KR101457513B1 - microRNAs for identification of human exposure to xylene and the method of identification using thereof - Google Patents

Abstract

The present invention relates to a microRNA for confirming the exposure of xylene to human body and a method for confirming the exposure of xylene to human body using the microRNA. More particularly, the present invention relates to a method for confirming exposure to xylene using human microRNA as a biomarker Can be used to monitor and determine xylene contamination in exposed human samples and can be used as a tool to identify xylene-induced toxic mechanisms.

Description

[0001] The present invention relates to a microRNA for detecting exposure to human xylene, and a method for identifying xylene using the microRNA,

The present invention relates to a microRNA whose expression is specifically changed by xylene exposure, and a method for confirming exposure of human to xylene using the same.

Recently, microRNA (miR, miRNA) has emerged as an important class of regulatory RNA involved in gene expression regulation. These small (typically 18-24 nucleotide-long) non-coding RNA molecules can regulate protein expression patterns by promoting RNA degradation, inhibiting mRNA translation, and influencing gene transcription. MicroRNAs regulate a variety of biological processes such as development and differentiation, cell proliferation control, and stress response. At present more than about 1000 human microRNAs are known.

MicroRNAs are transcribed by RNA polymerase II (pol II) or RNA polymerase III (pol III; see Qi, P. et al. Cell . Mol . Immunol . 3 , 411-419, 2006) Genes, introns of protein-encoding genes, or multiple individuals, poly-cistron transcripts that primarily encode closely related microRNAs. Transcription of miRNA genes by RNA pol II or pol III produces the initial transcripts, commonly referred to as pri-miRNAs, thousands of nucleotides in length. At the nucleus, pri-miRNAs are processed by RNAse, Drosha, to produce 70- to 100-nucleotide hairpin-shaped precursors (pre-miRNAs). After delivery to the cytoplasm, the hairpin pre-miRNA is further processed by the dicer to produce double-stranded miRNA. The mature miRNA strand is mixed into an RNA-induced silencing complex (RISC), where the mature miRNA binds its target mRNAs by base pair complementarity. In relatively infrequent cases where miRNA basepairs perfectly match mRNA targets, this promotes mRNA degradation. More generally, miRNAs form mismatched duplexes with target mRNAs, affecting mRNA translation.

The microRNA mechanism plays an important role in the development of cancer, cell senescence, organs growth. Therefore, microRNA-related research plays a pivotal role in the identification of causal relationships between life events such as cancer development and aging, It is expected to be a core field of biotechnology research in Korea that can be applied to the development of cell therapy using stem cells. Therefore, it is significant that the discovery of microRNA markers will be of great help in predicting the early diagnosis of diseases, including cancer.

In addition, microRNA markers are expected to be useful in predicting the exposure of certain environmentally hazardous substances. In recent years, research has been carried out on the relationship between changes in genes (mRNA) due to exposure to environmentally harmful substances and diseases, and in recent years, interest in relation to microRNAs has increased, The expression of microRNAs by exposure to harmful substances such as arsenic (arsenic) or RDX was observed, and microRNAs whose expression was specifically changed and their target genes were shown as markers for harmful substances (Baccarelli, A. & Bollati, V. Curr . Opin . Prediatr . 21 , 243-251, 2009). In addition, the extracted microRNA can be used not only as a marker for exposure prediction, but also as a marker for predicting toxic mechanisms by environmentally harmful substances as a regulator for regulating the expression of a target gene. Although the role of microRNA markers in predicting the exposure of environmentally harmful substances and their toxic mechanisms is very important, current microRNA-related research is limited mainly to finding markers for diagnosis of diseases, Studies on the expression of microRNAs by exposure to harmful substances such as chemical compounds are lacking. In addition, epigenetic changes are not so severe compared to the diversity of genetic changes, such as expression of genes, and therefore, gene expression profiling, which requires a large number of markers, epigenetic markers have the advantage of being able to diagnose disease or harmful substances at an early stage as well as noninvasive methods. It is expected that potential microRNA-controlled circuits will be enormous because each microRNA controls a variety of target genes and there are a thousand microRNAs in higher eukaryotes.

Xylene (CAS No. 1330-20-7) is one of the representative volatile organic compounds. It may be used as a solvent for some paints, paint thinners, lacquers, and inks, and may also be used as a solvent in the manufacture of plastics, polyester, and other chemicals. Generally, the most serious exposure sources of xylene are byproducts from petroleum, coal and styrene production processes, chemical intermediates, and cigarette smoke.

Xylene is volatile and its volatility in soil is faster than other compounds. It has fast half-life in the atmosphere and it is easy to move to a large area, and the movement from the surface water to the air is fast. Xylene is colorless and transparent flammable, has a similar smell to thinner, and has a property of sticking to fire. It is mainly used as a raw material when making basic chemicals, and used as a raw material for synthetic resins and synthetic fibers. Also, oil-based paints, adhesives, printing inks. It is also used as a solvent such as thinner and pesticide. According to the Ministry of Environment 's 2008 volume survey, the volume increased from 12,759 tons in 2006 to 13,169 tons in 2007. According to the 2006 Utilization Survey, the production volume is 4,836,000 tons, the import volume is 662,000 tons, and the export volume is 1,933 tons.

