CN113881777A - Kit applied to environmental pollution carcinogenic risk assessment - Google Patents

Abstract

The invention discloses a kit applied to environmental pollution carcinogenic risk assessment. The invention specifically discloses a specific methylation primer pair for detecting the methylation state or level of a CpG island in a MUC17 gene promoter region, and an application, a kit and a method thereof. The primer pair consists of a primer F3(SEQ ID No.8) and a primer R3(SEQ ID No. 9). The kit for detecting the DNA sequence methylation in the MUC17 gene can specifically detect the DNA methylation level related to the MUC17 gene, is conveniently and quickly used for aspects of environmental pollution carcinogenic risk assessment, early lung cancer screening, lung cancer risk prediction and the like, and the detection result can provide an evaluation method for early tumor diagnosis.

Description

Kit applied to environmental pollution carcinogenic risk assessment

Technical Field

The invention belongs to the technical field of molecular diagnosis, and particularly relates to a methylation marker kit applied to environmental pollution carcinogenic risk assessment.

Background

Lung Cancer is a global high-Incidence tumor, which is the first of malignant tumors in China, and the number of new lung Cancer cases is 220 million and 180 million in2020 world (Sun H, Ferlay J, Siegel RL, et al. Global Cancer statics 2020: GLOBOCAN Estimates of existence and Mortality Worldwide for36Cancers in 185countries. CA Cancer J Clin 2021, 71(3): 209) 249.). Despite the significant improvement in survival of lung cancer patients over the past decade through early diagnosis and cancer treatment by biomarkers, the global morbidity and mortality of lung cancer has further worsened with the increase of many risk factors. Among these, environmental air pollution and exposure to other respirable environmental pollutants are considered to be major factors in Lung Cancer incidence and mortality (Ettingger DS, Wood DE, Aggarwal C, et al. NCCN Guidelines instruments: Non-Small Cell Lung Cancer, Version 1.2020.Journal of the National Comprehensive Cancer Network: JNCCCN 2019, 17(12): 1464. outer 1472Turner MC, Andersen ZJ, Baccarlli A, et al. outer air pollution and Cancer: An overview of the current and clinical health indications. CA Cancer J Clin.). Research shows that the chronic lung inflammation induced by environmental carcinogens is closely related to the occurrence and development of lung cancer.

Many chemical and environmental factors can cause carcinogenesis in human bronchial epithelial cells. Phthalic Acid Esters (PAEs) are a class of plasticizers and solvents used in polyvinyl chloride (PVC) plastics, and are widely used in novel synthetic materials to improve mechanical properties such as flexibility and stretchability of the materials. PAEs in these new synthetic materials slowly migrate to air, particulate matter, dust and indoor surfaces and enter the body by mouth-in, inhalation and skin contact. PAEs are considered to be a class of genetically toxic organic pollutants that can cause reproductive toxicity, endocrine disorders and canceration (Li PH, Jia HY, Wang Y, et al. Characterisation of PM (2.5) -bound Pharmaceutical Acid Esters (PAEs) at a geographic background site in northern China: Long-range transport and risk assessment. the Science of the total environment 2019, 659: 140-. It has been reported that PAEs act as allergens to negatively regulate the immune system, leading to inflammation and asthma, and further possibly to develop respiratory diseases such as lung cancer (Guo H, Song N, Wang D, Ma J, Jia Q.A modulation approach for respiratory organic diseases: Application to soluble phase microorganisms of phthalate esters. Talanta 2019, 198: 277-) 283.).

DBP and DEHP are two major phthalate species, associated with micro-plastic contamination in the environment. Wherein di (2-ethylhexyl) phthalate (DEHP) is mainly used for PVC products and is taken as a representative of PAEs macromolecular compounds; di-n-butyl phthalate (DBP) is a low molecular PAEs, and is widely included as a soluble plasticizer in volatile substances such as varnish, cosmetics, perfume, and the like. Because the PAEs are not covalently bound to the polymer matrix, after a slight physicochemical process such as heating, ionization, acid treatment, etc., the PAEs can be released by evaporation or leaching from the plastic into the surrounding environment, causing environmental pollution. Meanwhile, there is also a research report on the contamination of phthalate ester in indoor dust. Due to the widespread use of PAEs and their ubiquitous presence in the environment, humans inevitably come into contact with phthalates. In recent years, the environmental health hazards caused by the compounds are receiving wide attention in the fields of environmental science and public health. The detected PAEs in the environment are more and more in types and higher in concentration, so that the method has important practical significance for associating the environmental pollution carcinogenic risk with human health and evaluating and predicting the environmental pollution carcinogenic risk.

Environmental stress factors (physical, chemical stimulation, etc.) can induce cellular DNA damage, and DNA damage sites are recognized by related damage receptors to influence corresponding repair signal pathways to carry out DNA repair. Current research shows that environmental pollutants can interfere with Epigenetic regulation patterns, and DNA damage repair participated by Epigenetic regulation pathways such as DNA methylation, chromatin remodeling, miRNA, acetylation and ubiquitination plays an important regulation role in the occurrence and development process of diseases and is one of the basic factors for triggering diseases (Kim D, Lee YS, Kim DH, Bae SC. Lung Cancer Staging and Associated Genetic and Epigenetic events. molecules and cells 2020, 43(1): 1-9.). The molecular basis of the lung cancer malignant progression caused by the air pollutants closely related to the occurrence and development of the lung cancer is deeply researched, so that the epigenetic biomarker can be used for early diagnosis of the tumor, screening of susceptible people and evaluation of the risk degree of chemicals, and the reversibility of the epigenetic gene also provides a target point for early prevention of the tumor. Therefore, the development of new markers for biological monitoring of human exposure to environmental factors has important scientific significance and clinical application value.

Disclosure of Invention

The invention aims to provide a kit applied to the assessment of the carcinogenic risk of environmental pollution and/or a kit, a primer pair, a method and application for detecting the methylation level of a MUC17 gene promoter. The technical problem to be solved is not limited to the technical subject as described, and other technical subject not mentioned herein may be clearly understood by those skilled in the art through the following description.

In order to achieve the above object, the present invention firstly provides a kit for assessing the risk of carcinogenesis due to environmental pollution, the kit comprising a primer pair for detecting the methylation level of the MUC17 gene promoter.

Further, the primer pair is a specific methylation primer pair for detecting the methylation level of the CpG island in the promoter region of the MUC17 gene.

The CpG island is positioned from 759bp (-759bp) of the upstream sequence of the MUC17 gene transcription start site (TSS site) to 321bp (-321bp) of the upstream sequence thereof. The nucleotide sequence of the CpG island can be SEQ ID No. 3.

In the kit, the primer pair consists of a primer F3 and a primer R3, the primer F3 is a single-stranded DNA with a nucleotide sequence of SEQ ID No.8, and the primer R3 is a single-stranded DNA with a nucleotide sequence of SEQ ID No. 9.

In the kit, the kit further comprises one or more of Taq DNA polymerase, dNTP and PCR buffer solution required by PCR amplification.

In the kit, the kit further comprises bisulfite.

Herein, the bisulfite may be sodium bisulfite (NaHSO)₃)。

The kit may also include a DNA methylation positive control and a negative control.

The kit can also comprise a detection reagent of an internal reference gene, and the internal reference gene can be a GAPDH gene and/or a beta-actin gene.

The various reagent components of the kit may be present in separate containers or may be pre-combined in whole or in part into a reagent mixture.

