, which imposes a serious health burden on the world in terms of morbidity, hospitalization and medical treatment. Particularly, the susceptibility risk screening and early diagnosis of the COPD at the present stage are very difficult, and no effective treatment scheme aiming at the COPD exists clinically. The condition of COPD is usually severe and progressive, and is a key and difficult point in the field of respiratory disease control. Therefore, the method has important significance in further research on pathogenesis and clinical features of COPD and further search for biological markers of early risk screening and clinical diagnosis medicines for COPD.

A large body of clinical epidemiological data and laboratory studies have found that accelerated lung aging is an important pathogenic mechanism of COPD. Epidemiological data clearly show that COPD is significantly more prevalent in the elderly over 65 years of age. Significant increases in senescence-associated cellular molecular signals, such as telomere shortening, cellular senescence and stem cell failure, and decreased expression of senescence-associated molecules, such as histone deacetylase and deacetylase, have been detected in COPD patients. The effective anti-aging intervention and elimination of lung aging cells can obviously improve the lung inflammation degree and the clinical progress of COPD, and the data strongly suggest that the expression and regulation abnormality of aging related genes participate in inducing the occurrence and development of COPD.

A recent series of evidence suggests that regulation of epigenetic mechanisms may be the primary means of regulation of senescence-associated gene expression. DNA methylation is the earliest and most important modification in epigenetic regulation, and is involved in complex gene/environment interactions, and is closely related to gene regulation, biological development and disease development. DNA methylation is mainly manifested by covalent binding of adenine or cytosine bases in a DNA sequence to a methyl group catalyzed by DNA methyltransferase, an epigenetic phenomenon that is transmitted to daughter cells during cell division, and generally occurs at CpG sites (cytosine-phosphate-guanine sites, i.e., sites in a DNA sequence immediately following cytosine by guanine). It has been proved that the level of DNA methylation is closely related to the onset of COPD, and the DNA methylation can be used as a biomarker for predicting the early risk of the onset of diseases such as tumors, COPD and the like. In addition, DNA methylation sequences are present in human serum in a highly stable form, and are resistant to digestion by RNases, proteases and various stress states of cells.

In conclusion, the incidence and fatality rate of COPD have been increasing year by year in recent years, and yet, it still cannot be predicted clinically at an early stage, and once diagnosis is progressively increased, it cannot be cured radically, which causes serious problems of family economic burden and social health. However, COPD is a complex disease caused by the interaction of polygenic inheritance and environmental factors, and no clinically effective risk prediction marker exists so far. Because serum is relatively easy to obtain, the DNA methylation biomarker in the circulating serum is expected to be used as a non-invasive risk marker for an early diagnosis medicament of COPD onset.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a COPD early diagnosis drug marker and a diagnosis drug kit comprising the marker. The method further verifies the variation rate in the blood of a COPD sample group and a healthy control sample by using the screened methylation sites, and the result shows that the methylation degrees of the screened 13 methylation sites in the control group and the COPD group have statistical significance (P <0.05), and the screened 13 methylation sites can be used as biomarkers to be applied to clinical diagnostic drugs for COPD (P < 0.01).

A COPD early diagnosis drug marker, which is one or a combination of any several of 8 senescence-associated genes differentially expressed in COPD, wherein the 8 senescence-associated genes differentially expressed in COPD are: FOXO3, TP53, TGF β 1, HDAC1, NUF2, ATG3, AREG, E2F 1.

Preferably, the marker is one or a combination of any several of 13 methylated CpG sites of 8 senescence-associated genes differentially expressed in COPD, which are: chr 6: 108879506, chr 20: 32274142, chr 3: 112281632, chr 20: 32274387, chr 3: 112280747, chr 6: 108879098, chr 1: 32757856, chr 19: 41858066, chr 6: 108880486, chr 6: 108879086, chr 3: 112280605, chrl: 163292017, chr 19: 41858034.

the application of the marker in the medicine for early diagnosis of COPD distinguishes a COPD population from a healthy population by the expression level of one or the combination of any more of 8 senescence-associated genes which are differentially expressed in COPD.

The application of the marker in the medicine for early diagnosis of COPD distinguishes a COPD population from a healthy population by the methylation level and the variation rate of one or any combination of sites of 13 methylation sites.

