CN113774131A - Molecular typing marker and kit for early diagnosis, treatment and prognosis detection of non-small cell lung cancer - Google Patents

Molecular typing marker and kit for early diagnosis, treatment and prognosis detection of non-small cell lung cancer Download PDF

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CN113774131A
CN113774131A CN202011517819.7A CN202011517819A CN113774131A CN 113774131 A CN113774131 A CN 113774131A CN 202011517819 A CN202011517819 A CN 202011517819A CN 113774131 A CN113774131 A CN 113774131A
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黄家强
林舒晔
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Beijing Chest Hospital
Beijing Tuberculosis and Thoracic Tumor Research Institute
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Beijing Tuberculosis and Thoracic Tumor Research Institute
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Abstract

The invention discloses a molecular typing marker for early diagnosis, treatment and prognosis detection of non-small cell lung cancer. The invention provides the following applications of a MUC22 gene methylation modification sequence as a molecular marker: tumor heterogeneity analysis of non-small cell lung cancer, molecular typing of squamous cell lung cancer and adenocarcinoma of lung (i.e., distinguishing squamous cell lung cancer from adenocarcinoma of lung in non-small cell lung cancer), diagnosis of non-small cell lung cancer, targeted therapy monitoring, and/or prognostic evaluation of drug use. The invention also provides a kit based on the detection of the DNA sequence methylation of the MUC22 gene, which can specifically detect the DNA methylation level related to the MUC22 gene and can also specifically and quantitatively detect the DNA methylation level related to the MUC22 gene. The invention can provide an evaluation method for the disease condition of the patient with the non-small cell lung cancer, the monitoring of the individual medication and the early diagnosis of the tumor, and also for the prognosis detection of the patient on the drug treatment.

Description

Molecular typing marker and kit for early diagnosis, treatment and prognosis detection of non-small cell lung cancer
Technical Field
The invention relates to the technical field of molecular diagnosis, in particular to a molecular typing marker and a kit for early diagnosis, treatment and prognosis detection of non-small cell lung cancer.
Background
Cancer is fatal to the prostate disease worldwide, and seriously affects the social and economic development. The mortality rate is extremely high due to the influence of sensitivity and tissue specificity of the detection marker and the limitation of application of the detection marker in treatment monitoring. Therefore, the reliable biomarkers are found to be used for accurate diagnosis and monitoring of tumors, and have important clinical application value for tumor diagnosis and prognosis detection.
Non-small cell lung cancer (NSCLC) is one of the most common high-grade tumors in China, and the fatality rate of NSCLC is in the front of various malignant tumors in the world and China. Due to the lack of effective detection means for early detection, most of the patients in the clinic are at the middle to late stage, have lost surgical opportunity or are treated with new adjuvant chemotherapy before surgery (Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA and Jemal A: Global Cancer standards 2018: GLOBOCAN animals of patients and patients for 36Cancer in 185 burners in Cancer. CA Cancer J Clin 68: 394; 424, 2018.). For patients with middle or advanced stage, chemotherapy-based clinical treatment is common, and a series of biological and clinical problems are caused by chemotherapy resistance and chemotherapy toxic and side effects (Relli V, Trerotola M, Guerra E and Alberti S: Absandoning the Notion of Non-Small Cell Lung cancer. trends in molecular medicine 25:585-594, 2019.). Therefore, the molecular pathway mechanism of tumor chemotherapy resistance is researched, so that the molecular target capable of inhibiting tumor cell proliferation and improving chemotherapy sensitivity is identified, and a new idea is provided for accurate diagnosis and individualized treatment of tumors (Rotow J and Bivona TG: evacuating and targeting resistance mechanisms in NSCLC. Nature reviews. cancer 17: 637-.
Non-small cell lung cancer is a heterogeneous disease caused by genetic and epigenetic changes, with high Tumor heterogeneity (Tumor heterogeneity), which is an important cause of cancer progression and resistance (Chen Z, Fillmore CM, Hammerman PS, Kim CF, Wong KK. non-small-cell cancer: a hepatogenes set of diseases. Nature reviews cancer.2014; 14:535 546.). Its development is a cumulative process of multifactorial and polygenic variation, in which the amplification or mutation of oncogenes and tumors are caused by genetic variation and epigenetic disorderThe silencing and function loss of the suppressor gene play an important role in the generation and the development of the non-small cell lung cancer. Epigenetics changes and regulates the expression of cell tissue specific genes through the modification of DNA methylation, histone and the like, and participates in a plurality of life activities of development and differentiation and functional metabolism of organisms. Different from gene mutation, the progressive phenotypic change caused by epigenetic change can be reversed under certain conditions, the characteristic opens up a new way for preventing and treating some diseases, particularly tumors, particularly abnormal changes of gene expression and function caused by abnormal hypermethylation of CpG islands in promoter regions of cancer suppressor genes and abnormal modification of histones, not only can be used as epigenetic markers and targets for molecular diagnosis, treatment, prognosis evaluation and the like of tumors, but also can be considered as one of main factors (Quintanal-villaloga) for causing diseases and treating drug resistance formation
Figure BDA0002848559500000021
and Molina-Pinelo S:Epigenetics of lung cancer:a translational perspective.Cellular oncology(Dordrecht)42:739-756,2019.)。
Lung Adenocarcinoma (Lung adenocarinoma, LUAD) and Squamous Cell Carcinoma (Lung Squalus Cell Carcinoma, LUSC for short) are two major subtypes thereof, have different genetic variation and functional heterogeneity, and thus require different intervention strategies and treatment measures, and there are acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) treatment (Roca E, Pozzari M, Vermi W, et al. Outcome of EGFR-mutated adenocarinoma NSCLC Patients with changed phenyl type to squamus Cell Carcino kinase enzyme linking inhibitors: A porous and adsorbed with amplified drug type, Lung cancer (Amdamm, Netherlands, 2019; 127: 12-18); with the progress of targeting therapy guided by accurate diagnosis, more convenient and definite markers need to be developed to distinguish the heterogeneity between lung adenocarcinoma and squamous carcinoma, and particularly to dynamically monitor whether and when patients develop resistance during targeting therapy.
Disclosure of Invention
The invention aims to provide a molecular typing marker and a kit for early diagnosis, treatment and prognosis detection of non-small cell lung cancer.
In a first aspect, the invention claims the use of a modified sequence (i.e. including the MUC22 gene sequence itself and modifications apparent thereto, including the degree of apparent modification, whether or not apparent, and/or not apparent) of the Mucin 22 (mucn 22, MUC22) gene, a novel member of the protein family, as a molecular marker, in any of:
p1, preparing products for molecular typing of lung squamous carcinoma and lung adenocarcinoma in non-small cell lung cancer, or molecular typing of lung squamous carcinoma and lung adenocarcinoma in non-small cell lung cancer.
