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CN118957064A - A methylation gene related to lung cancer and a detection kit thereof - Google Patents

A methylation gene related to lung cancer and a detection kit thereof Download PDF

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CN118957064A
CN118957064A CN202411004926.8A CN202411004926A CN118957064A CN 118957064 A CN118957064 A CN 118957064A CN 202411004926 A CN202411004926 A CN 202411004926A CN 118957064 A CN118957064 A CN 118957064A
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methylation
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lung cancer
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李丹
江南
刘代新
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Suzhou Huhu Health Technology Co ltd
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Abstract

本发明涉及一种与肺癌相关的甲基化基因及其检测试剂盒，具体涉及一种与肺癌相关的甲基化基因及其检测试剂盒，其中基因包括HOXA7、HOXA9、SCT以及SHOX2。检测试剂盒包括：检测HOXA7基因的试剂，含编号1‑1、1‑2序列所示的引物及编号1‑3序列所示的探针；检测HOXA9基因的试剂，含编号2‑1、2‑2序列所示的引物及编号2‑3序列所示的探针；检测SCT的试剂，含编号3‑1、3‑2序列所示的引物及编号3‑3序列所示的探针；以及含编号3‑4、3‑5序列所示的引物及编号3‑6序列所示的探针；检测SHOX2基因的试剂，含编号4‑1、4‑2序列所示的引物及编号4‑3序列所示的探针。The present invention relates to a methylation gene related to lung cancer and a detection kit thereof, and in particular to a methylation gene related to lung cancer and a detection kit thereof, wherein the genes include HOXA7, HOXA9, SCT and SHOX2. The detection kit includes: a reagent for detecting the HOXA7 gene, comprising primers shown in sequences numbered 1-1 and 1-2 and a probe shown in sequences numbered 1-3; a reagent for detecting the HOXA9 gene, comprising primers shown in sequences numbered 2-1 and 2-2 and a probe shown in sequences numbered 2-3; a reagent for detecting SCT, comprising primers shown in sequences numbered 3-1 and 3-2 and a probe shown in sequences numbered 3-3; and a reagent comprising primers shown in sequences numbered 3-4 and 3-5 and a probe shown in sequences numbered 3-6; a reagent for detecting the SHOX2 gene, comprising primers shown in sequences numbered 4-1 and 4-2 and a probe shown in sequences numbered 4-3.

Description

Methylation gene related to lung cancer and detection kit thereof
The application of the invention is a divisional application of an invention patent application based on the application date of 2019, 7, 30, 201910697653.2 and the name of a methylation gene related to lung cancer and a detection kit thereof.
Technical Field
The invention relates to a reagent for detecting genes, in particular to a methylation gene related to lung cancer and a detection kit thereof.
Background
Lung nodules are small focal, round-like shadows with increased imaging density, can be single or multiple, and do not accompany atelectasis, enlargement of the lung door and pleural effusion. The etiology of forming pulmonary nodules is numerous and is largely classified into benign nodules, which are benign tumors, tuberculosis, sarcoidosis, inflammatory nodules, etc., and malignant nodules. Malignant nodules include lung cancer, metastatic tumor and the like, and the occurrence of the lung cancer is subjected to a series of stages of hyperplasia-atypical hyperplasia-in-situ cancer-invasive cancer and the like, and the rule is that the malignant nodules initially develop slowly, and the speed of the malignant nodules is rapidly increased when the malignant nodules develop to a certain stage.
Lung cancer in malignant lung nodules is one of the most common malignant tumors in China, according to the latest cancer data of China issued by the national cancer center in 2 months of 2017, the incidence rate and the death rate of lung cancer are first (the death rate of lung cancer in large, medium and small cities is 40% -54%), the incidence rate and the death rate of lung cancer are about twice as high as those of other cancer in the second cancer, and the cancer is the first cancer in 2017.
For lung cancer, early diagnosis and early treatment are the most effective methods for improving prognosis, and if early treatment is carried out, early lung cancer patients can expect higher survival rate of 5 years, and the overall survival rate of 5 years of the lung cancer of stage I can reach 80 percent. However, the clinical outcome of patients with progressive lung cancer is much worse, even those with stage IV lung cancer, with an overall 5-year survival rate of only about 10%. Therefore, the differential diagnosis of the pulmonary nodules is effectively carried out, the benign and malignant nodules are quickly clarified, the malignant nodules are excised as early as possible, and meanwhile, unnecessary over-treatment is avoided, so that the differential diagnosis and treatment of the pulmonary nodules are key.
Recent advances in epigenetic research of lung cancer have profound implications for early diagnosis and treatment of lung cancer. There is evidence that the hypermethylation of the cancer suppressor gene promoter CpG island is an important mechanism of gene inactivation, and the transcriptional inactivation of different genes affects cell cycle, DNA repair, apoptosis and the like, thus being closely related to the occurrence and development of cancers. Gene methylation refers to the process of selectively adding methyl groups to cytosine (C) in CpG dinucleotides on a DNA molecule under the action of an enzyme to form 5' -methylcytosine. CpG island methylation of the promoter region of genes is a more common phenomenon in lung cancer patients. Clinically, the detection of methylation gene markers on tissue cells and body fluids (such as sputum, serum, plasma and the like) of a lung cancer patient is considered to be an effective auxiliary detection method for lung cancer, and has the value of improving the lung cancer detection rate. Studies have shown that DNA methylation can be detected from patient sputum 3 years prior to lung cancer diagnosis. If the method is combined with the detection of cytopathology, the efficiency of lung cancer diagnosis can be greatly improved, the detection period can be shortened, and the accuracy of diagnosis can be improved.