Because xylene is used as a raw material for many basic chemicals, it has a higher amount of release than other chemicals. It is discharged not only at the business site but also at construction sites using paints and adhesives. It is also contained in gasoline and is discharged into the air through automobile exhaust gas. Mainly air, which can be exposed in places such as traffic congestion areas, gas stations, or solvents, or refineries.

In addition, xylene is one of the substances that cause sick house syndrome. It is known that exposure to high concentrations can cause irritation to the eyes and throat and affect the central nervous system. Other areas, vomiting, headache, sleepy symptoms. Dizziness, stomach pain, chest pain, difficulty breathing, etc. Because of this risk, xylene has been designated as toxic substance by the Toxic Chemicals Control Act of the Ministry of Environment, and it is required to register for import and business registration. The indoor air quality control laws, such as multi-use facility to a comfortable indoor air in the expansion joint with the housing of the xylene standard recommendations (700g / m ³ or less).

Pollution data for xylene in domestic food and consumer products are insufficient data on the characteristics of volatile xylene. Domestic xylene-containing products are used in automobile raw materials, explosives, dyes, paints, inks, adhesives, artificial leather manufacturing, solvents, cosmetics, medicines, lacquer diluents, resins, detergents (10% less) and water (not detected), but the concentration of xylene is regulated only in the case of adhesive (less than 10%) and water (not detected). (Korea Food and Drug Administration).

In addition, studies on the degree of human toxicity and changes in gene expression of volatile organic compounds have been reported, but they are limited to studies on formaldehyde, benzene, etc., which are known as typical volatile organic compounds. Despite the potential risks to xylenes in humans, there is insufficient risk assessment data in the human body, and the method of screening for exposure is also known as GC-MS (Gas Chromatography-Mass Spectrometer) or HPLC (High Performance Liquid Chromatography) It is limited to the classical method. 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 microarray chip or a primer, It is an important task to identify and manage the exposure of xylene to human body by identifying and using molecular indices that can detect toxic effects, especially whether microRNA expression is involved in various diseases including cancer. would.

Since the first microRNAs were discovered in 1997, microRNAs from various species such as mammals and microorganisms have been rapidly identified and reported in the Sanger miRBASE database (http://www.mirbase.org/index.shtml). Genome-wide expression studies have been conducted to study the function of genes based on the microRNA data. Microarray analysis is performed in order to analyze the expression of thousands of genes in a single experiment (Schena, M, et al. Proc . Natl . Acad . Sci . USA 93 , 10614-10619,1996).

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 (Sellheyer K. et al., J. Am . acad. Dermatol. 51, 681-692, 2004). The oligonucleotide microarray is prepared 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 and the like (Sellheyer, K. et al. et. al. J. Am. Acad . Dermatol. 51, 681-692, 2004).

For analysis of gene expression, microRNAs are obtained from samples such as tissues to perform hybridization reaction with oligonucleotides in the microarray. The obtained microRNA is labeled with fluorescence or isotope.

Two methods for measuring gene expression through microarrays are two-dye and one-dye. When measuring the complementary binding reaction, two different fluorescence (for example, Cye3 and Cye5) were labeled on the control sample and the test sample, respectively, and the reaction was performed on the microarray. The two-dye microarray The method of measuring two microarrays by reacting two samples with the same fluorescence is called a one-dye microarray (Vivian, G. et al., Nature 21, 15- 19, 1999).

Recently, it has been applied to toxic genomics (high-throughput research), which is a cutting-edge technique using DNA microarray technology. It is expressed in specific tissues and cell lines by all chemical substances including representative environmental pollutants as well as drug candidates And the quantitative analysis of the pattern of expression of microRNAs. Thus, by analyzing the frequency of expression of specific microRNAs in specific cells, it is possible to identify genes related to adverse effects of drugs and harmful effects of environmental pollutants. Thus, harmful effects of environmental pollutants, actions of drugs and side effects Will be able to understand molecular mechanisms and will be able to search for and identify substances that cause toxicity and side effects.

Therefore, the present inventors observed and analyzed the microRNA expression profiles of xylenes using an oligomicroarray in which 887 human microRNAs were integrated and obtained blood samples from workers exposed to xylene. As a result, The present inventors completed the present invention by establishing a method of identifying microRNAs whose expression is specifically changed by the microRNAs and using the microRNAs as biomarkers to determine whether xylene is exposed.

It is an object of the present invention to provide microRNAs whose expression is specifically changed by exposure to human body of xylene and a method for confirming exposure of human to xylene using the biomarker.

In order to achieve the above object,

The present invention provides a microRNA biomarker selected from the following group that specifically induces an expression change by exposure of xylene:

MicroRNA registration number (miRbase): MIMAT0000063 (hsa-let-7b, Homo sapiens let-7b)

MicroRNA registration number: MIMAT0000099 (hsa-miR-101, Homo sapiens miR-101),

MicroRNA registration number: MI0000456 (hsa-miR-140-5p, Homo sapiens miR-140-5p),

The microRNA registration number MIMAT0000434 (hsa-miR-142-3p, Homo sapiens miR-142-3p),

MicroRNA registration number: MIMAT0000433 (hsa-miR-142-5p, Homo sapiens miR-142-5p),

MicroRNA registration number: MI0000460 (hsa-miR-144, Homo sapiens miR-144),

MicroRNA registration number: MIMAT0004600 (hsa-miR-144 *, Homo sapiens miR-144 *),

