KR101673197B1 - microRNAs for identification of exposure to particulate matter 2.5(PM2.5) and the method of identification using thereof - Google Patents

Abstract

The present invention relates to a microarray for confirming the exposure of particulate matter 2.5 (PM2.5) to less than 2.5 micrometer (㎛) and a method for confirming the microarray by using the microarray. More particularly, As a result of the exposure of the extract and the organic extract, one type of micro-overexpressing micro-type was selected, and the micro-type micro-RNA was used as a biomarker to monitor micro-dust below 2.5 micrometer, And a tool for identifying a toxic action mechanism caused by fine dust less than 2.5 micrometers

Description

[0002] MicroRNAs for confirming exposure to fine dusts (PM2.5) of less than 2.5 micrometers and methods for identifying them using microRNAs and identification methods thereof [

The present invention relates to microRNAs for confirming the exposure of particulate matter 2.5 (PM 2.5) to 2.5 micrometer (㎛) or less, and to a method of using the microRNAs.

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 modulate protein expression patterns through RNA degradation promotion, mRNA translation inhibition, and gene transcription. The microRNA regulates various biological processes such as development and differentiation, cell proliferation control, and stress response. It is now known that there are about a thousand microRNAs in higher eukaryotes.

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) Gene, an intron of a protein-encoding gene, or several, 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-like 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 has an important influence on cancer development, cell senescence, organs growth. Therefore, microRNA-related research plays a pivotal role in the identification of the causal relationship between life events such as cancer development and aging, and it can be applied to researches such as development and development of cell treatment agents using differentiation or stem cells. As a core area of Thus, the discovery of microRNA markers can be of great help in predicting the early diagnosis of many diseases, including cancer.

In addition, microRNA markers may be useful for predicting the exposure of certain environmentally hazardous substances. Up to now, studies have been conducted mainly on the pattern of changes in gene (mRNA) due to the exposure of environmentally harmful substances and their relation with diseases. Recently, as interest in microRNA has increased, several studies have observed changes in the expression of microRNAs following the exposure of harmful substances such as benzene, arsenic, or RDX, MicroRNAs and their target genes are presented 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 mainly limited to the discovery of marker for disease diagnosis, There has been little research on the expression of microRNAs by exposure to environmental agents such as fine dusts, which can be exposed at any time in everyday life. In addition, epigenetics changes are less severe than the variety of genetic alterations, such as gene expression, so that a single microRNA or DNA methylation, as compared to gene expression profiling, where multiple markers are required Early genetic markers can also be used to diagnose disease or toxic substances and diagnose them noninvasively. Each microRNA regulates a variety of target genes and in high eukaryotes, there are a thousand microRNAs, so it is expected that potential circuits that can be regulated by microRNAs will be enormous.

Dusts with particle sizes of 10 (= 0.001) or less are called fine dusts, and most of the fine dusts artificially generated by fuel combustion are classified as PM10 having a diameter smaller than 10 and PM2.5 having a diameter smaller than 2.5. Generally, the source of PM2.5, which can have a very serious effect on the human body, is mostly generated by fuel combustion and is a major source of emissions from boilers, automobiles and power generation facilities. In addition, dust scattering from construction sites and roads also occupies a large amount.

It is estimated that PM2.5 is moving faster in the air as the size is smaller, about 1/3 is due to long-distance movement, 1/3 is direct discharge from the area, and 1/3 is due to air-borne reaction. PM2.5 is characterized by its diameter smaller than 2.5, so it is difficult to distinguish it from the eyes and it penetrates deep into the alveoli. PM2.5 is mainly infused into the human body through respiration and is generated by fuel combustion, so it can be overexposed to factories, traffic congested roads, and power generation facilities.

PM2.5 is one of the substances that cause lung disease through the respiratory system. When it is exposed for a long time, the fine particles do not get caught through the nasal mucosa and penetrate directly into the alveoli when inhaled. Therefore, PM2.5 is a cause of diseases such as asthma, And can affect early mortality rates. Because of this risk multi-use facility Indoor Air Quality Act in the air quality standards for PM2.5 for comfortable indoor air in the expansion cavity, such as housing is placed below the annual average of 25g / m ³ or less per day, 50g / m ^3. The Ministry of Environment has announced that it will introduce a new environmental standard (annual average of 25 /) from 2015, because there is a growing number of reports that the health effect is greater by PM2.5.