Methylation gene markers are of great significance in the identification of benign and malignant lung nodules and the diagnosis of lung cancer. The methylation gene markers related to lung cancer, which are reported at present, have the defects of low sensitivity (35.5%) and low specificity (73%), so that the application of the methylation gene markers to clinical detection has a certain limit. Here, a brief introduction of related knowledge is needed: the methylation gene marker has high sensitivity to one or some cancers, which means that the missed diagnosis rate of the corresponding cancers is low; similarly, high specificity indicates a low misdiagnosis rate of the corresponding cancer. Therefore, the sensitivity and specificity of the detection of the methylation gene marker are two important indexes for measuring the reference value of the methylation gene marker. In summary, further research on methylation gene markers is conducted, and detection reagents of methylation genes and combinations thereof related to lung cancer with high sensitivity and high specificity are found, so that a more effective means is provided for identifying benign and malignant lung nodules or diagnosing lung cancer accurately and providing a good prognosis, and the problems to be solved in the current methylation gene marker detection technology are urgently needed.
Disclosure of Invention
The invention aims to solve the technical problem of providing a methylation gene related to lung cancer and a detection kit thereof, which can provide valuable reference information for benign and malignant lung nodule identification or lung cancer diagnosis through high sensitivity and high specificity.
In order to solve the technical problems, the invention provides a methylation gene related to lung cancer, which comprises one or more of HOXA7, HOXA9, SCT and SHOX2 genes, and the nucleotide sequence of the methylation gene is shown as sequence 1, sequence 2, sequence 3 and sequence 4 in a sequence table; wherein, the methylated cytosine sites of the genes HOXA7, HOXA9, SCT and SHOX2 are sequentially defined in sequence 1, sequence 2, sequence 3 and sequence 4.
In order to solve the technical problems, the invention provides a detection kit of the genes, which is used for detecting methylated genes in a sample to be detected and comprises one or more of the following reagents:
a reagent for detecting HOXA7 gene, comprising primers shown in the sequences of No. 1-1 and No. 1-2 and a probe shown in the sequence of No. 1-3;
a reagent for detecting HOXA9 gene, comprising primers shown in the sequences of numbers 2-1 and 2-2 and a probe shown in the sequence of numbers 2-3;
A reagent for detecting SCT gene, comprising primers shown in sequence numbers 3-1 and 3-2 and a probe shown in sequence number 3-3; comprises primers shown in the sequences of 3-4 and 3-5 and probes shown in the sequences of 3-6;
a reagent for detecting SHOX2 gene comprises primers shown in sequence numbers 4-1 and 4-2 and a probe shown in sequence number 4-3.
Preferably, the detection kit further comprises ACTB primers and probes for the detection of reference genes.
Preferably, the sample to be tested comprises one or more of human blood, alveolar lavage fluid, sputum and pulmonary tissue samples, and further comprises a bronchial brush sample.
Preferably, the primer sets corresponding to HOXA7, HOXA9, SCT and SHOX2 genes are respectively composed of primers and probes contained in respective reagents, and the concentrations of the respective components in the PCR reaction solution are as follows: 0.2 mu M of gene corresponding primer set, 0.6 mu MACTB primer and probe, 3.5mM MgCl 2, 0.25mM dNTP; in addition, DNA polymerase was contained in an amount of 100U, DNA extracted after the count of non-small cell HCC827 cell line for positive control, and sterilized water for negative control.
Preferably, any two of the reagents of the detection kit are combined, or any three of the reagents are combined, or four reagents are combined, respectively, to constitute a combined kit.
Preferably, the primer sets corresponding to HOXA7, HOXA9, SCT and SHOX2 genes are respectively composed of primers and probes contained in respective reagents, and the concentrations of the respective components in the PCR reaction solution are as follows: the primer sets corresponding to the genes in the combination are respectively set to be 0.2 mu M,0.6 mu MACTB primer and probe, 3.5mM MgCl 2 and 0.25mM dNTP; in addition, DNA polymerase was contained in an amount of 100U, DNA extracted after the count of non-small cell HCC827 cell line for positive control, and sterilized water for negative control.
In order to solve the above technical problems, the present invention provides a method for obtaining the gene as described above, comprising:
carrying out genome-wide methylation array analysis on a sample to be detected, and screening 159 candidate genes related to benign and malignant lung nodule identification or lung cancer;
Selecting target genes HOXA7, HOXA9, SCT and SHOX2 from candidate genes, designing corresponding primers and probes respectively aiming at methylated DNA specific fragments of the target genes, wherein the designed primers and probes are respectively equivalent to, complementary to or hybridized with at least 15 continuous nucleotides or complementary sequences thereof in sequence 1-4 of a sequence table, and acquiring and verifying performance indexes of the target genes meeting detection requirements.
Preferably, the performance indicators of the target genes mentioned in the method include: sensitivity, specificity, and area under receiver operating characteristics AUC values.
Preferably, the sample to be tested in the method comprises one or more of human blood, alveolar lavage fluid, sputum, pulmonary tissue and bronchial brush samples.
Because the reagent in the kit comprises one or more corresponding primer groups of HOXA7, HOXA9, SCT and SHOX2 methylation gene markers, the reagent kit can provide valuable reference information for identifying benign and malignant lung nodules and diagnosing lung cancer and for good prognosis by carrying out genome-wide methylation array analysis on detection samples and screening out methylation gene markers with special sites from 159 candidate genes, wherein the methylation gene markers have high sensitivity and specificity which are not possessed by the existing methylation gene detection, and the sensitivity and specificity of the methylation gene markers can be further improved by carrying out different combinations on the four methylation markers.