The application of the marker in the medicine for early diagnosis of COPD comprises the following steps:

(1) collecting peripheral blood from COPD patients and healthy control testers;

(2) evaluating the accuracy of diagnostic drugs for distinguishing COPD (chronic obstructive pulmonary disease) population from healthy population by 13 methylation sites through an ROC (rock-characteristic curve);

(3) assessing the optimal diagnostic drug threshold for the methylation site; evaluating the optimal diagnostic drug threshold value of 13 methylation sites by an ROC curve, and determining the optimal diagnostic drug threshold value of each methylation site according to the methylation expression level corresponding to the maximum value of the Johnson index (sensitivity + specificity-1); converting the methylation level of the methylation sites from continuous variables into classification variables according to the threshold value, namely determining whether the methylation level of each methylation site changes according to the optimal diagnostic drug threshold value;

(4) the rate of variation was evaluated for the changes in 13 methylation sites in each sample.

Preferably, the basis for judging whether the patients with COPD in the step (4) is that the variation rate is 0-23.08% and indicates that the patients with COPD are negative, the variation rate is 23.08% -61.54% and indicates that the patients with COPD are at high risk, and the variation rate is 61.54-100% and indicates that the patients with COPD are positive.

A kit for early diagnosis of the drug COPD, said kit comprising 13 methylation PCR primer pairs for amplification of the above markers.

Preferably, the kit contains a Taq enzyme polymerase chain reaction system and 13 methylation PCR primer pairs for amplifying the 13 methylation sites, and the Taq enzyme polymerase chain reaction system comprises: taq enzyme, PCR buffer, dNTPs.

Preferably, the kit comprises a Taq enzyme polymerase chain reaction system and primer pairs corresponding to the following methylation sites:

the kit comprises: taq enzyme 2.6. mu.l, PCR buffer 10.5. mu.l, dNTPs 19.5. mu.l, ddH₂Mu.l of O222. mu.l of each of the 10. mu.M upstream and downstream primers for the 13 methylation sites.

A method of screening for said marker comprising the steps of:

1. screening senescence-associated genes;

2. validating in large sample volumes senescence-associated genes differentially expressed in COPD patients;

3. performing second-generation methylation sequencing and differential CpG site analysis on senescence-associated genes differentially expressed in COPD patients; determining DNA methylation biomarkers that distinguish COPD populations from healthy populations;

4. the large sample size verifies the feasibility of using the methylation level and variation rate of 13 key differential CpG sites as biomarkers to distinguish COPD from healthy population.

Preferably, the screening for senescence-associated genes comprises the following specific steps:

s1, screening the healthy control peripheral blood mRNA gene expression profile chip aging related genes;

s2, transcriptome sequencing and differential gene analysis of healthy control and COPD peripheral blood;

s3, screening for senescence-associated genes differentially expressed in COPD.

Preferably, the step of S1 screening the healthy control peripheral blood mRNA gene expression profile chip for senescence-associated genes comprises:

(1) screening an aging database: in NCBI, using "aging" and "senescence" as keywords, the database with human species and peripheral blood/peripheral blood mononuclear cells as samples is searched. Exclusion criteria were: 1. lack of specific age information for the subject; 2. subjects had a definite disease or received some intervention during the study; 3. the sample size is less than 30. The 3 databases that were finally included were GSE58137, GSE65219, GSE74786, respectively.

(2) Screening of senescence-associated genes: differential expression genes accompanied with aging in the 3 databases are respectively analyzed through the adjusted linear regression model, and then the differential expression genes generated by the three databases are subjected to Wien drawing intersection to obtain the aging related genes.

Preferably, the transcriptome sequencing and differential gene analysis of the healthy control of S2 and COPD peripheral blood specifically comprises the following steps:

(1) collecting peripheral blood of patients with COPD and healthy control testers for clinically definite diagnosis;

(2) transcriptome sequencing and differential gene analysis of healthy controls and COPD peripheral blood.

Preferably, the transcriptome sequencing step is

Firstly, extracting RNA, and the specific steps are as follows:

a. cracking red blood cells in peripheral blood, centrifuging, adding Trizol and chloroform, shaking up and centrifuging;

b. taking the upper water phase of the centrifugate, adding isopropanol and centrifuging;

c. after alcohol washing and drying, the mixture is dissolved in ribozyme-free water, and the concentration of RNA is measured by an ultraviolet spectrophotometer.

② RNA quality detection, which comprises the following steps:

a. determination of concentration and total amount of RNA: the Qubit accurately quantifies the concentration, and the total amount is calculated according to the concentration and the volume;

b. determination of RNA purity: the ratios of OD260/280 and 260/230 were measured using a Nanodrop;

c. determination of RNA integrity: detecting the degradation degree of RNA by agarose gel electrophoresis and Agilent2100 Bioanalyzer;

the total amount of RNA sample is between 1 and 4 mu g, the 260/280 ratio is between 1.8 and 2.0, or the RIN ≧ 8 detected by Agilent2100Bioanalyzer indicates that the RNA amount is sufficient and the integrity is high.

