CN113881777B - Kit applied to environmental pollution and cancerogenic risk assessment - Google Patents

Kit applied to environmental pollution and cancerogenic risk assessment Download PDF

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CN113881777B
CN113881777B CN202111336575.7A CN202111336575A CN113881777B CN 113881777 B CN113881777 B CN 113881777B CN 202111336575 A CN202111336575 A CN 202111336575A CN 113881777 B CN113881777 B CN 113881777B
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primer
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dbp
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林舒晔
王海潮
黄家强
庞梦迪
赵聪
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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 kit applied to environmental pollution and cancerogenic risk assessment. The invention specifically discloses a specific methylation primer pair for detecting the methylation state or level of a CpG island of a MUC17 gene promoter region, and 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 methylation of the DNA sequence in the MUC17 gene can specifically detect the DNA methylation level related to the MUC17 gene, is convenient and quick to use in the aspects of environmental pollution and cancerogenic 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 and cancerogenic 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 and cancerogenic risk assessment.
Background
Lung cancer is the first high-grade tumor in China, the new cases of lung cancer in2020 are 220 ten thousand, and the death cases are 180 ten thousand (Sung H, ferly J, siegel RL, et al global Cancer Statistics 2020:GLOBOCAN Estimates of Incidence and Mortality Worldwide for36Cancers in 185Countries.CA Cancer J Clin 2021, 71 (3): 209-249.). Despite the significant increase in survival of lung cancer patients in past decade through early diagnosis and cancer treatment with biomarkers, the global morbidity and mortality of lung cancer is still further exacerbated with the increase in a number of risk factors. Among them, environmental air pollution and exposure to other inhalable environmental pollutants are considered to be major factors in lung cancer morbidity and mortality (Ettinger DS, wood DE, agarwal C, et al nccn Guidelines Insights: non-Small Cell Lung Cancer, version 1.2020.Journal of the National Comprehensive Cancer Network:JNCCN 2019, 17 (12): 1464-1472Turner MC,Andersen ZJ,Baccarelli A,et al.Outdoor air pollution and cancer:An overview of the current evidence and public health recommendations.CA Cancer J Clin2020.). Studies have shown that there is a close link between the induction of chronic inflammation in the lung by environmental carcinogens and the development and progression of lung cancer.
Many chemical and environmental factors can cause carcinogenesis to human bronchial epithelial cells. Phthalate esters (phthalic acid esters, PAEs) are a class of plasticizers and solvents used in polyvinyl chloride (PVC) plastics and are widely used in new synthetic materials to improve the mechanical properties such as flexibility and stretchability of the materials. The PAEs in these new synthetic materials migrate slowly to the air, particulate matter, dust fall and indoor surfaces and enter the body through oral, inhalation and skin contact. PAEs are considered to be a class of genotoxic organic contaminants that can lead to reproductive toxicity, endocrine disorders and canceration (Li PH, jia HY, wang Y, et al, research of PM (2.5) -bound Phthalic Acid Esters (PAEs) at regional background site in northern China: long-range transport and risk analysis. The Science of the total environment 2019, 659: 140-149.). PAEs have been reported to down regulate the immune system as allergens, leading to inflammation and asthma, and possibly to the development of respiratory diseases such as lung cancer (Guo H, song N, wang D, ma J, jia Q.A modulation approach for covalent organic frameworks: application to solid phase microextraction of phthalate esters. Talanta 2019, 198:277-283.).
DBP and DEHP are two major phthalate species, associated with microplastic contamination in the environment. Wherein di (2-ethylhexyl) phthalate (DEHP) is mainly used in PVC products as a representative of PAEs high molecular compounds; di-n-butyl phthalate (DBP) is a low molecular PAEs, which are widely used as soluble plasticizers in volatile substances such as varnishes, cosmetics and perfumes. Because the PAEs are not covalently bonded to the polymer matrix, the PAEs can be released by evaporation or leaching from the plastic into the surrounding environment after a slight physicochemical process such as heating, ionization, acid treatment, etc., resulting in environmental pollution. At the same time, there are studies reporting the contamination of phthalate esters in house dust. Due to the widespread use of PAEs and their ubiquitous presence in the environment, humans are inevitably exposed to phthalates. In recent years, environmental health hazards caused by such compounds have received widespread attention in the fields of environmental science and public health. The variety and concentration of PAEs detected in the environment are more and more, so that the environmental pollution and cancerogenic risk is related to the health of a human body, and the environmental pollution and cancerogenic risk assessment and prediction have important practical significance.
Environmental stress factors (physical, chemical stimulus, etc.) can induce cell DNA damage, and DNA damage sites are recognized by related damage receptors, so that corresponding repair signal paths are affected to carry out DNA repair. The current research shows that environmental pollutants can interfere with epigenetic regulation modes, DNA methylation, chromatin remodeling, miRNA, acetylation, ubiquitination and other apparent regulation pathways play an important regulation role in the occurrence and development of diseases, and the DNA damage repair plays an important role in the initiation of the disease (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 air pollutants closely related to the occurrence and development of lung cancer on the malignant progress of the lung is studied deeply, so that the epigenetic biomarker can be provided for early diagnosis of tumors, screening of susceptible people and risk evaluation of chemicals, and the reversibility of epigenetic biomarker also provides a target point for early prevention of the tumors. 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 environmental pollution cancerogenic risk assessment and/or a kit for detecting the methylation level of a MUC17 gene promoter, a primer pair, a method and application thereof. The technical problems to be solved are not limited to the technical subject matter as described, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.
In order to achieve the above object, the present invention provides a kit for environmental pollution cancerogenic risk assessment, 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 of the MUC17 gene promoter region.
The CpG island is located between 759bp (-759 bp) of the upstream sequence of the transcription initiation site (TSS site) of the MUC17 gene and 321bp (-321 bp) of the upstream sequence. 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, wherein the primer F3 is single-stranded DNA with a nucleotide sequence of SEQ ID No.8, and the primer R3 is single-stranded DNA with a nucleotide sequence of SEQ ID No. 9.
In the kit, the kit also comprises one or more of Taq DNA polymerase, dNTP and PCR buffer solution required by PCR amplification.
In the above kit, the kit further comprises bisulfite.
Herein, the bisulfite may be sodium bisulfite (NaHSO 3 )。
The kit may further comprise a DNA methylation positive control and a negative control.
The kit may further comprise an internal reference gene detection reagent, and the internal reference gene may 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.
The primer pairs described herein are also within the scope of the present invention.
The invention also provides any one of the following applications of the primer pair:
a1 Use of said primer pair in detecting the methylation level of the MUC17 gene promoter;
a2 Use of said primer pair in the preparation of a product for detecting the methylation level of the MUC17 gene promoter;
a3 Use of said primer pair in the preparation of a product for environmental pollution cancerogenic risk assessment;
a4 Use of said primer pair in the preparation of a product for lung cancer screening;
A5 Use of said primer pair for the preparation of a product for predicting the risk of having lung cancer.