In particular, the invention relates to the following:
1. A methylation gene related to lung cancer comprises one or more of genes HOXA7, HOXA9, SCT and SHOX2, and the nucleotide sequence of the methylation gene is shown as sequence 1, sequence 2, sequence 3 and sequence 4 in a sequence table; the method is characterized in that the methylation cytosine loci of the genes HOXA7, HOXA9, SCT and SHOX2 are sequentially marked in the sequence 1, the sequence 2, the sequence 3 and the sequence 4.
2. A kit for detecting a methylated gene in a sample according to claim 1, comprising one or more of the following reagents:
a reagent for detecting HOXA7 gene, comprising primers shown in the sequences of No. 1-1 and No. 1-2 and a probe shown in the sequence of No. 1-3;
a reagent for detecting HOXA9 gene, comprising primers shown in the sequences of numbers 2-1 and 2-2 and a probe shown in the sequence of numbers 2-3;
A reagent for detecting SCT gene, comprising primers shown in sequence numbers 3-1 and 3-2 and a probe shown in sequence number 3-3; comprises primers shown in the sequences of 3-4 and 3-5 and probes shown in the sequences of 3-6;
a reagent for detecting SHOX2 gene comprises primers shown in sequence numbers 4-1 and 4-2 and a probe shown in sequence number 4-3.
3. The test kit according to item 2, further comprising an ACTB primer and a probe for detecting a reference gene.
4. The test kit according to item 3, wherein the sample to be tested comprises one or more of human blood, alveolar lavage fluid, sputum and pulmonary tissue sample, and wherein the sample to be tested further comprises a bronchial brush sample.
5. The detection kit according to item 3 or 4, wherein the primer set for HOXA7, HOXA9, SCT and SHOX2 gene consists of the primer and probe contained in the respective reagents, respectively, and the concentration of each component in the PCR reaction solution is as follows: 0.2 mu M of gene corresponding primer set, 0.6 mu MACTB primer and probe, 3.5mM MgCl 2, 0.25mM dNTP; in addition, DNA polymerase was contained in an amount of 100U, DNA extracted after the count of non-small cell HCC827 cell line for positive control, and sterilized water for negative control.
6. The kit according to any one of items 2,3 and 4, wherein any two of the reagents are combined, or any three of the reagents are combined, or four of the reagents are combined, to constitute a combined kit, respectively.
7. The kit according to item 6, wherein the primer set for each of the HOXA7, HOXA9, SCT and SHOX2 genes comprises a primer and a probe contained in each of the detection reagents, and the concentrations of the components in the PCR reaction solution are as follows: the primer sets corresponding to the genes in the combination are respectively set to be 0.2 mu M,0.6 mu MACTB primer and probe, 3.5mM MgCl 2 and 0.25mM dNTP; in addition, DNA polymerase was contained in an amount of 100U, DNA extracted after the count of non-small cell HCC827 cell line for positive control, and sterilized water for negative control.
8. A method of obtaining the gene of claim 1, comprising:
carrying out genome-wide methylation array analysis on a sample to be detected, and screening 159 candidate genes related to benign and malignant lung nodule identification or lung cancer;
Selecting target genes HOXA7, HOXA9, SCT and SHOX2 from the candidate genes, respectively designing corresponding primers and probes for the methylation DNA specific fragments of the target genes, wherein the primers and the probes are respectively equivalent to, complementary to or hybridized with at least 15 continuous nucleotides or complementary sequences thereof in sequence 1-4 of a sequence table, and acquiring and verifying performance indexes of the target genes meeting detection requirements.
9. The method according to item 8, wherein the performance index of the target gene comprises: sensitivity, specificity, and area under receiver operating characteristics AUC values.
10. The method according to any one of claims 8 or 9, wherein the sample to be tested comprises one or more of human blood, alveolar lavage fluid, sputum, pulmonary tissue, and bronchial brush sample.
Drawings
FIG. 1 shows the ROC curve for detecting the methylation gene HOXA7 provided by the invention;
FIG. 2 shows the ROC curve for detecting the methylation gene HOXA9 provided by the invention;
FIG. 3 shows the ROC curve for detecting the methylation gene SCT provided by the invention;
FIG. 4 shows the ROC curve for detecting methylated gene SHOX2 provided by the invention.
Detailed Description
The technical scheme of the present invention will be described in detail with reference to the preferred embodiments. It should be noted that the following examples are given for the purpose of illustration and explanation of the present invention only and are not to be construed as limiting the invention in any way.
The invention carries out methylation array analysis on samples to be tested of patients with lung cancer and benign lung cancer, screens 159 candidate genes related to benign and malignant lung nodule identification or lung cancer, screens four target genes related to benign and malignant lung nodule identification or lung cancer from the candidate genes, and comprises HOXA7, HOXA9, SCT and SHOX2, wherein:
The HOXA7 gene (homeobox A7 gene), which is a member of the HOX gene family, is located on chromosome 7, 7P15-P14, contains 2961 pairs of bases and encodes 230 amino acids. The HOXA7 gene can be excessively high or excessively low expressed in tumors in a gene fusion, gene silencing and other modes, and can regulate the occurrence and development of tumor cells; involved in the occurrence of tumors, especially in the early and late stages of tumor development, and signal molecules (such as MEIS, protein kinase-C, etc.) involved in regulating biological differentiation and proliferation; interaction with a targeted gene (e.g., CAVE-OLIN-1, etc.) triggers gene silencing and inactivation.
The HOXA9 gene (homeobox A9 gene), a member of the HOX gene family, is located on chromosome 7, 7P15-P14, and contains 3 exons, encoding two different proteins. The HOXA9 gene as transcription regulator can act on downstream target gene to perform transcription activation or inhibition, and can interact with upstream regulation protein to regulate biological behavior of tumor. Studies prove that HOXA9 has abnormal expression in various tumors, can be used as a marker for diagnosis of various tumors, and is closely related to occurrence, invasion and metastasis and prognosis of the tumors.