MicroRNA registration number: MI0000069 (hsa-miR-15a, Homo sapiens miR-15a),

MicroRNA registration number: MIMAT0000256 (hsa-miR-181a, Homo sapiens miR-181a),

MicroRNA registration number: MI0000073 (hsa-miR-19a, Homo sapiens miR-19a),

MicroRNA registration number: MIMAT0000074 (hsa-miR-19b, Homo sapiens miR-19b),

MicroRNA registration number: MIMAT0000076 (hsa-miR-21, Homo sapiens miR-21),

MicroRNA registration number: MIMAT0000681 (hsa-miR-29c, Homo sapiens miR-29c),

MicroRNA registration number: MIMAT0000688 (hsa-miR-301a, Homo sapiens miR-301a),

MicroRNA registration number: MIMAT0000245 (hsa-miR-30d, Homo sapiens miR-30d),

MicroRNA registration number: MIMAT0005792 (hsa-miR-320b, Homo sapiens miR-320b),

MicroRNA registration number: MIMAT0015072 (hsa-miR-320e, Homo sapiens miR-320e),

MicroRNA registration number: MIMAT0000761 (hsa-miR-324-5p, Homo sapiens miR-324-5p),

MicroRNA registration number: MIMAT0000760 (hsa-miR-331-3p, Homo sapiens miR-331-3p),

MicroRNA registration number: MIMAT0000727 (hsa-miR-374a, Homo sapiens miR-374a),

MicroRNA registration number: MIMAT0000731 (hsa-miR-378 *, Homo sapiens miR-378 *),

MicroRNA registration number: MIMAT0003885 (hsa-miR-454, Homo sapiens miR-454), and

MicroRNA registration number: MIMAT0000252 (hsa-miR-7, Homo sapiens miR-7).

Also, the present invention provides a microarray chip for confirming exposure of xylene, in which the microRNA or its complementary strand molecules are integrated.

In addition, the present invention provides a kit for confirming exposure of xylene containing the microarray chip.

Also, the present invention provides a kit for confirming the exposure of xylene comprising a primer complementary to the microRNA and capable of amplifying the microRNA.

In addition,

1) separating each RNA from a blood sample separated from the subject to be tested in a test group and a blood sample separated from a normal subject as a control group;

2) labeling the RNA of the experimental group and the control group of step 1) with different fluorescent materials;

3) hybridizing the fluorescently labeled RNA of step 2) with the microarray chip;

4) analyzing the reacted microarray chip of step 3); And

5) checking the expression level of the microRNA integrated in the microarray chip by comparing the data analyzed in step 4) with the control group.

In addition,

1) separating each RNA from a blood sample separated from the subject to be tested in a test group and a blood sample separated from a normal subject as a control group;

2) synthesizing the RNA of the experimental group and the control group of step 1), respectively, with cDNA;

3) Real-time reverse transcript polymerase chain reaction (RT-PCR) using primers capable of amplifying the microRNAs integrated in the microarray chip of the cDNA of the test group and the control group of step 2), respectively; And

4) confirming the degree of expression of the amplification product of the experimental group of step 3) in comparison with the control group.

The microRNAs of the present invention, which are specifically expressed in response to xylene exposure, can be used specifically to monitor and determine the contamination of xylene, and can be used to identify toxic mechanisms caused by xylene .

Brief Description of the Drawings Fig. 1 is a diagram showing the results of analysis of microRNA expression patterns in xylene-exposed human blood samples using a microRNA microarray chip. Fig.
FIG. 2 is a graph showing the results of analysis of expression patterns of 23 kinds of microRNAs whose expression is specifically changed by xylene exposure.
FIG. 3 is a graph showing the results of comparative analysis of microRNA expression patterns of xylene-exposed human blood samples and unexposed human blood samples using PCA analysis.

Hereinafter, the present invention will be described in detail.

The present invention provides a biomarker for confirming microRNA expression against xylene, which is characterized in that the expression of xylene is changed by human exposure.

The biomarker is a microRNA expressed with a p-value < 0.05, which is composed of microRNAs whose expression is increased or decreased by 1.5 times or more by exposure to xylene.

The present invention provides a biomarker characterized in that it is selected from the group consisting of:

MicroRNA registration number (miRbase): MIMAT0000063 (hsa-let-7b, Homo sapiens let-7b)

MicroRNA registration number: MIMAT0000099 (hsa-miR-101, Homo sapiens miR-101),

MicroRNA registration number: MI0000456 (hsa-miR-140-5p, Homo sapiens miR-140-5p),

The microRNA registration number MIMAT0000434 (hsa-miR-142-3p, Homo sapiens miR-142-3p),

MicroRNA registration number: MIMAT0000433 (hsa-miR-142-5p, Homo sapiens miR-142-5p),

MicroRNA registration number: MI0000460 (hsa-miR-144, Homo sapiens miR-144),

MicroRNA registration number: MIMAT0004600 (hsa-miR-144 *, Homo sapiens miR-144 *),

MicroRNA registration number: MI0000069 (hsa-miR-15a, Homo sapiens miR-15a),

MicroRNA registration number: MIMAT0000256 (hsa-miR-181a, Homo sapiens miR-181a),

