CN118360407A - SNP (Single nucleotide polymorphism) marker related to lung cancer, kit and application thereof - Google Patents
SNP (Single nucleotide polymorphism) marker related to lung cancer, kit and application thereof Download PDFInfo
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Abstract
The invention discloses a SNP marker related to lung cancer, a kit and application thereof. The SNP markers selected by the invention comprise rs2239611、rs12196996、rs3798577、rs3179969、rs938682、rs7178270、rs17486278、rs708274、rs28362459、rs3745453、rs10485505、rs7266300. SNP molecular markers which can be used for early screening and diagnosis of lung cancer. The invention provides a novel tool for early diagnosis, treatment and prognosis evaluation of lung cancer.
Description
Technical Field
The invention belongs to the technical field of biomedicine, in particular to the technical field of SNP molecular markers, and particularly relates to a lung cancer-related SNP marker, a kit and application thereof.
Background
Lung cancer is a malignancy of the tracheal, bronchial or alveolar epithelial cells originating in the lung, and is one of the most common types of cancer worldwide. As one of the most common cancer types worldwide, lung cancer can be classified into different subtypes of Small Cell Lung Cancer (SCLC), adenocarcinoma, squamous cell carcinoma, and the like, according to histological type. Lung cancer is the result of the combined actions of environmental-lifestyle-genetic factors, and is found by preventive and early screening.
Through methods such as gene detection, imaging examination and the like, lung cancer susceptibility can be screened, early diagnosis and treatment are realized, and thus survival rate of patients is improved. There are no reports of Single Nucleotide Polymorphisms (SNPs) for lung cancer diagnosis. If SNP related to lung cancer susceptibility can be screened out as a biomarker, the method can help promote the development of early diagnosis of lung cancer. The method not only can effectively improve the accuracy and efficiency of early diagnosis of lung cancer, but also is expected to provide a new way and possibility for screening, evaluating the efficacy and targeted treatment of lung cancer treatment medicines.
Disclosure of Invention
In view of the shortcomings of the prior art, the inventors have found SNPs associated with breast cancer by studying single nucleotide polymorphisms in DNA of lung cancer patients, and developed corresponding diagnostic kits. Based on the current research results, the invention provides a SNP marker related to lung cancer, a kit and application thereof.
The technical scheme of the invention is as follows:
it is an object of the present invention to provide an SNP marker associated with lung cancer, comprising at least one of the following 12 SNP markers; the rs number of the SNP marker and the mutation type thereof are as follows:
Preferably, the SNP markers are rs12196996, rs3798577, rs28362459 and rs3745453.
It is another object of the present invention to provide a primer set for amplifying the SNP marker,
The nucleotide sequences of the primers for amplifying rs2239611 are shown as SEQ ID NO.1 and SEQ ID NO. 2;
The nucleotide sequences of the primers for amplifying rs12196996 are shown as SEQ ID NO.7 and SEQ ID NO. 8;
The nucleotide sequences of the primers for amplifying rs3798577 are shown as SEQ ID NO.9 and SEQ ID NO. 10;
The nucleotide sequences of the primers for amplifying rs3179969 are shown as SEQ ID NO.13 and SEQ ID NO. 14;
The nucleotide sequences of the primers for amplifying rs938682 are shown as SEQ ID NO.15 and SEQ ID NO. 16;
The nucleotide sequences of the primers for amplifying rs7178270 are shown as SEQ ID NO.17 and SEQ ID NO. 18;
the nucleotide sequences of the primers for amplifying rs17486278 are shown as SEQ ID NO.19 and SEQ ID NO. 20;
the nucleotide sequences of the primers for amplifying rs708274 are shown as SEQ ID NO.21 and SEQ ID NO. 22;
The nucleotide sequences of the primers for amplifying rs28362459 are shown as SEQ ID NO.23 and SEQ ID NO. 24;
the nucleotide sequences of the primers for amplifying rs3745453 are shown as SEQ ID NO.25 and SEQ ID NO. 26;
The nucleotide sequences of the primers for amplifying rs10485505 are shown as SEQ ID NO.27 and SEQ ID NO. 28;
The nucleotide sequences of the primers used for amplifying rs7266300 are shown as SEQ ID NO.29 and SEQ ID NO. 30.
The invention also aims to provide an application of the SNP marker or the primer pair thereof in preparing lung cancer detection products.
The fourth object of the present invention is to provide a lung cancer detection kit, which comprises at least one of the primer pairs, and can also comprise reagents commonly used in PCR technology, such as dNTPs, mgCl 2, double distilled water and Taq enzyme.