Primer pairs described herein are also within the scope of the invention.

The invention also provides any one of the following applications of the primer pair:

A1) the application of the primer pair in detecting the methylation level of the MUC17 gene promoter;

A2) the application of the primer pair in preparing a product for detecting the methylation level of the MUC17 gene promoter;

A3) the application of the primer pair in preparing a product for evaluating the carcinogenic risk of environmental pollution;

A4) the application of the primer pair in preparing a product for screening lung cancer;

A5) the application of the primer pair in preparing a product for predicting the risk of lung cancer.

In the above application, the product may be a reagent and/or a kit.

In the above application, the lung cancer may be non-small cell lung cancer.

The invention also provides a method for detecting the methylation level of the MUC17 gene promoter, which comprises the following steps:

B1) extracting DNA of a sample to be detected;

B2) subjecting said DNA to bisulfite conversion;

B3) carrying out PCR amplification by using the primer pair by using the converted DNA as a template to obtain a PCR amplification product;

B4) and carrying out methylation detection on the PCR amplification product.

In the above method, the sample to be tested may be a cell or a tissue.

Further, in the above method, the primer pair consists of a primer F3(SEQ ID No.8) and a primer R3(SEQ ID No. 9).

Further, B3) the reaction components of the PCR amplification comprise an upstream primer F3, a downstream primer R3, a DNA template, a PCR amplification enzyme, dNTPs, 10 XPCR Buffer and ddH₂O。

Further, B3) the reaction system for PCR amplification (8.00 μ L) was: ddH₂O4.90. mu.L, 10 XPCR Buffer 0.80. mu. L, dNTPs (10. mu.M), 0.80. mu. L, PCR amplimer (5U/. mu.L), 0.20. mu.L of primer F3(10 nmol/. mu.L), and 1.00. mu.L of primer R3(10 nmol/. mu.L) 0.20. mu. L, DNA template.

Further, B3) the reaction conditions for the PCR amplification are: 4min at 94 ℃; 94 ℃ 20sec,56 ℃ 30sec,72 ℃ 1min,45 cycles; 3min at 72 ℃.

Bisulfite can convert unmethylated cytosine into uracil, and then detect the change of DNA methylation level by the techniques of PCR, sequencing, chip hybridization and the like. In one embodiment of the invention, the bisulfite salt is sodium bisulfite (NaHSO)₃)。

In the above method, the methylation detection of the PCR amplification product in B4) further comprises an agarose gel electrophoresis detection step, a Shrimp Alkaline Phosphatase (SAP) reaction step, a T-cut/RNase A digestion reaction step, a resin purification step, and a chip spotting step.

In one embodiment of the present invention, the SAP reaction system (7.00. mu.L) is: RNase-free ddH₂O1.70. mu. L, SAP enzyme 0.30. mu. L, PCR amplification product 5.00. mu.L; the SAP reaction conditions were: 20min at 37 ℃ and 5min at 85 ℃;

the T cut/RNase A digestion reaction system (7.00. mu.L) was: RNase-free ddH₂O3.21. mu.L, 5 XT 7 Polymerase Buffer 0.89. mu.L, T Cleavage Mix 0.22. mu.L, 100mM Dithiothreitol (DTT) 0.22. mu. L, T7 RNA&DNA Polymerase 0.40. mu.L, RNase A0.06. mu.L and PCR/SAP mix 2.00. mu.L; the digestion reaction conditions of the T cutting/RNase A are as follows: incubating for 3h at 37 ℃;

the resin purification step comprises: the resin was uniformly filled in an 384/6MG Dimple plate and left to dry for 10 minutes. To each well of 384 sample plates was added 16. mu.l of water. The 384 sample plate was gently snapped onto the sample plate, flipped over and tapped to drop the resin into each well of the sample plate. Sealing the 384 sample plate by using a sealing film, placing the sealed plate in a turnover plate centrifuge, rotating at room temperature, and uniformly mixing for 30 minutes;

the chip sample application step is as follows: the MassARRAY nanodispenseRS 1000 sample applicator is started, and the product after resin purification is transferred to 384-well

On the bioarray, the spotted SpectroCHIP chip was analyzed by MALDI-TOF. Detection result is EpitYPER^TMSoftware obtains raw data and a circular diagram and checks the integrity and accuracy of the data file. And storing the result in a corresponding storage medium, submitting the result to a biological information room for analysis, and analyzing and calculating to obtain the methylation level of the DNA of the sample to be detected.

Further, PCR amplification using the above methylation primers (primer F3 and primer R3) and the DNA modified by sulfurization as a template yielded a 439bp fragment, which was detected by methylation using the Agena MassArray system. The methylated primer has Tm 59.99 of the upstream primer and GC content of 40.00 percent, and the Tm 60.32 of the downstream primer and GC content of 32 percent. Detection result is EpitYPER^TMThe software obtains original data, and the original data is obtained by comparing the areas of the peak containing G and the peak containing A, and the methylation degree and the non-methylation degree of the sample to be detected are calculated, and belong to the relative quantitative ratio of the two. If the value detected in the table is 0.43, this means that the degree of methylation of the corresponding methylation site in the sample is 43%. Taking the average of methylation values of all sites in the region, and if the methylation percentage is more than or equal to 60, regarding the methylation 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".

The invention also provides the kit and/or any one of the following applications of the method:

C1) the application of the promoter methylation level of the MUC17 gene is detected;

C2) the application in the evaluation of the carcinogenic risk of environmental pollution.

The above uses and methods may be for disease diagnosis purposes, disease prognosis purposes and/or disease treatment purposes, or they may be for non-disease diagnosis purposes, non-disease prognosis purposes and non-disease treatment purposes; their direct purpose may be to obtain information on the outcome of a disease diagnosis, prognosis of a disease and/or intermediate outcome of a disease treatment, and their direct purpose may be non-disease diagnosis, non-disease prognosis and/or non-disease treatment.

The research of the invention finds that the DNA hypermethylation of the MUC17 promoter region can influence the expression of MUC 17. Environmental pollutants can reduce the expression of MUC17 of the respiratory epithelial cells, and the expression silencing of MUC17 in the respiratory epithelial cells can be reversed by using a DNA methylation inhibitor, which indicates that the expression silencing of MUC17 genes in the cells is related to the hypermethylation modification of the DNA of the genes. Therefore, the DNA methylation modification of the MUC17 gene can be used as a molecular marker and a drug action target for early diagnosis of the development of the non-small cell lung cancer.

The invention provides a specific methylation primer pair for detecting the methylation state or level of a CpG island in a promoter region of a MUC17 gene. Using the methylation primer pair, DNA after sulfuration modification is used as a template for PCR amplification, a 439bp fragment is obtained, and methylation detection is carried out by using an Agena MassArray system. The upstream primer of the methylated primer pair has Tm 59.99 and GC content of 40.00 percent, and the downstream primer has Tm 60.32 and GC content of 32 percent. Detection result is EpitYPER^TMThe software obtains original data, and the original data is obtained by comparing the areas of the peak containing G and the peak containing A, and the methylation degree and the non-methylation degree of the sample to be detected are calculated, and belong to the relative quantitative ratio of the two. If the value detected in the table is 0.43, this means that the degree of methylation of the corresponding methylation site in the sample is 43%.