MicroRNA registration number: MI0000073 (hsa-miR-19a, Homo sapiens miR-19a),

MicroRNA registration number: MIMAT0000074 (hsa-miR-19b, Homo sapiens miR-19b),

MicroRNA registration number: MIMAT0000076 (hsa-miR-21, Homo sapiens miR-21),

MicroRNA registration number: MIMAT0000681 (hsa-miR-29c, Homo sapiens miR-29c),

MicroRNA registration number: MIMAT0000688 (hsa-miR-301a, Homo sapiens miR-301a),

MicroRNA registration number: MIMAT0000245 (hsa-miR-30d, Homo sapiens miR-30d),

MicroRNA registration number: MIMAT0005792 (hsa-miR-320b, Homo sapiens miR-320b),

MicroRNA registration number: MIMAT0015072 (hsa-miR-320e, Homo sapiens miR-320e),

MicroRNA registration number: MIMAT0000761 (hsa-miR-324-5p, Homo sapiens miR-324-5p),

MicroRNA registration number: MIMAT0000760 (hsa-miR-331-3p, Homo sapiens miR-331-3p),

MicroRNA registration number: MIMAT0000727 (hsa-miR-374a, Homo sapiens miR-374a),

MicroRNA registration number: MIMAT0000731 (hsa-miR-378 *, Homo sapiens miR-378 *),

MicroRNA registration number: MIMAT0003885 (hsa-miR-454, Homo sapiens miR-454), and

MicroRNA registration number: MIMAT0000252 (hsa-miR-7, Homo sapiens miR-7).

The microRNA registration number: MIMAT0000063 (hsa-let-7b, Homo sapiens let-7b)

MicroRNA registration number: MIMAT0000256 (hsa-miR-181a, Homo sapiens miR-181a),

MicroRNA registration number: MIMAT0000245 (hsa-miR-30d, Homo sapiens miR-30d),

MicroRNA registration number: MIMAT0005792 (hsa-miR-320b, Homo sapiens miR-320b),

MicroRNA registration number: MIMAT0015072 (hsa-miR-320e, Homo sapiens miR-320e),

MicroRNA registration number: MIMAT0000761 (hsa-miR-324-5p, Homo sapiens miR-324-5p),

MicroRNA registration number: MIMAT0000760 (hsa-miR-331-3p, Homo sapiens miR-331-3p), and

MicroRNA registration number: MIMAT0000731 (hsa-miR-378 *, Homo sapiens miR-378 *) showed increased expression for xylene exposure,

The microRNA registration number: MIMAT0000099 (hsa-miR-101, Homo sapiens miR-101)

MicroRNA registration number: MI0000456 (hsa-miR-140-5p, Homo sapiens miR-140-5p),

The microRNA registration number MIMAT0000434 (hsa-miR-142-3p, Homo sapiens miR-142-3p),

MicroRNA registration number: MIMAT0000433 (hsa-miR-142-5p, Homo sapiens miR-142-5p),

MicroRNA registration number: MI0000460 (hsa-miR-144, Homo sapiens miR-144),

MicroRNA registration number: MIMAT0004600 (hsa-miR-144 *, Homo sapiens miR-144 *),

MicroRNA registration number: MI0000069 (hsa-miR-15a, Homo sapiens miR-15a),

MicroRNA registration number: MI0000073 (hsa-miR-19a, Homo sapiens miR-19a),

MicroRNA registration number: MIMAT0000074 (hsa-miR-19b, Homo sapiens miR-19b),

MicroRNA registration number: MIMAT0000076 (hsa-miR-21, Homo sapiens miR-21),

MicroRNA registration number: MIMAT0000681 (hsa-miR-29c, Homo sapiens miR-29c),

MicroRNA registration number: MIMAT0000688 (hsa-miR-301a, Homo sapiens miR-301a),

MicroRNA registration number: MIMAT0000727 (hsa-miR-374a, Homo sapiens miR-374a),

MicroRNA registration number: MIMAT0003885 (hsa-miR-454, Homo sapiens miR-454) and

It is preferred that the microRNA registration number: MIMAT0000252 (hsa-miR-7, Homo sapiens miR-7) is decreased in expression upon xylene exposure.

In a specific embodiment of the present invention, the inventors of the present invention conducted a study to find out the biomarker for confirming microRNA expression against human exposure to xylenes, And metabolites were analyzed according to the Biological Exposure Index (BEI) and used as biomarkers of exposure to hazardous substances (see Table 1). In the case of xylene, methyl hippuric acid (MHA) was analyzed by biomarkers and urinary creatinine was analyzed for each value.

Total RNAs containing microRNAs were then isolated from the blood samples of unexposed and exposed workers exposed to xylene at the above concentrations and labeled with a fluorescent material (Cy3). The fluorescently labeled microRNAs were hybridized with Agilent Human miRNA array v14.0 (Agilent, USA), and fluorescence images were scanned to analyze differences in microRNA expression patterns (see FIG. 1). The microRNAs that showed significant mutations in expression compared to the unexposed group were selected for unpaired Welchs t-test, 211 microRNAs showing significant expression at p-value <0.05 through the Multiple Test Correction-Benjamini and Hochberg False Discovery Rate Respectively. The analysis revealed that there were 23 microRNAs with increased expression over 1.5 times and 15 species with reduced microRNAs (Table 2, Fig. 2). These microRNAs were found to be associated with toxicity There is no report that it is.