The invention has the beneficial effects that:
The invention screens out SNP markers related to lung cancer, and the related SNP molecular markers can be used for early screening and diagnosis of lung cancer. The invention provides a novel tool for early diagnosis, treatment and prognosis evaluation of lung cancer.
Detailed Description
In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.
Experimental example association study of genetic polymorphism and lung cancer genetic susceptibility
1. Materials and methods
(1) Study object
890 Subjects were enrolled in the Hainan general hospital from 1 month 2022 to 10 months 2023. The study subjects included 445 non-small cell lung cancer patients in the same phase and 445 non-cancer controls. Trend score matching (PSM) was used for a 1:1 match by gender and age between the patient group and the cancer-free group. The study was conducted according to the principles of helsinki and was approved by the ethical committee of the university of medical science, hainan (HYLL-2019-034). All participants in the study signed informed consent. Subjects with limited physical activity, difficulty in speech expression, severe psychological disorders, and mental illness resulting in unconsciousness were excluded from the study. The participants responded to a questionnaire containing questions about age, gender, exposure factors (e.g., smoking), etc. Smoking is defined as smoking at least one cigarette per day for at least six consecutive months at any time during the lifetime of a person; once smoked is defined as smoking at least one cigarette per day for at least six consecutive months at any time during the lifetime of a person, and no smoking at the time of investigation. Alcohol consumption is defined as at least one drink per week at least six consecutive months at any time during the lifetime of a person. 2ml of peripheral blood was collected from each study subject using a vacuum blood collection tube containing ethylenediamine tetraacetic acid (EDTA) and stored in a refrigerator at 4 ℃.
(2) DNA extraction and SNP genotyping
Venous blood DNA and RNA samples were extracted by Tris phenol-chloroform method. The OD 260/OD280 ratio of DNA concentration (between 1.8 and 2.0) was measured by the biophotometer-Plus nucleic acid protein assay. SNP genotyping was performed using the Massarray system.
(3) Statistical method
Quality control was performed using PLINK v1.9 (detection rate >90%, MAF >5%, HWE > 0.000001). The missing genotypes were filled in using the snp_ fastImpute function in the bigsnpr package. The condition logistic regression analysis before and after adjustment of potential confounding factors (such as gender, age, smoking and alcohol) was performed using R (4.2.1) to obtain P-value, OR-value, 95% CIs. Trend score matching (PSM) was applied to a 1:1 match between the medical record group and the cancer-free control group. The significance level for all statistical analyses was set to P <0.05.
2. Results
(1) Selected SNP information
50 Loci are screened in the early stage of the experiment, 40 SNP loci are studied after quality control, and 15 SNP loci are further screened. The following are 15 pieces of current SNP locus information and analysis results.
TABLE 1 data quality control results in 15 SNP loci
TABLE 2 amplification primers for 15 SNP loci
(2) Feature comparison of study objects before and after matching
The study used 1:1 tendency score match, caliper value set to 0.05, successfully match 445 pairs. Before matching, the total number of cases and the distribution situation of men and women in the control group are different, the total number of cases after matching is the same, and the number of men is 296 cases and 149 cases of women. The ages of the case group and the control group after matching are 61.51 +/-10.31 and 58.11+/-10.19 respectively, and the case group and the control group are balanced and comparable. Other baseline data are shown in table 3.