 The data on pollution degree of PM2.5 in domestic air are very mobility in the air and the distribution concentration and composition of each region are large, so that data on the characteristics of ultrafine dust are very insufficient. The Ministry of Environment, in order to provide effective measures, contributes to collecting data on the long-distance movement of PM2.5, regional analysis of each component,

In addition, some studies on the degree of human toxicity, component analysis and gene expression changes of yellow dust, fine dust PM10, etc. have been reported, but studies on ultrafine dust PM2.5 have been limited to component analysis.

Assuming that the PM2.5 risk assessment data are not sufficient and the WHO recommended concentration level of PM2.5 achieves the air quality standard despite the potential risks of PM2.5 to humans as described above, And the uncertainty and limitations of the analytical method that calculates the health benefits of expected early death reduction.

Therefore, a rapid and simple screening method, for example, a real-time reverse transcript polymerase chain reaction (PCR) using a microarray chip or a primer, Based on the technology for evaluating PM2.5 exposure in mouse model, molecular markers that can detect the change of microRNA expression involved in various diseases such as cancer are identified and utilized. Is an important task.

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). In order to study the function of the gene based on the microRNA data thus revealed, microarray analysis capable of analyzing the expression of thousands of genes is performed in a single experiment (Schena, M, et al. Proc . Natl . Acad . Sci . USA 93 , 10614-10619, 1996) genome-wide expression studies have been conducted.

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 Affymetrix, which is a direct synthesis method on a chip using photolithography. Agillent and the like are methods for immobilizing synthesized oligonucleotides and (Sellheyer, K. 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.

Recently, it has been combined with toxic genomics research, which is a cutting-edge technique using DNA macromolecule technology, and it can be applied to high-throughput drugs such as drugs and new drug candidates, as well as representative environmental pollutants, 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 It will be possible to understand the molecular mechanisms and to search for and identify substances that cause toxicity and side effects.

Accordingly, the present inventors have used a oligo microarray in which 1,247 mouse microRNAs have been integrated to determine the microRNA expression profile of PM2.5 from a mouse model exposed to two kinds of water soluble, organic soluble (PM2.5) As a result of analyzing the changes in microRNA expression by collecting lung tissue samples, it was found that microRNAs whose expression is commonly changed by PM2.5 exposure in a mouse model and microRNA expression can be detected at PM2.5 exposure The present inventors have completed the present invention by establishing a biomarker and a method of confirming exposure using the biomarker.

An object of the present invention is to provide a microarray chip for confirming exposure of particulate matter 2.5 (PM 2.5) below 2.5 micrometer (쨉 m) in which microRNAs (miRNA) or complementary strand molecules thereof are integrated It provides:

MicroRNA registration number (miRbase) MIMAT0017342 (Mm-miR-1943-3p).

Another object of the present invention is to provide a kit for confirming exposure of fine dusts of 2.5 micrometers or less comprising a primer complementary to the following microRNAs and capable of amplifying the microRNAs:

MicroRNA registration number (miRbase) MIMAT0017342 (Mm-miR-1943-3p).

Another object of the present invention is to provide

1) separating the respective RNAs from somatic cells isolated from the test body of the experimental group and somatic cells isolated from the normal individuals as the 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 a microRNA microarray chip;

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

5) Providing a micro-dust exposure confirmation method of less than 2.5 micrometers including the step of comparing the degree of expression of microRNA integrated in the microarray chip of the first claim of the experimental group with the control group in the analyzed data of step 4) will be.

Another object of the present invention is

1) separating the respective RNAs from somatic cells isolated from the test body of the experimental group and somatic cells isolated from the normal individuals as the 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 primers capable of amplifying the microRNAs integrated in the microarray chip of the above item 1, respectively, 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 product of the experimental group of step 3) in comparison with the control group.

In order to achieve the above object, the present invention provides a microarray chip for confirming exposure of particulate matter 2.5 (PM2.5) below 2.5 micrometer (㎛) in which microRNAs (miRNA) or complementary strand molecules thereof are integrated lt; RTI ID = 0.0 > microarray < / RTI &

MicroRNA registration number (miRbase) MIMAT0017342 (Mm-miR-1943-3p).

