CN115807095B - Primer composition for detecting methylation sites of lung adenocarcinoma and application of primer composition - Google Patents

Abstract

The invention discloses a gene SPATS2L, NSUN5 and ZNF207 which can be jointly detected for diagnosing lung adenocarcinoma, and whether a subject suffers from lung adenocarcinoma or not can be accurately known according to methylation levels of the SPATS2L, NSUN and the ZNF207 obtained by the joint detection. The invention also discloses a kit for diagnosing lung adenocarcinoma, which is a kit for simultaneously carrying out joint detection on the methylation levels of genes SPATS2L, NSUN5 and ZNF207 by fluorescence quantitative PCR reaction, and comprises forward primers, reverse primers, blocker and probe of SPATS2L, NSUN and ZNF207, and forward primers, reverse primers and probe of internal reference genes.

Description

Primer composition for detecting methylation sites of lung adenocarcinoma and application of primer composition

Technical Field

The invention belongs to the field of gene joint detection.

Background

Lung cancer is one of the malignant tumors with the highest morbidity and mortality increase and the greatest threat to the health and life of people, and the five-year survival rate is less than 20%. Lung cancer can be divided simply into small cell lung cancer and non-small cell lung cancer according to the pathological type, with lung adenocarcinoma (Lung Adenocarcinoma, LUAD) being the most prevalent type of non-small cell lung cancer, accounting for approximately half of all malignant lung tumors. If lung cancer can be diagnosed at an early stage, particularly non-small cell lung cancer (NSCLC) at stage IA, its relative survival rate for 5 years can reach 92%. Therefore, the lung cancer disease risk is predicted in advance, and early screening is very likely to improve the survival rate of lung cancer patients and reduce the death rate.

Currently, diagnosis of lung cancer is mainly based on imaging methods such as Low Dose Computed Tomography (LDCT) to find lung tumor. The application of LDCT can improve the detection rate of lung nodules and reduce the death rate of lung cancer. However, it is difficult to distinguish between malignant and benign nodules using only LDCT. According to the data of the National Lung Screening Test (NLST) test, the false positive rate of LDCT reaches 96.4%, possibly resulting in an increase of unnecessary treatment.

Current standard clinical diagnostic methods for lung cancer include transbronchopulmonary biopsy, percutaneous pulmonary needle biopsy, bronchoalveolar lavage (BALF) and fluid biopsy (blood or sputum). Transbronchopulmonary biopsy and percutaneous pulmonary needle biopsy are invasive diagnostic techniques and have limitations in terms of tumor sampling that are not visible under bronchoscopy. Bronchoalveolar lavage (BAL) can overcome these problems. BALFs sampling is a routine procedure when suspected lung cancer patients undergo bronchoscopy. BAL has the advantages of large sampling amount and multiple times of one-time operation cleaning, improves the specificity and sensitivity of lung cancer detection, is a simple and low-invasive diagnosis technology, and is an ideal method for diagnosing lung nodules of high-risk lung cancer groups. Liquid biopsies, particularly blood-based tests, are popular in early cancer screening and diagnosis due to their minimal/non-invasive nature, high compliance and simplicity of surgery. However, the lack of tissue specificity and low sensitivity is a great challenge for blood detection. Sputum detection can diagnose squamous cell carcinoma because sputum is mainly expectorated from the central atmospheric channel, but may not be suitable for detection of adenocarcinoma that frequently occurs around the lungs.

Through tumor markers, the prediction of the lung cancer disease risk and early screening can be rapidly and conveniently realized. The lung cancer markers clinically applied at present mainly comprise the following types of (1) autoantibodies; (2) other proteins than autoantibodies; (3) a metabolite; (4) RNA; (5) a sequence-altered DNA; (6) DNA which is methylated to different extents. The number of the markers in each type is numerous, and the marker has a certain reference value for auxiliary diagnosis of lung cancer, but due to individual differences of the testees, the existing marker combination is relied on for prediction and screening, and the sensitivity and the specificity still lack. Therefore, finding new tumor markers for lung cancer is of great importance.

Disclosure of Invention

In order to overcome the above problems, the present inventors have studied and found that methylation genes SPATS2L, NSUN, ZNF207 can be detected in combination to accurately diagnose lung adenocarcinoma, thereby completing the present invention.

Specifically, the invention aims to provide a gene combination for diagnosing lung adenocarcinoma, which consists of a gene SPATS2L, a gene NSUN5 and a gene ZNF 207.