The environmental pollutants can inhibit the expression of MUC17 which is an important component of human mucosal barrier through a methylation pathway, and the low expression of MUC17 can promote the malignant transformation of tumors. The advantage of using MUC17 methylation as an early marker is that the change in methylation proceeds progressively, based first on the predominance of methylation changes, which if diagnosed early can be reversed by methylation inhibitors. Secondly, modifications on DNA are an early event with respect to RNA transcription and protein translation, and if detected, can intervene early, allowing RNA transcription and protein translation to proceed normally. The MUC17 gene can be used as a molecular marker for diagnosing and detecting non-small cell lung cancer, and the molecular marker has the characteristics of strong sensitivity and high specificity; the kit based on the detection of the DNA sequence methylation in the MUC17 gene can specifically detect the DNA methylation level related to the MUC17 gene and can also specifically and quantitatively detect the DNA methylation level related to the MUC17 gene; therefore, the detection method and the kit can be conveniently and quickly used for the aspects of environmental pollution carcinogenic risk assessment, lung cancer risk prediction and the like, and the detection result can provide an evaluation method for the early diagnosis and detection of the non-small cell lung tumor.

Drawings

FIG. 1 is the analysis of MUC17 expression and biological function change after the respiratory tract epithelial cell BEAS-2B and the tumor cell A549 are treated by PAEs. FIG. 1A shows RT-qPCR analysis of mRNA expression of transmembrane mucins in BEAS-2B and A549 cells after DBP or DEHP exposure (1 μm, 60 h); FIG. 1B is the expression of MUC1 in BEAS-2B and A549 cells after immunofluorescence detection of DBP or DEHP exposure (1 μm, 60 h); FIG. 1, C is RT-qRCR to detect the expression of MUC17 at various time points (0, 12, 24, 36, 48 and 60h) after treatment of BEAS-2B and A549 cells with DBP or DEHP (1 μ M); d in FIG. 1 is RT-qRCR detection of expression of DBP or DEHP (60h) in BEAS-2B and A549 cells of MUC17 after treatment at different doses (0, 100nM, 1 μ Μ, 10 μ Μ, 100 μ Μ and 1 mM); e in figure 1 and F in figure 1 shows that A549 cells are stably transfected with MUC17 (upper) or shMUC17 (lower) and are treated with DBP or DEHP (1 mu m, 60h), and RT-qPCR is expressed; MTT indicates the viability of the cells.

FIG. 2 shows the analysis of the methylation of MUC17 gene in the respiratory epithelial cell BEAS-2B and the tumor cell A549. FIG. 2A is a schematic diagram of the MUC17 gene structure, wherein the amplification region of MassARRAY is marked, and each vertical line represents a CpG site. These numbers indicate the position relative to the Transcription Start Site (TSS). In FIG. 2, B is the PCR quality control chart after primer amplification. In FIG. 2, C is the percentage of methylation at each CpG site detected after MassARRAY detection. FIG. 2D shows RT-qPCR analysis of MUC17 expression in BEAS-2B and A549 cells treated with epigenetic modulating agent (5-Aza). 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.

FIG. 3 is the analysis of the relationship between the respiratory tract epithelial cell BEAS-2B and the tumor cell A549 treated with PAEs and methylase. A in FIG. 3 and B in FIG. 3 are the expression of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) and UHRF1 mRNA measured by RT-qPCR of BEAS-2B and A549 cells treated with DBP or DEHP (1 μm, 60 h); C-E in FIG. 3 shows that A549 cells were treated with DBP or DEHP after being stably transfected with shDNMT1 or a non-specific control (shNC) (1 μm, 60 h). RT-qPCR analysis of DNMT1 and MUC17 mRNA expression (C); detecting cell proliferation (D) by MTT method; cell invasion was detected by the Transwell method (E).

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.

Respiratory epithelial cells (human lung normal epithelial cells) BEAS-2B and human lung adenocarcinoma cells A549 (also called non-small cell lung cancer cell line A549) in the following examples were purchased from national biomedical experimental cell resource libraries and cultured and passaged under normal conditions in the applicant unit laboratories.

Example 1 expression and biological function of MUC17 Gene in Lung cancer cells

1、RT-PCR

1.1 cellular RNA extraction and reverse transcription Process

(1) The cell RNA extraction process comprises selecting cells with good growth state according to 1mL/10⁶Trizol reagent (Invitrogen, USA, cat # 15596026) was added to each cell.

(2) 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. Shaking vigorously for 15s, standing at room temperature for 5min, 12000g, and centrifuging at 4 deg.C for 15 min.

(3) 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.

(4) 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 ℃.

(5) 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 ℃.

1.2、qPCR

(1) qPCR primer sequences:

RT-qPCR uses an upstream primer: 5'-GGGCCAGCATAGCTTCGA-3', respectively; a downstream primer: 5'-GCTACAGGAATTGTGGGAGTTGA-3' are provided.

(2) The reaction systems for qPCR amplification are shown in table 1.

TABLE 1 reaction System for qPCR amplification

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; melting 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 melting curve ensures the specificity of the product.

3. Overexpression and interference with MUC17 Gene expression in A549 and BEAS-2B cells

Universal no-load plasmid pcDNA^TM3.1⁽⁺⁾Purchased from Thermo Fisher, usa, cat #: v79020.

The MUC17 overexpression sequence (MUC17 cDNA sequence) is 12435-13483 (Update Date8-Jul-2021) of GenBank Accession No. NM-001040105.1, and is synthesized from the Shanghai Jikai gene.

Universal negative control pSilencer^TM3.1-H1 neo distensiled, available from Thermo Fisher, usa under the trade designation: AM 5770.

The sequence of the MUC17 interference shRNA recognition region is designed as follows: 5'-UAUGGUUGGAGCUGAGAAU-3', respectively;

non-small cell lung cancer cell line a549 was selected for stable transfection. Inoculating cells into a 6-hole cell culture plate, adding 200 mu L of DMEM into 20nM plasmid expressing interference shRNA, plasmid expressing unloaded shRNA, plasmid expressing MUC17 and control plasmid expressing MUC17 when the cells grow to 60% of fusion degree, mixing uniformly, and standing at room temperature for 5 min. Then, 200. mu.L of DMEM was added to 6. mu.L of Lipofectamine2000 (Thermo Fisher Co., USA, Cat. No.: 11668019), and the mixture was mixed and left at room temperature for 5min to obtain Lipofectamine2000 diluted solution. 200mL Lipofectamine2000 diluent is added into 200 μ L plasmid solution, mixed evenly and placed for 20min at room temperature to obtain 400 μ L mixed solution. Adding 400 mu L of the mixed solution into a non-small cell lung cancer cell line A549 cell culture plate, carrying out serum-free culture at 37 ℃ for 8h, replacing 10% FBS DMEM, incubating at 37 ℃ for 24h, and detecting by RT-qPCR. The construction method of the overexpression plasmid and the interference slow plasmid comprises the following steps:

construction of overexpression plasmids: the MUC17 cDNA (12435-13483 (Update Date8-Jul-2021) of GenBank Accession No. NM-001040105.1) was cloned and constructed into expression vector pcDNA^TM3.1⁽⁺⁾The above. In addition, the empty vector plasmid was selected as a control for subsequent control experiments. The constructed cell lines were designated MUC17 cell line (transfected with a plasmid overexpressing MUC 17) and Vector cell line (transfected with a control plasmid overexpressing MUC 17), respectively. The overexpression plasmid was synthesized by the Shanghai Jikai Gene company.