The SCT gene (secretin gene), located on chromosome 11p15.5, encodes an endocrine hormone of 27 amino acids. The SCT gene expression product can prevent cell from reentering cycle division, inhibit cell infiltration, transfer and promote cell apoptosis. The SCT gene is highly methylated in lung cancer tissue and can be a potential biomarker for lung cancer and other types of solid tumors.
The SHOX2 gene (short homeobox gene) is a member of the homeobox gene family, and its gene expression regulation is closely related to organ development. It was found that the SHOX2 gene is expressed in mesoderm and ectoderm during embryonic stage and plays an important role in the development of bone, heart and nervous system. In addition, the methylation of the SHOX2 gene is related to tumors such as lung cancer, and the methylation level of the SHOX2 gene is used as a common transcription regulator to influence the tumorigenesis process.
The detection of the methylation level of the gene comprises the following steps: extracting DNA from a sample to be detected (one or more of human blood, alveolar lavage fluid, alveolar rinse fluid, sputum, lung tissue and bronchial brush sample), treating the sample with bisulfite, and performing fluorescent quantitative PCR reaction by using methylation specific primers and probes; wherein the bisulfite treatment causes deamination of the unmethylated cytosine residue C of the DNA duplex to uracil; while methylated cytosine residue C remains unchanged; thus, in the PCR reaction, the methylated cytosine residue C site on the template is paired as cytosine residue C with guanine residue G in the primer, while unmethylated cytosine residue C is paired as uracil residue U with adenine residue A in the primer.
In the process of selecting the sample type commonly used for clinical detection as the detection sample for diagnosis of benign and malignant lung nodules and lung cancer, the methylation gene detection is unexpectedly found by using the bronchus brushing detection sample, the sensitivity and the specificity of the lung cancer diagnosis reach 77% -95%, and compared with the prior clinical detection of cytological smear by using only the bronchus brushing detection sample, the sensitivity and the specificity of the lung cancer diagnosis are less than 35%, and the method has remarkable improvement. In view of this, the present invention particularly increases the types of test samples for diagnosis of benign and malignant lung nodules and lung cancer by using a bronchoscopy sample as a test sample in addition to the types of samples commonly used for clinical tests.
The invention designs a specific primer pair correspondingly aiming at each of the four genes to detect the methylation level of a target region in each gene, wherein the target region is respectively selected from fragments with the length of at least 15 continuous bases in the DNA shown in the sequences 1-4; the primer pair provided by the invention uses the methylation difference to detect the methylation level of a target region in a biomarker gene.
In the method of the present invention, a fluorescent probe is used to monitor the PCR reaction in real time. The reporter fluorophore at the 5' end of the probe used was one or more of Cy5, FAM, TET, rox, texas Red, HEX, cy3 and JOE.
The detection of the methylation level of a gene in the methods of the invention includes detecting the presence or absence of methylation in a biomarker gene, and qualitative and quantitative detection of the methylation.
The invention provides a kit for detecting methylation of genes related to lung cancer, which comprises a primer pair and a probe for performing methylation-specific PCR reaction on one or more of HOXA7, HOXA9, SCT and SHOX2 genes.
The technical scheme provided by the invention is further described in detail by specific examples.
Example 1 selection of methylation genes associated with Lung cancer
The invention screens 159 candidate genes related to benign and malignant lung nodule identification or lung cancer by carrying out whole genome 850K Illumina methylation array analysis on bronchoalveolar flushing fluid samples of patients with lung cancer and benign lung cancer, and specifically comprises the following steps:
NFAM1,BZRAP1,MIR142,C16ORF68,MIR365-1,SPNS1,LAT,SIX1,MAB21L1,MIR548,NBEA,BIN2,ALX1,CUX2,C11ORF21,TSPAN32,WDFY4,TRIM39,LOC728264,MIR145,HOXA9,STAB1,ARHGAP25,PRDM16,SCT,NDUFS2,FCER1G,SHOX2,ETV5,DPP10,PADI6,BAIAP2L1,MEIS1,DLGAP2,WDR66,JAM3,RBM24,RBM38,HOXD8,GBX2,DPP6,KCNIP4,SHANK2,CBLN1,SRGAP3,PCDHGA4,PCDHGC3,PCDHGA12,PCDHGA11,PCDHGA9,PCDHGA1,PCDHGB1,PCDHGC5,PCDHGB6,PCDHGB3,PCDHGB7,PCDHGA6,PCDHGA8,PCDHGA10,PCDHGA5,PCDHGB4,PCDHGC4,PCDHGA3,PCDHGA2,PCDHGA7,PCDHGB2,PCDHGC5,PCDHGB5,PCDHGC3,PCDHGB19P,MYCBPAP,HOXA7,FRR3,SPATA16,DRD5,PTCH1,SCN10A,C2CD4D,LOC100132111,YBX2,ELAVL4,GPR146,ZNF135,TFAP2E,HCN1,CMTM2,SLC12A7,TRIM58,LHX1,KCNC2,FRAT1,ESPN,RPH3AL,EN1,FAM135B,SCARF2,SLC32A1,PTCH1,LHX8,TBX1,FAT1,ARHGEF4,ZNF781,GPSM1,DUOX1,DLG4,ALX3,SYK,SLC32A1,SLC43A2,ASAM,EXOC3,SIX2,CDH4,CKB,IRX4,ZNF135,SNCB,HOXD10,RPH3AL,ISL2,HOXD3,CACNG8,ZNF529,LINC00554,GPR88,C2CD2,ZNF667,CRB2,WDR8,PRDM14,TAC1,NLFN1,MRI1,HOXD9,SRCIN1,GPR177,SLC38A1,DLX5,GPR88,CDH13,AKR7L,C4ORF39,TRIM61,PACRG,GPR6,FLJ45983,ZNF582,HLa-a,LAMA4,RGS12,HSF2BP,ZNF544,SDK1,ZNF382,MTUS1,CD52,UBXN11,LOC101929563.