In the above applications, 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 Performing bisulfite conversion of the DNA;
b3 Using the converted DNA as a template, and carrying out PCR amplification by using the primer pair to obtain a PCR amplification product;
b4 Methylation detection of 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 primer F3 (SEQ ID No. 8) and primer R3 (SEQ ID No. 9).
Further, B3) the reaction components of the PCR amplification include an upstream primer F3, a downstream primer R3, a DNA template, a PCR amplification enzyme, dNTPs, a 10 XPCR Buffer and ddH 2 O。
Further, B3) the reaction system (8.00. Mu.L) of the PCR amplification is: ddH 2 O4.90. Mu.L, 10 XPCR Buffer 0.80. Mu. L, dNTPs (10. Mu.M) 0.80. Mu. L, PCR amplification enzyme (5U/. Mu.L)0.10. Mu.L, primer F3 (10 nmol/. Mu.L) 0.20. Mu.L, primer R3 (10 nmol/. Mu.L) 0.20. Mu. L, DNA template 1.00. Mu.L.
Further, the reaction conditions for the PCR amplification of B3) are: 94 ℃ for 4min;94℃20sec,56℃30sec,72℃1min,45 cycles; and 3min at 72 ℃.
Bisulfite can convert unmethylated cytosine to uracil, and then detect DNA methylation level change by PCR, sequencing, chip hybridization and other techniques. In one embodiment of the invention, the bisulfite is sodium bisulfite (NaHSO 3 )。
In the above method, the methylation detection of the PCR amplification product of 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 invention, the SAP reaction system (7.00. Mu.L) is: RNase-free ddH 2 O1.70 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 ddH 2 O3.21. Mu.L, 5×T7 Polymerase Buffer 0.89.89. Mu.L, T clear 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 T-cut/RNase A digestion reaction conditions are as follows: incubating for 3h at 37 ℃;
The resin purification steps are as follows: resin was uniformly filled in 384/6MG simple plates and allowed to stand for 10 minutes to allow to air dry. To each well of 384 sample plates, 16 μl of water was added. The 384 sample plates were snapped onto the two plates and flipped and tapped to drop the resin into each well of the sample plates. Sealing the 384 sample plates by using a sealing film, and then placing the sealed 384 sample plates in a plate turnover centrifuge for uniformly rotating and mixing for 30 minutes at room temperature;
the chip sample application step comprises the following steps: starting MassARRAY NanodispenserRS sample application instrument, transferring the product after resin purification to 384-wellOn bioarray, the spotted SpectroCHIP chip was analyzed using MALDI-TOF. The test results used were EpiTYPER TM The software obtains the raw data and the circle map and checks the integrity and accuracy of the data file. Storing the result in a corresponding storage medium, submitting the result to analysis in a biological information room, and analyzing and calculating to obtain the methylation level of the DNA of the sample to be detected.
Further, PCR amplification was performed using the above-mentioned methylation primers (primer F3 and primer R3) and the sulfided modified DNA as a template, to obtain a 439bp fragment, and methylation was detected using the Agena Massary array system. The methylation primer has Tm 59.99 of the upstream primer, GC content 40.00%, tm 60.32 of the downstream primer and GC content 32%. The test results used were EpiTYPER TM The software obtains the original data, the original data is to calculate the methylation and unmethylation degree of the sample to be detected according to the area comparison of the peak containing G and the peak containing A, and the methylation and unmethylation degree belongs to the relative quantitative ratio of the peak containing G and the peak containing A. If the detection value in the table is 0.43, it represents a 43% methylation degree of the corresponding methylation site in the sample. Taking the average methylation value of all sites in the region, and considering the methylation percentage as high methylation if the methylation percentage is more than or equal to 60; a "partial methylation" is considered if the methylation percentage is 20-60% (inclusive); a "unmethylated" is considered if the methylation percentage is less than or equal to 20%.
The invention also provides the kit and/or any one of the following applications of the method:
c1 Use in detecting the methylation level of the MUC17 gene promoter;
c2 For use in environmental pollution cancerogenic risk assessment.
The purpose of the above-described applications and methods may be for disease diagnosis purposes, disease prognosis purposes and/or disease treatment purposes, as well as for non-disease diagnosis purposes, non-disease prognosis purposes and non-disease treatment purposes; their direct purpose may be information of intermediate results of obtaining disease diagnosis results, disease prognosis results and/or disease treatment results, and their direct purpose may be non-disease diagnosis purpose, non-disease prognosis purpose and/or non-disease treatment purpose.
The present inventors have found that hypermethylation of the DNA in the promoter region of MUC17 can affect the expression of MUC 17. Environmental contaminants can reduce the expression of MUC17 in airway epithelial cells, and the use of DNA methylation inhibitors can reverse the silencing of MUC17 expression in airway epithelial cells, indicating that the silencing of MUC17 gene expression in cells is associated with its genetic DNA hypermethylation modification. 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 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. By using the methylation primer pair, PCR amplification is carried out by taking vulcanized and modified DNA as a template, a 439bp fragment is obtained, and methylation detection is carried out by using an Agena MassArray system. The methylation primer pair has Tm 59.99 of the upstream primer, GC content 40.00%, tm 60.32 of the downstream primer and GC content 32%. The test results used were EpiTYPER TM The software obtains the original data, the original data is to calculate the methylation and unmethylation degree of the sample to be detected according to the area comparison of the peak containing G and the peak containing A, and the methylation and unmethylation degree belongs to the relative quantitative ratio of the peak containing G and the peak containing A. If the detection value in the table is 0.43, it represents a 43% methylation degree of the corresponding methylation site in the sample.
Environmental pollutants can inhibit the expression of MUC17, an important constituent of the human mucosal barrier, through the methylation pathway, while low expression of MUC17 can promote malignant transformation of tumors. The advantage of using MUC17 methylation as an early marker is that, first, methylation changes are progressive based on the advantage of methylation changes, which can be reversed with methylation inhibitors if diagnosed at an early stage. Secondly, with respect to transcription of RNA and translation of protein, modification on DNA is an early event, and if modification on DNA can be detected, intervention can be performed early, so that RNA transcription and protein translation are performed 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 for detecting the methylation of the DNA sequence in the MUC17 gene can specifically detect the methylation level of the DNA related to the MUC17 gene and can also specifically and quantitatively detect the methylation level of the DNA related to the MUC17 gene; therefore, the detection method and the kit can be conveniently and rapidly used for environmental pollution and cancerogenic risk assessment, lung cancer risk prediction and the like, and the detection result can provide an evaluation method for early diagnosis and detection of non-small cell lung tumor.