Construction of an interference plasmid: cloning and constructing the coding DNA molecule of the interference shRNA and the coding RNA molecule of the no-load shRNA contrast into a plasmid vector pSilencer^TM3.1-H1, respectively obtaining a plasmid for expressing the interference shRNA and a plasmid for expressing the idle shRNA, and using the plasmids to transfect cells to establish a cell line, wherein the constructed cell line is respectively named as an shMUC17 cell line (the plasmid for transfectioning and expressing the interference shRNA) and an shNC cell line (the plasmid for transfectioning and expressing the idle shRNA). The interfering plasmid was synthesized by the Shanghai Jikai Gene company.

4. Cell biological function experiment

The test cell lines were: MUC17 cell line, Vector cell line, shMUC17 cell line, shmnc cell line, MUC17+ DBP, MUC17+ DEHP, Vector + DBP, Vector + DEHP, shMUC17+ DBP, shMUC17+ DEHP, shmnc + DBP, shmnc + DEHP.

Wherein: MUC17+ DBP represents a DBP (1. mu.M, 24h) treated MUC17 cell line; MUC17+ DEHP represents DEHP (1 μ M, 24h) treated MUC17 cell line; vector + DBP represents DBP (1. mu.M, 24h) treated Vector cell line; vector + DEHP represents DEHP (1. mu.M, 24h) treated Vector cell line; shMUC17+ DBP represents a DBP (1. mu.M, 24h) treated shMUC17 cell line; shMUC17+ DEHP represents DEHP (1 μ M, 24h) treated shMUC17 cell line; shNC + DBP denotes a DBP (1. mu.M, 24h) treated shNC cell line; shNC + DEHP represents DEHP (1. mu.M, 24h) treated shNC cell line.

(1) MTT (3- [4, 5-dimethylthiozol-2-yl ] -2, 5-diphenyltetrazolium bromide) assay

Digesting and counting the test cell line in logarithmic growth phase and good growth condition, adjusting the concentration of cell suspension, and making each hole have good qualityAdding 100 μ L of the suspension into a 96-well cell culture plate, laying 2000 cells in each well, setting 8 multiple wells, and continuously observing for 4 days; mu.L of MTT (Sigma, USA; cat # M2128) was added to each well at a daily timing so that the final concentration was 5. mu.g/mL, and after incubating for 4 hours, the medium was discarded. After 4 days, 150. mu.L of dimethyl sulfoxide (DMSO, Sigma, cat # D2650) was added to each well, and after gentle shaking in the dark for 10min, the absorbance of each well was measured at OD490nm (detection wavelength) in an ELISA detector, and 570nm was used as a reference wavelength to eliminate nonspecific absorption_490nm/OD_{570 nm}"to demonstrate cell viability, the data obtained were analyzed.

(2) Statistical analysis

The experiments were repeated at least three times. The results of the experiments were all measured by a two-sided t-test, and the results were expressed as mean ± sd,. p <0.05 for statistically significant differences, and 0.01 for statistically significant differences.

5. Results and analysis

The human lung adenocarcinoma cells A549 and the human lung normal epithelial cells BEAS-2B were set in the Control group (Control), DBP group (DBP) and DEHP group (DEHP).

Control group (Control): human lung normal epithelial cells BEAS-2B were cultured in DMEM medium (Control) at 37 ℃ in 5% CO₂Culturing for 60 hours in a constant temperature incubator, and collecting cells which are named as Control-BEAS-2B; placing human lung adenocarcinoma cell A549 in DMEM medium (Control) at 37 deg.C and 5% CO₂Culturing for 60 hours in a constant temperature incubator, and collecting cells which are named as Control-A549;

DBP group (DBP): placing human lung adenocarcinoma cell A549 in DMEM +1 μ M DBP medium (medium obtained by adding DBP to DMEM medium to DBP content of 1 μ M) at 37 deg.C and 5% CO₂Culturing for 60 hours in a constant temperature incubator, and collecting cells, wherein the cells are named as 1 mu M DBP-A549; placing human lung normal epithelial cell BEAS-2B in DMEM +1 μ M DBP medium (medium obtained by adding DBP to DMEM medium to DBP content of 1 μ M) at 37 deg.C and 5% CO₂Culturing for 60 hours in the incubator, collecting the cells, and naming the cells as1μM DBP-BEAS-2B；

DEHP group (DEHP): human lung adenocarcinoma cells A549 were cultured in DMEM + 1. mu.M DEHP medium (medium obtained by adding DEHP to a DEHP content of 1. mu.M to DMEM medium) for 60 hours, and the cells were collected and named as 1. mu.M DEHP-A549.

5.1RT-PCR detection of expression of transmembrane mucins in BEAS-2B and A549 cells

Total RNA of the above groups of cells was extracted, and the mRNA expression levels of 8 transmembrane mucins, MUC1, MUC12, MUC13, MUC16, MUC17, MUC20, MUC21 and MUC22, were determined by RT-PCR using the following primer pairs according to the method of step 1. Wherein the mRNA expression level of the corresponding protein in the control group is 1. Wherein, the specific primer pair for detecting MUC1 by RT-PCR is F: 5- 'TACAGCTACCACAGCCCCTA-3', R: 5- 'AGCTGGGCACTGAACTTCTC-3'; the specific primer pair for detecting MUC12 by RT-PCR is F: 5- 'CTCGTGTATGGGATCGTGGG-3', R: 5- 'AGCTCTGTGCCAGAGTCAAC-3'; the specific primer pair for detecting MUC13 by RT-PCR is F: 5- 'AAGAATGTGGAACCCGCCAT-3', R: 5- 'CCCGGAGGCCAGATCTTTAC-3'; the specific primer pair for detecting MUC16 by RT-PCR is F: 5- 'CACCTGGGATGTCCACCTTG-3', R: 5- 'CGACGGTTATAACTGCTGGTGGT-3'; the specific primer pair for detecting MUC17 by RT-PCR is F: 5- 'GGGCCAGCATAGCTTCGA-3', R: 5- 'GCTACAGGAATTGTGGGAGTTGA-3'; the specific primer pair for detecting MUC20 by RT-PCR is F: 5- 'CAAGATCACAACCTCAGCGA-3', R: 5- 'ACCTCCATTTTCACCTGCAC-3'; the specific primer pair for detecting MUC21 by RT-PCR is F: 5- 'TAGCACCTCTGCCAACACTG-3', R: the specific primer pair of 5- 'GGTCACGCTGGACCCT-3' and MUC22 is F: 5- 'TGGCCTCTACTTCGGCCTTA-3', R: 5- 'GGTGGAGGCCACGATAGTTT-3'. The result is shown as A in FIG. 1.

5.2 immunofluorescence staining to detect the expression of MUC17 in BEAS-2B and A549 cells

The above groups of cells were subjected to immunofluorescence staining. The result is shown as B in FIG. 1. The method comprises the following specific steps:

dropping the cells with good growth state on a cover glass, putting the cover glass into a 6-hole cell culture plate for culturing for 24h, washing with 1 × PBS, and adding 4% paraformaldehyde for fixing for 10 min. After washing with 1 XPBS for three times, 0.5% Triton X-100 was added and left at room temperature for 5 min. 1 XPBS washing for three times, 5min each time. Add appropriate volume of 5% BSA for blocking at room temperature for 1 h. Incubating the primary antibody: Anti-MUC17 (Sigma-Arich Co., USA, Cat: HPA031634, dilution ratio: 1: 25), at 4 deg.C overnight. Wash three times with 1 XPBS for 5min each time. Adding a secondary antibody: goat anti-rabbit IgG-FITC (Proteitech, China, cat # SA00013-4, dilution ratio 1:100) was incubated at room temperature for 1 h. Wash three times with 1 XPBS for 5min each time. DAPI (Sigma-Arich, USA, Cat: D9542) staining for 2min, 1 x PBS washing three times, each time for 5 min. ddH₂After 2min of O-wash, 90% glycerol mounting, and intracellular localization of protein molecules was observed under a flow analyzer of the Vectra3 type (PerkinElmer, USA).