TABLE 3 comparison of subject characteristics before and after matching of the tendency scores
(2) Genotype distribution frequency of 15 target SNPs loci and correlation analysis result of genotype distribution frequency and lung cancer susceptibility
And analyzing the genotype distribution frequency of 15 target SNPs loci and the relation between the genotype distribution frequency and the lung cancer risk. The results show that the risk of lung cancer for the mutant (AA) carrier of rs2239611 gene is higher than that of wild type (GG) (coarse or=1.99, 95% ci=1.13-3.52, p=0.017), and that the risk of lung cancer for the mutant (AA) carrier of this site is higher than that of heterozygote (GA) and wild type (GG) in the recessive genetic model (coarse or= 0.32,95% ci=01.12-3.39, p=0.018), and that the risk of lung cancer for the a allele carrier of this site is higher than that of the G allele carrier in the allele model (coarse or= 1.28,95% ci=1.01-1.63, p=0.045); The heterozygous (GA) and mutant (AA) carriers of the rs3179969 gene are at higher risk of lung cancer than wild type (GG) (crude or=1.53, 95% ci=1.14-2.06, p=0.005), in the dominant genetic model, the heterozygous (GA) and mutant (AA) carriers of the locus are at higher risk of lung cancer than wild type (GG) (crude or= 1.48,95% ci=1.12-1.95, p=0.007), in the allele model, the a-allele carrier of the locus is at higher risk of lung cancer than the G-allele carrier (crude or= 1.32,95% ci=1.05-1.67, p=0.019); the risk of lung cancer for the mutant (GG) carrier of rs938682 gene is lower than that of wild type (AA) coarse or=0.68, 95% ci=0.46-0.99, p=0.045), in recessive genetic model, the risk of lung cancer for the mutant (GG) carrier of this site is lower than that of heterozygote (AG) and wild type (AA) (coarse or= 0.67,95% ci=0.48-0.94, p=0.020), in allele model, the risk of lung cancer for the G allele carrier of this site is lower than that of a allele carrier (coarse or= 0.80,95% ci=0.64-0.99, p=0.039); The risk of lung cancer for the mutant (GG) carrier of rs7178270 gene is higher than that of wild type (CC) (coarse OR= 1.69,95% CI=1.15-2.51, P=0.008), the risk of lung cancer for the heterozygous (CG) and mutant (GG) carrier of the locus is higher than that of wild type (CC) (coarse OR= 1.32,95% CI=1.01-1.71, P=0.040) in the dominant genetic model, the risk of lung cancer for the mutant (GG) carrier of the locus is higher than that of heterozygous (CG) and wild type (CC) (coarse OR= 1.55,95% CI=1.08-02.24 P=0.019) in the recessive genetic model, in the allele model, the G allele carrier at this site is at higher risk of lung cancer than the C allele carrier (coarse or= 1.39,95% ci=1.13-1.72, p=0.002); in the allele model at position rs17486278, the risk of lung cancer for the C allele carrier is higher than for the a allele carrier (coarse or= 1.26,95% ci=1.01-1.58, p=0.038); The risk of lung cancer for heterozygous (GT) carriers of rs708274 gene is lower than that of wild type (GG) (coarse or=0.02, 95% ci=0.01-0.06, p < 0.001), in dominant genetic model, the risk of lung cancer for heterozygous (GT) and mutant (TT) carriers of this site is lower than that of wild type (GG) (coarse or= 0.10,95% ci=0.06-0.16, p < 0.001), in allele model, T allele carrier has lower lung cancer risk than G allele carrier (coarse or= 0.23,95% ci=0.17-0.32, p < 0.001); The risk of lung cancer for the mutant (TT) carrier of rs10485505 gene is higher than that of wild type (CC) (coarse or=2.48, 95% ci=1.12-5.47, p=0.025), in the recessive genetic model, the risk of lung cancer for the mutant (TT) carrier of the locus is higher than that of heterozygote (CT) and wild type (CC) (coarse or= 2.33,95% ci=1.07-5.10, p=0.033), in the allele model, the risk of lung cancer for the T allele carrier of the locus is higher than that of the a allele carrier (coarse or= 1.32,95% ci=1.02-1.71, p=0.036); The mutant (TT) carrier of the rs7266300 gene is at higher risk for lung cancer than the wild type (AA) (crude or=2.35, 95% ci=1.02-5.45, p=0.046), however this relationship does not exist after correction of smoking and drinking factors.
The risk of lung cancer of a mutant (GG) carrier of rs12196996 gene is lower than that of a wild type (AA) (correction OR=0.00, 95% CI=0.00-0.00, P < 0.001), and the risk of lung cancer of the mutant (GG) carrier of the locus is lower than that of heterozygote (AG) and wild type (AA) (correction OR= 0.00,95% CI=0.00-0.00, P < 0.001) in a recessive genetic model; the risk of lung cancer in heterozygous (TC) and mutant (CC) carriers of rs3798577 gene is higher than that of wild type (TT) (corrected OR＝6.00×1021,1.20×1022,95％ CI＝1.10×1021～3.28×1022,1.09×1021～1.32×1023,P＜0.001,＜0.001), in dominant genetic model, heterozygous (TC) and mutant (CC) carriers of the locus are higher than that of wild type (TT) (corrected or=5.17×10 22,95％ CI＝4.70×1020～5.70×1022, P < 0.001), in rs28362459 allele model, C allele carrier of the locus has lung cancer risk lower than that of a allele carrier (corrected or= 0.07,95% ci=0.01-0.74, p=0.027) the mutant (GG) carrier of rs3745453 gene has lung cancer risk higher than that of wild type (AA) (coarse or=2.34, 95% ci=1.16-4.71, p=0.017, corrected or=2.96×10 18,95％ CI＝1.85×1017～4.73×1019, P < 0.001), in recessive genetic model, mutant (GG) carrier of the locus has lung cancer risk lower than that of heterozygous (AA) and wild type (AA) (coarse or=2.3% ci=0.01-0.74, p=0.027) the rest of rs3745453 gene is found to be significantly lower than that of wild type (AA) (coarse or=2.34, 95% ci=1.16-4.71, p=0.017), p=2.96×10.35, P < 0.001).