The present invention also provides a kit for confirming exposure of fine dusts of 2.5 micrometers or less comprising a primer complementary to the following microRNAs and capable of amplifying the microRNAs:

MicroRNA registration number (miRbase) MIMAT0017342 (Mm-miR-1943-3p).

In addition,

1) separating the respective RNAs from somatic cells isolated from the test body of the experimental group and somatic cells isolated from the normal individuals as the 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 a microRNA microarray chip;

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

5) Providing a microscopic dust exposure confirmation method of less than 2.5 micrometers including the step of comparing the degree of expression of microRNA integrated in the microarray chip of the first test item with the control group in the analyzed data of step 4) .

In addition,

1) separating the respective RNAs from somatic cells isolated from the test body of the experimental group and somatic cells isolated from the normal individuals as the 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 primers capable of amplifying the microRNAs integrated in the microarray chip of the above item 1, respectively, 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 product of the experimental group of step 3) in comparison with the control group.

A biomarker for confirmation of the expression of microRNA whose expression is commonly changed for two types of water soluble or organic soluble (PM2.5) micro-dusts of less than 2.5 micrometer in characteristics of the present invention And identification method using the same are useful for monitoring PM2.5 and determining the risk of PM2.5 by using reactive microRNAs selected through microRNA microarray chip as a biomarker and to identify the toxic action mechanism caused by PM2.5 It can be useful as a tool.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the extraction process route of the aqueous extract and the organic extract thereof in the fine dust-treating group of 2.5 micrometer (μm) or less.
FIG. 2 is an image showing the result of analysis of microRNA expression patterns in a mouse lung tissue sample exposed to PM2.5 using a microRNA microarray chip. FIG.

Hereinafter, the present invention will be described in detail.

The present invention is based on the surprising finding that the present invention is commonly expressed by two species of particulate matter 2.5 (PM2.5) (water soluble or organic soluble) A biomarker for confirming exposure of fine dust less than 2.5 micrometers.

It is preferable that the biomarker is composed of microRNAs whose expression is increased or decreased by 1.3 times or more by PM2.5 exposure.

MicroRNA registration number (miRbase) MIMAT0017342 (Mm-miR-1943-3p) (SEQ ID NO: 1).

The microRNA sequence consists of caggugccagcuccucccuuc.

The fine dust less than 2.5 micrometers is characterized by being a micro-dust water-soluble extract of less than 2.5 micrometers or a micro-dust organic extract of less than 2.5 micrometers.

The fine dust is preferably particulate matter 2.5 (PM2.5) of less than 2.5 micrometer (micrometer) including ultra fine particles (UFP), more preferably of 0.1 to 2.5 micrometers Most preferable is fine dust, but is not limited thereto.

The fine dust extract is preferably, but not limited to, prepared by a manufacturing method comprising the following steps:

1) collecting fine dust less than 2.5 micrometers;

2) extracting the collected fine dust in step 1) by adding an extraction solvent; And

3) filtering the extract of step 2).

In this method, the extraction solvent of step 2) is preferably water, alcohol, dichloromethane or a mixture thereof. The water is preferably purified or distilled, and it is more preferable to use tertiary distilled water. As the alcohol, C1 to C2 lower alcohol is preferably used, and as the lower alcohol, ethanol or methanol is preferably used. As the extraction method, it is preferable to use an ultrasonic pulverizer and a high volume air sampler, but it is not limited thereto. In addition, the ultrasonic pulverizer time is preferably 20 to 60 minutes, more preferably 30 minutes, but is not limited thereto.

In a specific example of the present invention, micro-dust extracts of less than 2.5 micrometer were processed into a BALB / C mouse model, total RNA containing microRNA was isolated from mouse lung tissue and labeled with a fluorescent material (Cy3). Fluorescently labeled microRNAs were hybridized with microarray chips, and fluorescence images were scanned for differences in gene expression patterns. When the signal intensity ratio of the experimental group and the control group was 1.3 times or more, it was classified as an increased expression gene. One microRNA overexpressed by the water-soluble extract of micro-dust below 2.5 micrometer and the organic extract was selected as follows.

MicroRNA registration number (miRbase): MIMAT0017342 (Mm-miR-1943-3p) (SEQ ID NO: 1).

