CN106191229B - micro-RNA for identifying exposure to particulate matter 2.5(PM2.5) and method for identifying using same - Google Patents
micro-RNA for identifying exposure to particulate matter 2.5(PM2.5) and method for identifying using same Download PDFInfo
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Abstract
The invention relates to the use of microRNAs for identifying exposure to particulate matter 2.5(PM2.5), and methods of identifying exposure to PM2.5 using the microRNAs. More precisely, when a mouse model was exposed to PM2.5 water soluble extract and PM2.5 organic soluble extract, it was confirmed that one microrna was overexpressed at a level 1.3 times the normal level. Thus, the selected micrornas can be used as biomarkers for monitoring PM2.5 and assessing risk of PM2.5, and as tools for examining the mechanisms of PM2.5 toxicity.
Description
Technical Field
The present invention relates to micrornas (micrornas) for identifying exposure (exposure) to particulate matter 2.5(PM2.5) and methods of identifying using the micrornas.
Background
Recently, micrornas (mirs, mirnas) have been raised as important regulatory RNAs involved in regulation of gene expression. Such small non-coding RNA molecules (typically consisting of 18-24 nucleotides) can regulate protein expression patterns by participating in RNA degradation, mRNA translation, and gene transcription. The micrornas modulate a variety of biological processes, such as development, differentiation, cell proliferation, stress response, and the like. Higher eukaryotes are known to contain about 1000 kinds of micrornas.
microRNAs are transcribed by RNA polymerase II (pol II) or RNA polymerase III (pol III; Qi, P, et al, cell. mol. Immunol.3, 411-419, 2006) and can be induced by such transcripts of each miRNA gene, introns of the gene encoding the protein, or polycistrons of closely related miRNAs. Transcription of miRNA genes by means of RNA pol II or pol III produces a first transcript of several thousand nucleotides in length, which is called primary miRNA transcript (pri-miRNA). In the nucleus, pri-mirnas are processed by rnases and Drosha to generate hairpin-type pre-mirnas, consisting of 70-100 nucleotides. Once transferred into the cytoplasm, the hairpin pre-miRNA is reprocessed by dicer to generate double stranded miRNA. The mature miRNA strand is incorporated into the RNA-induced silencing complex (RISC). Subsequently, the mature miRNA binds to the target mRNA by base pair complementarity in RISC. mRNA degradation is promoted when miRNA base pairs perfectly match mRNA targets (very rarely). In general, mirnas form incomplete heteroduplexes with target mrnas, which affect mRNA translation.
miRNA mechanisms have important effects on cancer development, cell aging, and organ growth, among others. Therefore, the research on miRNA is important not only for the research on cancer development, aging and its effect on human life span, but also applied in the research on screening cell therapy products using stem cells, and development, differentiation, which indicates that miRNA can be a core target for biological research in korea. Therefore, screening for microrna markers would be very helpful for the prediction or early diagnosis of a variety of diseases, including cancer.
It is also believed that microrna markers would be useful for predicting exposure to particular environmental hazardous substances. Until recently, studies have focused primarily on the modification of mRNA induced by exposure to environmentally harmful substances, and the association of the modified mRNA with disease. Based on recently proposed and interesting implications of micrornas with disease, the expression pattern of micrornas under exposure to harmful substances (e.g., benzene, arsenic or RDX) was investigated. As a result, micrornas and their target genes, the expression patterns of which are target-specifically altered, have been proposed as markers for such harmful substances (baccarlli, a. and Bollati, v.curr.opin.prediatr., 21, 243-. The identified micrornas are not only markers for prediction of exposure, but also gene expression regulators capable of regulating the expression of target genes, indicating that micrornas can also be advantageously used as markers for prediction of toxicity caused by environmentally harmful substances. Although micrornas play an important role in predicting exposure to environmentally harmful substances and toxicity resulting therefrom, research on micrornas has been limited to the use of micrornas to develop markers for disease diagnosis. In particular, the expression pattern of micrornas as a host of alterations when exposed to environmental substances (e.g., particulate matter generated from natural and environmental sources and which persists through exposure) has not been thoroughly studied. Epigenetic changes are not as extensive as genetic modifications (e.g., changes in gene expression patterns). Thus, epigenetic markers (e.g., microrna or DNA methylation) can simply utilize only one marker to facilitate early identification of exposure to harmful agents, and are advantageous for non-invasive methods, as compared to gene expression profiles that require a large number of markers. Each microrna regulates multiple target genes. There are thousands of micrornas in higher eukaryotes. Thus, a large number of potential circulating pathways (cycles) that can be regulated by micrornas are expected.