The invention selects the best target genes from the candidate genes, refers to a large number of documents, screens out the existing data to prove that the target genes of benign and malignant lung nodules and lung cancer can be effectively distinguished, further carries out small sample fluorescence quantitative PCR reaction test on the target genes screened out by the documents, detects the performance difference of clinical samples according to different target genes, and finally selects HOXA7, HOXA9, SCT and SHOX2 methylation genes from the target genes, such as those four with increased fonts in the candidate genes.
Through repeated experiments, the invention designs a primer group for detection aiming at HOXA7, HOXA9, SCT and SHOX2 gene methylation DNA specific fragments respectively, and comprises the following steps: HOXA7 primer set, HOXA9 primer set, SCT primer set 1 and SCT primer set 2, SHOX2 primer set; wherein each primer set comprises a respective upstream primer, downstream primer and probe:
HOXA7 primer set
Upstream primer (F): CGACGTTTACGGTAATTTGTTTTGC-sequence 1-1
Downstream primer (R): TCAACCGCGCCATACAACG-sequence 1-2
Probe (P): TCGTTAAAGGCGTTTGCGATAAGACGGAC-sequences 1 to 3
HOXA9 primer set
Upstream primer (F): CGGGCGTTTTTCGTTTTAGGC-sequence 2-1
Downstream primer (R): AAATCCGTCCCAAACGAAACCG-sequence 2-2
Probe (P): TAAATCCCCACAACTACC-sequence 2-3
SCT primer set 1
Upstream primer (F1): AGGGTTCGGCGATATTTAGAC-sequence 3-1
Downstream primer (R1): AACAACCGCTAAAACCGC-sequence 3-2
Probe (P1): CCCTCCCGCAAACGACTAAACTCGCTAA-sequence 3-3
SCT primer set 2
Upstream primer (F2): TCGTTATAAAGGGGTTTTGC-sequence 3-4
Downstream primer (R2): CCTAACGAACGACTCACCT-sequence 3-5
Probe (P2): TTCGGGGTCGTGGTCGTAGCGTTTAGT-sequence 3-6
SHOX2 primer set
Upstream primer (F): GTTTTTTGGATAGTTAGGTAAT-sequence 4-1
Downstream primer (R): TAACCCGACTTAAACGACGA-sequence 4-2
Probe (P): CTCGTACGACCCCGATCG-sequence 4-3
The primer sequences and probe sequence numbers in each of the above methylation gene primer sets are listed in tables 1 to 0.
In this example, 122 bronchoscopic patients were collected from bronchoalveolar lavage fluid as samples to be tested, 53 lung cancer patients and 69 benign lung cancer patients, DNA was extracted from these samples, and after bisulfite treatment, methylation levels of target regions in each biomarker gene of HOXA7, HOXA9, SCT and SHOX2 were detected by fluorescence PCR using corresponding primer sets, respectively, wherein the target regions were selected from continuous fragments of at least 15 bases in the DNA shown in sequences 1 to 4, respectively; thereby determining whether these target genes can differentiate lung cancer from benign lung cancer and good performance of the differentiation. The present invention utilizes fluorescent probes to monitor the PCR reaction in real time. The reporter fluorophore at the 5' end of the probe used was one or more of Cy5, FAM, TET, rox, texas Red, HEX, cy3 and JOE.
TABLE 1-0
Name of the name Sequence(s) Numbering device Action
HOXA7F CGACGTTTACGGTAATTTGTTTTGC 1-1 HOXA7 upstream primer
HOXA7R TCAACCGCGCCATACAACG 1-2 HOXA7 downstream primer
HOXA7P TCGTTAAAGGCGTTTGCGATAAGACGGAC 1-3 HOXA7 probe
HOXA9F CGGGCGTTTTTCGTTTTAGGC 2-1 HOXA9 upstream primer
HOXA9R AAATCCGTCCCAAACGAAACCG 2-2 HOXA9 downstream primer
HOXA9P TAAATCCCCACAACTACC 2-3 HOXA9 probe
SCTF1 AGGGTTCGGCGATATTTAGAC 3-1 SCT upstream primer
SCTR1 AACAACCGCTAAAACCGC 3-2 SCT downstream primer
SCTP1 CCCTCCCGCAAACGACTAAACTCGCTAA 3-3 SCT probe
SCTF2 TCGTTATAAAGGGGTTTTGC 3-4 SCT upstream primer
SCTR2 CCTAACGAACGACTCACCT 3-5 SCT downstream primer
SCTP2 TTCGGGGTCGTGGTCGTAGCGTTTAGT 3-6 SCT probe
SHOX2F GTTTTTTGGATAGTTAGGTAAT 4-1 SHOX2 upstream primer
SHOX2R TAACCCGACTTAAACGACGA 4-2 SHOX2 downstream primer
SH0X2P CTCGTACGACCCCGATCG 4-3 SHOX2 probe
The present invention uses IBM SPSS STATISTICS analysis software to analyze the detection results, compares the detection results of methylation of each gene in the samples of lung cancer patients and benign lung cancer patients by the receiver operating characteristic (ROC, receiver Operating Characteristic) curve, and the ROC curves of four methylated genes HOXA7, HOXA9, SCT, SHOX2 are shown in fig. 1 to 4 in sequence.