P2, preparation of a product for use in diagnosis (particularly early diagnosis), targeted therapy monitoring (particularly dynamic monitoring) and/or drug use prognosis evaluation of non-small cell lung cancer, or diagnosis (particularly early diagnosis), targeted therapy monitoring (particularly dynamic monitoring) and/or drug use prognosis evaluation of non-small cell lung cancer.
P3, preparing a product for tumor heterogeneity analysis of non-small cell lung cancer, or performing tumor heterogeneity analysis of non-small cell lung cancer.
Wherein, the MUC22 gene appearance modification sequence is a DNA sequence of the MUC22 gene, and preferably, the nucleotide sequence of the DNA sequence at least comprises a modification of an appearance modification site.
In a second aspect, the invention claims the use of a substance for detecting the apparent modification of the MUC22 gene in any one of:
p1, preparing a product for molecular typing of lung squamous carcinoma and lung adenocarcinoma in non-small cell lung cancer;
p2, preparation of a product for use in the diagnosis (particularly early diagnosis), targeted therapy monitoring (particularly kinetic monitoring) and/or prognostic assessment of non-small cell lung cancer;
p3, and preparing products for tumor heterogeneity analysis of non-small cell lung cancer.
In a third aspect, the invention claims the application of MUC22 gene epigenetic modification sequence as a drug action target in the preparation of products for treating and/or preventing non-small cell lung cancer (such as squamous cell lung carcinoma or lung adenocarcinoma).
In the present invention, the apparent modification is preferably a methylation modification. That is, in the first and third aspects, the MUC22 gene epigenetic modification sequence may specifically be a MUC22 gene methylation modification sequence; in the second aspect, the MUC22 gene epigenetic modification may specifically be a MUC22 gene methylation modification.
Further, in the first and third aspects, the MUC22 gene methylation modification sequence may be a methylation modification sequence of a promoter region or a regulatory region of the MUC22 gene. In a second aspect, the methylation modification of the MUC22 gene may be a methylation modification of the promoter region or the regulatory region of the MUC22 gene.
In each of the above aspects, the promoter region or regulatory region of the MUC22 gene is a DNA sequence located from 1281bp upstream to 1680bp downstream of the transcription start site of MUC22 gene.
Furthermore, the nucleotide sequence of the promoter region of the MUC22 gene is shown as SEQ ID No.1 (namely, the DNA sequence from the upstream 9bp to the downstream 155bp of the transcription start site of the MUC22 gene) or SEQ ID No.2 (namely, the DNA sequence from the upstream 1281bp to the upstream 999bp of the transcription start site of the MUC22 gene). The nucleotide sequence of the regulatory region of the MUC22 gene is shown as SEQ ID No.3 (namely, the DNA sequence from 1390bp to 1680bp downstream of the transcription start site of the MUC22 gene).
In the present invention, the substance for detecting methylation modification of the MUC22 gene is specifically a primer set or a primer pair set described in the fifth aspect or a kit described in the sixth aspect described later.
In a fourth aspect, the invention claims a molecular marker having the function of any one of P1 to P3 as follows:
p1, carrying out molecular typing on lung squamous carcinoma and lung adenocarcinoma in the non-small cell lung cancer;
p2, diagnosis (especially early diagnosis), targeted therapy monitoring (especially dynamic monitoring) and/or drug prognosis evaluation of non-small cell lung cancer;
p3, and carrying out tumor heterogeneity analysis on the non-small cell lung cancer.
The molecular marker claimed by the invention can be a MUC22 gene appearance modification sequence.
Wherein the apparent modification is preferably a methylation modification. That is, the MUC22 gene epigenetic modification sequence may be a MUC22 gene methylation modification sequence.
Further, the MUC22 gene methylation modification sequence may be a methylation modification sequence of the promoter region or the regulatory region of the MUC22 gene.
Still further, the promoter region or the regulatory region of the MUC22 gene is the DNA sequence located from 1281bp upstream to 1680bp downstream of the transcription start site of the MUC22 gene.
Furthermore, the nucleotide sequence of the promoter region of the MUC22 gene is shown as SEQ ID No.1 (namely, the DNA sequence from the upstream 9bp to the downstream 155bp of the transcription start site of the MUC22 gene) or SEQ ID No.2 (namely, the DNA sequence from the upstream 1281bp to the upstream 999bp of the transcription start site of the MUC22 gene). The nucleotide sequence of the regulatory region of the MUC22 gene is shown as SEQ ID No.3 (namely, the DNA sequence from 1390bp to 1680bp downstream of the transcription start site of the MUC22 gene).
In a fifth aspect, the invention claims a primer pair or primer pair set that can be used to detect methylation modification of the MUC22 gene.
The primer pair is a methylated primer pair or an unmethylated primer pair; the primer pair group consists of the methylated primer pair and the corresponding unmethylated primer pair; the methylated primer pair and the corresponding unmethylated primer pair are both designed based on the MUC22 gene methylation modification sequence (i.e., the primer pair is used to amplify the MUC22 gene methylation modification sequence).
Further, the MUC22 gene methylation modification sequence is a methylation modification sequence of a promoter region or a regulatory region of the MUC22 gene.
Furthermore, the promoter region or the regulatory region of the MUC22 gene is a DNA sequence located from 1281bp upstream to 1680bp downstream of the transcription start site of the MUC22 gene.
More specifically, the nucleotide sequence of the promoter region of the MUC22 gene is shown as SEQ ID No.1 (namely, the DNA sequence from 9bp upstream to 155bp downstream of the transcription start site of the MUC22 gene) or SEQ ID No.2 (namely, the DNA sequence from 1281bp upstream to 999bp upstream of the transcription start site of the MUC22 gene). The nucleotide sequence of the regulatory region of the MUC22 gene is shown as SEQ ID No.3 (namely, the DNA sequence from 1390bp to 1680bp downstream of the transcription start site of the MUC22 gene).
The primer pair or the primer pair group claimed by the invention has the following functions shown in any one of P1-P3:
p1, carrying out molecular typing on lung squamous carcinoma and lung adenocarcinoma in the non-small cell lung cancer;
p2, diagnosis (especially early diagnosis), targeted therapy monitoring (especially dynamic monitoring) and/or drug prognosis evaluation of non-small cell lung cancer;
p3, and carrying out tumor heterogeneity analysis on the non-small cell lung cancer.
In a specific embodiment of the invention, the methylation primer pair is composed of two single-stranded DNA molecules shown as SEQ ID No.4 and SEQ ID No.5 aiming at the promoter region of the MUC22 gene shown as SEQ ID No.1 (namely, the DNA sequence from 9bp upstream to 155bp downstream of the transcription start site of the MUC22 gene); the corresponding non-methylated primer pair consists of two single-stranded DNA molecules shown by SEQ ID No.6 and SEQ ID No. 7.