TABLE 3 distribution frequency of genotypes at 15 target SNPs loci and correlation analysis results of susceptibility to lung cancer
Note that: a. and (5) adopting a conditional logistic regression model to analyze and calibrate smoking, drinking, sex and age factors.
Example 1 SNP kit for lung cancer susceptibility diagnosis
Primers for detecting lung cancer-related SNPs are shown in table 2. Based on the primer pair, a kit for lung cancer susceptibility detection can be assembled. The kit comprises the primer and can also comprise conventional reagents required for PCR, such as dNTPs, mgCl 2, double distilled water, taq enzyme and the like. Specific concentrations and amounts of the components are shown below with reference to example 2.
The kit is based on the PCR technology, has a simple and accurate use method, can be used for rapidly determining the susceptibility of lung cancer, and helps to guide lung cancer diagnosis and early screening.
Example 2A method of detecting susceptibility to lung cancer
1) Extracting DNA of a sample to be detected;
2) Mixing 1 μl template DNA (20 ng/. Mu.l) with PCR reaction solution by using the sample DNA to be detected as a template, and performing PCR amplification reaction by using the primers shown in Table 2 to obtain amplified products;
3) After the PCR reaction is finished, the PCR product is treated with SAP (SHRIMP ALKALINE phosphotase, shrimp alkaline phosphatase); SAP reaction procedure: 20min at 37 ℃;85 ℃ for 5min;4 ℃ is infinity.
4) After the alkaline phosphatase treatment is finished, single-base extension reaction is carried out to obtain an extension product;
5) The final extension products were analyzed by a time of flight mass spectrometry system (TOF) and SNPs were typed according to the molecular weight differences of the different bases. And judging the risk of lung cancer according to the parting result and combining the research result.
TABLE 4 PCR reaction solution
Table 5 PCR reaction procedure
The above embodiments are only some of the embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention fall within the protection scope of the present invention.
Claims (6)
1. A SNP marker associated with lung cancer, characterized by comprising at least one of the following 12 SNP markers; the rs number of the SNP marker and the mutation type thereof are as follows:
2. The SNP marker associated with lung cancer according to claim 1, wherein the SNP markers are rs12196996, rs3798577, rs28362459 and rs3745453.
3. A primer set for amplifying the SNP marker according to claim 1, wherein,
The nucleotide sequences of the primers for amplifying rs2239611 are shown as SEQ ID NO.1 and SEQ ID NO. 2;
The nucleotide sequences of the primers for amplifying rs12196996 are shown as SEQ ID NO.7 and SEQ ID NO. 8;
The nucleotide sequences of the primers for amplifying rs3798577 are shown as SEQ ID NO.9 and SEQ ID NO. 10;
The nucleotide sequences of the primers for amplifying rs3179969 are shown as SEQ ID NO.13 and SEQ ID NO. 14;
The nucleotide sequences of the primers for amplifying rs938682 are shown as SEQ ID NO.15 and SEQ ID NO. 16;
The nucleotide sequences of the primers for amplifying rs7178270 are shown as SEQ ID NO.17 and SEQ ID NO. 18;
the nucleotide sequences of the primers for amplifying rs17486278 are shown as SEQ ID NO.19 and SEQ ID NO. 20;
the nucleotide sequences of the primers for amplifying rs708274 are shown as SEQ ID NO.21 and SEQ ID NO. 22;
The nucleotide sequences of the primers for amplifying rs28362459 are shown as SEQ ID NO.23 and SEQ ID NO. 24;
the nucleotide sequences of the primers for amplifying rs3745453 are shown as SEQ ID NO.25 and SEQ ID NO. 26;
The nucleotide sequences of the primers for amplifying rs10485505 are shown as SEQ ID NO.27 and SEQ ID NO. 28;
The nucleotide sequences of the primers used for amplifying rs7266300 are shown as SEQ ID NO.29 and SEQ ID NO. 30.
4. Use of the SNP marker of claim 1 or claim 2 in the preparation of a lung cancer detection product.
5. Use of the primer set of claim 3 in the preparation of a lung cancer detection product.
6. A lung cancer detection kit comprising at least one of the primer pairs of claim 3.
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