Therefore, it is confirmed that the biomarker according to the present invention is microRNA overexpressed specifically in fine dusts of less than 2.5 micrometers. Thus, it is possible to confirm whether or not microscopic dust is exposed below 2.5 micrometer, to monitor micro dust below 2.5 micrometer, It can be usefully used as a tool to determine the risk of sub-meter fine dust, and the mechanism of toxic action caused by fine dust less than 2.5 micrometers.

In addition, the present invention provides a microRNA microarray chip for confirming exposure to specific PM2.5 oligonucleotides or their complementary strand molecules synthesized / integrated with all or a part of the biomarker microRNA sequence.

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

The microRNA microarray chip for PM2.5 search of the present invention can be produced by a method known to a person skilled in the art. As a method for manufacturing the microarray chip, it is preferable to use an inkjet method or the like in order to immobilize the biomarker on a substrate using a probe microarray molecule, and a super print ink jet microdrop More preferably, but not exclusively, using a SurePrint inkjet micro dropping microarray.

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 is preferably selected from the group consisting of a slide glass, a plastic, a metal, a silicon, a nylon film, and a nitrocellulose membrane, and more preferably an epoxy coated slide glass , But is not limited thereto.

In addition, the present invention provides a method for confirming exposure of micro-RNA to PM2.5 exposure using the microarray chip.

The present invention

1) separating the respective RNAs from somatic cells isolated from the test body of the experimental group and somatic cells isolated from the normal individuals as the 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 a microRNA microarray chip;

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

5) Providing a microscopic dust exposure confirmation method of less than 2.5 micrometers including the step of comparing the degree of expression of microRNA integrated in the microarray chip of the first test item with the control group in the analyzed data of step 4) .

In the method for confirming the exposure, the somatic sample of step 1) is preferably obtained from each group of mice exposed or not exposed to PM2.5.

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.

The microRNA microarray chip of step 5) is preferably a mouse miRNA 8 X 60K (Rel 19.0) (Agilent, USA), but the present invention is not limited thereto, Among the microarray chips, microarray microarray chips can be used as long as they are microarray chips in which the microarrays having the commonly changed expression in the present invention (see Table 1) are mounted. Most preferably, microarray microarray chips manufactured by the present inventor are used.

Also, the analysis method of step 4) is preferably to use GeneSpring GX 12.6.1 software (Agilent, USA), but it is not limited thereto, and analysis software known to those skilled in the art may be used.

The method for confirming the expression of microRNA according to the present invention uses a microarray chip capable of confirming the distribution of microRNAs that are overexpressed or underexpressed specifically in fine dusts of 2.5 micrometers or less, , The monitoring of fine dust less than 2.5 micrometers, the determination of the risk of fine dust less than 2.5 micrometers, and the toxic action mechanism caused by fine dust less than 2.5 micrometers.

The present invention provides a kit for confirming exposure of fine dusts of 2.5 micrometer or less including a microarray chip.

The kit for confirming whether or not the micro dust exposure is less than 2.5 micrometers further includes mouse lung cells or lung tissue.

Also, the kit for confirming exposure of fine dusts below 2.5 micrometers includes a primer complementary to the following microRNAs and capable of amplifying the microRNAs:

MicroRNA registration number (miRbase) MIMAT0017342 (Mm-miR-1943-3p).

Preferably, the mouse model lung tissue sample is obtained from a group of mice exposed or not exposed to PM2.5.

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.

Therefore, it is confirmed that the kit according to the present invention is microRNA that is specifically overexpressed in fine dusts of 2.5 micrometers or less, which confirms whether or not fine dusts of 2.5 micrometers or less are exposed, monitoring fine dusts of 2.5 micrometers or less, And can be usefully used as a tool for determining the risk of fine dust and a toxic action mechanism caused by fine dust less than 2.5 micrometers.

In addition,

1) separating the respective RNAs from somatic cells isolated from the test body of the experimental group and somatic cells isolated from the normal individuals as the 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 primers capable of amplifying the microRNAs integrated in the microarray chip of the above item 1, respectively, 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 product of the experimental group of step 3) in comparison with the control group.

The somatic cell in the step 1) is characterized by being a mouse lung cell or lung tissue.