In a specific embodiment of the invention, the methylation primer pair is composed of two single-stranded DNA molecules shown as SEQ ID No.8 and SEQ ID No.9 aiming at a promoter region of the MUC22 gene shown as SEQ ID No.2 (namely a DNA sequence from 1281bp to 999bp upstream of the transcription start site of the MUC22 gene); the corresponding non-methylated primer pair consists of two single-stranded DNA molecules shown in SEQ ID No.10 and SEQ ID No. 11.
In a specific embodiment of the invention, the methylation primer pair consists of two single-stranded DNA molecules shown as SEQ ID No.12 and SEQ ID No.13 aiming at the regulatory region sequence of the MUC22 gene shown as SEQ ID No.3 (namely, the DNA sequence from 1390bp to 1680bp downstream of the transcription start site of the MUC22 gene); the corresponding non-methylated primer pair consists of two single-stranded DNA molecules shown in SEQ ID No.14 and SEQ ID No. 15.
In a sixth aspect, the invention claims a kit comprising a primer pair or primer pair set as described in the fifth aspect above.
The kit may also contain a methylated control DNA template and/or an unmethylated control DNA template for MSP amplification, as desired.
Wherein, the methylation control DNA template for MSP amplification can be DNA which is vulcanized and modified and shows hypermethylation of more than 90%. Preferably, the promoter region sequence or regulatory region sequence of MUC22 gene is modified by sulfurization to show more than 90% hypermethylation. Furthermore, the promoter region or the regulatory region sequence of the MUC22 gene is a DNA sequence located from 1281bp upstream to 1680bp downstream of the transcription start site of the MUC22 gene. Furthermore, the nucleotide sequence of the promoter region of the MUC22 gene is shown as SEQ ID No.1 (namely, the DNA sequence from the upstream 9bp to the downstream 155bp of the transcription start site of the MUC22 gene) or SEQ ID No.2 (namely, the DNA sequence from the upstream 1281bp to the upstream 999bp of the transcription start site of the MUC22 gene). The nucleotide sequence of the regulatory region of the MUC22 gene is shown as SEQ ID No.3 (namely, the DNA sequence from 1390bp to 1680bp downstream of the transcription start site of the MUC22 gene).
Wherein, the non-methylated control DNA template for MSP amplification can be normal tissue cell DNA. Preferably, the promoter region or regulatory region sequence of the MUC22 gene is modified by sulfurization to exhibit non-methylation. Furthermore, the promoter region or the regulatory region sequence of the MUC22 gene is a DNA sequence located from 1281bp upstream to 1680bp downstream of the transcription start site of the MUC22 gene. Furthermore, the nucleotide sequence of the promoter region of the MUC22 gene is shown as SEQ ID No.1 (namely, the DNA sequence from the upstream 9bp to the downstream 155bp of the transcription start site of the MUC22 gene) or SEQ ID No.2 (namely, the DNA sequence from the upstream 1281bp to the upstream 999bp of the transcription start site of the MUC22 gene). The nucleotide sequence of the regulatory region of the MUC22 gene is shown as SEQ ID No.3 (namely, the DNA sequence from 1390bp to 1680bp downstream of the transcription start site of the MUC22 gene).
Of course, the kit may further contain deionized water as a negative system control and/or PCR reagents required for MSP amplification.
In a seventh aspect, the invention claims any of the following methods:
the method A comprises the following steps: a method for detecting methylation modification of promoter region or regulatory region of MUC22 gene. Wherein, the promoter region or the regulatory region of the MUC22 gene can be a DNA sequence which is positioned from 1281bp upstream to 1680bp downstream of the transcription start site of the MUC22 gene. Further, the nucleotide sequence of the promoter region of the MUC22 gene can be specifically shown as SEQ ID No.1 or SEQ ID No. 2; the nucleotide sequence of the regulatory region of the MUC22 gene can be specifically shown as SEQ ID No. 3.
The method for detecting the methylation modification condition of the promoter region or the regulatory region of the MUC22 gene comprises the following steps: sulfurizing and modifying a DNA sample to be tested containing the promoter region or the regulatory region of the MUC22 gene, using the methylation primer pair and the corresponding non-methylation primer pair in the fifth aspect as templates to perform MSP amplification respectively, and determining the methylation modification condition of the promoter region or the regulatory region of the MUC22 gene in the DNA sample to be tested according to the amplification result as follows (A1) or (A2):
(A1) and (3) judging according to whether a target product is obtained: if the methylation primer pair is adopted for amplification to obtain a target product and the non-methylation primer pair is not adopted for amplification to obtain the target product, the target product is regarded as the promoter region or the regulation region of the MUC22 gene in the DNA sample to be detected to be hypermethylated; if the methylation primer pair is adopted for amplification to obtain a target product and the non-methylation primer pair is also adopted for amplification to obtain the target product, the target product is regarded as the partial methylation of the promoter region or the regulatory region of the MUC22 gene in the DNA sample to be detected; if the target product is obtained by adopting the non-methylation primer pair for amplification and the target product is obtained by adopting the methylation primer pair for non-amplification, the target product is regarded as the promoter region or the regulation region of the MUC22 gene in the DNA sample to be detected to be unmethylated;
(A2) the MSP amplification is methylation specific fluorescent quantitative PCR, and is carried out by 2-ΔΔCtCalculating the methylation percentage of the promoter region or the regulatory region of the MUC22 gene in the DNA sample to be detected; if the methylation percentage is more than or equal to 60, the methylation is regarded as high methylation; if the percentage methylation is 20-60% (excluding the end point), it is considered as "partial methylation"; if the percentage methylation is less than or equal to 20%, this is regarded as "unmethylated".
In method a, the test DNA sample can be non-small cell lung cancer (e.g., squamous lung cancer or adenocarcinoma lung cancer) cell and/or tissue DNA, including but not limited to blood or other human body secretions (e.g., sputum, bronchial lavage, etc.).
The method B comprises the following steps: a method of distinguishing between a squamous lung carcinoma sample and a lung adenocarcinoma sample in non-small cell lung cancer comprising the steps of: the methylation modification of the promoter region or regulatory region of MUC22 gene of a test sample was determined according to method A described above, and then the test sample was determined as a squamous cell lung carcinoma sample or a adenocarcinoma lung carcinoma sample as follows: the test sample that is "hypermethylated" or "partially methylated" of the promoter region or regulatory region of the MUC22 gene is or is candidate for being a squamous cell lung carcinoma sample; the test sample in which the promoter or regulatory region of the MUC22 gene is "unmethylated" is or is candidate for being a lung adenocarcinoma sample.
In the method B, the sample is a lung squamous carcinoma sample or a lung adenocarcinoma sample in non-small cell lung cancer, including but not limited to blood or other human body exudates (e.g. sputum, bronchial lavage, etc.).