5.3RT-PCR detection of the expression of MUC17 at different time points (0, 12, 24, 36, 48 and 60h) after DBP or DEHP (1 μ M) treatment of BEAS-2B and A549 cells.

Placing human lung normal epithelial cell BEAS-2B and human lung adenocarcinoma cell A549 in the above DMEM +1 μ M DBP culture medium and DMEM +1 μ M DEHP culture medium respectively at 37 deg.C and 5% CO₂The cells were collected after 0, 12, 24, 36, 48 and 60 hours of incubation in the incubator of (1), and the mRNA expression level of MUC17 in the cells was measured according to the method of 5.1. Wherein the mRNA expression level at 0h treatment was 1. The result is shown as C in FIG. 1.

5.4RT-PCR detection of expression of MUC17 by BEAS-2B and A549 cells treated with DBP or DEHP at different doses (0, 100nM, 1 μ M, 10 μ M, 100 μ M and 1mM) for 60 h.

Human lung normal epithelial cell BEAS-2B and human lung adenocarcinoma cell A549 were respectively cultured in the following medium at 37 deg.C and 5% CO₂The cells were collected after 60 hours of incubation in the incubator of (1), and the mRNA expression level of MUC17 in the cells was measured according to the method of 5.1. Wherein the mRNA expression level treated with the control medium was 1. The result is shown as D in FIG. 1.

Control medium (Control): DMEM medium.

DBP-100nM, 1. mu.M, 10. mu.M, 100. mu.M and 10mM medium: the resulting culture media were supplemented with DBP to DBP contents of 100nM, 1. mu.M, 10. mu.M, 100. mu.M and 10mM, respectively, in DMEM medium.

DEHP-100nM, 1. mu.M, 10. mu.M, 100. mu.M and 10mM medium: to DMEM medium, DEHP was added to a medium containing DEHP in an amount of 100nM, 1. mu.M, 10. mu.M, 100. mu.M and 10mM, respectively.

5.5RT-PCR assay of expression of MUC17 in cells treated with DBP or DEHP (1 μm) for 60h and MTT assay of proliferation potency of cells treated with DBP or DEHP (1 μm) for 60h

The mRNA expression level of MUC17 in the following cells was measured according to the method of 5.1: MUC17 cell line, Vector cell line, shMUC17 cell line, shmnc cell line, MUC17+ DBP, MUC17+ DEHP, Vector + DBP, Vector + DEHP, shMUC17+ DBP, shMUC17+ DEHP, shmnc + DBP, and shmc + DEHP. In the treatment of MUC17 cell line, Vector cell line, MUC17+ DBP, MUC17+ DEHP, Vector + DBP and Vector + DEHP, the expression level of mRNA of the Vector cell line is 1, and the result is shown in the left graph of E in FIG. 1; in the rest of the cell treatments, the expression level of mRNA of the shNC cell line was 1, and the result is shown in the left panel F of FIG. 1.

The following cells were tested for OD at 0, 24, 48, 72 and 96 hours according to the method of step 4 above_490nm/OD_{570 nm}: MUC17 cell line, Vector cell line, shMUC17 cell line, shmnc cell line, MUC17+ DBP, MUC17+ DEHP, Vector + DBP, Vector + DEHP, shMUC17+ DBP, shMUC17+ DEHP, shmnc + DBP, and shmc + DEHP. The results are shown on the right of E and F in FIG. 1.

The MUC17+ DBP, shMUC17+ DBP and shNC + DBP are obtained by adding MUC17 cell line, shMUC17 cell line and shNC cell line to DMEM + 1. mu.M DBP medium (medium obtained by adding DBP to DMEM medium to DBP content of 1. mu.M) at 37 ℃ with 5% CO₂The collected cells were cultured in the incubator of (1) for 60 hours.

The MUC17+ DEHP, shMUC17+ DEHP, and shNC + DEHP were prepared by adding MUC17 cell line, shMUC17 cell line, and shNC cell line to DMEM + 1. mu.M DEHP medium (a medium obtained by adding DEHP to 1. mu.M in DMEM medium) at 37 ℃ with 5% CO₂The collected cells were cultured in the incubator of (1) for 60 hours.

The results showed that expression of transmembrane-type mucins in BEAS-2B and A549 cells after exposure to DBP or DEHP (1 μm, 60h) was measured in a lung cancer cell line A549 and a lung epithelial cell line BEAS-2B. As shown in fig. 1, a, DBP or DEHP exposure can significantly reduce expression of transmembrane mucin MUC 17. Furthermore, expression of MUC1, 12 and 13 was promoted after DBP exposure, whereas DEHP exposure only up-regulated expression of MUC13 and MUC 16. Immunofluorescent staining also confirmed that DBP or DEHP exposure reduced the expression of MUC17 in a549 and BEAS-2B cells (B in fig. 1). Furthermore, the dose and time dependent decrease in DBP or DEHP exposure to MUC17 expression (C in fig. 1 and D in 1) indicates that MUC17 exhibits specific expression for DBP or DEHP exposure in human airway epithelial cells.

Further, the functional effect of MUC17 on airway epithelial cells was examined by stable overexpression of MUC17 in a549 cells. As shown in E and F in fig. 1, overexpression of MUC17 has an anti-proliferative effect, and can significantly inhibit DBP or DEHP-induced proliferation of a549 cells. Conversely, knock down of MUC17 resulted in a significant increase in cell proliferation. This proliferation-promoting effect is further enhanced when a549 cells are treated with DBP or DEHP. The results indicate that MUC17 plays an important role in PAEs-induced lung cell malignancies, suggesting that DBP or DEHP exposure may promote airway epithelial cell malignancy by down-regulating MUC17 expression.

Example 2 detection of MUC17 Gene methylation in Phthalate (PAEs) treated cell lines

1. Design and synthesis of MUC17 gene methylation detection primer

The sequence of the gene MUC17 to be detected (GenBank Accession No. NC-000007.14 (Update Date8-Jul-2021)) is inquired and downloaded, and the target sequence is input into the EpiDesigner software for primer design. And selecting and determining a proper primer design scheme according to the software operation result. Primers were synthesized, 5OD each.

The methylation detection primer has the following specific sequence:

the primer group 1 covers a DNA sequence (SEQ ID No.1) which is positioned at-3898 bp to-3400 bp of the MUC17 gene transcription start site;

primer set 1 consists of F1 and R1, and has the following sequence:

the upstream primer F1: 5'-aggaagagag TTGTAGTTGGGAAGGAGTTTGTTT-3' (SEQ ID No.4),

the downstream primer R1: 5'-cagtaatacgactcactatagggagaaggct CCTCCACCTAATCTCCCTACTTTAAT-3' (SEQ ID No. 5).