Drawings
FIG. 1 is an analysis of changes in MUC17 expression and biological function of airway epithelial cells BEAS-2B and tumor cells A549 after treatment with PAEs. FIG. 1A shows the RT-qPCR analysis of mRNA expression of transmembrane mucin in BEAS-2B and A549 cells after DBP or DEHP exposure (1. Mu.M, 60 h); FIG. 1B shows MUC1 expression in BEAS-2B and A549 cells after immunofluorescence detection of DBP or DEHP exposure (1 μΜ,60 h); FIG. 1C shows the detection of MUC17 expression at various time points (0, 12, 24, 36, 48 and 60 h) after DBP or DEHP (1 μΜ) treatment in BEAS-2B and A549 cells by RT-qRCR; FIG. 1D is a graph of RT-qRCR detection of DBP or DEHP (60 h) expression in BEAS-2B and A549 cells in MUC17 after treatment at different doses (0, 100nM, 1 μΜ, 10 μΜ, 100 μΜ and 1 mM); e in FIG. 1 and F in FIG. 1 are stable transfection of A549 cells into MUC17 (up) or shMUC17 (down) with DBP or DEHP treatment (1 μΜ,60 h), RT-qPCR expression; MTT shows the viability of cells.
FIG. 2 shows the methylation analysis of MUC17 gene in respiratory epithelial cells BEAS-2B and tumor cells A549. In FIG. 2, A is a schematic representation of the MUC17 gene structure, wherein the amplified region of Massarray is labeled, and each vertical line represents a CpG site. These numbers represent the position relative to the Transcription Start Site (TSS). FIG. 2B is a PCR quality control chart after primer amplification. In FIG. 2C is the percentage methylation of each CpG site after detection by Massarray. FIG. 2D shows the expression of MUC17 in BEAS-2B and A549 cells after treatment with epigenetic regulatory reagent (5-Aza) by RT-qPCR analysis. Cell treatment: cells were subjected to 5. Mu.M DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-2' -deoxyytidine) for 96 hours.
FIG. 3 is an analysis of the relationship between the respiratory epithelial cells BEAS-2B and tumor cells A549 after treatment with PAEs and methylases. FIGS. 3A and 3B show the detection of DNA methyltransferases (DNMT 1, DNMT3A and DNMT 3B) and UHRF1 mRNA expression by RT-qPCR using DBP or DEHP treated (1. Mu.M, 60 h) BEAS-2B and A549 cells; in FIG. 3, C-E is a treatment with DBP or DEHP (1. Mu.M, 60 h) after stable transfection of A549 cells with shDNMT1 or non-specific control (shNC). RT-qPCR analysis of DNMT1 and MUC17 mRNA expression (C); MTT assay to detect cell proliferation (D); the Transwell method detects cell invasion (E).
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Respiratory epithelial cells (normal epithelial cells of human lung) 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 under normal conditions in the applicant's unit laboratory for passage.
EXAMPLE 1 expression and biological Functions of the MUC17 Gene in Lung cancer cells
1、RT-PCR
1.1 cellular RNA extraction and reverse transcription Process
(1) The cellular RNA extraction process is to select cells with good growth state according to 1mL/10 6 Trizol reagent (Invitrogen, inc. of USA, cat# 15596026) was added to each cell.
(2) After 10min at room temperature, chloroform extraction was performed after sufficient lysis, and 0.2mL chloroform was added per 1mL Trizol. Shaking vigorously for 15s, standing at room temperature for 5min, centrifuging at 12000g at 4℃for 15min.
(3) The upper colorless liquid after delamination was transferred to a new centrifuge tube, precipitated with pre-chilled isopropanol, 0.5mL isopropanol was added per 1mL Trizol, and left on ice for 20min.12000g, centrifuging at 4 ℃ for 10min, and discarding the supernatant.
(4) The precipitate was washed with pre-chilled 75% ethanol, 1mL Trizol was added to 1mL 75% ethanol, 7500g, centrifuged at 4℃for 5min, and the supernatant discarded. Drying, adding proper amount of DEPC-H 2 O was dissolved, and 0.8% agarose gel electrophoresis confirmed, and the concentration of RNA was measured by NanoDrop and stored at-80 ℃.
(5) 1.0. Mu.g of RNA was reverse transcribed into cDNA using a TransScript II First-Strand cDNA Synthesis SuperMix kit (cat# AH 301-02) produced by Beijing full gold Biotechnology Co., ltd.): mu.L of Anchored Oligo (dT) 20, 10. Mu.L of 2 XS Reaction Mix and 1. Mu.L of RT/RI Enzyme Mix were added to DEPC-H 2 O to 20. Mu.L. The reaction conditions, 42℃30min,85℃5min, and the cDNA obtained by reverse transcription were allowed to stand at-20 ℃.
1.2、qPCR
(1) qPCR primer sequence:
the RT-qPCR uses the upstream primer: 5'-GGGCCAGCATAGCTTCGA-3'; a downstream primer: 5'-GCTACAGGAATTGTGGGAGTTGA-3'.
(2) The reaction system for qPCR amplification is shown in table 1.
TABLE 1 reaction System for qPCR amplification
Note that: 2 XSYBR-Green: zymo Research, usa, cat No. E2004.
(3) The PCR conditions were as follows:
2min at 50 ℃, 10min at 95 ℃ and 15s at 95 ℃;60 ℃ for 1min; collecting fluorescence in 40 cycles; a melting curve was prepared at 95℃for 15s,60℃for 1min,95℃for 15s, and 60℃for 15 s. Experimental results Using 2 -ΔΔCt The data are analyzed by a method, and the specificity of the product is ensured by a melting curve.
3. Overexpression and interference of MUC17 Gene expression in A549 and BEAS-2B cells
Universal empty plasmid pcDNA TM 3.1 (+) Available from Thermo Fisher company, usa, cat: v79020.
MUC17 over-expression sequence (MUC 17 cDNA sequence) was GenBank Accession No. NM-001040105.1 at positions 12435-13483 (Update Date 8-Jul-2021), and was synthesized from Shanghai Ji Kai gene.
Universal negative control pSilencer TM 3.1-H1 neo distominued, available from Thermo Fisher company, usa, cat: AM5770.