Wherein, the method A and the method B can be used as non-disease diagnosis and treatment methods, such as methylation detection and non-small cell lung cancer sample differentiation for simple and unrelated disease diagnosis. Of course, said method a and said method B may also be diagnostic therapeutic methods for diseases.
In the present invention, the diagnosis (especially early diagnosis) of non-small cell lung cancer includes not only distinguishing squamous cell lung cancer from adenocarcinoma of lung in non-small cell lung cancer, but also determining whether to shift from adenocarcinoma of lung in non-small cell lung cancer to squamous cell lung cancer (if the methylation degree of MUC22 gene of a patient with adenocarcinoma of lung is monitored to be increased, it is determined that the patient with adenocarcinoma of lung is most likely to shift from adenocarcinoma of lung to squamous cell lung cancer).
In the present invention, the targeted therapy monitoring of non-small cell lung cancer is as follows: an increasing number of cases report a new NSCLC phenotype, i.e., an "EGFR mutant lung adenocarcinoma transformed into squamous cell lung carcinoma" that develops acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) treatment, i.e., there is transformation of lung adenocarcinoma into squamous cell lung carcinoma during treatment, which suggests resistance. Dynamic monitoring needs a simple and easy method. The present invention helps to solve such problems.
The present invention finds that the DNA methylation of the Mucin 22(Mucin 22, MUC22) gene, a novel member of the Mucin family, is abnormally changed in NSCLC tissues and cells, namely, the promoter region of the DNA methylation gene shows relatively high methylation level (being hypermethylation or partial methylation) in lung squamous carcinoma tissue cells and relatively low methylation level (being unmethylated or partial methylation) in adenocarcinoma cells. The expression silencing of MUC22 in non-small cell lung cancer cells (mainly lung squamous carcinoma cells) can be reversed by using a chemotherapeutic medicament DNA methylation inhibitor, and the fact that the expression silencing of MUC22 gene in the non-small cell lung cancer cells (mainly lung squamous carcinoma cells) is apparently regulated by hypermethylation of gene DNA is shown. Therefore, the distinct apparent modification changes of the MUC22 gene in squamous cell lung carcinoma and adenocarcinoma, including DNA methylation modification, can be used as molecular markers and drug action targets for early diagnosis, treatment and prognosis detection of non-small cell lung carcinoma development, and are particularly used for molecular typing of squamous cell lung carcinoma and adenocarcinoma and non-small cell lung carcinoma tumor heterogeneity analysis.
The detection primer developed by the invention has the characteristics of simplicity, convenience, easiness, strong sensitivity and high specificity; the kit based on the detection of the DNA sequence methylation of the MUC22 gene can specifically detect the DNA methylation level of the MUC22 gene and can also specifically and quantitatively detect the DNA methylation level of the MUC22 gene; the detection result can be used for monitoring the condition of the patient with the non-small cell lung cancer and personalized medicine and early diagnosis of tumor, and also provides an evaluation method for the prognosis detection of the patient on the medicine treatment.
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FIG. 1 is a methylation analysis of the MUC22 gene in lung squamous carcinoma and lung adenocarcinoma cells and tissues. A is a structural schematic diagram of MUC22 gene, wherein an amplification region of MSP and MS-qPCR is marked, and an amplification region of RT-qPCR for verifying MUC22 gene mRNA expression is verified. Each bar represents a CpG site. These numbers indicate the position relative to the Transcription Start Site (TSS). And B, analyzing the methylation of the MUC22 gene in lung squamous carcinoma, lung adenocarcinoma cell line and immortalized bronchial epithelial cell BEAS-2B cells by MS-qPCR. And C is the MS-qPCR amplification data in the B picture which is presented by the percentage of methylation after being homogenized. D is the DNA methylation status of MUC22 gene in clinical tissue specimens analyzed by MSP for squamous lung carcinoma (n-24) and adenocarcinoma of lung (n-23) (U indicates the amplification result of non-methylated primers, M indicates the amplification result of methylated primers, T indicates tumor tissue). E is statistical analysis of the frequency of MUC22 gene methylation present in lung squamous carcinoma and lung adenocarcinoma tissue samples. Methylation (%) - (number of "hypermethylated" and "partially methylated" cases/total cases × 100%; non-methylated (%) - (number of "unmethylated" cases/total number of cases × 100%. F is RT-qPCR analysis of MUC22 expression in lung squamous carcinoma cells treated with epigenetic regulating agent. Cell treatment: cells were treated with 5. mu.M of the DNA methylation inhibitor 5-Aza-2 '-deoxycytidine (5-Aza-2' -deoxycytidine, 5-Aza) for 96 hours.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 design of primers for detecting methylation of MUC22 Gene
Selecting a CG-rich DNA region from a MUC22 gene sequence presented by NCBI, and determining a possible CpG island; according to the distribution characteristics of DNA methylation modification (Wang, Y., et al. Sci Rep 2016,6:22051.), a specific CG-rich DNA region (A in figure 1) in the sequence near the TSS site of the MUC22 gene is selected through manual alignment analysis, a plurality of Primer pairs are designed by using software Primer Express 3.0, and the following methylated primers and unmethylated primers are screened out. The target sequence of the gene sequence covers the MUC22 gene transcription start site-1282 bp- +1680 bp.
The specific sequences of the primers are as follows:
primer set 1Covering a DNA sequence (SEQ ID No.1) which is positioned at the transcription start site of MUC22 gene from-9 bp to +155 bp;
the methylation primers are as follows:
an upstream primer: 5'-TTAAAAATATAAAAATTAGTTGGCGT-3' (SEQ ID No.4),
a downstream primer: 5'-TAAAACGAAATCTCGCTCTATCG-3' (SEQ ID No. 5).
The unmethylated primers are as follows:
an upstream primer: 5'-TTAAAAATATAAAAATTAGTTGGTGT-3' (SEQ ID No. 6);
a downstream primer: 5'-TAAAACAAAATCTCACTCTATCAAC-3' (SEQ ID No. 7).
Primer set 2Covering a DNA sequence (SEQ ID No.2) positioned at-1281 bp to-999 bp of the transcription start site of the MUC22 gene;
the methylation primers are as follows:
an upstream primer: 5'-TGGATTTAGTATAATGATTTTTACGT-3' (SEQ ID No.8),
a downstream primer: 5'-CCTAAAATATTCTACACCCTACGAA-3' (SEQ ID No. 9).
The unmethylated primers are as follows:
an upstream primer: 5'-TGGATTTAGTATAATGATTTTTATGT-3' (SEQ ID No. 10);
a downstream primer: 5'-CCTAAAATATTCTACACCCTACAAA-3' (SEQ ID No. 11).