Therefore, the method of confirming the degree of expression of the amplification products using the microRNA and cDNA of the present invention in comparison with the control group is a microarray capable of confirming the distribution of microRNAs overexpressed or underexpressed specifically in fine dusts of 2.5 micrometers or less By using chips, it is possible to confirm the exposure of fine dust below 2.5 micrometer, to monitor fine dust below 2.5 micrometer, to determine the risk of fine dust below 2.5 micrometer, and to identify the toxic mechanism caused by fine dust below 2.5 micrometer It can be useful as a tool to do.

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.

< Example  1> Less than 2.5 micrometer fine dust Capture  And extraction

<1-1> Capture  Place selection

In order to identify biomarkers for microRNA expression in PM2.5 exposure, an average of 70,000 weekdays in November 2011 and an average of 6.9 million weekdays in December 2011 The top of the gymnasium of Korea Research Institute of Science and Technology (KIST) located in Haewok-dong, Seongbuk-gu, Seoul, which is adjacent to the circulation road, was selected as a sample collection site to collect fine dusts of 2.5 micrometers (㎛) or less. In order to carry out an in vitro test of fine dust less than 2.5 micrometers, a large amount of fine dust smaller than 2.5 micrometers is required. Low Volume Air Sampler is suitable for collecting fine dust less than 2.5 micrometer for testing in the laboratory because the amount of fine dust less than 2.5 micrometers obtained in operation for 24 hours is only a few milligrams (mg) The experiment was conducted by selecting a high-volume air sampler capable of capturing a large amount of water per day. Inlet (Tisch Instrument Company) of 2.5 micrometer or less micrometer size has a hydrodynamic size of 2.5 micrometer or less and can not overcome the inertia of its weight and stops by striking against oil, Are collected using a Teflon Coated Glass Fiber Filter.

<1-2> Capture  filter

Teflon Coated Glass Fiber Filter, which has the advantage of not being able to extract 8 × 10 inch (inch) size components in the filtration stage when using a high volume air sampler, was adopted. Distilled water (18.2 ㏁) And dichloromethane were used. In order to minimize the error due to moisture, the filter was completely removed from the filter for 48 hours by a decicator, and then the weight of the filter was weighed using a mettler Toledo Company (Mettler Toledo Company) capable of measuring up to 0.1 mg. After the inside of the filter is folded together, it is put in a dark room and a zipper bag with airtight effect. It is stored at -80 ° C until the sample is used. When moving to the collection place, use an ice box Respectively. The appropriate flow rate (1.13 m3 / min ± 10%) was maintained for fine dust collection of less than 2.5 micrometers.

<1-3> Capture  term

The fine dust less than 2.5 micrometers was collected using a high volume air sampler for 5 months. The first collection on November 1, 2011, and the last collection on March 28, 2012, has ended. The filter values showing the atmospheric concentration of the fine dust less than the reference value of 20 ㎍ / ㎥ were excluded and the total of 5 groups were grouped into 10 filters.

<1-4> Fine dust Collection sample  Extraction process

In order to analyze the components of the fine dust less than 2.5 micrometers, one filter was divided into two, and each of the two filters was composed of ultra pure water (distilled water) for extraction of water soluble components Dichloromethane was used for extraction of organic soluble components. 2.5 micrometer or less fine dust-soluble and 2.5 organic extracts were prepared according to the extraction process route (Fig. 1).

< Example  2> PM2 .5 Exposure model selection and mouse Lung tissue  Obtaining Samples

<2-1> PM2 .5 Exposure Models

In the case of PM2.5, which is an environmentally harmful substance, it is suspended in air. Particulate matter tends to enter the lungs and induce typical particle-induced inflammation. There are many reports of studies using BALB / C as animal models to sensitively measure these responses. In addition, due to the characteristics of BALB / C, it was selected as a suitable model in consideration of the characteristic of being highly susceptible to chronic pneumonia, highly sensitive to radiation, relatively gentle, and low in breast cancer incidence rate.

Specifically, PM2.5 collected in Example 1 was divided into two types of water soluble and ganic soluble groups, which were different in characteristics. Exposure group and non-exposed group were exposed to mouse model The exposed group was divided into 6 groups (Table 1). The exposed groups were exposed to low concentrations of PM2.5 and high concentrations of PM2.5.