The invention aims to provide a kit for diagnosing lung adenocarcinoma, which is used for simultaneously detecting respective methylation levels of genes SPATS2L, NSUN5 and ZNF207 through fluorescence quantitative PCR reaction and comprises the following components:

forward primer, reverse primer, blocker and probe of gene SPATS 2L;

forward primer, reverse primer, blocker and probe of gene NSUN 5;

forward primer, reverse primer, blocker and probe of gene ZNF 207.

The invention has the beneficial effects that:

according to the kit provided by the invention, the methylation level of SPATS2L, the methylation level of NSUN5 and the methylation level of ZNF207 of a subject can be accurately and jointly analyzed in one PCR reaction, so that whether the subject suffers from lung adenocarcinoma or not can be accurately known.

Drawings

Fig. 1 shows the methylation level differences of SPATS2L, NSUN and ZNF207 genes in BALF fluid samples from benign lung disease patients and lung adenocarcinoma patients, with the horizontal axis showing sample groupings and the vertical axis showing methylation levels of the genes in the respective groupings.

FIGS. 2 and 3 illustrate the efficacy of a predictive model in a training set and a validation set, the predictive model being used to distinguish between benign and malignant tissue samples in the training set and the validation set, the diagnostic efficacy of the model being calculated; the horizontal axis shows false positive rate, and the vertical axis shows true positive rate; as shown in fig. 2, the AUC of the prediction model in the training set reaches 0.996, as shown in fig. 3, the AUC of the prediction model in the verification set is 0.967, which is equivalent to the diagnostic efficiency of the training set, and can accurately distinguish the benign lung disease from the BALF fluid sample of the lung adenocarcinoma patient.

Detailed Description

The invention is further described in detail below by means of the figures and examples. The features and advantages of the present invention will become more apparent from the description.

The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments. Although various aspects of the embodiments are illustrated in the accompanying drawings, the drawings are not necessarily drawn to scale unless specifically indicated.

DNA methylation is an epigenetic modification that is of great importance in human development and disease. Aberrant DNA methylation may be involved in the progression of cancer through a variety of mechanisms, such as inactivation of tumor suppressor genes. With the development of high-sensitivity DNA methylation detection technology, the abnormal methylation state of CpG islands has become an attractive biomarker for cancer diagnosis.

SPATS2L (Spermatogenesis-associated serine-rich 2-like) is an intranuclear stress response protein involved in chromosomal organization, ribosomal biogenesis and translational control.

NSUN5 is a member of the NOL1/NOP 2/solar domain protein family, a gene encoding cytosine-5 RNA methyltransferase, which promotes proliferation and progression of colorectal cancer by regulating the cell cycle.

ZNF207 is a member of the zinc finger protein family and plays a vital role in self-renewal, pluripotency and differentiation of human embryonic stem cells.

The research of the invention discovers that the joint detection of the methylation gene SPATS2L, NSUN5 and ZNF207 has important significance for diagnosing lung adenocarcinoma.

In specific studies, methylation levels of SPATS2L, NSUN and ZNF207 genes in BALF fluid sample DNA were measured and statistically analyzed in samples from benign lung disease patients and lung adenocarcinoma patients.

Regarding the subject:

230 subjects from 2017, month 1 to 2021, general hospitals of the free army were included in the study of the present invention and were randomly divided into a set up group and a validation group.

Wherein, the selection standard of the patient group is that the lung adenocarcinoma patient is initially diagnosed and untreated by clear pathology, and the operation, the chemo-radiation and the chemo-radiation are not carried out before blood collection and the chemo-radiation are not carried out before the operation.

The criteria for the selection of the benign disease patient group are patients suffering from benign lesions such as pneumonia, tuberculosis, bulla, bronchiectasis, pneumothorax and/or chronic obstructive pulmonary disease, or benign lung diseases such as at least one of benign tumors such as pulmonary cyst, pulmonary hamartoma, pulmonary sclerosing hemangioma and pulmonary tuberculosis tumor.

Study procedure:

1. methylation level determination of SPATS2L, NSUN and ZNF207 genes in BALF liquid

Taking 10ml of the subject alveolar lavage fluid, centrifuging at 10000rpm for 5min, removing supernatant, and precipitating for DNA extraction, wherein the specific extraction step is described in a blood/cell/tissue genome DNA extraction kit (centrifugal column type) produced by Tiangen Biochemical technology Co., ltd.

The extracted DNA is directly subjected to sulfite modification, and specific steps are described in the instructions of ZYMO RESEARCH biological company EZ DNA Methylation-DirectTM kit.

2. Real-time PCR detection

(2-1), primer probes, and blocking Agents

The primer probe and blocker sequences for SPATS2L, NSUN5, ZNF207 and ACTB are shown in table 1 below.

