KR20170022361A - Biomarker for identification of genes related to inflammatory response after exposure to diesel exhaust particle and the method of identification using thesame - Google Patents
Biomarker for identification of genes related to inflammatory response after exposure to diesel exhaust particle and the method of identification using thesame Download PDFInfo
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Abstract
The present invention relates to an inflammatory reaction-related biomarker for confirming exposure of diesel exhaust particles, which is one of the fine particles exposed in the environment, and a method of using the same. More specifically, The present invention relates to a biomarker relating to an inflammatory reaction and a method for identifying specific exposure to diesel exhaust fine particles using the same. The biomarker of the present invention comprises a DNA microarray chip, Can be used to monitor and determine the contamination of diesel exhaust particulates in environmental samples and can be used as a tool to identify toxic action mechanisms specifically induced by diesel exhaust particulates.
Description
TECHNICAL FIELD The present invention relates to a biomarker for confirming the expression of an inflammatory reaction related gene upon exposure to diesel exhaust particles and a method for confirming expression of the biomarker by using the biomarker, The present invention relates to a method for confirming exposure to diesel exhaust particulates using a marker.
According to the National Statistical Office, according to the Korea National Statistical Office, the leading cause of death in Korea is cancer. Lung cancer in cancer has been the number one cause of death since 2000, and the death rate is the largest increase compared to 10 years ago. In addition to lung cancer, asthma and chronic bronchitis, such as chronic diseases and pneumonia, such as the incidence and mortality rate is increasing steadily.
On the other hand, diesel exhaust particles are mainly composed of incomplete combustion product particles of fuel, oxides and nitro compounds such as formaldehyde, which give stimulation to engine oil and unburned fuel and surrounding body, Of organic particles and sulphates and nitrates. Most of the particle size distribution of diesel exhaust particulate exists as nanoparticles with particle size of 100 mm or less. The smaller the grain size, the larger the number of particles and the larger the total surface area becomes. When the surface of the particle itself is toxic or adsorbs a substance which is toxic to the surface, the larger the surface area, the stronger the toxicity may be. It has been found that exposure to diesel exhaust particles exacerbates the allergic reaction, which has been shown to be due to an increase in histamine production due to hyperactivity of T cells and excessive release of toxic substances produced by eosinophils.
The nanoparticles of 100 mm or less increase in deposition in the lungs as the particle size decreases, reaching a peak of 20 mm and deposition of about 50% in the alveoli. Diesel exhaust particulate nanoparticles of 20 mm or less are more likely to enter the bloodstream than large particles and have a large surface area and are highly related to toxicity, so they are likely to cause inflammation, thrombosis, and affect organs such as the heart and liver. Studies on the toxicity of diesel particulate matter, which also plays a major part of fine particles, have been actively conducted, and studies on the mechanism of allergic rhinitis and allergic conjunctivitis have been studied. In addition, cardiac dysfunction was observed in experimental animal models exposed to diesel exhaust particulates, suggesting the possibility of aging. It has been reported that long-term diesel exhaust particulates are injected into the respiratory tract of experimental animals together with allergen, causing asthma together with airway eosinophilic inflammation (Ris C, 2007; Sagai et al., 1996). It has been reported that the bronchial asthma, which is a symptom of bronchial asthma, narrows widely when the diesel exhaust particles are administered to the mice together with the antigen, and the airway hyperresponsiveness is sensitively sensitized by the small stimulus and the mucous component is over secreted (Lim and Lee, 2002; Lim et al., 1998 Sagai et al., 1996).
Studies on the human health effects of airborne fine particles have been reported, but there are not many studies on the expression of inflammatory reaction related genes in diesel exhaust particulates. The inflammatory response in the airways is the most important cause of various lung diseases. When exposed to environmentally harmful substances such as airborne fine dusts, inflammatory cells in the airways are activated and various inflammatory reaction-related cytokines and chemokines, pro-inflammatory mediators, and proteolytic enzymes (et al., 1998; Hetland et al., 1998), which is associated with increased production of proteases and the like, resulting in oxidative stress, imbalance between proteolytic enzymes and anti-proteolytic enzymes ., 2004). Since changes in the expression of genes involved in inflammatory reactions are likely to lead to pulmonary disease induction, studies to observe changes in the expression of inflammatory response genes in lung cells following exposure of diesel exhaust particulates have shown that exposure of diesel exhaust particulates It is very important in terms of prediction of lung disease induction.
Although the risk of microparticles including diesel exhaust particulates has been increasing, studies on epidemiological studies and actual animal models have been carried out until now. In addition, human lung cell line system which can predict exposure and toxicity by more convenient method There are still few research reports. In addition, the method of searching for exposure to harmful substances is still limited to classical methods such as GC-MS (Gas Chromatography-Mass Spectrometer) or HPLC (High Performance Liquid Chromatography). Quantification is possible using GC-MS or HPLC method, but appropriate conditions for analysis are required and expensive equipment is required. Therefore, a quick and easy screening method, for example, real-time reverse transcript polymerase chain reaction (PCR) using a primer or a DNA microarray chip, And to identify the molecular markers that can be used to detect the toxicity and gene expression of the diesel exhaust particulate matter.
A genome sequencing project of 236 species of eukaryotes, 2602 species of bacteria, 167 species of highly bacterial species has been completed and reported in National Center for Biotechnology Information (NCBI). Genome-wide expression studies have been conducted to study the function of genes based on the vast amount of data thus obtained. DNA microarray analysis is performed to analyze the expression of thousands of genes in a single experiment (Schena, M., et al, 1996).
A microarray is a collection of cDNA (complementary DNA) or a set of oligonucleotides of 20-25 base pairs in length on glass. cDNA microarrays are produced either by in-house laboratories or by companies such as Agilent, Genomic Solutions, etc., by mechanically immobilizing cDNA collections onto chips or by using ink jetting (J. Am. Acad. Dermatol. 51: 681 -692, 2004). The oligonucleotide microarray is produced by a direct synthesis method on a chip using photolithography in Affymetrix, and the oligonucleotide is produced by a method of immobilizing a synthesized oligonucleotide in Agillent (J. Am. Acad Dermatol 51: 681-692, 2004).