PM2 .5 Exposure concentration and mouse experiment group composition Experimental group Exposure (absolute) Population ( BALB / C) Control group 0 24 grains Low capacity 20 24 grains High capacity 164 24 grains

&Lt; 2-2 > PM2 .5 Exposure Concentration Selection and Exposure

Exposure of the BALB / C mouse model using the existing PM2.5 was selected so that the amount of PM2.5 would not exceed 100 g based on the absolute amount. (Hans Hedrich, The laboratory mouse , 2004), and the control group (0 g), low-dose group (control group (low dose) (20 g) and high dose (164 g) were selected for the exposure experiment (Table 2).

 In the case of the PM2.5 sample, it is preferable to perform the inhalation experiment. However, it is impossible to expose the same concentration of the PM2. 5 were prepared as liquid samples and exposed to the BALB / C mouse model in groups by the following calculation method through tracheal instillation method.

How to Calculate PM2.5 Mouse Dose

Breathing capacity (ml) per minute of mouse = 33.5 to 47.5 ml / min

Breathing capacity (ml) of mouse for 1 week = 337,680 ~ 478,800 ml / week

When 20 ㎍ was administered

20 占 퐂 / 337,680 ml = 0.0000592277 占 퐂 / ml

20 占 퐂 / 478,800 ml = 0.0000417711 占 퐂 / ml

The daily air quality standard of PM 2.5 is 50 ㎍ / m ³

Human respiratory capacity per minute (ml) = 6000 ml

Human respiratory capacity (ml) per week = 6000 * 60 minutes * 24 hours * 7 weeks = 60,480,000 ml = 60 m ³

When exposed for one week from 25 ㎍ to 50 ㎍

25 * * 60 m ³ / 60,480,000 ml = 0.0000248016 / / ml

^{50 ㎍ * 60 m 3 / 60,480,000} ㎖ = 0.0000496032 ㎍ / ㎖

Therefore, when exposed to 20 ㎍ of mouse, it is 41 ~ 59 ㎍ / ㎖ ^-6 , and when compared with exposure dose of 24 ~ 49 ㎍ / ㎖ ^-6 when applied to humans atmospheric environment standard of PM2.5, Respectively. The reason for the low dose exposure is that cytotoxicity and histopathological effects do not appear, but changes at the gene level may occur.

On the other hand, 164 g of the drug was expected to be cytotoxic and histopathologically effective, and a high dose was set because it was expected that a change in gene level would occur.

<2-3> Lung tissue  sample

In order to prioritize the purpose of extraction of high quality total RNA containing efficient miRNA from the target organs in the BALB / C mouse model, the lung tissue of the most undamaged state was obtained. After harvesting, the cells were stored at -80 ° C. for 6 months in a light-shielded state at -20 ° C. and in Allprotect Solution (Qiagen), which can be stored for one year in a shade state at -80 ° C., before use.

< Example  3> Microarray  Experiment

<3-1> Target micro RNA Separation of fluorescent material and labeling

RNA extraction from PM2.5 exposed and unexposed blood samples was extracted using RNA extraction using a triazole and the RNeasy Mini kit (Cat NO.74106) from Qiagen &Lt; / RTI &gt; Genomic DNA was removed during RNA purification using an RNase-free DNase set (Qiagen, USA). The concentration of each total RNA sample was measured using a spectrophotometer NanoDrop ND 1000 spectrophotometer (NanoDrop Technologies Inc., USA) and the qualitative state of the RNA was measured using an Agilent 2100 Bioanalyzer ).

<3-2> Labeled  Micro RNA  Produce

For the oligomicroarray analysis, the fluorescent material was labeled on the total RNA of the PM2.5 exposed group obtained in the above Example <3-1>. Fluorescence labeling was performed using the manufacturer's method using the complete labeling and hybridization kit of Agilent's miRNA. 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 2l (100 ng) of the obtained total RNA, After incubation for 30 minutes at &lt; RTI ID = 0.0 &gt; 30 C &lt; / RTI &gt;, 2.8 L of 100% DMSO was added and reacted at 100% for 5-10 minutes and immediately transferred to ice. 1 μl of 10 × T4 RNA ligase buffer, 3 μl of cyanine 3-pCp (Cyanine 3-pCp), and 0.5 μl of T4 RNA ligation were added for dyeing and reacted at 16 ° C. for 2 hours . 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 deficient water. After adding 4.5 μl of 10 × GE Blocking Agent, 22.5 μl of 2 × Hi-RPM hybridization A buffer (hybridization buffer) was added. The prepared sample 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.