Dust having a size of 10 μm or less (1 μm ═ 0.001mm) is called particulate matter. The particulate matter artificially generated by the combustion of fuel is roughly divided into two groups: PM10 is 10 μm or less in diameter, PM2.5 is 2.5 μm or less in diameter, and PM2.5 is referred to as fine particulate matter. Generally, PM2.5, which can be a serious hazard to humans, is produced by the combustion of fuel. Boilers, automobiles, and power plants are also major sources. Dust dispersed from construction sites and roads also constitutes a large part of PM 2.5.
The speed of movement in air is faster due to the smaller size of PM 2.5. It is speculated that 1/3 PM2.5 comes from a long distance migration, 1/3 is generated locally directly, 1/3 is due to reactions in air. Since PM2.5 has a diameter of 2.5 μm or less, it is extremely difficult to distinguish by naked eyes. PM2.5 can invade deeply into the alveoli. PM2.5 is inhaled into the human body mainly by breathing. PM2.5 is produced by fuel combustion and therefore, particularly at manufacturing plants, heavily trafficked roads and power plants, etc., the risk of exposure to PM2.5 is high.
PM2.5 is one of the substances that induce lung diseases by the respiratory system. Since the particles are very small, PM2.5 can invade the lung without being filtered by the nasal mucosaBubbles, prolonged exposure to PM2.5, may be responsible for a variety of diseases, including asthma, allergy (atopy), and lung cancer, among others, and may increase the rate of premature death. Due to this risk, according to the indoor air quality management law, the ambient air quality standard for indoor air quality of new apartment houses is strictly limited to an annual average of 25 μ g/m for PM2.53The daily average value is 50. mu.g/m3The following. Recent reports indicate that PM2.5 is a significant threat to our health, and thus a new environmental standard (annual average 25 μ g/m)3) Effective since 2015.
Since PM2.5 has characteristics of fine particulate matter (e.g., high fluidity; and, depending on regions, distribution, concentration, and composition have high diversity and difference), korean recorded information on the pollution level of PM2.5 is insufficient. The korean ministry of the environment is eagerly under investigation in an attempt to provide an effective plan by collecting information on the long-distance migration of PM2.5 and the difference in the composition of the area (with particular attention paid to facilities that produce a large amount of PM 2.5).
Although some studies report toxicity of PM10 to humans, the composition of PM10, and changes in gene expression patterns caused by PM10, the study of PM2.5 is currently limited to its composition.
While PM2.5 has a potential risk to humans, the risk assessment data for PM2.5 is still insufficient, and there are uncertainties and limitations in the analytical methods that calculate the health benefits (resulting from the reduction in premature death achieved by improving the PM2.5 concentration, assuming that the WHO recommended PM2.5 concentration meets the air quality criteria).
Therefore, there is a strong urgent need for rapid assessment of human risk by using microarray chips or real-time PCR with primers to develop molecular markers for screening human toxicity and expression patterns of micrornas involved in the development of various diseases, particularly including cancer; and there is a very urgent need for methods based on the evaluation of PM2.5 exposure in a mouse model, using such markers to formulate and manage appropriate countermeasures against exposure to PM 2.5.
Since the first identification of microRNAs in 1997, a large number of microRNAs in mammals and microorganisms have been rapidly identified and reported to the Sanger miRBASE database (http:// www.mirbase.org/index. Genome-wide expression studies (Schena, M et al, proc.natl.acad.sci.usa., 93, 10614-.
Microarrays are prepared by integrating cDNA (complementary DNA) or sets of oligonucleotides 20-25 base pairs in length onto glass. Laboratories in schools or companies, including Agilent or Genomic Solutions, have prepared cDNA microarrays by mechanically immobilizing cDNA pools on a chip or by ink jet (ink jet) (Sellheyer K. et al, J.Am.Acad.Dermatol., 51, 681-692, 2004). Oligonucleotide microarrays prepared by Affymetrix co. used a method of synthesizing oligonucleotides directly on a chip by means of photolithography (photolitography). Oligonucleotide microarrays prepared by Agilent Co. utilize a method of immobilizing presynthesized oligonucleotides on a chip (Sellheyer, K. et al, J.Am.Acad.Dermatol., 51, 681-692, 2004).
To analyze gene expression, micrornas obtained from a sample (e.g., tissue) are hybridized to oligonucleotides on a microarray. The obtained micro RNA is labeled with fluorescence or isotope.
With the aid of toxicological genomics (advanced technology using DNA microarrays), quantitative and high-throughput analysis of the expression pattern of micrornas expressed in specific tissues or cell lines induced by chemicals, including drugs and new drug candidates as well as contaminants, is enabled. Therefore, it is now possible to identify genes specifically associated with the side effects of drugs and the human toxicity of contaminants by analyzing cell-specific microrna expression, thereby understanding the molecular mechanisms of the side effects and the toxicity of contaminants and further enabling the screening and identification of substances causing the toxicity and side effects.