Thus, the microRNAs of the present invention that specifically alter expression of xylene exposure can be used specifically for monitoring and determining xylene contamination, and are tools for identifying xylene-induced toxic mechanisms Can be usefully used.

Also, the present invention provides a microarray chip for confirming exposure of xylene, in which oligonucleotides containing all or a part of the microRNA sequence of the present invention or complementary strand molecules thereof are integrated.

The oligonucleotide or its complementary strand molecule comprises 15 to 20 nucleic acids of the biomarker microRNA.

The microRNA microarray chip for xylene search of the present invention can be produced by a method known to a person skilled in the art. A method of fabricating the microarray chip is as follows. It is preferable to use an inkjet method or the like in order to immobilize the biomarker on a substrate using a probe microRNA molecule. However, the present invention is not limited thereto. In a preferred embodiment of the present invention, a SurePrint inkjet micro dropping microarray Respectively. The substrate of the DNA microarray chip is coated with a single active group selected from the group consisting of epoxy, 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. However, the substrate is not limited thereto. In a preferred embodiment of the present invention, Slide glass was used.

Also, the present invention provides a method for confirming whether a human is exposed to xylene using the biomarker.

The present invention provides a method for determining whether or not xylene is exposed to a human body comprising the steps of:

1) separating each RNA from a blood sample separated from the subject to be tested in a test group and a blood sample separated from a normal subject as a control group;

2) labeling the RNA of the experimental group and the control group of step 1) with different fluorescent materials;

3) hybridizing the fluorescently labeled RNA of step 2) with the microarray chip of the present invention;

4) analyzing the reacted microarray chip of step 3); And

5) Identifying the degree of expression of the microRNA integrated in the microarray chip of the present invention by comparing the analyzed data with the control group in step 4).

In the method for confirming the exposure, it is preferable to use a blood sample obtained in step 1) obtained from a group of workers exposed or not exposed to xylene.

Wherein the fluorescent material of step 2) 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.

In the method for confirming the exposure, it is preferable to use Agilent Human miRNA array v14.0 (Agilent, USA) or the like for the microRNA microarray chip of step 3), but the present invention is not limited thereto. The present invention can be used as a microarray chip on which the commonly expressed microRNAs (see Table 2) are mounted, and it is most preferable to use the microRNA microarray chip manufactured by the present inventor. In addition, it is preferable to use GeneSpring GX 11 software (Agilent, USA) for the analysis in step 4), but it is not limited thereto, and analysis software known to those skilled in the art may be used.

The present invention also provides a method for identifying human exposure to xylene comprising the steps of:

1) separating each RNA from a blood sample separated from the subject to be tested in a test group and a blood sample separated from a normal subject as a control group;

2) synthesizing the RNA of the experimental group and the control group of step 1), respectively, with cDNA;

3) Real-time reverse transcript polymerase chain reaction (RT-PCR) was performed using the primers capable of amplifying the microRNAs integrated in the microarray chip of the present invention with the cDNAs of the experimental group and the control group of step 2) step; And

4) confirming the degree of expression of the amplification products of the experimental group of step 3) in comparison with the control group.

In the method for confirming the exposure, it is preferable to use a blood sample obtained in step 1) obtained from a group of workers exposed or not exposed to xylene.

Any primer capable of amplifying the microRNA can be used as long as it can amplify the microRNA of the present invention.

In addition, the present invention provides a kit for confirming the exposure of a human xylene containing the microRNA microarray chip prepared in the present invention.

In addition to the confirmation kit, a kit for confirming microRNA expression against human exposure to xylenes containing a human blood sample is provided. Preferably, the human blood sample is obtained from a group of workers exposed or not exposed to xylene.

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 However, the present invention is not limited thereto. In the preferred embodiment of the present invention, Cy3 is used.

In addition to the kit, the reaction reagent may include a buffer solution used for hybridization, a labeling reagent such as a chemical inducer of a fluorescent dye, a washing buffer solution, and the like, but is not limited thereto. Any reaction reagent necessary for the hybridization reaction of the microRNA microarray chip can be included.

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 present invention is not limited to the following examples.

&Lt; Example 1 > Study Population and Blood Sampling

<1-1> Research population

Xylene exposure group and non - exposed workers were recruited by researchers at the Institute of Occupational Health, Catholic University Medical School. The population of this study consisted of workers (N = 30) who were exposed or not exposed to xylen from ship manufacturers handling xylene (Table 1). These samples were selected regardless of factors such as gender, age distribution, working experience, lifestyle such as smoking or drinking, and average working hours. Drug users were excluded. Prior to the experiment, this study was evaluated and certified by the IRB committee (approval number CUMC10U066) and all participants read and signed the agreement prior to the experiment.