<3-3> Hybridization  reaction

Hybridization and washing were carried out according to the instructions of this Biogen Co., Ltd. Hybridization was carried out in a 55 ° C oven for 20 hours. As a DNA microarray chip, mouse miRNA 8 X 60K (Rel 19.0) (Agilent, USA) was used.

<3-4> Fluorescence image acquisition

Hybridization images on the slides were scanned with an Agilent C scanner (Agilent Technologies, USA) and the extracted data was analyzed using an Agilent GeneSpring GX 12.6.1 (Agilent technologies) After normalization, the expression pattern of each microRNA was analyzed.

As a result, as shown in FIG. 2, approximately 1,247 microRNAs present on the oligonucleotide were verified by comparing the microRNA expression patterns of exposed and unexposed groups using GeneSpring GX 12.6.1 software (Fig. 2). As a result, one microRNA whose expression was increased by more than 1.3 times was measured, and a reduced microRNA was not found (Table 2). There is no report that the microRNA is associated with toxicity upon PM2.5 exposure in a mouse model.

PM2 .5 micro-organisms whose expression changes by exposure RNA
Registration Number

Gene name
 Change in drainage control Water soluble _ Water soluble _ Organic _ Organic __ MIMAT0017342 Mm-miR-1943-3p 3.18 1.87 1.73 1.33 Increase

<110> Korea Institute of Science and Technology <120> MicroRNAs for identification of exposure to particulate matter          2.5 (PM2.5) and the method of identification using it <130> 14P-09-019 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> MIMAT0017342 / Mm-miR-1943-3p <400> 1 caggugccag cuccucccuu c 21

Claims (11)

(Microarray (microarray) chip (microarray) for confirming exposure of particulate matter 2.5 (PM2.5) in which the lung cells or lung tissue-derived microRNAs (miRNA) of the following mice or their complementary strand molecules are integrated chip:
MicroRNA registration number (miRbase) MIMAT0017342 (Mm-miR-1943-3p) (SEQ ID NO: 1).
[3] The micro-dust according to claim 1, wherein the fine dust less than 2.5 micrometer (micrometer) is a micro-dust water-soluble extract of less than 2.5 micrometer or a fine dust organic extract of less than 2.5 micrometer. Array chip.
A kit for confirming exposure of fine dust below 2.5 micrometer (占 퐉) comprising the microarray chip of claim 1.
4. The kit according to claim 3, wherein the kit further comprises mouse lung cells or lung tissue.
Kit for confirming exposure of fine dust below 2.5 micrometer including a primer capable of complementing microRNA derived from pulmonary cells or lung tissues of the following mouse and amplifying said microRNA:
MicroRNA registration number (miRbase) MIMAT0017342 (Mm-miR-1943-3p).
1) separating the respective RNAs from the tissues separated from the test body of the experimental group and the tissues separated from the normal individuals as the control group;
2) The RNA of the experimental group and the control group of step 1) was incubated with Cy3 (Cyanine 3), Cy5, polyL-lysine-fluorescein isothiocyanate (FITC), rhodamine- A fluorescent substance selected from the group consisting of rhodamine-B-isothiocyanate (RITC) and rhodamine;
3) hybridizing the fluorescently labeled RNA of step 2) with a microRNA microarray chip;
4) analyzing the reacted microRNA microarray chip of step 3); And
5) A method for confirming exposure of fine dust below 2.5 micrometers comprising the step of comparing the degree of expression of microRNA integrated in the microarray chip of claim 1 with the control group in the analyzed data of step 4).
The method according to claim 6, wherein the tissue of step 1) is lung cells or lung tissue of a mouse.
[8] The method according to claim 7, wherein the lung tissue is a BALB / C mouse lung tissue.
delete 1) separating the respective RNAs from the tissues separated from the test body of the experimental group and the tissues separated from the normal individuals as the 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 primers capable of amplifying the microRNAs integrated in the microarray chip of the above item 1, respectively, with the cDNAs of the experimental group and the control group of step 2) step; And
4) confirming the extent of expression of the amplification product in the experimental group of step 3) compared with the control group.
11. The method according to claim 10, wherein the tissue of step 1) is mouse lung cells or lung tissue.

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