Disclosure of Invention
The present inventors analyzed PM2.5 microrna expression profiles using an oligonucleotide microarray having integrated probes for detecting 1,247 mouse micrornas; lung tissue samples were taken from mice exposed to two different types of PM2.5 (water soluble and organic soluble) and subsequently analyzed for expression patterns of micrornas. As a result, the inventors identified micrornas whose expression was altered in mice exposed to two different types of PM 2.5. Subsequently, the present inventors determined a biomarker for identifying exposure to PM2.5 and a method for identifying the exposure using the biomarker, thereby completing the present invention.
One object of the present invention is to provide a use of a microarray chip on which cdnas of the following micro rnas (mirnas) are integrated for identifying exposure to particulate matter 2.5(PM 2.5):
microRNA accession number (miRbase) MIMAT0017342(Mm-miR-1943-3 p).
It is another object of the invention to provide the use of a kit comprising primers complementary to and capable of amplifying the cDNA of a microrna:
microRNA accession number (miRbase) MIMAT0017342(Mm-miR-1943-3 p).
It is a further object of the present invention to provide a method for identifying exposure to PM2.5, said method comprising the steps of:
1) isolating RNA from somatic cells obtained from experimental subjects and somatic cells obtained from control normal subjects;
2) labeling each of the RNAs obtained from the experimental group and the control of step 1) with a different fluorescent substance;
3) hybridizing the RNA marked with the fluorescent substance in the step 2) with a micro RNA microarray chip;
4) analyzing the micro RNA microarray chip reacted in the step 3); and
5) using the data analyzed in step 4), the expression of Mm-miR-1943-3p in the experimental group and the control was compared.
It is a further object of the present invention to provide a method for identifying exposure to PM2.5, said method comprising the steps of:
1) isolating RNA from somatic cells obtained from experimental subjects and somatic cells obtained from control normal subjects;
2) synthesizing cDNA from each RNA of the experimental group and each RNA of the control;
3) performing RT-PCR (real-time polymerase chain reaction) using the cDNAs of the experimental group and the control of step 2) using primers capable of amplifying the cDNA of Mm-miR-1943-3 p; and
4) comparing the expression of the amplification products of the experimental group and the control of step 3).
To achieve the above object, the present invention provides a use of a microarray chip for identifying exposure to particulate matter 2.5(PM2.5), the microarray chip having integrated thereon cDNA of the following micro rna (mirna):
microRNA accession number (miRbase) MIMAT0017342(Mm-miR-1943-3 p).
The invention also provides the use of a kit comprising primers complementary to and capable of amplifying the cDNA of a microrna:
microRNA accession number (miRbase) MIMAT0017342(Mm-miR-1943-3 p).
The present invention also provides a method for identifying exposure to PM2.5, the method comprising the steps of:
1) isolating RNA from somatic cells obtained from experimental subjects and somatic cells obtained from control normal subjects;
2) labeling each of the RNAs obtained from the experimental group and the control of step 1) with a different fluorescent substance;
3) hybridizing the RNA marked with the fluorescent substance in the step 2) with a micro RNA microarray chip;
4) analyzing the micro RNA microarray chip reacted in the step 3); and
5) using the data analyzed in step 4), the expression of Mm-miR-1943-3p in the experimental group and the control was compared.
Furthermore, the present invention provides a method for identifying exposure to PM2.5, said method comprising the steps of:
1) isolating RNA from somatic cells obtained from experimental subjects and somatic cells obtained from control normal subjects;
2) synthesizing cDNA from each RNA of the experimental group and each RNA of the control;
3) performing RT-PCR (real-time polymerase chain reaction) using the cDNAs of the experimental group and the control of step 2) using primers capable of amplifying the cDNA of Mm-miR-1943-3 p; and
4) comparing the expression of the amplification products of the experimental group and the control of step 3).
Advantageous effects
Biomarkers for identifying exposure to PM2.5, the expression pattern of which changes upon exposure to two different types of PM2.5 (water soluble or organic soluble), and methods of identifying exposure to PM2.5 using the biomarkers, are useful for monitoring and assessing PM2.5 risk, since they use the only reactive microrna biomarkers selected from microrna microarray chips, and thus, they can be useful tools for studying PM2.5 toxicity mechanisms.
Drawings
The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:
fig. 1 is a diagram showing an extraction process of a water-soluble extract and an organic-soluble extract from the environment.
Fig. 2 is a graph showing the results of analysis of microrna expression patterns of mouse lung tissue samples exposed to PM2.5, performed using a microrna microarray chip.