To explain fig. 1-4, the meanings of ROC curve, about log index, and AUC values will be explained. The ROC curve is a comprehensive index reflecting sensitivity and specificity continuous variables, the ordinate "sensitivity" is used for representing the true positive rate, and the abscissa "1-specificity" is used for representing the false positive rate. The about log index, which is equal to the sum of sensitivity and specificity minus 1, is used for indicating the total capacity of a diagnosis test to find a real patient (true positive) and a non-patient (false positive), and the value range is 0-1, and the larger the value is, the stronger the authenticity of diagnosis is. AUC value refers to the Area Under the Curve (AUC, area unit Curve) size. Auc=0.5, referring to the area under the line at the 45 degree angle on the ROC plot. For a methylation gene marker, AUC > 0.5 indicates that it has detection value.
Returning to fig. 1-4. According to the invention, the sensitivity and the specificity of the corresponding methylation genes to benign and malignant lung nodule identification or lung cancer diagnosis are calculated by selecting the abscissa and the ordinate corresponding to the point with the maximum about dengue index on the ROC curve, such as star points (x) marked on the curves in fig. 1-4; meanwhile, AUC value, 95% confidence interval and P value (P < 0.001, showing that the statistical difference of marker detection is significant, and can effectively distinguish benign and malignant nodules and cancer patients or not). The performance indicators for these four methylation genes as human alveolar lavage fluid are listed in Table 1-1.
TABLE 1-1
The results of Table 1 are seen in conjunction with FIGS. 1-4. First, it can be seen intuitively from each figure that the ROC curve for each gene is far to the upper left of auc=0.5, indicating that the respective AUC values are high overall. Referring again to Table 1, the AUC values of methylation levels of the four biomarker genes in lung cancer samples are all greater than 0.5, between 0.69 and 0.85, and all have P < 0.001, relative to benign lung disease, which is sufficient to demonstrate that the detection of methylation levels of the four genes HOXA7, HOXA9, SCT, SHOX2 provided by the invention has a trusted reference value for identifying lung cancer and benign and malignant lung nodules.
Example 2 verification of designed primer set
This example uses real-time fluorescent PCR as an example to detect the methylation level of biomarker genes. The detection targets are HOXA7, HOXA9, SCT and SHOX2 genes. In this example, the DNA subjected to bisulfite treatment was used as a template for detection by real-time fluorescent PCR amplification.
It should be noted that designing a primer set of good quality is a key factor in detecting success or failure of a methylated gene. The invention is different from the common gene detection in design principle, and the primer sequence is required to contain at least more than 1 CpG site, preferably a plurality of CpG sites, so that the specificity of the primer can be ensured, and the detection rate of methylated gene bases can be improved.
For HOXA7, HOXA9, SCT and SHOX2 genes, a plurality of sets of primer and probe combinations can be designed, and the performance of each set of probe primer combinations can be different, so that the selection and verification of the advantages and disadvantages are required through experiments.
In this example, the primers and probes designed and selected in accordance with the present invention were verified using a methylated gene template and a unmethylated gene template. The invention aims at a plurality of primers and probes designed by HOXA7, HOXA9, SCT and SHOX2 genes, which are respectively equivalent to, complementary to or hybridized with at least 15 continuous nucleotides in sequence 1-4 of a sequence table or complementary sequences thereof, and the design of the primers and probes meets the requirement that the size of PCR amplified products is in the range of 80-350bp base pairs.
Gene amplification was performed by real-time fluorescent PCR using the sequences of each of the methylated and unmethylated genes as a template for each primer set designed (five sets listed in example 1), and three sets of experiments were performed in total, and the amplification results were expressed as Ct values as shown in Table 2. In the table, the Ct values are positive for methylation detection, while the countless Ct values are negative for methylation detection.
TABLE 2
As can be seen from the results of Table 2, in the three sets of experiments, each primer set had a value for amplifying Ct value for the methylated gene template, which was detected as positive, and none for amplifying Ct value for the unmethylated gene template, which was detected as negative. This shows that each primer set designed and selected for four methylation genes of the invention is highly specific for methylation gene templates.
Example 3 sensitivity and specificity of testing methylation Gene detection Using a bronchial brush sample
In this example 167 bronchoscopic patients were selected as the test samples, and 95 patients with lung cancer and 72 patients with benign lung disease were selected. DNA was extracted from these samples, after bisulfite treatment, the aforementioned HOXA7 primer set, HOXA9 primer set, SCT primer set 2 and SHOX2 primer set were selected, methylation levels (Ct values) of the four genes were detected by fluorescence PCR, respectively, and methylation detection results of each gene in respective samples of a lung cancer patient and a benign lung cancer patient were compared by ROC curve, sensitivity, specificity, and area under the curve (AUC value) were calculated by selecting the point with the maximum about log index on the curve. The test results are shown in Table 3.
TABLE 3 Table 3
The test results in Table 3 show that the performance index of diagnosing benign and malignant lung nodules or lung cancer by using a bronchus brushing sample is very superior, and the AUC values of methylation levels of four biomarker genes detected are all larger than 0.88 and far higher than 0.5 of the excellent line, so that the four methylation genes provided by the invention have very high detection value, and can provide very useful information for diagnosing benign and malignant lung nodules and diagnosing lung cancer.