Xylenes for blood samples (n = 30) Metabolite ( MHA ) Analysis Sample number
(sample No.) Occupation fair Metabolite analysis results Xylene MHA (g / gCr)






Xylene
Unexposed group (n = 19) A0-001 C / L E / R CLEANING 0.01 A0-006 manager Field management 0 A0-007 manager Field management 0 A0-009 manager Field management 0.01 A0-011 manager Field management 0 A0-013 manager Field management 0 A0-016 manager Field management 0.14 A0-017 manager Field management 0 B0-001 Possession B / L 0 B0-002 Possession B / L 0 B0-003 Possession B / L 0 B0-004 Possession B / L 0 B0-006 Possession B / L 0 B0-007 Possession B / L 0 B0-010 Possession B / L 0 B0-012 Possession Water cleaning 0 C0-005 stamp material 0 D0-001 stamp material 0.07 D1-002 stamp B / L 0

Xylene
Exposure group (n = 11) A1-019 T / UP E / R T / UP 0.22 A1-033 T / UP E / R T / UP 0.15 A1-039 T / UP E / R T / UP 0.20 A1-042 T / UP E / R T / UP 0.24 B1-030 Possession C / L 0.14 C1-005 stamp SPRAY Auxiliary 0.19 C1-007 stamp SPRAY SHOOTER 0.59 D1-004 stamp S / P 0.19 D1-014 stamp T / UP 0.51 D1-015 stamp T / UP 0.21 D1-016 stamp T / UP 0.26

<1-2> Identification of the test compound in the study population

Metabolites were analyzed according to the Biological Exposure Index (BEI) and used as biomarkers of xylene. To date, the criteria for biological exposure indicators in Korea have not been clear, so the standard BEI of the American Conference of Governmental Industrial Hygienists (ACGIH), which is widely used internationally, was used as a metabolic measurement standard. In the case of xylene, methyl hippuric acid (MHA) was analyzed as a biomarker. Urine collected for urinary analysis of urine was analyzed for urinary creatinine As shown in Equation (1).

&Lt; 1-3 >

After receiving the consent of the above Example <1-1>, blood samples were obtained by a research nurse using a PAXgene Blood RNA tube, stored at -20 ° C for 24 hours and then at -80 ° C.

&Lt; Example 2 > Microarray experiment

<2-1> Isolation of target microRNA and labeling of fluorescent substance

RNA extraction from xylene exposed and unexposed blood samples was performed using human blood RNA kit (Qiagen, USA). Genomic DNA was removed using an RNase-free DNase set (Qiagen, USA) during RNA purification. The concentration of each total RNA sample was measured using a spectrophotometer, NanoDrop ND 1000 spectrophotometer (NanoDrop Technologies Inc., USA), and purity was confirmed by Experion (Automated Electrophoresis station, Bio-Rad).

<2-2> Preparation of labeled microRNA

For oligomicroarray analysis, the fluorescent substance was labeled on the total RNA of xylene exposed group obtained in the above Example <2-1>. The fluorescent labeling was performed using the manufacturer's method using the Agilent miRNA complete labeling and hybridization kit. 0.4 μl of 10 × CIP buffer, 1.1 μl of nuclease-free water, and 0.5 μl of Calf Intestinal Phosphatase were added to 2 μl (100 ng) of the obtained total RNA, followed by reaction at 37 ° C. for 30 minutes. Then, 2.8 μl 100% DMSO was added to react at 100% for 5-10 minutes and immediately transferred to ice. 1 μl of 10 × T4 RNA Ligase buffer, 3 μl of Cyanine3-pCp, and 0.5 μl of T4 RNA ligase were added to the reaction mixture at 16 ° C for 2 hours to attach the dye (Dye). The labeled sample was purified using a Micro Bio-Spin 6 column. The purified RNA was completely dried in a vacuum concentrator at 55 ° C and dissolved in 18 μl of nuclease-free water. 4.5 μl of 10 × GE Blocking Agent was added, and 22.5 μl of 2 × Hi-RPM Hybridization buffer was added. Was reacted at 100 ° C for 5 minutes, immediately transferred to ice, allowed to react for 5 minutes, and then used for the hybridization reaction.

<2-3> Hybridization reaction

Hybridization and washing procedures were carried out according to the instructions of ZinoCheat Co., Hybridization was carried out in a 55 ° C oven for 20 hours. As a DNA microarray chip, 860 k human miRNA array v14.0 (Agilent, USA) was used.

<2-4> Fluorescence image acquisition

Hybridization images on the slides were scanned using an Agilent C scanner (Agilent Technologies, USA). The extracted data were normalized using Agilent GeneSpring GX 11 (Agilent Technologies) and the expression pattern of each microRNA was analyzed.

As a result, as shown in FIG. 1, among the 887 microRNAs present on the oligonucleotide, microRNAs showing significant expression mutations in the xylene exposure group compared to the unexposed group were analyzed by unpaired Welchs t-test, Multiple Test Correction - 211 strains of microRNAs that were significantly expressed at p-value <0.05 through Benjamini and Hochberg False Discovery Rate were selected (Fig. 1).

In addition, the microRNA expression patterns of xylene exposure group and non-exposed group were compared using GeneSpring GX 11 software (FIG. 3). As a result of analysis, there were 23 kinds of microRNAs with expression increased by more than 1.5 times and 8 kinds of reduced microRNAs and 15 kinds of reduced microRNAs (Table 2, Fig. 2). These microRNAs showed toxicity There is no report that it is related.