Detailed Description
The present invention will be specifically explained below.
The present invention provides biomarkers for identifying exposure to PM2.5, the expression of which is altered upon exposure to both different types of PM2.5 (water soluble or organic soluble).
The biomarker preferably consists of such micro-RNAs: the microrna is up-regulated by at least 1.3 fold or down-regulated by at least 1.3 fold when exposed to PM 2.5.
Microrna accession number (miRbase) MIMAT0017342(Mm-miR-1943-3p) (seq. id. No.: 1).
The sequence of the microRNA consists of CAGGUGCCAGCUCCUCCCUUC.
PM2.5 herein is preferably a PM2.5 water soluble extract or a PM2.5 organic soluble extract.
The particulate matter herein is preferably under 2.5 μm in diameter (including ultrafine particulate matter (UFP)), and more preferably 0.1 to 2.5 μm in diameter, but is not always limited thereto.
The particulate extract is preferably prepared by a process comprising the following steps, but is not always limited thereto:
1) collecting particles with the size below 2.5 mu m;
2) extracting the particles collected in step 1) with an extraction solvent; and
3) filtering the extract obtained in step 2).
In the above process, the extraction solvent of step 2) is preferably water, alcohol, dichloromethane or a mixture of the above solvents. The water herein is preferably purified water (purified water) or distilled water, more preferably water which has been subjected to tertiary distillation. The alcohol herein is preferably C1-C2The lower alcohol, herein preferably ethanol or methanol. The extraction method preferably uses an ultrasonic generator and a high volume air sampler (high volume air sampler), but is not always limited thereto. The operation time of the ultrasonic generator is preferably 20 to 60 minutes, more preferably 30 minutes, but is not always limited thereto.
In a preferred embodiment of the invention, a BALB/C mouse model is treated with a PM2.5 extract. Subsequently, total RNA containing microrna was extracted from mouse lung tissue and labeled with a fluorescent substance (Cy 3). After hybridization of fluorescently labeled micrornas to the microarray chip, the fluorescence image was scanned to study gene expression patterns. When the ratio of the signal intensity of the experimental group to that of the control is at least 1.3-fold different, the gene expression is considered to be increased.
Selected micrornas that showed overexpression due to PM2.5 water soluble extract or PM2.5 organic soluble extract are shown below:
microrna accession number (miRbase) MIMAT0017342(Mm-miR-1943-3p) (seq. id. No.: 1).
Thus, the biomarker of the present invention was confirmed to be a microrna specifically overexpressed due to PM 2.5. Thus, the biomarkers of the invention can be effective tools for identifying exposure to PM2.5, monitoring PM2.5, assessing risk of PM2.5, and studying toxicity mechanisms of PM 2.5.
The present invention also provides a micro RNA microarray chip for specific identification of exposure to PM2.5, on which oligonucleotides comprising part or all of the sequence of the cDNA of the biomarker micro RNA are synthesized/integrated.
The oligonucleotide comprises 15-20 nucleotides of the cDNA of the biomarker microrna.
The micro RNA microarray chip for identifying PM2.5 of the present invention can be prepared using conventional methods well known to those skilled in the art. The method for preparing the microarray chip is preferably an inkjet method in which cDNA of a selected biomarker is immobilized on a chip as a probe for a microrna molecule, and more preferably a preprint inkjet microdroplet microarray (preprint inkjet microarray), but is not always limited thereto.
The support plate (board) of the DNA microarray chip is preferably coated with one of the reactive groups selected from the group consisting of: epoxides (epoxy), aminosilanes, poly-L-lysine, and aldehydes, but are not always limited thereto. The chip support plate is preferably selected from the group consisting of: glass slides, plastic, metal, silicon, nylon membranes, and nitrocellulose membranes, more preferably, but not always limited to, glass slides coated with epoxy.
The invention also provides the use of a micro-RNA microarray chip on which oligonucleotides comprising part or all of the sequence of the cDNA of the biomarker micro-RNA are synthesized/integrated for specific identification of exposure to PM 2.5.
The oligonucleotide comprises 15-20 nucleotides of the cDNA of the biomarker microrna.
The micro RNA microarray chip for identifying PM2.5 of the present invention can be prepared using conventional methods well known to those skilled in the art. The method for preparing the microarray chip is preferably an inkjet method in which cDNA of the selected biomarker is immobilized on the chip as a probe for a microrna molecule, and more preferably a preprint inkjet microdroplet microarray, but is not always limited thereto.
The support plate of the DNA microarray chip is preferably coated with one of the reactive groups selected from the group consisting of: epoxides, aminosilanes, poly-L-lysine, and aldehydes, but are not always limited thereto. The chip support plate is preferably selected from the group consisting of: glass slides, plastic, metal, silicon, nylon membranes, and nitrocellulose membranes, more preferably, but not always limited to, glass slides coated with epoxy.