Recently, it has been applied to toxic genomics research which is a cutting-edge technique using DNA microarray technology, and it can be expressed in a large amount (high throughput) in a specific tissue or cell line due to all chemical substances, including representative drug candidates, And the quantitative analysis of the expression patterns of the genes. Thus, by analyzing the frequency of expression of a specific gene in a specific cell, it is possible to identify a gene related with adverse effects of drugs and harmful effects of environmental pollutants, and thereby, harmful effects of environmental pollutants, And will be able to search for and identify substances that cause toxicity and side effects.
The present inventors observed and analyzed the gene expression pattern of diesel exhaust microparticles in the A549 cell line derived from human lung cancer tissue using an oligonucleotide microarray in which 44,000 human genes were integrated, The present inventors have completed the present invention by establishing a biomarker capable of specifically detecting diesel exhaust particulates and a method of confirming exposure using the biomarker capable of detecting specifically overexpressed or underexpressed inflammatory reaction related genes.
It is an object of the present invention to provide a biomarker related to an inflammation reaction which is overexpressed or underexpressed by exposure of diesel exhaust fine particles and a method for confirming whether or not the biomarker is specifically exposed to diesel exhaust fine particles.
In order to accomplish the above object, the present invention provides an inflammatory reaction-related biomarker for detecting specific exposure of diesel exhaust particulates, which causes an expression change by exposure of diesel exhaust particulates.
The present invention also provides a DNA microarray chip for confirming the expression of an inflammatory reaction-related gene for exposure to diesel exhaust microparticles in which nucleic acid sequences of the following genes or complementary strand molecules thereof are integrated:
GenBank NM_002986 (CCL11, CC motif ligand 11), GenBank NM_014707 (Histone deacetylase 9), GenBank NM_198389 (PDPN, Podoplanin), Gene registration number (GenBank) NM_000065 (C6, Complement component 6), GenBank NM_015364 (LY96, Lymphocyte antigen 96), GenBank NM_000698 (ALOX5, Arachidonate 5-lipoxygenase), GenBank NM_080706 (TRPV1, transient receptor potential cation channel, subfamily V, member 1), GenBank NM_138938 (REG3A, Regenerating islet-derived 3 alpha), GenBank NM_004991 (MECOM, MDS1 and EVI1 complex locus) , GenBank NM_002558 (P2RX1, Purinergic receptor P2X, ligand-gated ion channel, 1), GenBank NM_000756 (CRH, Corticotropin releasing hormone), GenBank NM_022468 (MMP25, Matrix metallopeptidase 25), GenBank NM_000612 (IGF2, Insulin-like growth factor 2 (somatomedin A)), GenBank NM_138284 IL17D, Interleukin 17D), GenBank NM_002982 (CCL2, CC motif ligand 2), GenBank NM_001570 (IRAK2, Interleukin-1 receptor-associated kinase 2), GenBank (KDM6B, Lysine (K) -specific demethylase 6B), GenBank NM_030754 (SAA2, Serum (SEQ ID NO: 2)), NM_002852 (PTX3, rapidly induced by IL-1 beta), GenBank NM_001080424 amyloid A2), GenBank NM_001718 (BMP6, Bone morphogenetic protein 6), GenBank NM_007115 (TNFAIP6, Tumor necrosis factor, alpha-induced protein 6), GenBank NM_000331 (SAA1 , Serum amyloid A1), GenBank NM_000204 (CFI, Complement factor I), GenBank NM_002985 (CCL5, Chemokine (C-C motif) ligand 5), GenBank NM_002984 (CCL4, Chemokine (C-C motif) ligand 4).
Further, according to the present invention,
1) separating the respective RNAs from the experimental group suspected of exposing the diesel exhaust particulates and the somatic cells of the normal control group;
2) labeling the experimental group and the control group with different fluorescent materials while synthesizing the RNA of the experimental group and the control group of the step 1) with cDNA;
3) hybridizing the cDNA labeled with the different fluorescent material of step 2) with the DNA microarray chip of the present invention;
4) analyzing the results of the reacted DNA microarray chip of step 3); And
5) The step of analyzing the DNA microarray chip of the present invention by comparing the degree of expression of the genes integrated in the DNA microarray chip of the present invention with that of the control group in the step 4) .
Further, according to the present invention,
1) separating the respective RNAs from the experimental group suspected of exposing the diesel exhaust particulates and the somatic cells of the normal control group;
2) Real-time reverse transcript polymerase chain reaction (RT-PCR) is performed using a pair of primers complementary to the following genes of the above step 1) step:
GenBank NM_014707 (HDAC9, Histone deacetylase 9), GenBank NM_015364 (LY96, Lymphocyte antigen 96), GenBank NM_022468 (MMP25, Matrix metallopeptidase 25), GenBank ), GenBank NM_002982 (CCL2, CC motif ligand 2), GenBank NM_030754 (SAA2, Serum amyloid A2), and the like), NM_000612 (Insulin-like growth factor 2 (somatomedin A) , GenBank NM_001718 (BMP6, Bone morphogenetic protein 6), GenBank NM_000331 (SAA1, Serum amyloid A1), GenBank NM_002984 (CCL4, Chemokine (CC motif) ligand 4) ; And
3) comparing the gene product of step 2) with that of the control, and confirming the expression level of the gene product.
Also, the present invention provides a kit for confirming the expression of an inflammatory reaction-related gene for exposure to diesel exhaust microparticles including the DNA microarray chip of the present invention.
In addition, the present invention provides a kit for confirming the expression of an inflammatory reaction-related gene for exposure to diesel exhaust fine particles containing a pair of primers complementary to the following genes and capable of amplifying the following genes:
GenBank NM_014707 (HDAC9, Histone deacetylase 9), GenBank NM_015364 (LY96, Lymphocyte antigen 96), GenBank NM_022468 (MMP25, Matrix metallopeptidase 25), GenBank ), NM_002982 (CCL2, CC motif ligand 2), GenBank NM_030754 (SAA2, Serum amyloid A2), gene registration number (GenBank) (BMP6, Bone morphogenetic protein 6), GenBank NM_000331 (SAA1, Serum amyloid A1), GenBank NM_002984 (CCL4, Chemokine (CC motif) ligand 4).