The primer group 2 covers a DNA sequence (SEQ ID No.2) positioned at the transcription start site-2844 bp to-2358 bp of the MUC17 gene;

primer set 2 consists of F2 and R2, and has the following sequence:

the upstream primer F2: 5'-aggaagagag GGGAATTTTAGAGGAAGTGGAGTAG-3' (SEQ ID No.6),

the downstream primer R2: 5'-cagtaatacgactcactatagggagaaggct TACTAACCCAAAACCAACCCTATCT-3' (SEQ ID No. 7).

The primer group 3 covers a DNA sequence (SEQ ID No.3) positioned at the transcription start site of the MUC17 gene of-759 bp to-321 bp;

primer set 3 consists of F3 and R3, and has the following sequence:

the upstream primer F3: 5'-aggaagagag GGAAAGGAGGAGGGTATTATTTGTA-3' (SEQ ID No.8),

the downstream primer R3: 5'-cagtaatacgactcactatagggagaaggct CCCAAACATAACCATATCTTCAAAA-3' (SEQ ID No. 9).

The position in the gene corresponding to the primer set 3 is shown as A in FIG. 2.

All primers were synthesized by Huada corporation (Beijing).

The methylation primers are used, DNA subjected to vulcanization modification is used as a template for PCR amplification, a fragment with the size of 499bp is obtained by amplification with the primer group 1, a fragment with the size of 487bp is obtained by amplification with the primer group 2, a fragment with the size of 439bp is obtained by amplification with the primer group 3, and methylation detection is carried out by using an Agena MassArray system. The methylated primer has Tm 59.99 of the upstream primer and GC content of 40.00 percent, and the Tm 60.32 of the downstream primer and GC content of 32 percent. Detection result is EpitYPER^TMThe software obtains original data, and the original data is obtained by comparing the areas of the peak containing G and the peak containing A, and the methylation degree and the non-methylation degree of the sample to be detected are calculated, and belong to the relative quantitative ratio of the two. If the value detected in the table is 0.43, this means that the degree of methylation of the corresponding methylation site in the sample is 43%. Taking the average of methylation values of all sites in the region, and if the methylation percentage is more than or equal to 60, regarding the methylation as high methylation; when the percentage of methylation is 20 to 60% (excluding the end points), "partial methyl group" is regarded asMelting "; if the percentage methylation is less than or equal to 20%, this is regarded as "unmethylated".

2. Methylation detection

The primer set 3 in step 1 will be described as an example.

Test cells: BEAS-2B, A549, BEAS-2B + DBP, BEAS-2B + DEHP, A549+ DBP and A549+ DEHP

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 for3 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) Adding 100. mu.L of pH 8.0TE solution to dissolve DNA, collecting 1. mu.L of the DNA solution, measuring the DNA concentration with a NanoDrop 2000c ultra-micro nucleic acid analyzer, and storing the rest of the sample in a refrigerator at-20 ℃.

2. Quality inspection of DNA sample quality and concentration

The DNA methylation positive and negative control samples were Human Methylated purchased from Zymo Research, USA&Non-Methylated DNA Set (cat # D5014), Human Methylated DNA is also called in vitro Methylated DNA (IVD). Detecting the concentration and purity of the DNA by OD detection and gel electrophoresis; total amount of DNA (sample to be tested, DNA methylation positive control and negative control sample): 3-5ug, high concentrationAt 50 ng/. mu.l. Further use of NaHSO₃The DNA sample to be detected was treated so that all cytosines in the unmethylated region were converted to thymines while cytosines in the methylated region remained unchanged, and was manipulated using the EZ DNA Methylation Gold Kit from Zymo Research, USA (cat # D5006).

3. Methylation detection step of Agena MassArray system

The operation was carried out using the EpitYPER Reagent and SpectroCHIP kit of Agena, USA (cat # 11377D)

(1) PCR amplification reaction

Subjecting the sample to NaHSO₃After treatment, the cells were centrifuged briefly at low speed for further use. PCR reaction solutions were prepared as shown in Table 2 (reaction system preparation was performed on ice to prevent inactivation by prolonged exposure to high temperature).

TABLE 2 methylation detection of amplification PCR reaction components for the Agena MassArray System

(2) The procedure for the amplification PCR reaction is shown in Table 3

TABLE 3 PCR reaction procedure

And (3) mixing 3. mu.l of PCR product with 1. mu.l of 6 loading buffer, detecting by using 1.5% agarose gel electrophoresis, observing the result (B in figure 2) after 160V and 20min, and continuing the subsequent experiment if the result is good.

(3) Shrimp Alkaline Phosphatase (SAP) reaction

The PCR amplification product was passed through an SAP reaction to remove excess deoxyribonucleoside triphosphates (dNTP/dUTP), as shown in Table 4:

TABLE 4 methylation detection of SAP reaction components by MassArray System

Reagent	Final volume
		RNase-free ddH2O	1.70μL
SAP enzymes	0.30μL
		PCR product	5.00μL
Total volume	7.00μL

Mu.l of SAP reaction was added to each well of 384 plates, the plates were carefully covered with 384 well plates and each well was firmly pressed to prevent evaporation during the PCR procedure, and the following reaction procedure was performed after centrifugation (Table 5).

TABLE 5 SAP reaction procedure

37℃	20min
		85℃	5min
4℃	∞

(4) T-cut/RNase A digestion reaction

The RNA fragment is cut into small fragments carrying CpG sites by utilizing the characteristic that RNase A can specifically recognize and cut the U3' end in RNA. The T-cut/RNase A digestion reaction system is shown in Table 6:

TABLE 6T-cut/RNase A digestion reaction components

T cutting/RNase A digestion reaction condition: incubate at 37 ℃ for3 h.

4. Resin purification

The resin was uniformly filled in an 384/6MG Dimple plate and left to dry for 10 minutes. To each well of 384 sample plates was added 16. mu.l of water. The 384 sample plate was gently snapped onto the sample plate, flipped over and tapped to drop the resin into each well of the sample plate. And sealing the 384 sample plate by using a sealing film, and then placing the plate in a turnover plate centrifuge to rotate and mix uniformly for 30 minutes at room temperature.

5. Chip sample application

The MassARRAY nanodispenseRS 1000 sample applicator is started, and the product after resin purification is transferred to 384-well

In bioarray. The spotted SpectroCHIP chip was analyzed by MALDI-TOF. Detection result is EpitYPER^TMSoftware obtains raw data and a circular diagram and checks the integrity and accuracy of the data file. Storing the result in corresponding storage medium and submitting the result to biological information room for analysis.

6. Results

The results are shown in fig. 2B and C, and the cells were subjected to MassARRAY detection using IVD as methylation control, NL as non-methylation control, and deionized water as negative system control.

Among them, BEAS-2B (percentage of methylation 38.29%), BEAS-2B + DBP (percentage of methylation 83.29%) and BEAS-2B + DEHP (percentage of methylation 77.86%) were present in normal human lung epithelial cells. While in lung adenocarcinoma cell lines a549 (methylation percentage 80.01%), a549+ DBP (methylation percentage 89.29%) and a549+ DEHP (methylation percentage 95.42%). That is, according to the method for determining the result given in step 1 of example 2, the results of the cell line measurements were all "hypermethylated" except that BEAS-2B was "partially methylated".

The results indicate that PAEs-type environmental pollutants (such as DBP and DEHP) can increase the methylation degree of the MUC17 promoter region.