The MUC17 interference shRNA recognition region sequence is designed as follows: 5'-UAUGGUUGGAGCUGAGAAU-3';
the non-small cell lung cancer cell line a549 was selected for stable transfection. Cells were inoculated into 6-well cell culture plates, and when the cells grew to 60% confluence, 200 μl DMEM was added to 20nM of plasmid expressing interfering shRNA, plasmid expressing empty shRNA, plasmid overexpressing MUC17, and control plasmid overexpressing MUC17, respectively, and after mixing, they were left at room temperature for 5min. 200. Mu.L of DMEM was added to 6. Mu.L of Lipofectamine 2000 (Thermo Fisher Co., U.S.A., cat# 11668019), and the mixture was left at room temperature for 5 minutes after mixing, to obtain a dilution of Lipofectamine 2000. The 200mL Lipofectamine 2000 dilution was added to 200. Mu.L of the plasmid solution, and the mixture was left at room temperature for 20 minutes after mixing to obtain 400. Mu.L of a mixture. 400. Mu.L of the mixture was added to a non-small cell lung cancer cell line A549 cell culture plate, and after 8 hours of serum-free culture at 37℃the mixture was replaced with 10% FBS DMEM and incubated at 37℃for 24 hours, and then detected by RT-qPCR. The construction method of the over-expression plasmid and the interference slow plasmid is as follows:
Construction of the over-expression plasmid: cloning and constructing MUC17 cDNA (GenBank Accession No. NM-001040105.1 at 12435-13483 (Update Date 8-Jul-2021)) into expression vector pcDNA TM 3.1 (+) And (3) upper part. In addition, empty plasmid is selected as a control for subsequent control experiments. The constructed cell lines were designated as MUC17 cell line (plasmid transfected over-expressing MUC 17) and Vector cell line (control plasmid transfected over-expressing MUC 17), respectively. The over-expression plasmid was synthesized by Shanghai Ji Kai Gene company.
Construction of interfering plasmids: cloning and constructing encoding DNA molecules of interfering shRNA and encoding RNA molecules of empty shRNA contrast into a plasmid vector pSilencer TM 3.1-H1 to obtain plasmid for expressing interfering shRNA and plasmid for expressing empty shRNA, respectively, and the plasmid is used for transfecting cells to establish cell lines, and the constructed cell lines are named shMUC17 cell line (plasmid for transfecting the interfering shRNA) and shNC cell line (plasmid for transfecting the empty shRNA). The interfering plasmid was synthesized by Shanghai Ji Kai gene company.
4. Cell biological function experiment
The test cell lines were: MUC17 cell line, vector cell line, shMUC17 cell line, shNC cell line, muc17+dbp, muc17+dehp, vector+dbp, vector+dehp, shmuc17+dbp, shmuc17+dehp, shnc+dbp, shnc+dehp.
Wherein: muc17+dbp represents DBP (1 μm,24 h) treated MUC17 cell line; muc17+dehp represents DEHP (1 μm,24 h) -treated MUC17 cell line; vector+DBP means DBP (1. Mu.M, 24 h) treated Vector cell line; vector+dehp represents DEHP (1 μm,24 h) -treated Vector cell line; shMUC17+DBP represents DBP (1. Mu.M, 24 h) treated shMUC17 cell line; shMUC17+DEHP represents the DEHP (1. Mu.M, 24 h) -treated shMUC17 cell line; shNC+DBP means DBP (1. Mu.M, 24 h) treated shNC cell line; shNC+DEHP represents a DEHP (1. Mu.M, 24 h) -treated shNC cell line.
(1) MTT (3- [4, 5-dimethylazol-2-yl ] -2,5-diphenyl-tetrazolium bromide) experiment
Performing digestion count on a tested cell line in a logarithmic growth phase and with good growth condition, adjusting the concentration of a cell suspension, adding 100 mu L of the cell suspension into a 96-well cell culture plate in each well, paving 8 compound wells in 2000 cells in each well, and continuously observing for 4 days; mu.L of MTT (product number: M2128 of sigma Co., USA) was added to each well at a fixed time every day to give a final concentration of 5. Mu.g/mL, and after co-culturing for 4 hours, the medium was discarded. After 4 days, 150. Mu.L of dimethyl sulfoxide (dimethyl sulfoxide, DMSO, sigma Co., USA, cat# D2650) was added to each well, and after light-shielding and light-shaking for 10min, the absorbance of each well was measured at OD 490nm (measurement wavelength) of the ELISA, and for eliminating nonspecific absorbance, 570nm was used as reference wavelength, and "OD" was used 490nm /OD 570 nm "to represent cell viability, and the resulting data was analyzed.
(2) Statistical analysis
The experiment was repeated at least three times. The experimental results were all tested using a two-sided t-test, the results being expressed as mean ± standard deviation, p <0.05 representing significant statistical differences and p <0.01 representing very significant statistical differences.
5. Results and analysis
Both human lung adenocarcinoma cells A549 and human lung normal epithelial cells BEAS-2B were set up in the Control group (Control) and in the DBP group (DBP) and in the DEHP group (DEHP).
Control group (Control): human lung normal epithelial cells BEAS-2B were placed in DMEM medium (Control) at 37℃with 5% CO 2 Culturing for 60 hours in a constant temperature incubator, collecting cells, which are named Control-BEAS-2B; human lung adenocarcinoma cells A549 were placed in DMEM medium (Control) at 37℃with 5% CO 2 Culturing in a constant temperature incubator for 60 hours to collect cells, which are named Control-A549;
DBP group (DBP): human lung adenocarcinoma cells A549 were placed in DMEM+1μm DBP medium (medium obtained by adding DBP to DMEM medium to a DBP content of 1 μm) at 37℃with 5% CO 2 Is cultured in a constant temperature incubator for 60 hours, and cells are collected, and the cells are named as 1 mu M DBP-A549; human lung normal epithelial cells BEAS-2B were placed in DMEM+1μm DBP medium (medium obtained by adding DBP to DMEM medium to a DBP content of 1 μm) at 37℃with 5% CO 2 Is cultured for 60 hours in a constant temperature incubator, and cells are collected, and the cells are named as 1 mu M 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 DMEM medium to a DEHP content of 1. Mu.M) for 60 hours, and cells were collected and designated as 1. Mu.M DEHP-A549.
5.1RT-PCR detection of transmembrane mucin expression in BEAS-2B and A549 cells
Total RNA of each of the above groups of cells was extracted, and mRNA expression levels of 8 transmembrane mucins, MUC1, MUC12, MUC13, MUC16, MUC17, MUC20, MUC21 and MUC22, were detected 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 was 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 for 5- 'GGTCACGCTGGACCCT-3' and MUC22 is F:5- 'TGGCCTCTACTTCGGCCTTA-3', R:5- 'GGTGGAGGCCACGATAGTTT-3'. The results are shown as A in FIG. 1.