TABLE 1 primer probe genes in SPATS2L, NSUN5, ZNF207 and ACTB methylation fluorescent quantitative PCR reactions

(2-2) PCR detection

Each sample was subjected to 3-fold PCR reactions, the reaction system is shown in table 2 below. The total volume of each reaction system was 40ul, to which was added 19.25ul of PCR pre-reaction solution, 0.75 ul of EpiTaqHS polymerase and 20ul of the above sulfite-modified DNA.

Wherein the PCR pre-reaction solution comprises two parts, (1) raw materials necessary for providing a general PCR reaction, including 10xEpiTaqPCR Buffer,dNTP Mixture (dATP, dTP, dCTP, dGTP), mgCl ₂ Etc.; (2) primer probes and blocking agents selected from: forward primers, reverse primers, blockers, probes for amplifying methylated SPATS2L gene as shown in table 1; forward primers, reverse primers, blockers, probes for amplifying the methylated NSUN5 gene as shown in table 1; forward primers, reverse primers, blockers, probes for amplifying the methylated ZNF207 gene, as shown in table 1; alternatively, forward primers, reverse primers, probes for amplifying methylated ACTB gene are shown in table 1. Wherein the detailed ratios are shown in Table 2 below, 19.25ul of PCR pre-reaction solution was prepared at the final concentrations shown in Table 2.

In one PCR reaction, four PCR detection of SPATS2L gene, NSUN5 gene, ZNF207 gene and reference B-Actin (ACTB) gene (reference is used for evaluating whether DNA is sufficient in the BALF treatment process and whether the PCR reaction is effective or not, namely, the whole PCR reaction is judged to be ineffective if the reference is ineffective, and the methylation level of the SPATS2L gene, NSUN5 gene and ZNF207 gene can be evaluated if the reference is effective).

The PCR reaction system is shown in Table 2 below

TABLE 2 PCR reaction System

Wherein EpiTaqHS, mgCl, 10xEpiTaqPCR Buffer and dNTP mix are purchased from TaKaRa under the trade name R110A.

The reaction procedure: pre-denaturation at 95℃for 10min; cycling at 95 ℃ for 30s,57 ℃ for 35s,72 ℃ for 30s,35 cycles; extending at 72deg.C for 5min.

After the PCR reaction is finished, the analysis is carried out by using ABI7500SDS software V2.0.5 which is adapted by an ABI7500 fluorescence quantitative PCR instrument, the analysis can set the baseline to be 10-22, and the thresholds of the targets are ACTB threshold 25000, SPAT 2L threshold 2500, NSUN5 threshold 25000 and ZNF207 threshold 25000 in sequence, so that the detection Ct values of 4 genes of each sample can be obtained.

In fluorescent quantitative PCR reactions, the Ct value of each template is linearly related to the logarithm of the starting copy number of the template, the more the starting copy number, the smaller the Ct value. Theoretically, the initial copy number of an unknown sample can be calculated as long as the Ct value of the sample is obtained. Thus, when a sample is tested for the size of the SPATS2L Ct value, it reflects how much of the template amount of the methylated SPATS2L gene in the free DNA in the plasma, i.e., the level of methylation of the SPATS2L gene.

3. Statistical analysis of experimental data

Statistical analysis of data biomarker levels for patients with benign lung disease and lung adenocarcinoma were statistically analyzed using the Mann-Whitney U test or t test. In order to establish a predictive model for lung adenocarcinoma identification, variables were screened using a forward logistic regression method. The diagnostic performance of the single marker was compared to the diagnostic performance of the model using the area under the subject's operating characteristic curve (ROC) with 95% Confidence Interval (CI). In addition, the cut-off value of the predictive model is determined using the about-log index of the training set. The diagnostic performance of the model in the different subgroups was analyzed by comparing AUCs of ROC curves. Other descriptive statistics, such as sensitivity, specificity, positive Predictive Value (PPV), negative Predictive Value (NPV), standard Deviation (SD), were analyzed using software such as SPSS 24.0, graphPad Prism 5.0, medCalc (version 11.4.2.0), microsoft Excel, etc. The P-value is two-tailed. The difference is statistically significant, with p-values less than 0.05.

4. Results

In the study of the present invention, all the group-entering populations were randomly divided into training and validation groups. The model had 161 subjects (80 lung benign disease patients and 81 lung adenocarcinoma patients), and the remaining 35 lung benign disease patients and 34 lung adenocarcinoma patients were randomly selected as the validation cohort.