Example 3 DNA methylation inhibitors can reverse MUC17 expression silencing in airway epithelial cells and non-Small cell Lung cancer cells

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 growth density of the cells (A549 and BEAS-2B) reaches 30%, 5-Aza is added until the content of the cells in the culture system is 5 mu M, the culture medium is replaced every 24h, and the 5-Aza is continuously added until the content of the cells in the culture system is 5 mu M, so that the total action time is 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

(1) qPCR primer sequences:

the RT-qPCR primer sequence covers the amplification product of 91bp from +11727 to +11817bp (positioned in the reading frame of MUC17 gene) from the transcription start site of the MUC17 gene.

An upstream primer: 5'-GGGCCAGCATAGCTTCGA-3', respectively;

a downstream primer: 5'-GCTACAGGAATTGTGGGAGTTGA-3' are provided.

Wherein the internal reference primer for homogenization is a primer taking Beta-Aactin as an internal reference,

an upstream primer: 5'-TTAGTTGCGTTACACCCTTTC-3', respectively;

a downstream primer: 5'-ACCTTCACCGTTCCAGTTT-3' are provided.

(2) The reaction systems for qPCR amplification are shown in table 7.

TABLE 7 reaction System for qPCR amplification

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; melting 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 melting curve ensures the specificity of the product.

3. Results and analysis

The expression silencing of MUC17 in lung normal human epithelial cells was reversible using the epigenetic drug DNA methylation inhibitor 5-Aza-2 '-deoxycytidine (5-Aza-2' -deoxycytidine, 5-Aza), indicating that MUC17 expression silencing in lung normal human epithelial cells is epigraphically regulated, as shown at D in FIG. 2.

Because the apparent drug DNA methylation inhibitor is applied to clinical tumor treatment, the MUC17 methylation site can be used as a potential molecular target for preventing and treating the lung cancer induced by environmental factors.

Example 4 DBP or DEHP Exposure inhibits the expression of MUC17 by promoting UHRF1-DNMT1

1、qPCR

(1) qPCR primer sequences:

the RT-qPCR primer sequence covers +800 to +903bp (positioned in the reading frame of DNMT1 gene) of an amplification product of 104bp from the transcription start site of the DNMT1 gene.

An upstream primer: 5'-CCAAAGAACCAACACCCAAAC-3', respectively;

a downstream primer: 5'-CTCATCTTTCTCGTCTCCATCTTC-3' are provided.

The RT-qPCR primer sequence covers the amplification product of 95bp from +1047 to +1141bp (positioned in the reading frame of DNMT3A gene) of the transcription start site of the DNMT3A gene.

An upstream primer: 5'-ACGATTGCTAGACTGGGATAATG-3', respectively;

a downstream primer: 5'-AGTAAGCAGGCCAGGTAGA-3' are provided.

The RT-qPCR primer sequence covers +4127 to +4224bp (positioned in the reading frame of DNMT3B gene) of an amplification product of 98bp from the transcription starting site of the DNMT3B gene.

An upstream primer: 5'-GGAGCCACGACGTAACAAATA-3', respectively;

a downstream primer: 5'-GTAAACTCTAGGCATCCGTCATC-3' are provided.

The RT-qPCR primer sequence covers + 3622- +3729bp (positioned in the reading frame of UHRF1 gene) of an amplification product of 108bp from the transcription start site of the UHRF1 gene.

An upstream primer: 5'-AAATGGCCTCAAGGGGACTC-3', respectively;

a downstream primer: 5'-CACTTGCACGTGACTTCGTG-3' are provided.

Wherein the internal reference primer for homogenization is a primer taking Beta-Aactin as an internal reference,

an upstream primer: 5'-TTAGTTGCGTTACACCCTTTC-3', respectively;

a downstream primer: 5'-ACCTTCACCGTTCCAGTTT-3' are provided.

(2) The qPCR reaction system and reaction conditions were the same as in example 3.

2. Interference with DNMT1 gene expression in A549 cells

Universal negative control pSilencer^TM3.1-H1 neo distensiled, available from Thermo Fisher, usa under the trade designation: AM 5770.

DNMT1 interference shRNA recognition region sequence:

5’-CUGCUGGGACUGGAGUUUAUA-3’；

non-small cell lung cancer cell lines A549 and BEAS-2B were selected for transient transfection. Inoculating the cells into a 6-hole cell culture plate, adding 200 mu L of DMEM into 20nM plasmid expressing the interference shRNA and the plasmid expressing the unloaded shRNA when the cells grow to 60% of fusion degree, mixing uniformly, and standing at room temperature for 5 min. Then 200. mu.L of DMEM is respectively added into 6. mu.L of Lipofectamine2000, and the mixture is uniformly mixed and placed for 5min at room temperature. 200mL of a dilution of Lipofectamine2000 (Thermo Fisher, USA, cat # 11668019) was added to 200. mu.L of the interfering plasmid solution, mixed well and left at room temperature for 20 min. Adding 400 mu L of the mixed solution into a cell culture plate, carrying out serum-free culture at 37 ℃ for 8h, then replacing 10% FBS DMEM, incubating at 37 ℃ for 24h, and then detecting by RT-qPCR.

Construction of an interference plasmid: cloning and constructing the coding RNA molecule of the interference shRNA and the coding RNA molecule of the no-load shRNA contrast into a plasmid vector pSilencer^TM3.1-H1, respectively obtaining a plasmid for expressing the interference shRNA and a plasmid for expressing the idle shRNA, and using the plasmids to transfect cells to establish a cell line, wherein the constructed cell line is respectively named as an shDNMT1 cell line (the plasmid for transfectioning the interference shRNA) and an shNC cell line (the plasmid for transfectioning the idle shRNA).

The interfering plasmid was synthesized by the Shanghai Jikai Gene company.

3. Cell biological function experiment

The test cell lines were: shDNMT1 cell line, shNC cell line, shDNMT1+ DBP and shDNMT1+ DEHP respectively constructed aiming at A549.

Wherein: shDNMT1+ DBP represents a DBP (1. mu.M, 24h) treated shDNMT1 cell line; sh DNMT1+ DEHP represents DEHP (1. mu.M, 24h) treated shDNMT1 cell line;

(1) MTT (3- [4, 5-dimethylthiozol-2-yl ] -2, 5-diphenyltetrazolium bromide) assay

Digesting and counting a test cell line in a logarithmic growth phase and with good growth condition, adjusting the concentration of cell suspension, adding 100 mu L of the test cell line into each hole of a 96-hole cell culture plate, laying 2000 cells in each hole, arranging 8 multiple holes, and continuously observing for 4 days; mu.L of MTT (Sigma, USA; cat # M2128) was added to each well at a daily timing so that the final concentration was 5. mu.g/mL, and after incubating for 4 hours, the medium was discarded. After 4 days, 150. mu.L of dimethyl sulfoxide (DMSO, Sigma, cat # D2650) was added to each well, and after gentle shaking in the dark for 10min, the absorbance of each well was measured at OD490nm of an ELISA detector, and the data obtained was analyzed.

(2) Migration experiment

The test cell lines were separately digested and counted, and then placed in the upper chambers of a Transwell chamber (from corning, USA: CLS3422) with a pore size of 8.0 μm, 200. mu.L of serum-free cell suspension was added to each upper chamber, and 2X 10 cells were added to each well⁴For each cell, 600. mu.L of complete medium was added to the lower chamber; culturing in a 37 ℃ cell culture box, culturing the cells for 24h, and carefully washing with 1 XPBS for a plurality of times; fixing with 4% paraformaldehyde for 30min, and washing with 1 × PBS for three times; 0.5% crystal violet for 30min, rinsed until the rinse is colorless, the upper chamber cells were carefully removed with a cotton swab, and placed under a microscope (100 ×) to count the number of migrating cells.