5.2 immunofluorescent staining to detect the expression of MUC17 in BEAS-2B and A549 cells
Immunofluorescent staining was performed on each of the above groups of cells. The results are shown as B in fig. 1. The method comprises the following specific steps:
cells with good growth state are dripped on a cover glass and placed into a 6-hole cell culture plate for culture for 24 hours, and 4% paraformaldehyde is added for fixation for 10min after 1 XPBS is washed. After three washes with 1 XPBS, 0.5% Triton X-100 was added and left at room temperature for 5min. The cells were washed three times with 1 XPBS for 5min each. The mixture was blocked for 1h at room temperature by adding an appropriate volume of 5% BSA. Incubating primary antibodies: anti-MUC17 (Sigma-Arich Co., U.S.A., cat# HPA031634, dilution ratio: 1:25), overnight at 4 ℃. The cells were washed three times with 1 XPBS for 5min each. Adding a secondary antibody: goat anti-rabbit IgG-FITC (China protein end company, cat# SA00013-4, dilution ratio 1:100) was incubated for 1h at room temperature. The cells were washed three times with 1 XPBS for 5min each. DAPI (Sigma-Arich Co., USA, cat# D9542) was stained for 2min, and washed three times with 1 XPBS for 5min each. ddH 2 After 2min of O-washing, 90% glycerol seal, vectra3 class flow analyser (Perki, USAnElmer company) to observe the localization in cells of the protein molecule.
5.3RT-PCR detection of the expression of MUC17 at different time points (0, 12, 24, 36, 48 and 60 h) after treatment of BEAS-2B and A549 cells with DBP or DEHP (1. Mu.M).
Human lung normal epithelial cells BEAS-2B and human lung adenocarcinoma cells A549 were placed in the DMEM+1μm DBP medium and DMEM+1μm DEHP medium at 37℃with 5% CO, respectively 2 Cells were collected by culturing for 0, 12, 24, 36, 48 and 60 hours, respectively, 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 at 0h was 1. The results are shown as C in FIG. 1.
5.4RT-PCR detection of the expression of MUC17 by BEAS-2B and A549 cells after 60h treatment with DBP or DEHP at different doses (0, 100nM, 1. Mu.M, 10. Mu.M, 100. Mu.M and 1 mM).
The normal epithelial cells BEAS-2B of human lung and the adenocarcinoma cells A549 of human lung were placed in the following media at 37℃and 5% CO, respectively 2 Cells were collected by culturing them in a constant temperature incubator for 60 hours, 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 results are 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: DBP was added to the DMEM medium to give a DBP content of 100nM, 1. Mu.M, 10. Mu.M, 100. Mu.M and 10mM, respectively.
DEHP-100nM, 1. Mu.M, 10. Mu.M, 100. Mu.M and 10mM medium: DEHP was added to DMEM medium to give medium with DEHP contents of 100nM, 1 μm, 10 μm, 100 μm and 10mM, respectively.
5.5RT-PCR detection of expression of MUC17 in cells treated with DBP or DEHP (1. Mu.m) for 60h and MTT detection of proliferation ability in cells treated with DBP or DEHP (1. Mu.m) 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, shNC cell line, muc17+dbp, muc17+dehp, vector+dbp, vector+dehp, shmuc17+dbp, shmuc17+dehp, shnc+dbp, and shnc+dehp. Wherein, in the treatment of MUC17 cell line, vector cell line, MUC17+DBP, MUC17+DEHP, vector+DBP and vector+DEHP, the mRNA expression level of Vector cell line is 1, and the result is shown in the left graph of FIG. 1E; in the rest of the cell treatments, the mRNA expression level was 1 in the shNC cell line, and the results are shown in the left panel of F in fig. 1.
OD at 0, 24, 48, 72 and 96 hours was measured for the following cells according to the procedure of step 4 above 490nm /OD 570 nm : MUC17 cell line, vector cell line, shMUC17 cell line, shNC cell line, muc17+dbp, muc17+dehp, vector+dbp, vector+dehp, shmuc17+dbp, shmuc17+dehp, shnc+dbp, and shnc+dehp. The results are shown in the right hand graph of fig. 1E and F.
The above MUC17+DBP, shMUC17+DBP and shNC +DBP are obtained by placing MUC17 cell line, shMUC17 cell line and shNC cell line in DMEM+1μm DBP medium (medium obtained by adding DBP to DBP content of 1 μm in DMEM medium) at 37deg.C, 5% CO 2 Cells collected by culturing in a constant temperature incubator for 60 hours.
The above MUC17+ DEHP, shMUC17+ DEHP and shNC + DEHP were obtained by placing MUC17 cell line, shMUC17 cell line and shNC cell line in DMEM + 1. Mu.M DEHP medium (medium obtained by adding DEHP to DEHP content of 1. Mu.M in DMEM medium) at 37deg.C, 5% CO 2 Cells collected by culturing in a constant temperature incubator for 60 hours.
The results show that in lung cancer cell line A549 and lung epithelial cell line BEAS-2B, the expression of transmembrane mucin in BEAS-2B and A549 cells after DBP or DEHP exposure (1 μΜ,60 h) was measured. As shown in fig. 1 a, DBP or DEHP exposure can significantly reduce the expression of transmembrane mucin MUC 17. In addition, DBP exposure promotes the expression of MUCs 1, 12 and 13, while DEHP exposure upregulates the expression of MUC13 and MUC16 only. Immunofluorescent staining also demonstrated that DBP or DEHP exposure reduced MUC17 expression in A549 and BEAS-2B cells (B in FIG. 1). Furthermore, DBP or DEHP exposure was dose and time dependent on reduced MUC17 expression (C and D in fig. 1), indicating that MUC17 exhibited specific expression for DBP or DEHP exposure in human airway epithelial cells.
Further, the functional impact of MUC17 on airway epithelial cells was examined by stably over-expressing MUC17 in a549 cells. As shown in E and F in fig. 1, MUC17 overexpression has an antiproliferative effect and can significantly inhibit DBP or DEHP-induced proliferation of a549 cells. In contrast, knockdown of MUC17 resulted in a significant increase in cell proliferation. This proliferation-promoting effect was further enhanced when the a549 cells were treated with DBP or DEHP. The results indicate that MUC17 plays an important role in pae-induced lung cell malignancy, suggesting that DBP or DEHP exposure may promote malignant changes in airway epithelial cells by down-regulating MUC17 expression.
EXAMPLE 2 MUC17 Gene methylation detection of Phthalate (PAEs) treated cell lines
1. Design and synthesis of MUC17 gene methylation detection primer
The MUC17 sequence of the gene to be detected (GenBank Accession No. NC-000007.14 (Update Date 8-Jul-2021)) is queried and downloaded, and the target sequence is input into EpiDesigner software for primer design. And selecting and determining a proper primer design scheme according to a software running result. Primers were synthesized, 5OD each.
The specific sequence of the methylation detection primer is as follows:
the primer group 1 covers a DNA sequence (SEQ ID No. 1) of-3898 bp to-3400 bp at the transcription initiation site of the MUC17 gene;
primer set 1 consists of F1 and R1, and its sequence is as follows:
upstream primer F1:5'-aggaagagag TTGTAGTTGGGAAGGAGTTTGTTT-3' (SEQ ID No. 4),
downstream primer R1:5'-cagtaatacgactcactatagggagaaggct CCTCCACCTAATCTCCCTACTTTAAT-3' (SEQ ID No. 5).