Primer and method for producing the sameGroup 3Covering a DNA sequence (SEQ ID No.3) positioned at the transcription start site of the MUC22 gene from +1390bp to +1680 bp;
the methylation primers are as follows:
an upstream primer: 5'-AGTGGTTGTTATTAGAGTGGTTCGT-3' (SEQ ID No.12),
a downstream primer: 5'-AAAACCTATATTCCAAATTCTCGCT-3' (SEQ ID No. 13).
The unmethylated primers are as follows:
an upstream primer: 5'-AGTGGTTGTTATTAGAGTGGTTTGT-3' (SEQ ID No. 14);
a downstream primer: 5'-AAAACCTATATTCCAAATTCTCACT-3' (SEQ ID No. 15).
All primers were synthesized by Huada corporation (Beijing).
MSP amplification is carried out by utilizing the methylation primer and taking DNA subjected to vulcanization modification as a template, and taking SEQ ID No.1 as an example, if the promoter region of the MUC22 gene shown in the SEQ ID No.1 is methylated, a 164bp fragment can be obtained; if the MUC22 gene promoter region shown in SEQ ID No.1 is not methylated, no amplification product is produced. Based on this, the present invention refers to the primer as a methylated primer. The methylated primer has Tm 56.29 and GC content of an upstream primer of the methylated primer being 34.62 percent, and Tm 59.9 and GC content of a downstream primer being 60.87 percent.
MSP amplification is carried out by using the DNA subjected to vulcanization modification as a template by using the non-methylation primer, and if the promoter region of the MUC22 gene shown in SEQ ID No.1 is not methylated, a 164bp fragment is obtained; if the promoter region of MUC22 gene shown in SEQ ID No.1 is methylated, no amplification product is produced. Based on this, the present invention refers to the primer as a non-methylated primer. The non-methylated primer has Tm 52.51 and GC content of an upstream primer of the non-methylated primer being 34.62 percent, and Tm 53.97 and GC content of a downstream primer being 60.0 percent.
If the 164bp fragments amplified using the methylated primers and the unmethylated primers are both present in the PCR amplification product, partial methylation of the MUC22 gene is suggested.
The interpretation method of the results is as follows (taking SEQ ID No.1 as an example):
the first method, based on whether the target product is obtained:
the methylation primer is used for amplification, 164bp amplification products exist, the non-methylation primer is used for amplification, and the samples without 164bp amplification products are judged to be hypermethylated (the methylation level is relatively high); using methylation primers and non-methylation primers for amplification, wherein the samples with 164bp amplification products are judged as partial methylation (the methylation level is relatively low);
the samples were amplified with unmethylated primers, 164bp amplified products were present, and methylated primers were used for amplification, and samples without 164bp amplified products were judged as "unmethylated".
The second method, determination by amplification using methylation-specific fluorescent quantitative PCR (MS-qPCR):
through 2-ΔΔCtMethod calculation (PfaffMW, Horgan GW, Dempfl L.relative expression software tool (REST) for group-wise comparison and static analysis of relative expression in real-time PCR. nucleic Acids Res.2002May 1; 30(9): e 36.). Ct values obtained with amplification with the methylated primer pair and Ct values obtained with amplification with the unmethylated primer pair are presented as percent methylation after homogenization (percent methylation ═ methylated/methylated + unmethylated) × 100%). If the methylation percentage is more than or equal to 60, the methylation is regarded as high methylation; if the percentage methylation is 20-60% (excluding the end point), it is considered as "partial methylation"; if the percentage methylation is less than or equal to 20%, this is regarded as "unmethylated".
Example 2 detection of MUC22 Gene methylation in Lung squamous cell carcinoma and Lung adenocarcinoma cell lines in non-Small cell Lung cancer
This example will be described as example 1 "Primer set 1"is described as an example.
Lung squamous carcinoma cells tested: NCl-H1703, NCl-H2170 and SK-MES-1.
Lung adenocarcinoma cells tested: HCC-H827, NCl-H1395 and NCl-H522.
Human normal lung epithelial cells as controls: BEAS-2B.
The cells can be purchased from national biomedical experimental cell resource libraries and cultured and passaged under normal conditions in the unit laboratory of an applicant.
1. Cell DNA extraction procedure
(1) Each of the test cells in good condition at the logarithmic growth phase was removed from the medium, and the cells were gently washed twice with ice-cold 1 × PBS.
(2) Adding pancreatin 1mL, digesting for 1min, adding RPMI 1640 2mL, neutralizing, gently blowing to remove suspended cells, transferring into a 15mL centrifuge tube, centrifuging at 1000rpm for 5min, discarding supernatant, and collecting cells.
(3) Adding 2mL of DNA extract and 100 μ L of proteinase K (10mg/mL), blowing, stirring, and placing in a 50 deg.C constant temperature water bath for 3 h.
(4) It is removed, cooled to room temperature, added with an equal volume of phenol/chloroform, mixed by inversion, centrifuged at 4200rpm for 15min, and the supernatant liquid is carefully transferred to a new 15mL centrifuge tube.
(5) Adding 1/10 volume of 7.5mol/L ammonium acetate and 2 times volume of absolute ethyl alcohol, gently mixing, centrifuging at 10000rpm for 20min, and discarding the supernatant.
(6) Adding 500 μ L70% ethanol, washing precipitate twice, centrifuging at 13000rpm for 5min, discarding supernatant, and air drying.
(7) mu.L of the DNA was dissolved by adding 100. mu.L of a TE solution having a pH of 8.0, 1. mu.L of the DNA solution was subjected to DNA concentration measurement using a NanoDrop2000c ultra-micro nucleic acid analyzer, and the remaining sample was stored in a freezer at-20 ℃.
2. The positive and negative control samples for DNA methylation were Human Methylated & Non-Methylated DNAset (cat # D5014), also known as in vitro Methylated DNA (IVD), purchased from Zymo Research, USA.
3. Cell DNA sample sulfurization
(1) Mu.g of genomic DNA (sample to be tested, positive control for DNA methylation and negative control sample) was added to a 0.2ml centrifuge tube and ddH was used2O diluted to 20. mu.l. The operation was carried out using the EZ DNA Methylation Gold Kit (cat # D5006) of Zymo Research, USA.
(2) Adding 130 mul of CT solution into 20 mul of DNA sample in a centrifuge tube, putting the sample into a PCR instrument, carrying out pre-cooling at 98 ℃ for 10min and 64 ℃ for 2.5h and 4 ℃ and then transferring the sample into an adsorption column.
(3) Add 600. mu.l Binding Buffer and mix by inversion. Centrifuging at 12000rpm for 30s, and discarding the waste liquid.
(4) Add 100. mu.l of Wash Buffer, centrifuge at 12000rpm for 30s, and discard the waste.
(5) Adding 200. mu.l of depletion Buffer, standing at room temperature for 20min, centrifuging at 12000rpm for 30s, and discarding the waste liquid. Add 200. mu.l of Wash Buffer, centrifuge at 12000rpm for 30s, discard the waste solution, repeat twice.