The biomarker for confirming the exposure of the diesel exhaust fine particles of the present invention and the confirmation method using the biomarker are useful for monitoring the monitoring and the risk of diesel exhaust particulates by using a reaction marker related to the inflammation reaction selected through the DNA microarray chip as a biomarker, It can be usefully used as a tool to identify the specific toxic action mechanism of inflammation caused by diesel exhaust particulate matter.
1 is a graph showing cytotoxicity of diesel exhaust microparticles in human lung cancer tissue-derived cell lines.
FIG. 2 is a graph showing the results of analysis of gene expression patterns of human lung cancer tissue-derived cell lines treated with diesel exhaust microparticles using a microarray chip.
FIG. 3 is a graph showing the results of microarray chip results and real-time reverse transcript polymerase chain reaction (RT-PCR) of each gene for nine genes that were increased or decreased under the condition of treating diesel exhaust microparticles, Increase / decrease of the market.
Hereinafter, the present invention will be described in detail.
The present invention provides a biomarker for confirming the expression of an inflammatory reaction-related gene which causes a change in expression by exposure of diesel exhaust fine particles.
The biomarker is a gene whose expression is increased or decreased by 2.0 times or more, and is composed of 25 kinds of inflammation reaction related genes whose expression is changed by diesel exhaust fine particles.
The biomarker related to the inflammation reaction which causes the expression change by the exposure of the diesel exhaust fine particles is preferably, but not limited to, the following genes:
GenBank NM_002986 (CCL11, CC motif ligand 11), GenBank NM_014707 (Histone deacetylase 9), GenBank NM_198389 (PDPN, Podoplanin), Gene registration number (GenBank) NM_000065 (C6, Complement component 6), GenBank NM_015364 (LY96, Lymphocyte antigen 96), GenBank NM_000698 (ALOX5, Arachidonate 5-lipoxygenase), GenBank NM_080706 (TRPV1, transient receptor potential cation channel, subfamily V, member 1), GenBank NM_138938 (REG3A, Regenerating islet-derived 3 alpha), GenBank NM_004991 (MECOM, MDS1 and EVI1 complex locus) , GenBank NM_002558 (P2RX1, Purinergic receptor P2X, ligand-gated ion channel, 1), GenBank NM_000756 (Corticotropin releasing hormone), GenBank NM_022468 (MMP25, Matrix metallopeptidase 25), gene registration number (GenBank ), NM_000612 (IGF2, Insulin-like growth factor 2 (somatomedin A)), GenBank NM_138284 (IL17D, Interleukin 17D), GenBank NM_002982 (CCL2, CC motif ligand 2) GenBank NM_001570 (IRAK2, Interleukin-1 receptor-associated kinase 2), GenBank NM_002852 (PTX3, Pentraxin-related gene, rapidly induced by IL-1 beta) GenBank NM_030754 (SAA2, Serum amyloid A2), GenBank NM_001718 (BMP6, Bone morphogenetic protein 6), GenBank (GenBank), GenBank NM_001080424 (KDM6B, Lysine ), NM_007115 (TNFAIP6, Tumor necrosis factor, alpha-induced protein 6), GenBank NM_000331 (SAA1, Serum amyloid A1), GenBank NM_000204 (CFI, Complement factor I) GenBank NM_002985 (CCL5, Chemokine (CC motif) ligand 5), GenBank NM_002984 (CCL4, Chemokin e (CC motif) ligand 4).
In a specific embodiment of the present invention, the inventors of the present invention have found that a diesel exhaust particulate reference material purchased from National Institute of Standards and Technology (NIST) can be used for the detection of inflammation-related biomarkers for confirming exposure of diesel exhaust particulates to human lung cancer tissues A549 cell line to confirm cytotoxicity. As a result, it was confirmed that the diesel exhaust fine particles had toxicity to human lung cancer tissue-derived cell lines (see FIG. 1), and the concentration of diesel exhaust fine particles was determined based on the experiment.
In the present invention, the diesel exhaust microparticles were treated with the above determined concentrations to a cell line derived from a human lung cancer tissue, the mRNA was isolated from the cell line treated with the substance and labeled with a fluorescent material (Cy5) while cDNA was synthesized, And labeled with Cy3 in the control group. The fluorescence-labeled cDNA was hybridized using Agilent Human GE V2 4X44K (Agilent, USA), and fluorescence images of the microarray chip were analyzed to analyze differences in gene expression patterns (see FIG. 2). When the ratio of Cy5 / Cy3 was 2.0 times or more, it was classified as an increased expression gene. When the ratio was 0.5 times or less, it was classified as a reduced expression gene. For the significance of the experiment, three replicate experiments were carried out. For the analysis, the median of the results of three replicates was used, and only the t-test p value of 0.05 was used for the analysis. As a result of the analysis, it was confirmed that 1.91% (653 out of 34,127 genes) increased the expression level and 0.47% (159 out of 34,127 genes) decreased the expression level. As a result of classification of these genes by function, 25 genes involved in inflammation reaction were included. Although the above genes have been reported to be involved in inducing diseases related to lung toxicity by other chemicals in the existing invention, when the diesel exhaust fine particles used in the present invention are treated, they are related to toxicity in human lung cancer tissue-derived cells There is no report.
The present inventors have identified five genes over-expressed, four genes with low expression (GenBank NM_014707 (HDAC9, Histone deacetylase 9), GenBank NM_015364 (LY96, Lymphocyte antigen 96) GenBank NM_022468 (MMP25, Matrix metallopeptidase 25), GenBank NM_000612 (IGF2, Insulin-like growth factor 2 (somatomedin A)), NM_002982 (CCL2, Chemokine (CC motif) ligand 2) GenBank NM_030754 (SAA2, Serum amyloid A2), GenBank NM_001718 (BMP6, Bone morphogenetic protein 6), GenBank NM_000331 (SAA1, Serum amyloid A1), GenBank ) NM_002984 (CCL4, Chemokine (CC motif) ligand 4)) was confirmed by real time quantitative PCR method. As a result, it was confirmed that the expression patterns of the nine genes appeared similar to those of the oligomicroarray chip (see FIG. 3).