The primer group 2 covers a DNA sequence (SEQ ID No. 2) of-2844 bp to-2358 bp at the transcription initiation site of the MUC17 gene;
primer set 2 consists of F2 and R2, and its sequence is as follows:
the upstream primer F2:5'-aggaagagag GGGAATTTTAGAGGAAGTGGAGTAG-3' (SEQ ID No. 6),
downstream primer R2:5'-cagtaatacgactcactatagggagaaggct TACTAACCCAAAACCAACCCTATCT-3' (SEQ ID No. 7).
The primer group 3 covers a DNA sequence (SEQ ID No. 3) of-759 bp to-321 bp at the transcription initiation site of the MUC17 gene;
primer set 3 consisted of F3 and R3, and its sequence was as follows:
upstream primer F3:5'-aggaagagag GGAAAGGAGGAGGGTATTATTTGTA-3' (SEQ ID No. 8),
downstream primer R3:5'-cagtaatacgactcactatagggagaaggct CCCAAACATAACCATATCTTCAAAA-3' (SEQ ID No. 9).
The positions in the corresponding genes of primer set 3 are shown in FIG. 2A.
All primers were synthesized by Huada (Beijing).
And (3) performing PCR amplification by using the vulcanized and modified DNA as a template, amplifying by using a primer group 1 to obtain a fragment with 499bp, amplifying by using a primer group 2 to obtain a fragment with 487bp, amplifying by using a primer group 3 to obtain a fragment with 439bp, and performing methylation detection by using an Agena Massary array system. The methylation primer has Tm 59.99 of the upstream primer, GC content 40.00%, tm 60.32 of the downstream primer and GC content 32%. The test results used were EpiTYPER TM The software obtains the original data, the original data is to calculate the methylation and unmethylation degree of the sample to be detected according to the area comparison of the peak containing G and the peak containing A, and the methylation and unmethylation degree belongs to the relative quantitative ratio of the peak containing G and the peak containing A. If the detection value in the table is 0.43, it represents a 43% methylation degree of the corresponding methylation site in the sample. Taking the average methylation value of all sites in the region, and considering the methylation percentage as high methylation if the methylation percentage is more than or equal to 60; a "partial methylation" is considered if the methylation percentage is 20-60% (inclusive); a "unmethylated" is considered if the methylation percentage is less than or equal to 20%.
2. Methylation detection
The "primer set 3" in step 1 is exemplified.
Test cells: BEAS-2B, A549, BEAS-2B+DBP, BEAS-2B+DEHP, A549+DBP, and A549+DEHP
1. Cell DNA extraction procedure
(1) The test cells in the log phase and in good condition were gently washed twice with ice-cold 1 XPBS after removing the medium.
(2) Adding 1mL of pancreatin, digesting for 1min, adding 2mL of RPMI 1640 complete medium for neutralization, gently blowing suspension cells, transferring into a 15mL centrifuge tube, centrifuging at 1000rpm for 5min, discarding the supernatant, and collecting the cells.
(3) 2mL of DNA extract and 100. Mu.L of proteinase K (10 mg/mL) were added, and after mixing by blowing, the mixture was placed in a constant temperature water bath at 50℃for 3 hours.
(4) Take out, cool to room temperature, add equal volume of phenol/chloroform, mix upside down, centrifuge at 4200rpm for 15min, carefully transfer the supernatant into a new 15mL centrifuge tube.
(5) 1/10 volume of 7.5mol/L ammonium acetate and 2 times volume of absolute ethyl alcohol are added, the mixture is gently mixed, the mixture is centrifuged at 10000rpm for 20min, and the supernatant is discarded.
(6) 500 μl of 70% ethanol was added, the precipitate was washed twice, centrifuged at 13000rpm for 5min, the supernatant was discarded, and the pellet was air-dried.
(7) 100. Mu.L of TE solution with pH 8.0 was added to dissolve DNA, 1. Mu.L of the DNA solution was taken, the DNA concentration was measured by a Nanodrop 2000c ultra-micro nucleic acid analyzer, and the remaining sample was stored in a-20℃refrigerator.
2. Quality and concentration detection of DNA samples
DNA methylation positive and negative control samples were Human methyl purchased from Zymo Research Inc., USA&Non-methylated DNA Set (cat# D5014), human Methylated DNA is also known as 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, with a concentration greater than 50 ng/. Mu.l. Further use of NaHSO 3 The DNA sample to be tested was treated so that all cytosines in the unmethylated region were converted to thymines, while cytosines in the methylated region remained unchanged, and the procedure was performed using EZ DNA Methylation Gold Kit from Zymo Research, inc., U.S.A. (cat# D5006).
3. Methylation detection step of Agena MassArray system
Operation was performed using EpiTYPER Reagent and SpectroCHIP kit from Agena, U.S. (cargo number: 11377D)
(1) PCR amplification reaction
Sample NaHSO 3 After treatment, the mixture is centrifuged briefly at a low speed for standby. The PCR reaction solutions were prepared as shown in Table 2 (the reaction system preparation process was completed on ice, preventing deactivation by long-term exposure to high temperature).
Table 2.Agena MassArray System methylation detection amplification PCR reaction Components
(2) The amplification PCR reaction procedure is shown in Table 3
TABLE 3 PCR reaction procedure
3 μl of PCR product was mixed with 1 μl of 6-loading buffer, and detected by 1.5% agarose gel electrophoresis, and the results were observed after 160V for 20min (B in FIG. 2), if the results were good, the subsequent experiments were continued.
(3) Shrimp Alkaline Phosphatase (SAP) reactions
The PCR amplified product was reacted with SAP to remove excess deoxyribonucleoside triphosphates (dNTPs/dUTPs), and the SAP reaction system was as shown in Table 4:
TABLE 4 methylation detection of SAP reactive Components by MassArray System
Reagent(s) Final volume
RNase-free ddH 2 O 1.70μL
SAP enzyme 0.30μL
PCR products 5.00μL
Total volume of 7.00μL
7 μl of SAP reaction solution was added to each well of 384 reaction plates, 384 Kong Fengban films were carefully covered and each well was firmly pressed to prevent evaporation and the like during 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 cleavage/RNase A digestion reaction
The characteristic that RNase A can specifically recognize and cleave the U3' end of RNA is utilized to cleave RNA fragments into small fragments carrying CpG sites. The T-cut/RNase A digestion reaction system is shown in Table 6:
TABLE 6T cleavage reaction/RNase A digestion reaction Components
T-cut/RNase A digestion reaction conditions: incubate at 37℃for 3h.