Example 4 sensitivity and specificity of testing methylation Gene combinatorial detection
In this example, bronchoalveolar lavage fluid from 122 patients undergoing bronchoscopy was used as the test sample, 53 lung cancer patients and 69 benign lung diseases. Extracting DNA from the samples, and performing bisulfite treatment; combining HOXA7, HOXA9, SHOX2, SCT genes, respectively, and testing the performance of methylation gene combination assays, including:
(one) selecting 2 of the HOXA7, HOXA9, SHOX2, SCT genes for combination and detecting methylation levels of each gene combination by fluorescent PCR, comprising:
combining: HOXA7 and HOXA9 gene combinations;
and (2) combining two: HOXA7 and SHOX2 gene combinations;
And (3) combining three: HOXA7 and SCT gene combinations, wherein SCT gene employs SCT primer set 1;
combination four: HOXA9 and SHOX2 gene combinations;
And (5) combining: HOXA9 and SCT gene combinations, wherein the SCT gene employs SCT primer set 2;
And (3) combining six: SCT and SHOX2 genes, wherein the SCT gene employs SCT primer set 1.
The test results of methylation detection of each gene combination in the lung cancer patient and benign lung cancer patient samples are compared by ROC curve, the sensitivity, specificity of benign and malignant lung nodule discrimination or lung cancer diagnosis is calculated by selecting the point on the ROC curve where the about log index is the largest, and the area under the curve (AUC value) is calculated. The test results are shown in Table 4.
TABLE 4 Table 4
Combination one Combination II Combined three Combined four Combined five Combined six
Sensitivity (%) 77.78 74.07 74.07 61.11 77.78 61.11
Specificity (%) 75.36 84.06 78.26 91.30 75.36 91.30
AUC values 0.809 0.828 0.807 0.785 0.809 0.791
As shown in the results of Table 4, the combination of two of the four methylation genes is used as a sample, the performance index of the four methylation genes is also superior to that of the four methylation genes for assisting in identifying benign and malignant lung nodules or diagnosing lung cancer, compared with benign lung diseases, the methylation level of four biomarker genes is detected in the sample of the pulmonary alveolus flushing liquid of a patient with lung cancer, and the AUC values are between 0.785 and 0.828 and are higher than the values listed in Table 1, so that the accuracy of detection is improved after the four methylation genes are combined two by two, and the detection result can provide accurate and valuable information for identifying benign and malignant lung nodules and diagnosing lung cancer.
(II) selecting 3 of HOXA7, HOXA9, SHOX2 and SCT genes to be combined, and detecting methylation level of the combined genes by fluorescence PCR, wherein the method comprises the following steps:
combination seven: HOXA7, HOXA9 and SCT gene combinations, wherein SCT gene employs SCT primer set 1;
Combination eight: HOXA7, HOXA9 and SHOX2 gene combinations;
Combination nine: HOXA9, SCT and SHOX2 gene combinations, wherein SCT gene employs SCT primer set 2;
combination ten: HOXA7, SCT and SHOX2 genes were combined, wherein SCT gene employed SCT primer set 2.
The test results of methylation detection of each gene combination in the lung cancer patient and benign lung cancer patient samples are compared by ROC curve, the sensitivity, specificity of benign and malignant lung nodule discrimination or lung cancer diagnosis is calculated by selecting the point on the ROC curve where the about log index is the largest, and the area under the curve (AUC value) is calculated. The test results are shown in Table 5.
TABLE 5
Seven combinations Combination eight Combined nine Combined ten
Sensitivity (%) 70.37 64.81 61.11 68.52
Specificity (%) 88.41 91.30 95.65 91.30
AUC values 0.839 0.838 0.805 0.839
As shown in the results of Table 5, the four methylation genes are combined to identify benign and malignant lung nodules or diagnose lung cancer by taking the alveolar lavage fluid as a sample, and the performance indexes of the pulmonary carcinoma are very good, compared with benign lung diseases, the pulmonary carcinoma patient can detect the methylation level of four biomarker genes in the alveolar lavage fluid sample, the AUC value of the pulmonary carcinoma patient is between 0.805 and 0.839, which is far higher than the optimal line of 0.5, and is far higher than the value listed in Table 1, which indicates that the accuracy of detection is further improved after three of the four methylation genes are combined, and the detection result can provide accurate and reliable information for identifying benign and malignant lung nodules and diagnosing lung cancer.
(III) selecting and combining HOXA7, HOXA9, SHOX2 and SCT genes, and detecting methylation level of the combined genes by fluorescent PCR, wherein the SCT genes adopt an SCT primer group 1.
The test results of methylation detection of four gene combinations in lung cancer patient and benign lung cancer patient samples are obtained through an ROC curve, the sensitivity and specificity of benign and malignant lung nodule identification or lung cancer diagnosis are calculated by selecting the point with the maximum about dengue index on the ROC curve, and the area under the curve (AUC value) is calculated. The test results are shown in Table 6.
TABLE 6
HOXA7、HOXA9、SCT、SHOX2
Sensitivity (%) 74.07
Specificity (%) 88.41
AUC values 0.851
The results in Table 6 show that the four methylation genes are combined to assist in identifying benign and malignant lung nodules or diagnosing lung cancer by taking the alveolar flushing fluid as a sample, compared with benign lung diseases, the pulmonary carcinoma patient has the advantages that the methylation level of four biomarker genes is detected in the alveolar flushing fluid sample of the lung cancer patient, the AUC value is 0.851, and the AUC value is far higher than the U.S. Pat. No. 0.5, so that the detection accuracy is improved after the four methylation genes are combined, and the detection result can provide accurate and reliable information for identifying benign and malignant lung nodules and diagnosing lung cancer.
EXAMPLE 5 methylation Gene associated with Lung cancer and kit for detecting the corresponding Gene
A methylation gene related to lung cancer comprises one or more of HOXA7, HOXA9, SCT and SHOX2 genes, and the nucleotide sequence of the methylation gene is shown as sequence 1, sequence 2, sequence 3 and sequence 4 in a sequence table; wherein, the methylated cytosine sites of the genes HOXA7, HOXA9, SCT and SHOX2 are sequentially marked in the sequence 1, the sequence 2, the sequence 3 and the sequence 4.