(3) Statistical analysis

The experiments were repeated at least three times. The results of the experiments were all measured by a two-sided t-test, and the results were expressed as mean ± sd,. p <0.05 for statistically significant differences, and 0.01 for statistically significant differences.

4. Results and analysis

Since 5-Aza was able to demethylate the MUC17 promoter region to restore its expression, we examined the expression of DNA methyltransferases (DNMTs) including DNMT1, DNMT3A and DNMT 3B. The RT-qPCR results showed that DNMT1 was most upregulated in DBP or DEHP exposed a549 cells and BEAS-2B cells among 3 DNMTs and was associated with the critical regulatory protein UHRF1 for DNA methylation (B in a and 3 in fig. 3). Interference with DNMT1 in a549 cells promoted expression of MUC17 and inhibited epigenetic silencing after DBP or DEHP exposure (C in fig. 3). The above results indicate that DBP or DEHP-induced epigenetic silencing of MUC17 is regulated by UHRF1-DNMT 1. It was further found that DBP or DEHP exposure did not promote cell growth and migration after interfering with DNMT1 (D in fig. 3 and E in fig. 3). The expression of MUC17 can be restored by interfering DNMT1, so that the carcinogenic effect of cells after DBP or DEHP exposure is inhibited, and the fact that MUC17 plays an important role in a specific epigenetic pathway of promoting the malignant transformation of human airway epithelial cells by low-dose PAEs is suggested.

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.

SEQUENCE LISTING

<110> Beijing thoracic Hospital affiliated to capital medical university

Research institute of tuberculosis and breast tumor in Beijing

<120> kit applied to environmental pollution carcinogenic risk assessment

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 499

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

ctgcagttgg gaaggagctt gcttttggcg gggaagggaa ggcaggtcaa tctcacaaag 60

aaagagtgag ggacgtgagg ttggcagggc tttgggaatg gggtgggggg ttgaggggct 120

ttggccgccc accttttttt gtgagacaga gtctcactct gtcacccagg ctggagtaca 180

gtggcacaat cttggctcac tgcaacttct gtctcccagg ttcaagcgat tctcctgccc 240

tcagcctccc tagtagctgt gattacaggc atgccaccac gcgtggctaa tttttgtatt 300

tttagtagag actgtagaga cagggtttca tcatgttggc caggctggtc tcgaactcca 360

gaccttaggt gatccgcccg ccttggcctc ccaaagtgct gggattacag gcgtgaacca 420

ccatgcccgg tcacattttg aatttttttt actcaaaagg ccattcaggg agcactaaag 480

cagggagacc aggtggagg 499

<210> 2

<211> 487

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

gggaacccca gaggaagtgg agcagcctca gggggtgggg gcagctcgca gctgagtcac 60

ctctgtgcca gagtcaacag tctccagggt gggccggaag ttgttgtagg cgttctcaag 120

gccgaatctg ctctccctca gattctggtc tgtagggaga ggccgtgatg agtgatggga 180

gccttggtag ggggaggact agggggcaga ggggatttcc tctggcaaaa ggaagagtca 240

gcccggcatc tgccctacgc cctttcccac tgccgaccac ccctccgacc ctgcatcact 300

gggctgcggg taagtgcaga gcctgcctgg ggagccgccc gctggacccc ggaacaggag 360

ggagcggagc agaagccccc cacatgcacg gggtggaagc gggggccacg gaggagcgtg 420

cgttcagccc tggactcagg ggtgaggtca gagtggagcg ggagacaggg ctggctctgg 480

gtcagca 487

<210> 3

<211> 439

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

ggaaaggagg agggtaccac ctgcatttga ccctaacccc tagcagggac accccctttg 60

tctcttttct aacccttctt attggccggg tatggtggct cacacctgta atcccagcac 120

tttgggaggc tgacgcggga agatcacttg agatgaggag tttgagacca acctggccaa 180

catgatgaaa caccatctct actaaaaata caaaaatcag ccagacttgg tggcgggcgc 240

ctgtaatccc agctactcag gaggctgagg caggagaatc gcttgaacct gggaggcgga 300

ggttgcagag agccaagatc gtgcctccgt actccagcct gggcaacaga gcaagactct 360

gtctcggcaa aaataaataa ataaaataac ccttcttatt gtcctatgag tatgccctga 420

agacatggcc atgcctggg 439

<210> 4

<211> 34

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

aggaagagag ttgtagttgg gaaggagttt gttt 34

<210> 5

<211> 57

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

cagtaatacg actcactata gggagaaggc tcctccacct aatctcccta ctttaat 57

<210> 6

<211> 35

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

aggaagagag gggaatttta gaggaagtgg agtag 35

<210> 7

<211> 56

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

cagtaatacg actcactata gggagaaggc ttactaaccc aaaaccaacc ctatct 56

<210> 8

<211> 35

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

aggaagagag ggaaaggagg agggtattat ttgta 35

<210> 9

<211> 56

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

cagtaatacg actcactata gggagaaggc tcccaaacat aaccatatct tcaaaa 56

Claims (10)

1. A kit for assessing the risk of carcinogenesis due to environmental pollution, which comprises a primer pair for detecting the methylation level of the MUC17 gene promoter.

2. The kit according to claim 1, wherein the primer pair consists of a primer F3 and a primer R3, the primer F3 is a single-stranded DNA having a nucleotide sequence of SEQ ID No.8, and the primer R3 is a single-stranded DNA having a nucleotide sequence of SEQ ID No. 9.

3. The kit of claim 1 or 2, wherein the kit further comprises one or more of Taq DNA polymerase, dntps, PCR buffer required for PCR amplification.

4. The kit of any one of claims 1 to 3, wherein the kit further comprises bisulfite.

5. The primer set according to claim 1 or 2.

6. The primer set according to claim 5, wherein any one of the following applications:

A1) use of the primer pair of claim 5 for detecting the level of methylation of the MUC17 gene promoter;

A2) use of the primer pair of claim 5 for the preparation of a product for detecting the methylation level of the MUC17 gene promoter;

A3) use of a primer pair as claimed in claim 5 in the manufacture of a product for the assessment of the risk of carcinogenesis due to environmental contamination;

A4) use of a primer pair as defined in claim 5 in the preparation of a product for lung cancer screening;

A5) use of a primer pair as claimed in claim 5 in the manufacture of a product for predicting the risk of lung cancer.

7. Use according to claim 6, the product being a reagent and/or a kit.

8. A method for detecting the methylation level of the promoter of MUC17 gene, which comprises the following steps:

B1) extracting DNA of a sample to be detected;

B2) subjecting said DNA to bisulfite conversion;

B3) carrying out PCR amplification by using the transformed DNA as a template and the primer pair as claimed in claim 5 to obtain a PCR amplification product;

B4) and carrying out methylation detection on the PCR amplification product.

9. The method of claim 8, wherein the test sample is a cell or tissue.

10. The kit of any one of claims 1 to 4, and/or any one of the following uses of the method of claim 8 or 9:

C1) the application of the promoter methylation level of the MUC17 gene is detected;

C2) the application in the evaluation of the carcinogenic risk of environmental pollution.

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