4. Resin purification
Resin was uniformly filled in 384/6MG simple plates and allowed to stand for 10 minutes to allow to air dry. To each well of 384 sample plates, 16 μl of water was added. The 384 sample plates were snapped onto the two plates and flipped and tapped to drop the resin into each well of the sample plates. And sealing the 384 sample plates by using a sealing film, and placing the sealed 384 sample plates in a plate turnover centrifuge for uniformly rotating and mixing for 30 minutes at room temperature.
5. Chip sample application
Starting MassARRAY NanodispenserRS sample application instrument, transferring the product after resin purification to 384-wellOn bioarray. The spotted SpectroCHIP chip was analyzed using MALDI-TOF. The test results used were EpiTYPER TM The software obtains the raw data and the circle map and checks the integrity and accuracy of the data file. The results are saved to the corresponding storage medium and submitted for analysis in the bioinformatics room.
6. Results
The results are shown in fig. 2B and C, where the control system was established with IVD as the methylation control, NL as the non-methylation control, deionized water as the negative system control, and the cell samples were individually subjected to MassARRAY detection with the methylation primers.
Wherein BEAS-2B (methylation percentage 38.29%), BEAS-2B+DBP (methylation percentage 83.29%) and BEAS-2B+DEHP (methylation percentage 77.86%) were found 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 result judgment method given in step 1 of example 2, except that BEAS-2B was "partially methylated", the cell line detection results were all "hypermethylated".
The results indicate that PAEs environmental pollutants (such as DBP and DEHP) can increase the methylation level of the MUC17 promoter region.
EXAMPLE 3 DNA methylation inhibitors reversible silencing of MUC17 expression in respiratory epithelial cells and non-Small cell lung cancer cells
1. Cellular RNA extraction and reverse transcription process
(1) DNA methylase inhibitor 5-Aza-2 '-deoxyytidine (5-Aza, 5-Aza-2' -Deoxycytidine, sigma-Aldrich, USA, cat# 189825) was dosed: when the growth density of the cells (A549 and BEAS-2B) reached 30%, 5-Aza was added to a content of 5. Mu.M in the culture system, the medium was changed every 24 hours, and 5-Aza was further added to a content of 5. Mu.M in the culture system for 96 hours.
(2) Selecting cells with good growth state according to 1mL/10 6 Trizol reagent (Invitrogen, inc. of USA, cat# 15596026) was added to each cell. After 10min at room temperature, chloroform extraction was performed after sufficient lysis, and 0.2mL chloroform was added per 1mL Trizol.
(3) Shaking vigorously for 15s, standing at room temperature for 5min, centrifuging at 12000g at 4℃for 15min.
(4) The upper colorless liquid after delamination was transferred to a new centrifuge tube, precipitated with pre-chilled isopropanol, 0.5mL isopropanol was added per 1mL Trizol, and left on ice for 20min.12000g, centrifuging at 4 ℃ for 10min, and discarding the supernatant.
(5) The precipitate was washed with pre-chilled 75% ethanol, 1mL Trizol was added to 1mL 75% ethanol, 7500g, centrifuged at 4℃for 5min, and the supernatant discarded. Drying, adding proper amount of DEPC-H 2 O was dissolved, and 0.8% agarose gel electrophoresis confirmed, and the concentration of RNA was measured by NanoDrop and stored at-80 ℃.
(6) 1.0. Mu.g of RNA was reverse transcribed into cDNA using a TransScript II First-Strand cDNA Synthesis SuperMix kit (cat# AH 301-02) produced by Beijing full gold Biotechnology Co., ltd.): mu.L of Anchored Oligo (dT) 20, 10. Mu.L of 2 XS Reaction Mix and 1. Mu.L of RT/RI Enzyme Mix were added to DEPC-H 2 O to 20. Mu.L. The reaction conditions, 42℃30min,85℃5min, and the cDNA obtained by reverse transcription were allowed to stand at-20 ℃.
2、qPCR
(1) qPCR primer sequence:
the RT-qPCR primer sequence covers the amplified product 91bp of +11727 to +11817bp (located in the frame of MUC17 gene) from the transcription start site of MUC17 gene.
An upstream primer: 5'-GGGCCAGCATAGCTTCGA-3';
a downstream primer: 5'-GCTACAGGAATTGTGGGAGTTGA-3'.
Wherein the internal reference primer for homogenization is a primer taking Beta-Aactin as an internal reference,
an upstream primer: 5'-TTAGTTGCGTTACACCCTTTC-3';
a downstream primer: 5'-ACCTTCACCGTTCCAGTTT-3'.
(2) The reaction system for qPCR amplification is shown in table 7.
TABLE 7 reaction System for qPCR amplification
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Note that: 2 XSYBR-Green: zymo Research, usa, cat No. E2004.
(3) The PCR conditions were as follows:
2min at 50 ℃, 10min at 95 ℃ and 15s at 95 ℃;60 ℃ for 1min; collecting fluorescence in 40 cycles; a melting curve was prepared at 95℃for 15s,60℃for 1min,95℃for 15s, and 60℃for 15 s. Experimental results Using 2 -ΔΔCt The data are analyzed by a method, and the specificity of the product is ensured by a melting curve.
3. Results and analysis
The use of the apparent drug DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-2' -deoxyytidine) can reverse the silencing of MUC17 expression in normal human lung epithelial cells, indicating that MUC17 expression silencing is apparent regulated in normal human lung epithelial cells, as shown in D in figure 2.
Because the use of apparent drug DNA methylation inhibitors has been applied to clinical tumor therapy, the MUC17 methylation site can be a potential molecular target for the prevention and treatment of environmental factors-induced lung cancer.
EXAMPLE 4 inhibition of MUC17 expression by UHRF1-DNMT1 by DBP or DEHP exposure
1、qPCR
(1) qPCR primer sequence:
the RT-qPCR primer sequence covers the DNMT1 gene transcription initiation site by +800 to +903bp (located in the DNMT1 gene reading frame) amplification product 104bp.
An upstream primer: 5'-CCAAAGAACCAACACCCAAAC-3';
A downstream primer: 5'-CTCATCTTTCTCGTCTCCATCTTC-3'.
The RT-qPCR primer sequence covers the amplified product of +1047 to +1141bp (located inside the frame of DNMT3A gene) from the transcription start site of DNMT3A gene.
An upstream primer: 5'-ACGATTGCTAGACTGGGATAATG-3';
a downstream primer: 5'-AGTAAGCAGGCCAGGTAGA-3'.
The RT-qPCR primer sequence covers +4127- +4224bp (located inside the reading frame of DNMT3B gene) amplified product 98bp from the transcription start site of DNMT3B gene.
An upstream primer: 5'-GGAGCCACGACGTAACAAATA-3';
a downstream primer: 5'-GTAAACTCTAGGCATCCGTCATC-3'.
The RT-qPCR primer sequence covers 108bp of amplified product of +3622 to +3729bp (located in the reading frame of UHRF1 gene) from the transcription start site of UHRF1 gene.