Sorting and purifying mRNA and fragmenting mRNA:

by utilizing the mechanism that the magnetic beads with oligo-dT are specifically combined with mRNA with a poly (A) structure and two rounds of purification of the magnetic beads with oligo-dT, the non-specific combination of rRNA and sorted magnetic beads is reduced to the maximum extent, and the effects of enriching and purifying mRNA from total RNA are achieved.

After the second round of magnetic bead purification, a buffer solution consisting of an eluent, fragmented mRNA and random primers is added, so that the random primers in the next step of cDNA synthesis can be conveniently combined with the fragmented mRNA. (fragmentation of mRNA: incubation of magnetic beads at 94 ℃ C., elution of bound mRNA and simultaneous thermal fragmentation, distribution of fragments between 100 and 300bp, optimization of fragmentation Effect by adjusting incubation time depending on RNA quality)

Synthesizing first strand cDNA:

first strand cDNA synthesis was performed using short fragment mRNA fragmented and bound with random reverse transcription primers as template, under the action of Invitrogen reverse transcriptase SuperScript IV (SS IV).

Synthesizing second strand cDNA:

adding a second strand synthesis premixed system (buffer solution, dNTPs, RNase H and DNA polymerase I) into the system for synthesizing the first strand cDNA, hydrolyzing and digesting the RNA on the RNA/cDNA hybrid strand, taking the first strand cDNA as a template, synthesizing the second strand cDNA by using DNA polymerase, and purifying by AgencourtAMpure XP magnetic beads to finally obtain Double-strand cDNA (Double-strand cDNA, ds cDNA);

sixthly, double-stranded cDNA tail end repair reaction:

to the purified ds cDNA, a terminal-filling system was added, which contained a combination of enzymes that repaired the irregular ends of the cDNA at both ends, as follows: digesting the single-stranded protrusion at the 3 'end by using Exonuclease (Exonuclease) activity, and supplementing the protrusion at the 5' end by using Polymerase (Polymerase) activity; meanwhile, phosphate groups necessary for subsequent ligation reaction are added to the 5 'end of Phosphokinase (PNK), and the mixture is purified by Agencourt AMpure XP magnetic beads to finally obtain a flat-end ds cDNA short fragment library containing the phosphate groups at the 5' end;

adding ' A ' at the 3 ' end for reaction:

to the above system was added a 3' end "A" buffer reaction system (buffer, RNA terminal adenylyltransferase, ATP). Adding single adenosine A at the 3 'end of both sides of the ds cDNA after the end modification to prevent the blunt end self-connection between cDNA fragments, and can be matched with the single T protrusion at the 5' end of the sequencing joint in the next step for accurate connection, thereby effectively reducing the self-series connection between the library fragments;

connecting sequencing connectors:

adding a connecting buffer solution, T4 DNA ligase and a double-stranded sequencing adaptor into the reaction system, and connecting the illumina sequencing adaptor to both ends of the library DNA by using T4 DNA ligase.

Ninthly, screening library fragments:

for the linker-added library, fragment size screening was performed using the Agencour SPRISELect nucleic acid fragment screening kit while purifying the library. A two-step screening (Double Size selection) is adopted, wherein a small Left fragment (Left-side Size selection) of a target region is removed by SPRI magnetic beads, and a large Right-side fragment (Right-side Size selection) of the target region is removed. Finally, screening the original library with the fragment peak value of 300bp for the next PCR amplification. The library after purification removes excessive sequencing joints in the system and products of self-connection of the joints, avoids ineffective amplification in the PCR amplification process and eliminates the influence on-machine sequencing;

r PCR amplification cDNA library:

the original library was amplified using high fidelity polymerase in a 50 μ L reaction to ensure sufficient library inventory on the sequencer. Meanwhile, only the DNA fragments with the joints at both ends can be amplified, and the fragments only connected with the single-ended joints are removed. The number of PCR amplification cycles was controlled between 12-15 (typically 1. mu.g of starting total RNA, and the final library amplification was controlled at 15 cycles). And on the premise of ensuring that the product is enough, the bias introduced due to the overlarge number of amplification cycles is reduced. Purifying the amplified library by magnetic beads to obtain a sequencing library which can be operated on a computer;

quality testing of the library:

and (4) carrying out quality detection and quantification on the constructed sequencing library. And (3) accurately quantifying the concentration of the library by using the Qubit, and being used for accurately mixing samples to ensure that the data volume of each sample is properly balanced. Determining the size distribution of the library fragments by using an Agilent2100Bioanalyzer, and evaluating suitability for computer installation;

mixing samples and performing machine sequencing:

diluting the qualified samples, mixing a plurality of samples according to the equimolar number, and loading the samples on a sample loader. The library was sequenced using the illumina Hiseq platform, 2 × 150 paired-end sequencing strategy.