(6) The DNA was eluted with 10. mu.l of Elution Buffer and stored at-20 ℃.
4. MS-qPCR detection
Each set of PCR reactions included one positive control, one negative control, and no template (water) control.
(1) MS-qPCR detection primer sequence:
methylation primer sequences:
an upstream primer: 5'-TTAAAAATATAAAAATTAGTTGGCGT-3' (SEQ ID No.4),
a downstream primer: 5'-TAAAACGAAATCTCGCTCTATCG-3' (SEQ ID No. 5).
Unmethylated primer sequence:
an upstream primer: 5'-TTAAAAATATAAAAATTAGTTGGTGT-3' (SEQ ID No. 6);
a downstream primer: 5'-TAAAACAAAATCTCACTCTATCAAC-3' (SEQ ID No. 7).
(2) qPCR amplification
The reaction system for amplification is shown in Table 1.
TABLE 1 reaction System for MS-qPCR amplification
Figure BDA0002848559500000111
Note: 2 × SYBR-Green: thermo fisher corporation, usa, cat # 4334973.
The PCR reaction conditions were as follows: 2min at 50 ℃, 10min at 95 ℃ and 15s at 95 ℃; 1min at 60 ℃; collecting fluorescence in 40 cycles; the dissolution curves were prepared at 95 ℃ for 15s, 60 ℃ for 1min, 95 ℃ for 15s, and 60 ℃ for 15 s. Results of the experimentBy using 2-ΔΔCtThe data were analysed by methods (see in particular the relevant steps of example 1) and the solubility curve ensured the specificity of the product.
5. Results
The results are shown in B and C in FIG. 1, IVD is used as methylation control, NL is used as non-methylation control, deionized water is used as negative system control to establish a control system, and the methylation primer and the non-methylation primer are respectively used for MS-qPCR detection of the non-small cell lung cancer cell sample.
Among them, the lung squamous carcinoma cell line was NCI-H1703 (percentage of methylation of 72%), NCI-H2170 (percentage of methylation of 69%) and SK-MES-1 (percentage of methylation of 73%). And HCC-827 (methylation percentage of 16%), NCI-H1395 (methylation percentage of 11%), and NCI-H522 (methylation percentage of 6%) in lung adenocarcinoma cell line. That is, according to the result judgment method given in example 1, all the lung squamous cancer cell line test results were "hypermethylation", and all the lung adenocarcinoma cell line test results were "unmethylated".
Using the catalyst of example 1 "Primer sets 2 and 3"a technical effect substantially in accordance with the present embodiment can be obtained.
Example 3 detection of the methylation State of the MUC22 Gene in Lung squamous carcinoma and Lung adenocarcinoma clinical tissue specimens
This example will be described as example 1 "Primer set 1"is described as an example.
1. Tissue DNA extraction procedure
(1) Frozen tissues (lung squamous carcinoma and lung adenocarcinoma clinical tissue specimens) in a liquid nitrogen tank are taken, 200mg to 1g are cut, and the tissue specimens are ground by a precooled mortar.
(2) Adding 1mL of DNA extracting solution and 100 mu L of 10mg/mL proteinase K into each 100mg of tissue specimen, gently blowing, beating, uniformly mixing, placing in a 50 ℃ constant-temperature water bath kettle, digesting overnight, and reversely mixing for several times within 1h from the beginning of digestion.
(3) The subsequent steps are the same as the cell line DNA extraction protocol (see step 1 of example 2).
2. Tissue DNA sample sulfurization
See example 2, step 3.
3. Methylation-specific PCR (MSP)
(1) MSP detection primer sequence
Methylation primer sequences:
an upstream primer: 5'-TTAAAAATATAAAAATTAGTTGGCGT-3' (SEQ ID No.4),
a downstream primer: 5'-TAAAACGAAATCTCGCTCTATCG-3' (SEQ ID No. 5).
Unmethylated primer sequence:
an upstream primer: 5'-TTAAAAATATAAAAATTAGTTGGTGT-3' (SEQ ID No. 6);
a downstream primer: 5'-TAAAACAAAATCTCACTCTATCAAC-3' (SEQ ID No. 7).
(2) Reaction system for PCR amplification
As shown in table 2.
TABLE 2 MSP amplification reaction System
Figure BDA0002848559500000131
Note: 2 XZymoTaq PreMix: zymo Research, Inc., USA, Cat No. E2004
(3) The PCR reaction conditions were as follows:
5min at 95 ℃; 35 cycles of 95 ℃/30s, 60 ℃/30s, 72 ℃/30 s; 7 min at 72 ℃; 4 ℃ is prepared.
(4) Gel electrophoresis: MSP products were electrophoresed on a 2% agarose gel.
4. Results
The results show that: the MSP method detects the methylation state of the MUC22 gene promoter region shown in SEQ ID No.1 in clinically confirmed squamous cell lung carcinoma (n-24) and adenocarcinoma (n-23) tissue specimens, as shown in fig. 1D, tissue samples were analyzed by agarose gel electrophoresis for MSP: according to the result judgment standard given in example 1, the methylated primers amplified 164bp amplification products, while the unmethylated primers amplified no 164bp amplification products, and the specimen was judged as "hypermethylated"; the methylation primers and the non-methylation primers are amplified, 164bp amplification products exist, and the specimen is judged to be partially methylated. The non-methylated primer is amplified, 164bp of amplification products exist, the methylated primer is amplified, 164bp of amplification products do not exist, and the specimen is judged to be unmethylated. As shown in fig. 1E, the promoter region of MUC22 gene shown in SEQ ID No.1 is 96% methylated (i.e., "hypermethylated" + "partial methylation") in squamous cell lung carcinoma tissue; more than 70% of the lung adenocarcinoma tissue specimens show a non-methylation state (namely, "non-methylation"), which indicates that the MUC22 gene promoter region shown in SEQ ID No.1 has a methylation degree which is remarkably different between squamous cell lung carcinoma patients and lung adenocarcinoma patients.
The results indicate that the methylation of the MUC22 gene promoter region shown in SEQ ID No.1 can be used as a molecular marker for early diagnosis and prognosis of the non-small cell lung cancer.
Using the catalyst of example 1 "Primer sets 2 and 3"a technical effect substantially in accordance with the present embodiment can be obtained.
Table 3 shows basic information of patients with squamous cell lung carcinoma (n-24) and adenocarcinoma of lung (n-23) according to the present example.
TABLE 3 clinical information of non-small cell Lung cancer samples
Figure BDA0002848559500000132
Figure BDA0002848559500000141
Figure BDA0002848559500000151
Using the catalyst of example 1 "Primer sets 2 and 3"a technical effect substantially in accordance with the present embodiment can be obtained.