Therefore, the inflammation-related gene of the present invention can be usefully used as a biomarker for confirming exposure of diesel exhaust fine particles by causing an expression change by exposure to diesel exhaust fine particles.
The present invention also provides a DNA microarray chip for confirming the expression of an inflammatory reaction-related gene for exposure to diesel exhaust microparticles in which nucleic acid sequences of the following genes or complementary strand molecules thereof are integrated:
GenBank NM_002986 (CCL11, CC motif ligand 11), GenBank NM_014707 (Histone deacetylase 9), GenBank NM_198389 (PDPN, Podoplanin), Gene registration number (GenBank) NM_000065 (C6, Complement component 6), GenBank NM_015364 (LY96, Lymphocyte antigen 96), GenBank NM_000698 (ALOX5, Arachidonate 5-lipoxygenase), GenBank NM_080706 (TRPV1, transient receptor potential cation channel, subfamily V, member 1), GenBank NM_138938 (REG3A, Regenerating islet-derived 3 alpha), GenBank NM_004991 (MECOM, MDS1 and EVI1 complex locus) , GenBank NM_002558 (P2RX1, Purinergic receptor P2X, ligand-gated ion channel, 1), GenBank NM_000756 (CRH, Corticotropin releasing hormone), GenBank NM_022468 (MMP25, Matrix metallopeptidase 25), GenBank NM_000612 (IGF2, Insulin-like growth factor 2 (somatomedin A)), GenBank NM_138284 IL17D, Interleukin 17D), GenBank NM_002982 (CCL2, CC motif ligand 2), GenBank NM_001570 (IRAK2, Interleukin-1 receptor-associated kinase 2), GenBank (KDM6B, Lysine (K) -specific demethylase 6B), GenBank NM_030754 (SAA2, Serum (SEQ ID NO: 2)), NM_002852 (PTX3, rapidly induced by IL-1 beta), GenBank NM_001080424 amyloid A2), GenBank NM_001718 (BMP6, Bone morphogenetic protein 6), GenBank NM_007115 (TNFAIP6, Tumor necrosis factor, alpha-induced protein 6), GenBank NM_000331 (SAA1 , Serum amyloid A1), GenBank NM_000204 (CFI, Complement factor I), GenBank NM_002985 (CCL5, Chemokine (C-C motif) ligand 5), GenBank NM_002984 (CCL4, Chemokine (C-C motif) ligand 4).
The DNA microarray chip for diesel exhaust fine particle exposure search of the present invention can be manufactured by a method known to a person skilled in the art.
A method of fabricating the microarray chip is as follows.
A micropipetting method using a piezo electric method or a pin-type spotter for immobilizing the searched biomarker as a probe DNA molecule on a substrate of a DNA chip, But the present invention is not limited thereto. In a preferred embodiment of the present invention, a micro array, which is a pin-shaped spotter, is used.
The substrate of the DNA microarray chip is preferably coated with one activator selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde, But is not limited thereto.
The substrate may be selected from the group consisting of a slide glass, a plastic, a metal, a silicon, a nylon film, and a nitrocellulose membrane, but the present invention is not limited thereto. In a preferred embodiment of the present invention, Coated slide glass was used.
In addition, the present invention provides a method for confirming the expression of an inflammatory reaction-related gene for exposure of diesel exhaust fine particles using the biomarker of the present invention.
The present invention provides a method for confirming the expression of an inflammatory reaction-related gene in diesel exhaust particulate exposure comprising the steps of:
1) separating the respective RNAs from the experimental group suspected of exposing the diesel exhaust particulates and the somatic cells of the normal control group;
2) labeling the experimental group and the control group with different fluorescent substances while synthesizing the RNA of the experimental group and the control group of step 1) with cDNA;
3) hybridizing the cDNA labeled with the fluorescent substance of step 2) with the DNA microarray chip of the present invention;
4) analyzing the reacted DNA microarray chip; And
5) confirming the degree of expression of the gene of the present invention in comparison with the control group in the analyzed data.
In the method for confirming the exposure, it is preferable that the somatic cell of step 1) is an A549 cell line, which is a cell derived from human lung cancer tissue, but it is not limited thereto. Any cell derived from human lung or human lung cancer tissue can be used It is possible.
Wherein the fluorescent material of step 3) is selected from the group consisting of Cy3, Cy5, FITC (poly L-lysine-fluorescein isothiocyanate), RITC (rhodamine-B-isothiocyanate) and rhodamine But it is not limited thereto, and fluorescent materials known to those skilled in the art are all usable.
The DNA microarray chip of step 4) is preferably a Whole Human Genome Oligo Microarray (Agilent, USA), but the present invention is not limited thereto. In the present invention, among the human genomes, Or a microarray chip on which a low-level inflammatory reaction-related gene is mounted, and it is most preferable to use the DNA microarray chip manufactured by the present inventor.
It is preferable to use Agilent Feature Extraction 10.7.3.1 (Agilent Technologies, CA, USA) or Agilent GeneSpring GX 12.6.1 (Agilent technologies, CA, USA) for the analysis method of step 4) Analytical software known to those skilled in the art may be used.