Micro-organisms whose expression changes by xylene exposure RNA Accession no. name Fold change Regulation p-value MIMAT0000063 hsa-let-7b 1.56 up 0.00 MIMAT0000099 hsa-miR-101 2.36 down 0.00 MI0000456 hsa-miR-140-5p 1.78 down 0.00 MIMAT0000434 hsa-miR-142-3p 2.58 down 0.00 MIMAT0000433 hsa-miR-142-5p 2.28 down 0.00 MI0000460 hsa-miR-144 2.89 down 0.01 MIMAT0004600 hsa-miR-144 * 4.80 down 0.00 MI0000069 hsa-miR-15a 1.76 down 0.00 MIMAT0000256 hsa-miR-181a 1.58 up 0.00 MI0000073 hsa-miR-19a 2.88 down 0.00 MIMAT0000074 hsa-miR-19b 2.05 down 0.00 MIMAT0000076 hsa-miR-21 2.19 down 0.00 MIMAT0000681 hsa-miR-29c 1.92 down 0.00 MIMAT0000688 hsa-miR-301a 1.64 down 0.00 MIMAT0000245 hsa-miR-30d 1.57 up 0.00 MIMAT0005792 hsa-miR-320b 1.53 up 0.00 MIMAT0015072 hsa-miR-320e 1.51 up 0.00 MIMAT0000761 hsa-miR-324-5p 1.96 up 0.00 MIMAT0000760 hsa-miR-331-3p 1.72 up 0.00 MIMAT0000727 hsa-miR-374a 2.04 down 0.00 MIMAT0000731 hsa-miR-378 * 1.54 up 0.00 MIMAT0003885 hsa-miR-454 1.65 down 0.00 MIMAT0000252 hsa-miR-7 1.57 down 0.00

Claims (8)