High throughput transcriptome sequencing data analysis:

and (3) performing bioinformatics analysis on the sequencing result, comparing the sequence after quality control with a reference genome by using tophat2, performing expression quantification on the known gene and the transcript by using the analytical process of cufflinks, and analyzing the differentially expressed gene by using cuffdiff software. mRNA was considered significantly differentially expressed when adj.p-value < 0.05.

Preferably, the S3 screens senescence-associated genes differentially expressed in COPD, comprising the specific steps of:

and screening the intra-lung expression abundance of the senescence-associated genes through NCBI to obtain the senescence-associated genes with high intra-lung expression abundance, and then taking intersection of the senescence-associated genes and the genes differentially expressed in COPD through a Wien diagram to determine the senescence-associated genes differentially expressed in COPD.

The large sample size verifies senescence-associated genes differentially expressed in COPD patients, with the specific steps as follows:

(1) collecting peripheral blood of COPD patients and healthy control testers according to the method in step 2;

(2) extracting peripheral blood RNA according to the method in the step 2;

(3) reverse transcription: the operation was carried out using a reverse transcription kit from Sigma, and the specific procedure was as follows: taking 1 μ gRNA, calculating the volume of RNA to be added according to the measured RNA concentration, preparing 12 μ l reaction system by using 1 μ l Oligo primer of random primer and 1 μ l ribozyme-free water, and denaturing at 65 ℃ for 5 min. Reverse transcription reaction is carried out by using AMV reverse transcriptase and RNase inhibitor at 25 deg.C for 5min, 42 deg.C for 60min, and 70 deg.C for 5 min.

(4) Real-Time qPCR, the concrete steps are as follows:

designing a qPCR amplification primer;

preparing a qPCR reaction system;

and carrying out PCR reaction.

Preferably, the second-generation methylation sequencing and differential CpG site analysis of the senescence-associated genes comprise the following specific steps:

(1) second-generation methylation sequencing:

collecting peripheral blood of COPD patients and healthy control testers according to the method in the step 2;

extracting DNA by a peripheral blood genome DNA extraction kit;

③ the quality control of the sample comprises the following steps: detecting the integrity of the genome DNA by agarose gel electrophoresis; nanodrop 2000 detects the quality of genomic DNA;

selecting CpG island in the promoter near the target gene;

designing primers and optimizing single-site PCR conditions: designing a sequencing primer based on software, selecting a primer which can obtain a clear single band by using a human genome treated by bisulfite as a template and amplifying the template for a subsequent experiment;

sixthly, optimizing the multiple PCR primer panel: mixing the primers optimized in the step (iv) into a multiplex PCR primer panel, amplifying by using a multiplex PCR technology and taking a standard human genome as a template, judging whether each pair of primers in a multiplex system is efficiently and specifically amplified or not based on a special method of capillary electrophoresis, and optimizing the primer composition and concentration in the multiplex PCRpanel according to the regulation;

and (c) bisulfite treatment:

carrying out sample treatment by using an EZ DNA Methylation-Gold Kit to convert cytosine C of genome DNA which is not subjected to Methylation modification into thymine U;

eighthly, carrying out multiple PCR reaction on the sample target segment:

performing multiple PCR amplification by using the optimized multiple PCR primer panel and taking the transformed sample genome as a template; after quality control, mixing the amplification products of all the multiple PCR primers panel which take the genome DNA of the same sample as a template, and ensuring that the amount of the amplification products of the primers at each site is equivalent;

ninthly, adding a specific tag sequence to the sample:

introducing a specific tag sequence compatible with the illumina platform into the end of the library by PCR amplification by using a primer with an Index sequence;

and (3) performing on-machine sequencing after the quantification of the Ribs:

mixing the sample Index PCR amplification products in equal amount, and obtaining a final MethylTarget sequencing library through tapping recovery, wherein the fragment length distribution of the library is verified by an Agilent2100 Bioanalyzer; after the molar concentration of the library is accurately quantified, performing high-throughput sequencing on an Illumina Hiseq/Nextseq platform in a 2 x 150bp double-end sequencing mode to obtain FastQ data;

preferably, the differential CpG site analysis specifically comprises: analyzing a sequencing result, comparing the sequencing data after quality control with a reference genome, counting the enrichment efficiency of a target fragment, the effective reads and salinization efficiency of the fragment, and analyzing the methylation proportion of each CpG locus in a sample; and (3) performing differential significance analysis on all methylation results according to grouping information of the samples by using a T test, univariate logistic regression and multivariate logistic regression, and considering that the methylation of the CpG sites is significantly and differentially expressed when the P value is less than 0.05.