Example 4 DNA methylation inhibitors can reverse MUC22 expression silencing in non-Small cell Lung cancer cells
First, experiment method
1. Cell RNA extraction and reverse transcription process
(1) DNA methylase inhibitor 5-Aza-2 '-Deoxycytidine (5-Aza, 5-Aza-2' -Deoxycytidine, Sigma-Aldrich, USA, Cat. No. 189825) was dosed: when the cell (NCl-H1703, NCl-H2170 or SK-MES-1) growth density reached 30%, 5. mu.M (final concentration) 5-Aza was added for treatment, the medium was changed every 24 hours, and 5-Aza (final concentration 5. mu.M) was added for 96 hours.
(2) Selecting cells with good growth state according to 1mL/106Trizol reagent (Invitrogen, USA, cat # 15596026) was added to each cell. After the mixture was allowed to stand at room temperature for 10min and sufficiently lysed, the mixture was extracted with chloroform, and 0.2mL of chloroform was added to 1mL of Trizol.
(3) Shaking vigorously for 15s, standing at room temperature for 5min, 12000g, and centrifuging at 4 deg.C for 15 min.
(4) The upper layer colorless liquid after layering was transferred to a new centrifuge tube, precipitated with pre-cooled isopropanol, 0.5mL isopropanol per 1mL Trizol and left on ice for 20 min. 12000g, centrifuge at 4 ℃ for 10min, and discard the supernatant.
(5) The precipitate was washed with pre-cooled 75% ethanol, 1mL of Trizol was added with 1mL of 75% ethanol, 7500g, centrifuged at 4 ℃ for 5min, and the supernatant was discarded. Drying, adding appropriate amount of DEPC-H2O dissolution, 0.8% agarose gel electrophoresis confirmation, NanoDrop determination of RNA concentration, preservation at-80 ℃.
(6) 1.0. mu.g of RNA was reverse-transcribed into cDNA using TransScript II First-Strand cDNA Synthesis SuperMix kit (cat # AH301-02) produced by Beijing Quanyu Biotech Co: mu.L of adsorbed Oligo (dT)20, 10. mu.L of 2 XTS Reaction Mix and 1. mu.L of RT/RI Enzyme Mix were added to DEPC-H2O to 20. mu.L. Under the reaction condition, the cDNA obtained by reverse transcription is placed at the temperature of minus 20 ℃ for 30min at the temperature of 42 ℃ and 5min at the temperature of 85 ℃.
2、qPCR Qualitative
(1) qPCR primer sequences:
the RT-qPCR primer sequence covers the amplification product 239bp from +19739 to +19877bp (positioned in the reading frame of MUC22 gene) of the transcription start site of the MUC22 gene.
An upstream primer: 5'-TGGCCTCTACTTCGGCCTTA-3', respectively;
a downstream primer: 5'-GGTGGAGGCCACGATAGTTT-3' are provided.
(2) Reaction system for qPCR amplification
As shown in table 4.
TABLE 4 reaction System for qPCR amplification
Figure BDA0002848559500000161
Note: 2 × SYBR-Green: zymo Research, Inc., USA, Cat No. E2004.
(3) The PCR reaction conditions were as follows:
2min at 50 ℃, 10min at 95 ℃ and 15s at 95 ℃; 1min at 60 ℃; collecting fluorescence in 40 cycles; the dissolution curves were prepared at 95 ℃ for 15s, 60 ℃ for 1min, 95 ℃ for 15s, and 60 ℃ for 15 s. Experimental results 2-ΔΔCtThe method analyzes data, and a dissolution curve ensures the specificity of the product.
Second, results and analysis
The results show that: the expression silencing of MUC22 in lung squamous carcinoma cells can be reversed by using the apparent drug DNA methylation inhibitor 5-Aza-2 '-deoxycytidine (5-Aza-2' -deoxycytidine, 5-Aza), which indicates that the expression silencing of MUC22 in lung squamous carcinoma tissue cells is apparently regulated, and is shown as F in figure 1.
Since the apparent drug DNA methylation inhibitor is applied to clinical tumor treatment, MUC22 gene methylation can be a potential molecular target for treating non-small cell lung cancer.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
<110> the university of capital medical department affiliated to the Beijing thoracic hospital; research institute of tuberculosis and breast tumor in Beijing
<120> molecular typing marker and kit for early diagnosis, treatment and prognosis detection of non-small cell lung cancer
<130> GNCLN203014
<160> 15
<170> PatentIn version 3.5
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atggaaggta ccattgagac tccttgtgat gaaaataact ctacttgaat tgggggtaaa 180
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Claims (10)

  1. Use of a modified sequence apparent from the MUC22 gene as a molecular marker in any one of:
    p1, preparing a product for molecular typing of squamous cell lung carcinoma and lung adenocarcinoma in the non-small cell lung cancer, or molecular typing of squamous cell lung carcinoma and lung adenocarcinoma in the non-small cell lung cancer;
    p2, preparing a product for diagnosing, targeted therapy monitoring and/or medication prognosis evaluation of the non-small cell lung cancer, or diagnosing, targeted therapy monitoring and/or medication prognosis evaluation of the non-small cell lung cancer;
    p3, preparing a product for tumor heterogeneity analysis of non-small cell lung cancer, or performing tumor heterogeneity analysis of non-small cell lung cancer.
  2. 2. Use of a substance for detecting an apparent modification of the MUC22 gene in any one of:
    p1, preparing a product for molecular typing of lung squamous carcinoma and lung adenocarcinoma in non-small cell lung cancer;
    p2, preparation of products for diagnosis, targeted therapy monitoring and/or drug-induced prognosis evaluation of non-small cell lung cancer;
    p3, and preparing products for tumor heterogeneity analysis of non-small cell lung cancer.
  3. The application of the MUC22 gene appearance modification sequence as a drug action target in preparing products for treating and/or preventing non-small cell lung cancer.
  4. 4. Use according to any one of claims 1 to 3, characterized in that: the method of claims 1 and 3 wherein the MUC22 gene epigenetic modification is a MUC22 gene methylation modification; the method of claim 2 wherein said MUC22 gene epigenetic modification is a MUC22 gene methylation modification;
    further, in claims 1 and 3, the MUC22 gene methylation modification sequence is a methylation modification sequence of a promoter region or a regulatory region of the MUC22 gene; the methylation modification of the MUC22 gene of claim 2 being a methylation modification of the promoter region or the regulatory region of the MUC22 gene.
  5. 5. Use according to claim 4, characterized in that: the promoter region or the regulatory region of the MUC22 gene is a DNA sequence located from 1281bp upstream to 1680bp downstream of the transcription start site of the MUC22 gene;
    further, the nucleotide sequence of the promoter region of the MUC22 gene is shown as SEQ ID No.1 or SEQ ID No. 2; the nucleotide sequence of the regulatory region of the MUC22 gene is shown as SEQ ID No. 3;
    and/or
    The means for detecting methylation modification of MUC22 gene is the primer pair or primer pair group or kit of any one of claims 7-9.