All of the following microRNAs or their complementary strand molecules are all integrated Micro array chip for xylene exposure confirmation:
MicroRNA registration number (miRbase): MIMAT0000063 (hsa-let-7b, Homo sapiens let-7b)
MicroRNA registration number: MIMAT0000099 (hsa-miR-101, Homo sapiens miR-101),
MicroRNA registration number: MI0000456 (hsa-miR-140-5p, Homo sapiens miR-140-5p),
The microRNA registration number MIMAT0000434 (hsa-miR-142-3p, Homo sapiens miR-142-3p),
MicroRNA registration number: MIMAT0000433 (hsa-miR-142-5p, Homo sapiens miR-142-5p),
MicroRNA registration number: MI0000460 (hsa-miR-144, Homo sapiens miR-144),
MicroRNA registration number: MIMAT0004600 (hsa-miR-144 *, Homo sapiens miR-144 *),
MicroRNA registration number: MI0000069 (hsa-miR-15a, Homo sapiens miR-15a),
MicroRNA registration number: MIMAT0000256 (hsa-miR-181a, Homo sapiens miR-181a),
MicroRNA registration number: MI0000073 (hsa-miR-19a, Homo sapiens miR-19a),
MicroRNA registration number: MIMAT0000074 (hsa-miR-19b, Homo sapiens miR-19b),
MicroRNA registration number: MIMAT0000076 (hsa-miR-21, Homo sapiens miR-21),
MicroRNA registration number: MIMAT0000681 (hsa-miR-29c, Homo sapiens miR-29c),
MicroRNA registration number: MIMAT0000688 (hsa-miR-301a, Homo sapiens miR-301a),
MicroRNA registration number: MIMAT0000245 (hsa-miR-30d, Homo sapiens miR-30d),
MicroRNA registration number: MIMAT0005792 (hsa-miR-320b, Homo sapiens miR-320b),
MicroRNA registration number: MIMAT0015072 (hsa-miR-320e, Homo sapiens miR-320e),
MicroRNA registration number: MIMAT0000761 (hsa-miR-324-5p, Homo sapiens miR-324-5p),
MicroRNA registration number: MIMAT0000760 (hsa-miR-331-3p, Homo sapiens miR-331-3p),
MicroRNA registration number: MIMAT0000727 (hsa-miR-374a, Homo sapiens miR-374a),
MicroRNA registration number: MIMAT0000731 (hsa-miR-378 *, Homo sapiens miR-378 *),
MicroRNA registration number: MIMAT0003885 (hsa-miR-454, Homo sapiens miR-454), and
MicroRNA registration number: MIMAT0000252 (hsa-miR-7, Homo sapiens miR-7).
The method according to claim 1, wherein the microRNA registration number: MIMAT0000063 (hsa-let-7b, Homo sapiens let-7b)
MicroRNA registration number: MIMAT0000256 (hsa-miR-181a, Homo sapiens miR-181a),
MicroRNA registration number: MIMAT0000245 (hsa-miR-30d, Homo sapiens miR-30d),
MicroRNA registration number: MIMAT0005792 (hsa-miR-320b, Homo sapiens miR-320b),
MicroRNA registration number: MIMAT0015072 (hsa-miR-320e, Homo sapiens miR-320e),
MicroRNA registration number: MIMAT0000761 (hsa-miR-324-5p, Homo sapiens miR-324-5p),
MicroRNA registration number: MIMAT0000760 (hsa-miR-331-3p, Homo sapiens miR-331-3p), and
MicroRNA registration number: MIMAT0000731 (hsa-miR-378 *, Homo sapiens miR-378 *) showed increased expression for xylene exposure,
The microRNA registration number: MIMAT0000099 (hsa-miR-101, Homo sapiens miR-101)
MicroRNA registration number: MI0000456 (hsa-miR-140-5p, Homo sapiens miR-140-5p),
The microRNA registration number MIMAT0000434 (hsa-miR-142-3p, Homo sapiens miR-142-3p),
MicroRNA registration number: MIMAT0000433 (hsa-miR-142-5p, Homo sapiens miR-142-5p),
MicroRNA registration number: MI0000460 (hsa-miR-144, Homo sapiens miR-144),
MicroRNA registration number: MIMAT0004600 (hsa-miR-144 *, Homo sapiens miR-144 *),
MicroRNA registration number: MI0000069 (hsa-miR-15a, Homo sapiens miR-15a),
MicroRNA registration number: MI0000073 (hsa-miR-19a, Homo sapiens miR-19a),
MicroRNA registration number: MIMAT0000074 (hsa-miR-19b, Homo sapiens miR-19b),
MicroRNA registration number: MIMAT0000076 (hsa-miR-21, Homo sapiens miR-21),
MicroRNA registration number: MIMAT0000681 (hsa-miR-29c, Homo sapiens miR-29c),
MicroRNA registration number: MIMAT0000688 (hsa-miR-301a, Homo sapiens miR-301a),
MicroRNA registration number: MIMAT0000727 (hsa-miR-374a, Homo sapiens miR-374a),
MicroRNA registration number: MIMAT0003885 (hsa-miR-454, Homo sapiens miR-454), and
MicroRNA regulatory number: MIMAT0000252 (hsa-miR-7, Homo sapiens miR-7) is characterized by decreased expression upon xylene exposure.
A kit for confirming exposure of xylene containing the microarray chip of claim 1.
4. The kit according to claim 3, wherein the kit further comprises human blood.
A kit for confirming exposure to xylene comprising all the primers complementary to each of the following microRNAs and capable of amplifying the respective microRNAs:
MicroRNA registration number (miRbase): MIMAT0000063 (hsa-let-7b, Homo sapiens let-7b)
MicroRNA registration number: MIMAT0000099 (hsa-miR-101, Homo sapiens miR-101),
MicroRNA registration number: MI0000456 (hsa-miR-140-5p, Homo sapiens miR-140-5p),
The microRNA registration number MIMAT0000434 (hsa-miR-142-3p, Homo sapiens miR-142-3p),
MicroRNA registration number: MIMAT0000433 (hsa-miR-142-5p, Homo sapiens miR-142-5p),
MicroRNA registration number: MI0000460 (hsa-miR-144, Homo sapiens miR-144),
MicroRNA registration number: MIMAT0004600 (hsa-miR-144 *, Homo sapiens miR-144 *),
MicroRNA registration number: MI0000069 (hsa-miR-15a, Homo sapiens miR-15a),
MicroRNA registration number: MIMAT0000256 (hsa-miR-181a, Homo sapiens miR-181a),
MicroRNA registration number: MI0000073 (hsa-miR-19a, Homo sapiens miR-19a),
MicroRNA registration number: MIMAT0000074 (hsa-miR-19b, Homo sapiens miR-19b),
MicroRNA registration number: MIMAT0000076 (hsa-miR-21, Homo sapiens miR-21),
MicroRNA registration number: MIMAT0000681 (hsa-miR-29c, Homo sapiens miR-29c),
MicroRNA registration number: MIMAT0000688 (hsa-miR-301a, Homo sapiens miR-301a),
MicroRNA registration number: MIMAT0000245 (hsa-miR-30d, Homo sapiens miR-30d),
MicroRNA registration number: MIMAT0005792 (hsa-miR-320b, Homo sapiens miR-320b),
MicroRNA registration number: MIMAT0015072 (hsa-miR-320e, Homo sapiens miR-320e),
MicroRNA registration number: MIMAT0000761 (hsa-miR-324-5p, Homo sapiens miR-324-5p),
MicroRNA registration number: MIMAT0000760 (hsa-miR-331-3p, Homo sapiens miR-331-3p),
MicroRNA registration number: MIMAT0000727 (hsa-miR-374a, Homo sapiens miR-374a),
MicroRNA registration number: MIMAT0000731 (hsa-miR-378 *, Homo sapiens miR-378 *),
MicroRNA registration number: MIMAT0003885 (hsa-miR-454, Homo sapiens miR-454), and
MicroRNA registration number: MIMAT0000252 (hsa-miR-7, Homo sapiens miR-7).
1) separating each RNA from a blood sample separated from the subject to be tested in a test group and a blood sample separated from a normal subject as a control group;
2) labeling the RNA of the experimental group and the control group of step 1) with different fluorescent materials;
3) hybridizing the fluorescently labeled RNA of step 2) with the microarray chip of claim 1;
4) analyzing the reacted microarray chip of step 3); And
5) confirming the degree of expression of all microRNAs integrated in the microarray chip of claim 1 in comparison with the control group in the analyzed data of step 4).
The method according to claim 6, wherein the fluorescent material of step 2) is selected from the group consisting of Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-non-isothiocyanate rhodamine-B-isothiocyanate (RITC), and rhodamine (Rhodamine).
1) separating each RNA from a blood sample separated from the subject to be tested in a test group and a blood sample separated from a normal subject as a control group;
2) synthesizing the RNA of the experimental group and the control group of step 1), respectively, with cDNA;
3) Real-time reverse transcript polymerase chain reaction (RT-PCR) was performed using all the primers capable of amplifying each microRNA integrated in the microarray chip of the above item 1, ; And
4) confirming the degree of expression of all the amplification products in the experimental group of step 3) in comparison with the control group.

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