Group-entering populations were grouped according to tumor size and pathology type, and subjects were divided into two groups, "no smoking" and "smoking" according to smoking history. "non-smoking" refers to those who have never smoked, while the "smoking" group includes both current and previous smokers.

Depending on the nodule size, the maximum diameter is determined by LDCT or surgery. For analysis of solid components in nodules, if containing non-solid components, the nodules are transformed into ground glass opaque solid (GGO) nodules; in contrast, solid nodules have only a solid component. The basic information is shown in Table 3 below.

Table 3 subject information

Results as shown in fig. 1, the levels of individual biomarkers in patients with benign lung disease and patients with lung adenocarcinoma were evaluated in the modeling and validation groups, respectively. Wherein, the methylation level of SPATS2L gene, NSUN5 gene and ZNF207 gene of the established lung adenocarcinoma patients is significantly higher than that of the lung benign disease patients (p < 0.05). The case of the validation set is similar.

By simultaneously detecting the levels of three biomarkers, a prediction model is established according to the analysis result of the optimal LASSO Cox regression model, and the result shows that all three measured biomarkers are incorporated, and finally, a combined detection diagnosis prediction model is obtained as follows

Diagnostic model predictor = 0.45 x SPATS2L-0.147 x NSUN5-0.28 x ZNF207

Wherein SPATS2L represents the methylation level of SPATS2L gene in BALF of the person to be tested (the carried-in value is Ct value when methylation specific Real-time PCR reaction is carried out on the SPATS2L gene of the person to be tested);

NSUN5 represents the methylation level of NSUN5 gene in BALF of the tester (the carry-in value is Ct value when methylation specific Real-time PCR reaction is carried out on NSUN5 gene of the tester);

ZNF207 represents the methylation level of the gene in BALF of the subject (the carry-over value is Ct value when methylation-specific Real-time PCR reaction is performed on ZNF207 gene of the subject).

The ROC curve is plotted with the predicted values in the modeling set (i.e., training set) and the cut-off value of the diagnosis is determined to be 0.02 based on the about log index value. Namely, when the predicted value of the diagnostic model is less than or equal to 0.02, the possibility that the tested person is a lung adenocarcinoma patient is considered to be low; when the model predictive value is >0.02, the likelihood that the subject is a lung adenocarcinoma patient is considered to be high.

The BALF sample of benign lung disease patients and lung adenocarcinoma patients can be accurately distinguished in a training set based on the diagnosis models of the 3 genes, the sensitivity is 92%, the specificity is 97%, and the area under the curve (Area under the Curve of ROC) reaches 0.996; sensitivity was 94%, specificity was 96%, AUC was 0.967 in the validation set, and BALF samples from benign and malignant patients could be accurately distinguished, as shown in fig. 2 and 3. Sensitivity increases mainly with increasing phase status.

The invention has been described above in connection with preferred embodiments, which are, however, exemplary only and for illustrative purposes. On this basis, the invention can be subjected to various substitutions and improvements, and all fall within the protection scope of the invention.

Claims (5)

1. A kit for distinguishing lung adenocarcinoma from benign lung disease, which is a kit for the joint detection of methylation levels of genes SPATS2L, NSUN5 and ZNF207 by fluorescent quantitative PCR reactions, comprising:

forward primer, reverse primer, blocker and probe of gene SPATS 2L;

forward primer, reverse primer, blocker and probe of gene NSUN 5;

forward primer, reverse primer, blocker and probe of gene ZNF 207;

the forward primer, the reverse primer, the blocker and the probe of the gene SPATS2L are respectively shown in SEQ NO. 1-4;

the forward primer, the reverse primer, the blocker and the probe of the gene NSUN5 are respectively shown in SEQ NO. 5-8;

the forward primer, the reverse primer, the blocker and the probe of the gene ZNF207 are respectively shown as SEQ NO. 9-12;

the benign lung disease is at least one of pneumonia, tuberculosis, bulla, bronchiectasis, pneumothorax and/or chronic obstructive pulmonary disease, or lung cyst, pulmonary hamartoma, pulmonary sclerosing hemangioma, and pulmonary tuberculosis tumor benign tumor.

2. The kit of claim 1, further comprising:

forward primer, reverse primer and probe of reference gene.

3. The kit of claim 2, wherein the reference gene is ACTB.

4. The kit of claim 1, further comprising: 10xEpiTaqPCR Buffer dNTP mix, and MgCl ₂ 。

5. A kit according to claim 3, wherein:

the forward primer, reverse primer, blocker and probe of reference gene ACTB are shown in SEQ NO.13-15 respectively.