Further, according to the present invention,
1) separating the respective RNAs from the experimental group suspected of exposing the diesel exhaust particulates and the somatic cells of the normal control group;
2) Real-time reverse transcript polymerase chain reaction (RT-PCR) using primer pairs complementary to the following genes and capable of amplifying the following genes:
GenBank NM_014707 (HDAC9, Histone deacetylase 9), GenBank NM_015364 (LY96, Lymphocyte antigen 96), GenBank NM_022468 (MMP25, Matrix metallopeptidase 25), GenBank ), GenBank NM_002982 (CCL2, CC motif ligand 2), GenBank NM_030754 (SAA2, Serum amyloid A2), and the like), NM_000612 (Insulin-like growth factor 2 (somatomedin A) , GenBank NM_001718 (BMP6, Bone morphogenetic protein 6), GenBank NM_000331 (SAA1, Serum amyloid A1), GenBank NM_002984 (CCL4, Chemokine (CC motif) ligand 4) ; And
3) comparing the gene product of step 2) with that of the control, and confirming the expression level of the gene product.
The primer pair in step 2) is preferably a forward primer pair and a reverse primer pair having a length of 18 to 30 m, which is capable of amplifying the gene of step 2), more preferably selected from the group consisting of primer pair 1 to 9 But are not limited to:
Primer pair 1: a forward primer described in SEQ ID NO: 1 and a reverse primer described in SEQ ID NO: 2;
Primer pair 2: the forward primer described in SEQ ID NO: 3 and the reverse primer described in SEQ ID NO: 4;
Primer pair 3: a forward primer described in SEQ ID NO: 5 and a reverse primer described in SEQ ID NO: 6;
Primer pair 4: a forward primer described in SEQ ID NO: 7 and a reverse primer described in SEQ ID NO: 8;
Primer pair 5: a forward primer described in SEQ ID NO: 9 and a reverse primer described in SEQ ID NO: 10; And
Primer pair 6: the forward primer described in SEQ ID NO: 11 and the reverse primer set forth in SEQ ID NO: 12.
Primer pair 7: a forward primer represented by SEQ ID NO: 13 and a reverse primer represented by SEQ ID NO: 14;
Primer pair 8: a forward primer described in SEQ ID NO: 15 and a reverse primer described in SEQ ID NO: 16; And
Primer pair 9: the forward primer described in SEQ ID NO: 17 and the reverse primer set forth in SEQ ID NO: 18.
Therefore, the biomarker of the present invention can be used to monitor and determine the contamination of diesel exhaust particulates in environmental samples, since gene expression is specifically increased or decreased in diesel exhaust particulates.
In addition, the present invention provides a kit for confirming the specific exposure of diesel exhaust fine particles comprising the DNA microarray chip fabricated in the present invention.
The kit preferably includes but is not limited to human somatic cells.
The human somatic cell is preferably A549, but is not limited thereto. Any cell derived from human lung cells or human lung cancer cells and tissues can be used.
The kit may further comprise a fluorescent material, wherein the fluorescent material is selected from the group consisting of a strepavidin-like phosphatease conjugate, a chemiluminescent substance and a chemiluminescent substance But it is not limited thereto. In a preferred embodiment of the present invention, Cy3 and Cy5 are used.
The reaction reagent may further include a buffer solution used for hybridization, a reverse transcriptase for synthesizing cDNA from RNA, cNTPs and rNTP (pre-mixed or separate feed type), a chemical inducer for fluorescent dye And a washing buffer solution. However, the present invention is not limited thereto. Any reaction reagent necessary for hybridization reaction of a DNA microarray chip known to a person skilled in the art can be included.
The biomarker of the present invention can be used to monitor and determine the contamination of diesel exhaust particulates in environmental samples because the gene expression is increased or decreased by the exposure of the diesel particulate exhaust particulates. Can be used as a tool to identify the toxic mechanism of inflammation reaction induced.
Further, according to the present invention,
There is provided a kit for confirming the expression of an inflammatory reaction-related gene for exposure of diesel exhaust fine particles containing a pair of primers complementary to the following genes and capable of amplifying the following genes:
GenBank NM_014707 (HDAC9, Histone deacetylase 9), GenBank NM_015364 (LY96, Lymphocyte antigen 96), GenBank NM_022468 (MMP25, Matrix metallopeptidase 25), GenBank ), GenBank NM_002982 (CCL2, CC motif ligand 2), GenBank NM_030754 (SAA2, Serum amyloid A2), and the like), NM_000612 (Insulin-like growth factor 2 (somatomedin A) , GenBank NM_001718 (BMP6, Bone morphogenetic protein 6), GenBank NM_000331 (SAA1, Serum amyloid A1), GenBank NM_002984 (CCL4, Chemokine (CC motif) ligand 4) .
The primer pair of the confirmation kit is preferably selected from the group consisting of the following primer pairs 1 to 9, but is not limited thereto, and it is preferable that the amplification product of the biomarker gene is 15 to 50 mPa Both forward and reverse primer pairs of length, preferably 15 to 30 mer long, more preferably 18 to 25 mer long, can be used:
Primer pair 1: a forward primer described in SEQ ID NO: 1 and a reverse primer described in SEQ ID NO: 2;
Primer pair 2: the forward primer described in SEQ ID NO: 3 and the reverse primer described in SEQ ID NO: 4;
Primer pair 3: a forward primer described in SEQ ID NO: 5 and a reverse primer described in SEQ ID NO: 6;
Primer pair 4: a forward primer described in SEQ ID NO: 7 and a reverse primer described in SEQ ID NO: 8;
Primer pair 5: a forward primer described in SEQ ID NO: 9 and a reverse primer described in SEQ ID NO: 10; And
Primer pair 6: the forward primer described in SEQ ID NO: 11 and the reverse primer set forth in SEQ ID NO: 12.
Primer pair 7: a forward primer represented by SEQ ID NO: 13 and a reverse primer represented by SEQ ID NO: 14;
Primer pair 8: a forward primer described in SEQ ID NO: 15 and a reverse primer described in SEQ ID NO: 16; And
Primer pair 9: the forward primer described in SEQ ID NO: 17 and the reverse primer set forth in SEQ ID NO: 18.
In addition to the above-mentioned confirmation kit, it is preferable to include human somatic cells but not limited thereto.
The human somatic cell is preferably an A549 cell line, which is a cell derived from human lung cancer tissue, but not limited thereto, and any cell derived from human lung cells or human lung cancer cells and tissues can be used.