A kit for detecting the aforementioned genes, for detecting methylated genes in a sample to be tested, comprising one or more of the following reagents:
A reagent for detecting HOXA7 methylation genes, which comprises primers shown in sequences No. 1-1 and No. 1-2 and a probe shown in sequences No. 1-3;
a reagent for detecting HOXA9 methylation gene, which comprises primers shown in the sequences of numbers 2-1 and 2-2 and a probe shown in the sequence of numbers 2-3;
A reagent for detecting the SCT methylation gene, which comprises primers shown in sequences 3-1 and 3-2 and a probe shown in sequence 3-3; comprises primers shown in the sequences of 3-4 and 3-5 and probes shown in the sequences of 3-6;
A reagent for detecting SHOX2 methylated genes comprises primers shown in sequences No. 4-1 and 4-2 and a probe shown in sequence No. 4-3.
The detection kit further comprises an ACTB primer and a probe for detecting the reference gene.
In the above-described detection kit, the primer set for each of the HOXA7, HOXA9, SCT and SHOX2 genes was composed of the primer and probe contained in each reagent, and the concentrations of each component in the PCR reaction solution in this example were as follows: 0.2. Mu.M HOXA7 primer set, 0.6. Mu MACTB primer and probe, 3.5mM MgCl2, 0.25mM dNTP; in addition, DNA polymerase was contained in an amount of 100U, and the DNA extracted after counting the non-small cell HCC827 cell line used for positive control; sterilized water for negative control.
Of course, the PCR reaction solution exemplified in this example may contain other methylation gene primer sets, such as HOXA9 primer set, SCT primer set, and SHOX2 primer set, depending on the detection object, and the concentration thereof in the PCR reaction solution may be set to 0.2. Mu.M.
In the above kit, the concentration of each component in the PCR reaction solution containing the four primer sets is as follows: 0.2. Mu.M HOXA7 primer set, 0.2. Mu.M HOXA9 primer set, 0.2. Mu.M SCT primer set 1, 0.2. Mu.M SHOX2 primer set, 0.5. Mu. MACTB primer and probe, 3.0mM MgCl 2, 0.3mM dNTP; in addition, DNA polymerase was contained in an amount of 500U, and DNA extracted after counting the non-minicell H1299 cell line used as a positive control; sterilized water for negative control.
Similarly, the PCR reaction solution exemplified in this example may contain other types of combinations of methylation gene primer sets depending on the combination to be detected, and the concentration thereof in the PCR reaction solution may be set to 0.2. Mu.M, as in the combination exemplified in example 4.
In the kit of the invention, the sample to be tested comprises one or more of human blood, alveolar lavage fluid, sputum, lung tissue and bronchial brush test sample.

Claims (10)

1. A detection kit for detecting a methylation gene related to lung cancer, which is used for detecting the methylation gene in a sample to be detected, wherein the detection kit comprises a reagent for detecting a HOXA7 gene, and the reagent for detecting the HOXA7 gene comprises primers shown in sequences of numbers 1-1 and 1-2 and a probe shown in sequences of numbers 1-3.
2. The test kit according to claim 1, wherein the test kit further comprises a reagent for detecting the SHOX2 gene, the reagent for detecting the SHOX2 gene comprising primers represented by sequences No. 4-1, 4-2 and a probe represented by sequence No. 4-3.
3. The test kit of claim 1, wherein the test kit further comprises reagents for detecting SCT genes, the reagents for detecting SCT genes comprising:
Primers shown in sequences No. 3-1 and 3-2 and a probe shown in sequence No. 3-3; or (b)
Primers shown in the sequences No. 3-4 and No. 3-5 and probes shown in the sequences No. 3-6.
4. The detection kit according to any one of claims 1 to 3, wherein the detection kit further comprises a reagent for detecting the HOXA9 gene, the reagent for detecting the HOXA9 gene comprising a primer represented by the sequence nos. 2-1 and 2-2 and a probe represented by the sequence nos. 2-3;
5. the test kit according to any one of claims 1-4, wherein the test kit further comprises ACTB primers and probes for the detection of a reference gene.
6. The test kit according to any one of claims 1 to 5, wherein the sample to be tested comprises one or more selected from the group consisting of human blood, alveolar lavage, sputum, pulmonary tissue sample and bronchial brush sample.
7. The test kit of any one of claims 1-6, wherein the components included in the test kit are used to formulate a PCR reaction solution comprising:
0.2. Mu.M of the corresponding primers and probes,
3.5mM MgCl2，
0.25mM dNTPs，
100U DNA polymerase
Optionally 0.6 mu MACTB primer and probe,
Optionally, DNA extracted after counting of non-minicell HCC827 cell line for positive control, and
Sterilized water for negative control.
8. A method for preparing the detection kit of any one of claims 1-7, comprising:
(1) Carrying out genome-wide methylation array analysis on a sample to be detected, and screening 159 candidate genes related to benign and malignant lung nodule identification or lung cancer;
(2) Selecting target genes HOXA7, HOXA9, SCT and/or SHOX2 from the candidate genes, respectively designing corresponding primers and probes aiming at methylated DNA specific fragments of the target genes, respectively, wherein the primers and the probes are shown in tables 1-0, and verifying that the primers and the probes meet the performance indexes of the target genes required by detection.
9. The method of claim 8, wherein the performance index of the target gene is selected from the group consisting of: sensitivity, specificity, and AUC values.
10. The method according to claim 8 or 9, wherein the sample to be tested comprises one or more selected from the group consisting of human blood, alveolar lavage, sputum, pulmonary tissue and bronchial brush.
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