  6. 6. A molecular marker having a function as any one of P1 to P3, wherein: the molecular marker is an MUC22 gene appearance modification sequence;
    p1, carrying out molecular typing on lung squamous carcinoma and lung adenocarcinoma in the non-small cell lung cancer;
    p2, diagnosis, targeted therapy monitoring and/or prognostic evaluation of drugs for non-small cell lung cancer;
    p3, carrying out tumor heterogeneity analysis on the non-small cell lung cancer;
    further, the MUC22 gene appearance modification sequence is a MUC22 gene methylation modification sequence;
    still further, the methylation modification sequence of the MUC22 gene is the methylation modification sequence of the promoter region or the regulatory region of the MUC22 gene;
    furthermore, the promoter region or the regulatory region of the MUC22 gene is a DNA sequence located from 1281bp upstream to 1680bp downstream of the transcription start site of the MUC22 gene;
    more specifically, the nucleotide sequence of the promoter region of the MUC22 gene is shown as SEQ ID No.1 or SEQ ID No. 2; the nucleotide sequence of the regulatory region of the MUC22 gene is shown as SEQ ID No. 3.
  7. 7. A primer pair or primer pair group having a function as any one of P1 to P3 as follows: the primer pair is a methylated primer pair or an unmethylated primer pair; the primer pair group consists of the methylated primer pair and the corresponding unmethylated primer pair; the methylated primer pair and the corresponding unmethylated primer pair are both designed according to a methylation modification sequence of the MUC22 gene;
    further, the MUC22 gene methylation modification sequence is a methylation modification sequence of a promoter region or a regulatory region of the MUC22 gene;
    furthermore, the promoter region or the regulatory region of the MUC22 gene is a DNA sequence located from 1281bp upstream to 1680bp downstream of the transcription start site of the MUC22 gene;
    more specifically, the nucleotide sequence of the promoter region of the MUC22 gene is shown as SEQ ID No.1 or SEQ ID No. 2; the nucleotide sequence of the regulatory region of the MUC22 gene is shown as SEQ ID No. 3;
    p1, carrying out molecular typing on lung squamous carcinoma and lung adenocarcinoma in the non-small cell lung cancer;
    p2, diagnosis, targeted therapy monitoring and/or prognostic evaluation of drugs for non-small cell lung cancer;
    p3, and carrying out tumor heterogeneity analysis on the non-small cell lung cancer.
  8. 8. Primer pair or primer pair group according to claim 7, characterized in that: the methylation primer pair consists of two single-stranded DNA molecules shown by SEQ ID No.4 and SEQ ID No. 5; the corresponding non-methylation primer pair consists of two single-stranded DNA molecules shown by SEQ ID No.6 and SEQ ID No. 7; or
    The methylation primer pair consists of two single-stranded DNA molecules shown in SEQ ID No.8 and SEQ ID No. 9; the corresponding non-methylation primer pair consists of two single-stranded DNA molecules shown in SEQ ID No.10 and SEQ ID No. 11; or
    The methylation primer pair consists of two single-stranded DNA molecules shown in SEQ ID No.12 and SEQ ID No. 13; the corresponding non-methylated primer pair consists of two single-stranded DNA molecules shown in SEQ ID No.14 and SEQ ID No. 15.
  9. 9. A kit comprising a primer pair or a primer pair set according to claim 7 or 8;
    further, the kit also contains a methylation control DNA template and/or an unmethylated control DNA template for MSP amplification;
    wherein the methylation control DNA template for MSP amplification is DNA which is subjected to sulfurization modification and shows hypermethylation of more than 90%; the non-methylated control DNA template for MSP amplification is normal tissue cell DNA;
    still further, the kit may further comprise deionized water as a negative system control and/or PCR reaction reagents required for performing the MSP amplification.
  10. 10. Any one of the following methods:
    the method A comprises the following steps: a method for detecting methylation modification of a promoter region or a regulatory region of MUC22 gene, comprising the steps of: sulfurizing and modifying a test DNA sample containing the promoter region or the regulatory region of the MUC22 gene, performing MSP amplification by using the methylation primer pair and the corresponding non-methylation primer pair of claim 7 or 8 as a template, and determining the methylation modification of the promoter region or the regulatory region of the MUC22 gene in the test DNA sample according to the following (A1) or (A2) according to the amplification result:
    (A1) and (3) judging according to whether a target product is obtained: if the methylation primer pair is adopted for amplification to obtain a target product and the non-methylation primer pair is not adopted for amplification to obtain the target product, the target product is regarded as the promoter region or the regulation region of the MUC22 gene in the DNA sample to be detected to be hypermethylated; if the methylation primer pair is adopted for amplification to obtain a target product and the non-methylation primer pair is also adopted for amplification to obtain the target product, the target product is regarded as the partial methylation of the promoter region or the regulatory region of the MUC22 gene in the DNA sample to be detected; if the target product is obtained by adopting the non-methylation primer pair for amplification and the target product is obtained by adopting the methylation primer pair for non-amplification, the target product is regarded as the promoter region or the regulation region of the MUC22 gene in the DNA sample to be detected to be unmethylated;
    (A2) the MSP amplification is methylation specific fluorescent quantitative PCR, and is carried out by 2-ΔΔCtCalculating the methylation percentage of the promoter region or the regulatory region of the MUC22 gene in the DNA sample to be detected; if the methylation percentage is more than or equal to 60, the methylation is regarded as high methylation; if the percentage methylation is 20-60% (excluding the end point), it is considered as "partial methylation"; if the percentage methylation is less than or equal to 20%, the product is regarded as "unmethylated";
    further, the promoter region or the regulatory region of the MUC22 gene is a DNA sequence located from 1281bp upstream to 1680bp downstream of the transcription start site of the MUC22 gene;
    furthermore, the nucleotide sequence of the promoter region of the MUC22 gene is shown as SEQ ID No.1 or SEQ ID No. 2; the nucleotide sequence of the regulatory region of the MUC22 gene is shown as SEQ ID No. 3;
    the method B comprises the following steps: a method of distinguishing between a squamous lung carcinoma sample and a lung adenocarcinoma sample in non-small cell lung cancer comprising the steps of: detecting methylation modification of a promoter region or a regulatory region of MUC22 gene of a test sample according to the method A, and determining whether the test sample is a squamous cell lung carcinoma sample or a adenocarcinoma lung carcinoma sample according to the following steps: the test sample that is "hypermethylated" or "partially methylated" of the promoter region or regulatory region of the MUC22 gene is or is candidate for being a squamous cell lung carcinoma sample; the test sample in which the promoter or regulatory region of the MUC22 gene is "unmethylated" is or is candidate for being a lung adenocarcinoma sample.
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