In addition, the kit may further comprise a reaction reagent, and the reaction reagent may be a labeling reagent such as reverse transcriptase, cNTPs and rNTP (pre-mixed or separate feed type) for synthesizing cDNA from RNA, chemical inducer of fluorescent dye, Washing buffer solution, and the like, but not limited thereto, and may include all reaction reagents necessary for RT-PCR reaction known to those skilled in the art.
Hereinafter, the present invention will be described in detail with reference to examples.
However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.
< Example 1> Cell culture and chemical treatment
<1-1> Cell culture
A549 cells (Korean Cell Line Bank), a human lung cancer cell line, were cultured to a growth of about 80% under RPMI medium (Gibro-BRL, USA) supplemented with 10% FBS in a 100 mm dish.
<1-2> Cytotoxicity Test MTT assay ) And chemical treatment
MTT assay using A549 cell line was performed according to the method of Mossman et al. Specifically, the diesel exhaust particulate reference material purchased from NIST was treated and cultured for 48 hours under conditions of 2.5 × 10 5 cells / well in RPMI medium (Gibro-BRL, USA) using a 24-well plate, And 5 mg / ml of MTT (3-4,5-dimethylthiazol-2,5-diphenyltetra zolium bromide) were mixed and further cultured at 37 ° C for 3 hours. Then, the medium was removed and the formazan crystal formed was dissolved in 500 占 퐇 of DMSO. The lysate was transferred to a 96-well plate and aliquoted and the OD value measured at 540 nm absorbance.
As a result, as shown in Fig. 1, the concentration (IC20) showing an 80% survival rate against cytotoxicity by diesel exhaust microparticles in the A549 cell line was 68 占 퐂 / ml (Fig. 1) Experiments were performed.
< Example 2> Microarray Experiment
<2-1> Target RNA detach
A549 cells were dispensed into a 6-well plate at a concentration of 2.5 x 10 < 5 > cells / ml, and the diesel exhaust particulate standards purchased from NIST were treated for 48 hours. Then, total RNA was extracted from the fine dust-treated cells according to the manufacturer's method using trizol (Invitrogen life technologies, USA) and purified using an RNeasy mini kit (Qiagen, USA). During RNA purification, the DNA genome was completely removed using RNase-free DNase set (Qiagen, USA). The amount of total RNA sample was measured with a spectrophotometer, and its concentration was confirmed by ND-1000 Spectrophotometer (Thermo Fisher Scientific Inc., USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA).
≪ 2-2 > Labeled cDNA Produce
For the DNA microarray analysis, the total RNA of the diesel exhaust fine particle treated group and the control group obtained in Example 1 was prepared from cDNA. 30 μg of the obtained total RNA and 2 μg (1 μg / μl) of an oligo (dT) primer were mixed and reacted at 65 ° C. for 10 minutes and immediately annealed by ice. For the reverse transcript enzyme reaction of the annealed RNA, reagents were mixed and prepared as shown in Table 1 below. The untreated control group was labeled with Cy3-dUTP (green), and the experimental group treated with diesel exhaust microparticles was labeled with Cy5-dUTP (red). At this time, the two samples were mixed and purified using a Microcon YM-30 column (Millipore, USA).
<2-3> Hybridization ( hybridization ) reaction
Hybridization and washing were carried out according to the instructions of BIOJEN. The hybridization reaction and the washing process were carried out according to the instructions of Biogen. Specifically, the Transcription Master Mix as shown in Table 2 below was prepared, mixed and reacted at 40 ° C for 2 hours. Then, the labeled cDNA was purified, and 600 ng of the Cy3 or Cy5-labeled cDNA Followed by reaction at 60 ° C for 30 minutes to perform a fragmentation process. The cDNA was mixed with 2x GEx Hybridization Buffer HI-RPM, placed on a microarray chip, and hybridized in an oven at 65 ° C for 17 hours. The microarray chip was then washed twice (1 minute with GE Wash Buffer 1 and 1 minute with GE Wash Buffer 2) and centrifuged at 800 rpm for 3 minutes.
<2-4> Fluorescence image acquisition
Hybridization images on slides were scanned with an Agilent C scanner (Agilent Technologies, CA, USA). At this time, the green fluorescence image shows the activity of the gene specifically expressed in the control group, and the red fluorescence image shows the specific activity of the gene only in the experimental group. The yellow fluorescence image means green and red complementary colors, and the expression of the two groups is not significantly different. The scanned images were analyzed with Agilent Feature Extraction 10.7.3.1 (Agilent technologies, CA, USA) to obtain gene expression ratios. The extracted data were normalized by using Agilent GeneSpring GX 12.6.1 (Agilent technologies, CA, USA) and the expression pattern of each gene was analyzed. From the thus obtained data, marker genes for diesel exhaust fine particles were selected.
As a result, as shown in FIG. 2, out of the approximately 44,000 genes existing on the oligosaccharide, among the 34,127 genes excluding the genes having no or overlapping expression signals, It was confirmed that 1.91% (653 out of 34,127 genes) showed an increase in gene expression with an expression ratio of 2.0 times or more, and 0.47% (159 out of 34,127 genes) decreased the expression level.
As shown in Table 3, 25 genes were involved in inflammation-related genes (Table 3). Although the above genes have been reported to be involved in inducing diseases related to lung toxicity by other chemicals in the existing invention, when the diesel exhaust fine particles used in the present invention are treated, they are related to toxicity in human lung cancer tissue-derived cells There is no report.
Registration Number
< Example 3> Real time RT - PCR dose
The inventors of the present invention have found that 9 genes (5 overexpressed genes, 4 low-expressed genes) (GenBank), which are expressed by the diesel exhaust fine particles from the microarray results, NM_014707 (HDAC9, Histone deacetylase 9), GenBank NM_015364 (LY96, Lymphocyte antigen 96), GenBank NM_022468 (MMP25, Matrix metallopeptidase 25), GenBank NM_000612 (IGF2, Insulin GenBank NM_002982 (CCL2, CC motif ligand 2), GenBank NM_030754 (SAA2, Serum amyloid A2), GenBank (GenBank) (BMP6, Bone morphogenetic protein 6), GenBank NM_000331 (SAA1, Serum amyloid A1), GenBank NM_002984 (CCL4, Chemokine (CC motif) ligand 4) To investigate and quantify My IQ real-time PCR (My IQ Real -time PCR) (Bio-Rad, USA) was used for quantitative real-time RT-PCR.