The micro-RNA microarray chip of the invention can detect miRNA specifically up-regulated or down-regulated due to PM2.5, thereby being used as a useful tool for identifying exposure to PM2.5, monitoring PM2.5, evaluating PM2.5 risk and researching PM2.5 toxicity mechanism.
The invention also provides a method for identifying exposure to PM2.5 by using the microarray chip.
The present invention also provides a method for identifying exposure to PM2.5, the method comprising the steps of:
1) isolating RNA from somatic cells obtained from experimental subjects and somatic cells obtained from control normal subjects;
2) labeling each of the RNAs obtained from the experimental group and the control of step 1) with a different fluorescent substance;
3) hybridizing the RNA marked with the fluorescent substance in the step 2) with a micro RNA microarray chip;
4) analyzing the micro RNA microarray chip reacted in the step 3); and
5) using the data analyzed in step 4), the expression of Mm-miR-1943-3p in the experimental group and the control was compared.
In the above method for identifying exposure to PM2.5, the somatic cell sample of step 1) is preferably obtained from each group of mice exposed or not exposed to PM 2.5.
In the above method for identifying exposure to PM2.5, the fluorescent substance of step 2) is preferably selected from the group consisting of: cy3, Cy5, FITC (poly L-lysine-fluorescein isothiocyanate), RITC (rhodamine-B-isothiocyanate) and rhodamine, but are not always limited thereto, and any fluorescent substance known to those skilled in the art may be used.
In the above-described method for identifying exposure to PM2.5, the micro RNA microarray chip of step 5) is preferably a mouse miRNA 8 × 60K (Rel 19.0) (Agilent, USA), but is not always limited thereto. Any microarray chip capable of detecting micrornas whose expression patterns are altered by the action of two different PM2.5 (see table 1) may be used, however, the microrna microarray chips constructed by the present inventors are most preferred.
In step 4) of the above method, GeneSpring GX 12.6.1 software (Agilent, USA) is preferably used, but is not always limited thereto, and any analysis software known to those skilled in the art may be used.
The method of identifying miRNA expression of the present invention uses a micro-RNA microarray chip that facilitates identification of down-regulation or up-regulation of micro-RNA specifically induced by PM2.5, and thus, the method of the present invention can be used as a useful tool for identifying exposure to PM2.5, monitoring PM2.5, assessing PM2.5 risk, and studying PM2.5 toxicity mechanisms.
The present invention provides a kit for identifying exposure to PM2.5, comprising the microarray chip.
The kit of the invention for identifying exposure to PM2.5 may further comprise mouse lung cells or tissues.
The kit of the invention for identifying exposure to PM2.5 further comprises a primer complementary to a cDNA of a microrna or to a complementary strand molecule of said cDNA and capable of amplifying said cDNA:
microRNA accession number (miRbase) MIMAT0017342(Mm-miR-1943-3 p).
Mouse model lung tissue samples are preferably obtained from a group of mice that are or are not exposed to PM 2.5.
The kit may further comprise a fluorescent substance. The fluorescent substance herein is preferably selected from the group consisting of: streptavidin-like phosphatase conjugate, chemiluminescent substance, and chemiluminescent substance, but are not always limited thereto. In a preferred embodiment of the invention, Cy3 is used.
The kit may further comprise a reaction reagent, and in this case, the reaction reagent is preferably composed of a hybridization buffer, a labeling reagent (e.g., a chemical inducer of a fluorescein dye), and a washing buffer, but is not always limited thereto, and may include any reaction reagent known to those skilled in the art necessary for hybridization of the micro RNA microarray chip.
Thus, the kit of the present invention was demonstrated to comprise micrornas specifically overexpressed due to PM2.5, enabling as an effective tool to identify exposure to PM2.5, monitor PM2.5, assess PM2.5 risk, and study the toxicity mechanism of PM 2.5.
The invention also provides the use of the kit for identifying exposure to PM2.5, wherein the kit comprises the microarray chip.
The kit of the invention for identifying exposure to PM2.5 may further comprise mouse lung cells or tissues.
The kit of the invention for identifying exposure to PM2.5 further comprises a primer complementary to a cDNA of a microrna or to a complementary strand molecule of said cDNA and capable of amplifying said cDNA:
microRNA accession number (miRbase) MIMAT0017342(Mm-miR-1943-3 p).
Mouse model lung tissue samples are preferably obtained from a group of mice that are or are not exposed to PM 2.5.