An upstream primer: 5'-AAATGGCCTCAAGGGGACTC-3';
a downstream primer: 5'-CACTTGCACGTGACTTCGTG-3'.
Wherein the internal reference primer for homogenization is a primer taking Beta-Aactin as an internal reference,
an upstream primer: 5'-TTAGTTGCGTTACACCCTTTC-3';
a downstream primer: 5'-ACCTTCACCGTTCCAGTTT-3'.
(2) The qPCR reaction system and reaction conditions were the same as in example 3.
2. Interfering with DNMT1 Gene expression in A549 cells
Universal negative control pSilencer TM 3.1-H1 neo distominued, available from Thermo Fisher company, usa, cat: AM5770.
DNMT1 interferes with shRNA recognition region sequences:
5’-CUGCUGGGACUGGAGUUUAUA-3’；
non-small cell lung cancer cell lines A549 and BEAS-2B were selected for transient transfection. Cells were inoculated into 6-well cell culture plates, and when the cells were grown to 60% confluence, 200. Mu.L of DMEM was added to 20nM of plasmid expressing interfering shRNA and plasmid expressing empty shRNA, respectively, and after mixing, they were left at room temperature for 5min. 200. Mu.L of DMEM was added to 6. Mu.L of Lipofectamine2000, and the mixture was left at room temperature for 5min after mixing. 200mL Lipofectamine2000 (Thermo Fisher, inc. of America, cat# 11668019) was added to 200. Mu.L of the interfering plasmid solution, and the mixture was left at room temperature for 20 minutes after mixing. mu.L of the mixture was added to the cell culture plate, and after 8 hours of serum-free culture at 37℃the cell culture plate was incubated at 37℃for 24 hours with 10% FBS DMEM and then detected by RT-qPCR.
Construction of interfering plasmids: cloning and constructing the coding RNA molecules of the interfering shRNA and the coding RNA molecules of the empty shRNA contrast into a plasmid vector pSilencer TM 3.1-H1 to obtain plasmid for expressing interfering shRNA and plasmid for expressing empty shRNA, which are used for transfecting cells to establish cell lines, and the constructed cell lines are named shDNMT1 cell line (plasmid for transfecting interfering shRNA) and shNC cell line (plasmid for transfecting empty shRNA).
The interfering plasmid was synthesized by Shanghai Ji Kai gene company.
3. Cell biological function experiment
The test cell lines were: shDNMT1 cell line, shNC cell line, shDNMT1+DBP and shDNMT1+DEHP constructed for A549 respectively.
Wherein: shDNMT1+DBP means DBP (1. Mu.M, 24 h) treated shDNMT1 cell line; sh DNMT1+DEHP represents a DEHP (1. Mu.M, 24 h) -treated shDNMT1 cell line;
(1) MTT (3- [4, 5-dimethylazol-2-yl ] -2,5-diphenyl-tetrazolium bromide) experiment
Performing digestion count on a tested cell line in a logarithmic growth phase and with good growth condition, adjusting the concentration of a cell suspension, adding 100 mu L of the cell suspension into a 96-well cell culture plate in each well, paving 8 compound wells in 2000 cells in each well, and continuously observing for 4 days; mu.L of MTT (product number: M2128 of sigma Co., USA) was added to each well at a fixed time every day to give a final concentration of 5. Mu.g/mL, and after co-culturing for 4 hours, the medium was discarded. After 4 days, 150. Mu.L of dimethyl sulfoxide (dimethyl sulfoxide, DMSO, sigma Co., USA, cat# D2650) was added to each well, and after shaking for 10min in the dark, the absorbance of each well was measured at OD490nm of the ELISA, and the data obtained was analyzed.
(2) Migration experiment
The test cell lines were digested and counted separately and added to the upper chambers of a Transwell cell (product number: CLS3422, co., ltd.) having 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 well 4 Cells were inoculated and 600. Mu.L of complete medium was added to the lower chamber; culturing in a 37 ℃ cell culture box, culturing cells for 24 hours, and carefully cleaning the cells with 1 XPBS for several times; fixing 4% paraformaldehyde for 30min, and cleaning with 1×PBS three times; 0.5% crystal violet staining for 30min, rinsing to colorless, carefully removing upper chamber cells with cotton swab, and counting the number of migrating cells under microscope (100×).
(3) Statistical analysis
The experiment was repeated at least three times. The experimental results were all tested using a two-sided t-test, the results being expressed as mean ± standard deviation, p <0.05 representing significant statistical differences and p <0.01 representing very significant statistical 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. RT-qPCR results showed that DNMT1 was up-regulated most in DBP or DEHP exposed A549 cells and BEAS-2B cells among 3 DNMTs and correlated with the key regulatory protein UHRF1 for DNA methylation (FIGS. 3A and 3B). Interfering with DNMT1 in a549 cells promoted the expression of MUC17 and inhibited epigenetic silencing following DBP or DEHP exposure (C in fig. 3). The above results indicate that DBP or DEHP induced epigenetic silencing of MUC17 is mediated 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 inhibition of the oncogenic effects of cells after DBP or DEHP exposure by interfering with DNMT1 may restore the expression of MUC17 suggests that low doses of PAEs play an important role in the specific epigenetic pathway of human airway epithelial malignant transformation of MUC 17.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
SEQUENCE LISTING
<110> Beijing chest Hospital affiliated with university of capital medical science
Beijing city tuberculosis chest tumor institute
<120> a kit for environmental pollution cancerogenic 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 (2)

1. Use of a primer pair consisting of primer F3 and primer R3, wherein primer F3 is single-stranded DNA having the nucleotide sequence of SEQ ID No.8 and primer R3 is single-stranded DNA having the nucleotide sequence of SEQ ID No.9, for the preparation of a product for predicting the risk of non-small cell lung cancer.
2. The use according to claim 1, wherein the product is a kit.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014082083A1 (en) * 2012-11-26 2014-05-30 Caris Science, Inc. Biomarker compositions and methods
CN113186287A (en) * 2021-05-10 2021-07-30 深圳康华君泰生物科技有限公司 Biomarker for non-small cell lung cancer typing and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014082083A1 (en) * 2012-11-26 2014-05-30 Caris Science, Inc. Biomarker compositions and methods
CN113186287A (en) * 2021-05-10 2021-07-30 深圳康华君泰生物科技有限公司 Biomarker for non-small cell lung cancer typing and application thereof

Non-Patent Citations (2)

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Title
DNA methylation and histone H3-K9 modifications contribute to MUC17 expression;Sho Kitamoto等;Glycobiology;第21卷(第2期);摘要、材料与方法 *
Epigenetic downregulation of MUC17 by H. pylori infection facilitates NF-κB-mediated expression of CEACAM1-3S in human gastric cancer;Shuye Lin等;Gastric Cancer;第22卷(第5期);第941-954页 *

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