The correlation analysis of the methylation level of the differential CpG sites and the clinical parameters of the COPD patients comprises the following specific methods:

based on the grouping information of the samples, the Benjamin-Hochberg method was used to control the False Discovery Rate (FDR), and Pearson correlation was used to assess the correlation of methylation levels of differential CpG sites with clinical parameters of COPD patients.

The invention is further explained below: the existence of varying degrees of lung aging in COPD patients, we thought that this accelerated lung aging could be due to aberrant DNA methylation regulation of aging-associated genes. Thus, methylation of key CpG sites of senescence-associated genes and their methylation levels may be involved in and constitute the pathogenesis of COPD.

To obtain a non-invasive serum marker for COPD as an early diagnostic drug for the risk of COPD onset, we examined the methylation expression levels of candidate CpG sites of senescence-associated genes differentially expressed in COPD patients and healthy control populations and evaluated the correlation between the DNA methylation levels of the differential CpG sites and clinical parameters of COPD patients. And then, further detecting the methylation level and the methylation variation rate of key difference CpG sites of the senescence-associated genes in a large-sample clinical specimen, and verifying the feasibility of using the methylation level and the variation rate of the key difference CpG sites of the senescence-associated genes as biomarkers to distinguish COPD (COPD) people from healthy people.

We performed a primary screening of senescence-associated genes by 3 senescence-associated databases of Gene Expression Omnibus (GEO), a secondary screening thereof by peripheral blood transcriptome sequencing of COPD patients to determine senescence-associated genes differentially expressed in COPD patients, and verified the differential Expression of these senescence-associated genes in COPD patients at the mRNA level. On this basis, we evaluated the candidate CpG sites of the senescence-associated genes and their methylation levels, as well as the correlations between the DNA methylation levels of the differential CpG sites and clinical parameters of COPD patients, including FEV1, FEV 1%, FEV/FVC, mrrc score, frequency of acute episodes and CAT score. The results show that the presence of 13 methylation sites in the DNA regulatory region of differentially expressed senescence-associated genes correlates with clinical parameters of COPD patients. Finally, we collected large sample size clinical specimens to examine the methylation level and methylation variation rate of the above 13 methylation sites in COPD patients. The result shows that the methylation variation rate of 13 methylation sites of the aging related gene with differential expression can be used as a biomarker to effectively distinguish the COPD population from the healthy population. In conclusion, the methylation sites of the senescence-associated genes as the biomarkers of COPD early diagnosis drugs are: chr 6: 108879506, chr 20: 32274142, chr 3: 112281632, chr 20: 32274387, chr 3: 112280747, chr 6: 108879098, chr 1: 32757856, chr 19: 41858066, chr 6: 108880486, chr 6: 108879086, chr 3: 112280605, chr 1: 163292017, chr 19: 41858034.

therefore, the serum DNA methylation biomarker spectrum consisting of the 13 methylation sites provided by the invention can be applicable to early diagnosis medicines of COPD. In addition, the COPD patients are subjected to hierarchical comparison according to age and sex, and the clinical factors are shown to be irrelevant to the methylation level of the aging-related genes (P > 0.05), so the 13 methylation site spectrum can be used as the biomarker of early diagnosis drugs of COPD.

According to the experimental data of the present invention, the differential methylation site spectrum of the senescence-associated genes is a biomarker of COPD early diagnosis drugs, and the above-mentioned DNA methylation site spectrum of the senescence-associated genes can also be used for preparing COPD early diagnosis drug kits.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a COPD early diagnosis drug marker and a kit, and test results show that the methylation degree of 13 screened methylated loci in a control group and a COPD group has statistical significance (P <0.05), and the variation rate of the 13 screened methylated loci can be used as a biomarker applied to clinical COPD diagnosis drugs (P < 0.01).

Drawings

FIG. 1 is a graph showing the results of differential expression of 8 senescence-associated genes of the present invention in the peripheral blood of COPD patients.

FIG. 2 is a graph of the results of the present invention in assessing the accuracy of diagnostic drugs for differentiating a COPD population from a healthy population by 13 methylation sites via the ROC curve.

Detailed Description

The present invention will be described in detail with reference to examples. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.