The kit may further comprise a fluorescent substance. The fluorescent substance herein is preferably selected from the group consisting of: streptavidin-like phosphatase conjugate, chemiluminescent substance, and chemiluminescent substance, but are not always limited thereto. In a preferred embodiment of the invention, Cy3 is used.
The kit may further comprise a reaction reagent, and in this case, the reaction reagent is preferably composed of a hybridization buffer, a labeling reagent (e.g., a chemical inducer of a fluorescein dye), and a washing buffer, but is not always limited thereto, and may include any reaction reagent known to those skilled in the art necessary for hybridization of the micro RNA microarray chip.
Thus, the kit of the present invention was demonstrated to comprise micrornas specifically overexpressed due to PM2.5, enabling as an effective tool to identify exposure to PM2.5, monitor PM2.5, assess PM2.5 risk, and study the toxicity mechanism of PM 2.5.
In addition, the present invention provides a method for identifying exposure to PM2.5, the method comprising the steps of:
1) isolating RNA from somatic cells obtained from experimental subjects and somatic cells obtained from control normal subjects;
2) synthesizing cDNA from each RNA of the experimental group and each RNA of the control;
3) performing RT-PCR (real-time polymerase chain reaction) using the cDNAs of the experimental group and the control of step 2) using primers capable of amplifying the cDNA of Mm-miR-1943-3 p; and
4) comparing the expression of the amplification products of the experimental group and the control of step 3).
In the above method, the somatic cells of step 1) are in particular mouse lung cells or tissues.
The method for identifying exposure to PM2.5 by comparing the expression of amplified products using the microrna and cDNA of the present invention uses a microarray chip useful for studying the distribution of micrornas that are specifically up-or down-regulated due to PM2.5, and thus can be effectively used as a tool for identifying exposure to PM2.5, monitoring PM2.5, assessing risk of PM2.5, and studying toxicity mechanism of PM 2.5.
Practical and presently preferred embodiments of the present invention shown in the following examples are illustrative.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make variations and modifications within the spirit and scope of the present invention.
Examples
Example 1: PM2.5 Collection and extraction
<1-1> selection of Collection sites
To screen for biomarkers for identifying exposure to PM2.5, the roof of the Korean Institute of Science and Technology (KIST) stadium (hawollgk-dong, Sungbuk-gu, seoul) was selected as the collection site for collecting PM2.5, which was located adjacent to the Naebu expressway located near the wolfok station in the northern city (Sungbuk-gu), where the traffic flow was as high as 70,000 vehicle passes on a working day in november and 69,000 vehicle passes on a working day in december (2011). For in vitro testing, a considerable amount of PM2.5 is required. Since the low-capacity air sampler can only obtain mg of PM2.5 by operating for 24 hours, the low-capacity air sampler is not suitable for collection of PM 2.5. Thus, for the in vitro test of PM2.5, a high volume air sampler is used in order to collect a high volume of particulate matter. PM2.5 was collected by the PM2.5 specific inlet (Tisch instruments) by using a teflon coated glass fiber filter that only allowed PM2.5 to pass, such that particles above 2.5 μm were blocked by oil due to mass inertia (weight inertia).
<1-2> Collection Filter
For the high volume air sampler, a teflon coated glass fiber filter was chosen that did not allow any particles larger than 8 x 10 inches to pass through. Distilled water (18.2 M.OMEGA.) and methylene chloride were used as extraction solvents. To minimize errors due to moisture (moisture), the filter was completely dried in a desiccator for 48 hours. Subsequently, the filter was weighed using a high precision balance (mettler toledo) capable of weighing to a weight of 0.1 mg. The filter was folded in half with the insides facing each other, and placed in a light-tight and sealed bag with a zipper. The filter in the zipped bag was stored at-80 ℃. When it is desired to transfer it to a collection site, it is carried with an ice bin. To collect PM2.5, maintain a suitable flow rate (1.13 m)3/min±10％)。
<1-3> Collection time
PM2.5 was collected for 5 months using a high volume air sampler. The first collection was completed at 11/2011 and the last collection (50 th collection) was completed at 28/3/2012. The filter showed 20. mu.g/m on rainy days3The following are providedAir PM concentration. A total of 10 filters were divided into the same group and a total of 5 groups of filters were prepared.
<1-4> extraction of collected particulate matter
To analyze the composition of the collected PM2.5, a filter was divided into two halves. Ultrapure water (distilled water) for extracting water-soluble components and methylene chloride for extracting organic-soluble components were added to each half filter, respectively. Thus, a PM2.5 water-soluble extract and a PM2.5 organic-soluble extract were prepared by the extraction process (fig. 1).