Specifically, cDNA was synthesized by performing reverse transcription using an oligo dT primer and Superscript kit (Omniscipt ™ kit, Qiagen, Co., USA). 0.2 μl of the cDNA, 3.8 μl of water, 0.5 μl of a sense primer, 0.5 μl of an antisense primer, and 5 μl of CyberGreen I Dye Supersmix (Bio-rad, USA) were mixed and placed in a PCR tube
Step 1: 95 [deg.] C, 3 min;
Step 2 (45 repeats):
Step 2-1: 95 ° C, 10 seconds;
Step 2-2: Reaction to gene HDAC9, gene SAA1 is 45 seconds at 58.9 ° C, reaction to gene MMP25 is 45 seconds at 59 ° C, reaction to gene LY96, gene IGF2, gene SAA2, gene BMP6 occurs at 60 ° C 45 sec, reaction to gene CCL2 at 45 ° C for 45 sec, reaction to gene CCL4 at 63 ° C for 45 sec;
Step 3: 95 캜, 1 min;
Step 4: 55 [deg.] C, 1 min;
Step 5 (repeated 80 times): The reaction was performed in a My IQ real-time PCR machine designed at 55 ° C for 10 seconds. The PCR products were stained with SYBR Green I staining (Bio-rad, USA). CyberGreen I staining binds to double helix DNA. As the double helix DNA is generated during the PCR process, the fluorescence intensity is increased. First, primer for the target gene used for PCR and primer for endogenous control (GAPDH) was added to the cyber green master mix and subjected to PCR, followed by a primer optimization process Respectively. The synthesized cDNA sample and each primer (Table 4) were mixed and PCR was performed after addition of Cyber Green Mastermix and analyzed using quantitative software.
As a result, as shown in FIG. 3, the genetic code of GenBank NM_014707 (HDAC9, Histone deacetylase 9), GenBank NM_015364 (LY96, Lymphocyte antigen 96), GenBank NM_022468 (MMP25, Matrix (GenBank) NM_002982 (CCL2, Chemokine (CC motif) ligand 2), Gene Accession Number (GenBank), GenBank Accession Number (SAA2, Serum amyloid A2), GenBank NM_001718 (BMP6, Bone morphogenetic protein 6), GenBank NM_000331 (SAA1, Serum amyloid A1), GenBank NM_002984 (CCL4 , And chemokine (CC motif) ligand 4) genes were very similar to those of the microarray chip of Example <2-4> (FIG. 3).
Microarray chips or kits containing inflammatory response-related biomarkers that exhibit altered expression relative to diesel exhaust particulate exposure can be usefully used to monitor and determine the risk of diesel exhaust particulates.
<110> Korea Institute of Science and Technology <120> Biomarker for identification of genes related to inflammatory response after exposure to diesel exhaust particles and the method of identification using thesame <130> 2015P-07-016 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> HDAC9_sense <400> 1 attcaggacc atcgtgaagc ctgt 24 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> HDAC9_antisense <400> 2 ggtgtggcct tccatgcatc aaa 23 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LY96_sense <400> 3 agctctgaag ggagagactg tgaa 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LY96_antisense <400> 4 ggtgtaggat gacaaactcc aagc 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MMP25_sense <400> 5 gtagctggct gtttcgtggc attt 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MMP25_antisense <400> 6 tgaggctgga cagcaactta ggaa 24 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> IGF2_sense <400> 7 ccgtgcttcc ggacaactt 19 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IGF2_antisense <400> 8 ctgcttccag gtgtcatatt gg 22 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CCL2_sense <400> 9 cattgtggcc aaggagatct g 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CCL2_antisense <400> 10 cttcggagtt tgggtttgct t 21 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SAA2_sense <400> 11 tggccgatca ggctgccaat aaat 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SAA2_antisense <400> 12 agcagagtga agaggaagct cagt 24 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BMP6_sense <400> 13 agagccggca gtccagaagt taat 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BMP6_antisense <400> 14 taacacgtga aggcaatgct cacc 24 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SAA1_sense <400> 15 tggctgatca ggctgccaat gaat 24 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SAA1_antisense <400> 16 agcagagtga agaggaagct cagt 24 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CCL4_sense <400> 17 cgcctgctgc ttttcttaca c 21 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CCL4_antisense <400> 18 cagacttgct tgcttctttt gg 22
Claims (13)
GenBank NM_002986 (CCL11, CC motif ligand 11), GenBank NM_014707 (Histone deacetylase 9), GenBank NM_198389 (PDPN, Podoplanin), Gene registration number (GenBank) NM_000065 (C6, Complement component 6), GenBank NM_015364 (LY96, Lymphocyte antigen 96), GenBank NM_000698 (ALOX5, Arachidonate 5-lipoxygenase), GenBank NM_080706 (TRPV1, transient receptor potential cation channel, subfamily V, member 1), GenBank NM_138938 (REG3A, Regenerating islet-derived 3 alpha), GenBank NM_004991 (MECOM, MDS1 and EVI1 complex locus) , GenBank NM_002558 (P2RX1, Purinergic receptor P2X, ligand-gated ion channel, 1), GenBank NM_000756 (Corticotropin releasing hormone), GenBank NM_022468 (MMP25, Matrix metallopeptidase 25), gene registration number (GenBank ), NM_000612 (IGF2, Insulin-like growth factor 2 (somatomedin A)), GenBank NM_138284 (IL17D, Interleukin 17D), GenBank NM_002982 (CCL2, CC motif ligand 2) GenBank NM_001570 (IRAK2, Interleukin-1 receptor-associated kinase 2), GenBank NM_002852 (PTX3, Pentraxin-related gene, rapidly induced by IL-1 beta) GenBank NM_030754 (SAA2, Serum amyloid A2), GenBank NM_001718 (BMP6, Bone morphogenetic protein 6), GenBank (GenBank), GenBank NM_001080424 (KDM6B, Lysine ), NM_007115 (TNFAIP6, Tumor necrosis factor, alpha-induced protein 6), GenBank NM_000331 (SAA1, Serum amyloid A1), GenBank NM_000204 (CFI, Complement factor I) GenBank NM_002985 (CCL5, Chemokine (CC motif) ligand 5), GenBank NM_002984 (CCL4, Chemokin e (CC motif) ligand 4).