Example 2: selection of PM2.5 Exposure model and acquisition of mouse Lung tissue samples
<2-1> PM2.5 Exposure model
PM2.5 is suspended particulate matter in the air, which is one of the environmentally harmful substances. Once the particulate matter enters the lungs, typical particle-induced inflammation is induced. According to previous reports, the BALB/C model has a high sensitivity for measuring such responses. BALB/C is prone to chronic pneumonia and has high sensitivity to radiation. The animals are more domesticated and mild and show lower incidence of breast cancer. Therefore, the animal was finally selected as a suitable model.
Specifically, PM2.5 collected in < example 1> was divided into two different groups, i.e., a water-soluble group and an organic-soluble group. The mouse model was also divided into two groups, one of which was exposed to PM2.5 and the other was not exposed to PM 2.5. The exposure group was exposed to either a low dose of PM2.5 or a high dose of PM 2.5. Thus, a total of 6 groups were prepared, including the unexposed group and the exposed group exposed to different doses of PM2.5 (table 1).
[ Table 1]
PM2.5 Exposure dose and mouse grouping
| Experimental group | Dosage (ug) | Quantity (BALB/C) |
| Control | 0 | 24 |
| Low dose | 20 | 24 |
| High dose | 164 | 24 |
<2-2> determination of PM2.5 dose for mouse model exposure
In a prior study investigating exposure of a BALB/C mouse model to PM2.5, it was determined that the exposure dose did not exceed 100 μ g on an absolute basis. The present inventors conducted exposure tests with three different PM concentrations (0 μ g (control), 20 μ g (low dose) and 164 μ g (high dose)) selected by referring to The study methods and results reported previously (Hans heirich, The laboratory, 2004), mouse mortality and concentration of PM2.5 distributed in The real environment, etc. (table 2).
For the exposure method, it is preferred that PM2.5 is inhaled. However, when PM2.5 is indeed inhaled, it is almost impossible to expose all different mice equally to the desired PM2.5 concentration. Thus, the two collected groups of PM2.5 were made into a liquid phase, which was directly inserted via the airways of the BALB/C mouse model (tracheal instillation). Calculation of exposure dose for each group is shown below.
Calculation of PM2.5 dose for each mouse group
Respiratory capacity per minute (ml) of mice is 33.5-47.5ml/min
Mice had a weekly respiratory volume (ml) of 337,680-478,800 ml/week
Exposure to a dose of 20 μ g:
20μg/337,680ml＝0.0000592277μg/ml
20μg/478,800ml＝0.0000417711μg/ml
daily atmospheric environmental standard for PM 2.5: 50 μ g/m3
The respiratory capacity (ml) per minute of human being is 6000ml
Human respiratory capacity per week (ml) 6000 × 60 min × 24 hr × 7 day 60,480,000ml 60m3
Exposure to 25. mu.g/m3-50μg/m3For one week:
25μg/m3×60m3/60,480,000ml＝0.0000248016μg/ml
50μg/m3×60m3/60,480,000ml＝0.0000496032μg/ml
thus, when mice were exposed to a 20 μ g dose of PM2.5, the calculated value was (41-59). times.10-6μ g/ml, by comparing the dose with the exposure dose (24-49). times.10 obtained using PM2.5 atmospheric environmental standards for humans-6The dosage is judged to be an administrable dosage by comparing μ g/ml. The reason for this low dose is to minimize cytotoxicity to avoid any possible genetic transformation (even if no histopathological changes were observed).
At a dose of 164 μ g, cytotoxic and histopathological changes are expected. In addition, genetic modifications are also expected to occur. Therefore, this is considered to be a high dose of exposure.
<2-3> Lung tissue sample
The most important objective of experiments using the BALB/C mouse model is to efficiently extract high quality total RNA containing microRNA. To achieve this, a non-invasive lung tissue sample is obtained. After extraction of lung tissue samples, they were stored in Allprotect solution (Qiagen) which allowed them to be stored at-20 ℃ for 6 months and at-80 ℃ for 1 year in the dark. The lung tissue samples were stored at-80 ℃ prior to use.
Example 3: microarray assay
<3-1> isolation of target microRNA and labeling with fluorescent substance
RNA was extracted from blood samples of PM2.5 exposed and PM2.5 unexposed groups using trizol. The extracted RNA was purified using RNeasy Mini kit (Cat NO.74106, Qiagen). During RNA purification, genomic DNA was removed using RNase-free DNase Set (Qiagen, USA). The concentration of each total RNA was measured with a NanoDrop ND 1000 spectrophotometer (NanoDrop technologies inc., USA). The quality of the RNA was measured using an Agilent 2100 bioanalyzer.