2) labeling the experimental group and the control group with different fluorescent materials while synthesizing the RNA of the experimental group and the control group of the step 1) with cDNA;
3) hybridizing the cDNA labeled with the different fluorescent material of step 2) with the DNA microarray chip of claim 1;
4) analyzing the reacted DNA microarray chip; And
5) checking the degree of expression of the genes integrated in the DNA microarray chip of claim 1 in comparison with the control group, and confirming the exposure of the diesel exhaust microparticles.
2) Real-time reverse transcript polymerase chain reaction (RT-PCR) using primer pairs complementary to the following genes and capable of amplifying the following genes:
GenBank NM_014707 (HDAC9, Histone deacetylase 9), GenBank NM_015364 (LY96, Lymphocyte antigen 96), GenBank NM_022468 (MMP25, Matrix metallopeptidase 25), GenBank ), GenBank NM_002982 (CCL2, CC motif ligand 2), GenBank NM_030754 (SAA2, Serum amyloid A2), and the like), NM_000612 (Insulin-like growth factor 2 (somatomedin A) , GenBank NM_001718 (BMP6, Bone morphogenetic protein 6), GenBank NM_000331 (SAA1, Serum amyloid A1), GenBank NM_002984 (CCL4, Chemokine (CC motif) ligand 4) ; And
3) comparing the gene product of step 2) with that of the control group to confirm the expression level of the diesel exhaust fine particles.
Primer pair 1: a forward primer described in SEQ ID NO: 1 and a reverse primer described in SEQ ID NO: 2;
Primer pair 2: the forward primer described in SEQ ID NO: 3 and the reverse primer described in SEQ ID NO: 4;
Primer pair 3: a forward primer described in SEQ ID NO: 5 and a reverse primer described in SEQ ID NO: 6;
Primer pair 4: a forward primer described in SEQ ID NO: 7 and a reverse primer described in SEQ ID NO: 8;
Primer pair 5: a forward primer described in SEQ ID NO: 9 and a reverse primer described in SEQ ID NO: 10; And
Primer pair 6: the forward primer described in SEQ ID NO: 11 and the reverse primer set forth in SEQ ID NO: 12.
Primer pair 7: a forward primer represented by SEQ ID NO: 13 and a reverse primer represented by SEQ ID NO: 14;
Primer pair 8: a forward primer described in SEQ ID NO: 15 and a reverse primer described in SEQ ID NO: 16; And
Primer pair 9: the forward primer described in SEQ ID NO: 17 and the reverse primer set forth in SEQ ID NO: 18.
GenBank NM_014707 (HDAC9, Histone deacetylase 9), GenBank NM_015364 (LY96, Lymphocyte antigen 96), GenBank NM_022468 (MMP25, Matrix metallopeptidase 25), GenBank ), GenBank NM_002982 (CCL2, CC motif ligand 2), GenBank NM_030754 (SAA2, Serum amyloid A2), and the like), NM_000612 (Insulin-like growth factor 2 (somatomedin A) , GenBank NM_001718 (BMP6, Bone morphogenetic protein 6), GenBank NM_000331 (SAA1, Serum amyloid A1), GenBank NM_002984 (CCL4, Chemokine (CC motif) ligand 4) .
Primer pair 1: a forward primer described in SEQ ID NO: 1 and a reverse primer described in SEQ ID NO: 2;
Primer pair 2: the forward primer described in SEQ ID NO: 3 and the reverse primer described in SEQ ID NO: 4;
Primer pair 3: a forward primer described in SEQ ID NO: 5 and a reverse primer described in SEQ ID NO: 6;
Primer pair 4: a forward primer described in SEQ ID NO: 7 and a reverse primer described in SEQ ID NO: 8;
Primer pair 5: a forward primer described in SEQ ID NO: 9 and a reverse primer described in SEQ ID NO: 10; And
Primer pair 6: the forward primer described in SEQ ID NO: 11 and the reverse primer set forth in SEQ ID NO: 12.
Primer pair 7: a forward primer represented by SEQ ID NO: 13 and a reverse primer represented by SEQ ID NO: 14;
Primer pair 8: a forward primer described in SEQ ID NO: 15 and a reverse primer described in SEQ ID NO: 16; And
Primer pair 9: the forward primer described in SEQ ID NO: 17 and the reverse primer set forth in SEQ ID NO: 18.
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| KR20180114770A (en) * | 2017-04-11 | 2018-10-19 | 한국과학기술연구원 | Biomarker for identification of genes related to inflammatory response after exposure to particulate matter 2.5 using human placenta cell line and the identification method using thereof |
| WO2019199128A1 (en) * | 2018-04-12 | 2019-10-17 | 서울대학교산학협력단 | Composition for identifying intranasal exposure of environmentally harmful substance diesel exhaust particles |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20180114770A (en) * | 2017-04-11 | 2018-10-19 | 한국과학기술연구원 | Biomarker for identification of genes related to inflammatory response after exposure to particulate matter 2.5 using human placenta cell line and the identification method using thereof |
| WO2019199128A1 (en) * | 2018-04-12 | 2019-10-17 | 서울대학교산학협력단 | Composition for identifying intranasal exposure of environmentally harmful substance diesel exhaust particles |
| KR20190120054A (en) * | 2018-04-12 | 2019-10-23 | 동국대학교 산학협력단 | Compositions for identification of exposure to diesel exhaust particle at nasal cavity |
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