<3-2> preparation of labeled microRNA
For the oligonucleotide microarray assay, total RNA obtained from the PM2.5 exposed group and PM2.5 unexposed group of example <3-1> was labeled with a fluorescent substance. The labeling was performed using miRNA complete labeling and hybridization kit (Agilent) according to the manufacturer's protocol. The obtained total RNA (100ng) was mixed with 0.4. mu.l of 10 XCAP buffer, 1.1. mu.l of nuclease-free water and 0.5. mu.l of calf intestinal phosphatase, followed by reaction at 37 ℃ for 30 minutes. Subsequently, 2.8. mu.l of 100% DMSO was added thereto, followed by reaction at 100 ℃ for 5 to 10 minutes. The reaction mixture was immediately transferred to ice. For labeling, 1. mu.l of 10 XT 4 ligase buffer, 3. mu.l of cyanine-3-pCp and 0.5. mu. l T4RNA ligase were added to the above mixture, followed by reaction at 16 ℃ for 2 hours. The labeled samples were purified using a micro bio-spin 6 column. The purified RNA was completely dried in a vacuum concentrator at 55 ℃ and then dissolved in 18. mu.l nuclease-free water. To the resulting solution was added 4.5. mu.l of 10 XGE blocking agent, to which was added 22.5. mu.l of 2 XHi-RPM hybridization buffer. The prepared sample was reacted at 100 ℃ for 5 minutes, then immediately transferred to ice again, and then reacted for 5 minutes. This sample was then used for the next hybridization.
<3-3> hybridization
The hybridization and washing procedures were performed as described in EBIOGEN Inc. Hybridization was induced at 55 ℃ for 20 hours. Mouse miRNA 8X 60K (Rel 19.0) (Agilent, USA) was used as a DNA microarray chip.
<3-4> acquisition of fluorescence image
The hybridization image on the slide was scanned using an Agilent C scanner (Agilent technologies, USA). The extracted data were normalized using Agilent GeneSpring GX 12.6.1(Agilent technologies) and the expression pattern of each microrna was subsequently studied.
As a result, as shown in fig. 2, the expression pattern of about 1,247 micrornas was investigated on an oligonucleotide chip using GeneSpring GX 12.6.1 software, and the expression pattern of micrornas between the exposed group and the unexposed group was compared (fig. 2). As a result, only one microrna was confirmed to be up-regulated by at least 1.3 fold compared to the normal expression level. Meanwhile, in none of the four cases, the expression of the micrornas was down-regulated (table 2). There is no report mentioning that the above micrornas correlate with the toxicity observed upon exposure of the mouse model to PM 2.5.
[ Table 2]
microRNAs with altered expression due to exposure to PM2.5
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. It will also be appreciated by those skilled in the art that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.
Claims (12)
1. Use of a microarray chip having integrated thereon cDNA of a microrna of:
miRNA Mm-miR-1943-3p, the microrna accession number of Mm-miR-1943-3p in miRbase being MIMAT0017342, the sequence of said Mm-miR-1943-3p consisting of seq.id no: 1, the method is shown in the specification,
wherein the use is for non-diagnostic purposes.
2. Use of the microarray chip of claim 1 for identifying exposure to PM2.5, wherein the PM2.5 is a water-soluble extract of PM2.5 or an organic-soluble extract of PM 2.5.
3. Use of a kit for identifying exposure to PM2.5, said kit comprising a microarray chip having integrated thereon cDNA of a microrna as follows:
miRNA Mm-miR-1943-3p, the microrna accession number of Mm-miR-1943-3p in miRbase being MIMAT0017342, the sequence of said Mm-miR-1943-3p consisting of seq.id no: 1, the method is shown in the specification,
wherein the use is for non-diagnostic purposes.
4. Use of a kit according to claim 3 for identifying exposure to PM2.5, wherein the kit further comprises mouse lung cells or tissue.
5. A method for identifying exposure to PM2.5, the method comprising the steps of:
1) isolating RNA from somatic cells obtained from experimental subjects and somatic cells obtained from control normal subjects;
2) labeling each of the RNAs obtained from the experimental group and the control of step 1) with a different fluorescent substance;
3) hybridizing the RNA marked with the fluorescent substance in the step 2) with a micro-RNA microarray chip, wherein the micro-RNA microarray chip is integrated with cDNA of the following micro-RNA:
miRNA Mm-miR-1943-3p, the microrna accession number of Mm-miR-1943-3p in miRbase being MIMAT0017342, the sequence of said Mm-miR-1943-3p consisting of seq.id no: 1 is shown;
4) analyzing the micro RNA microarray chip reacted in the step 3); and
5) comparing the expression of Mm-miR-1943-3p in the experimental group and the control group by using the data analyzed in the step 4),
wherein the method is used for non-diagnostic purposes.
6. The method for identifying exposure to PM2.5 as recited in claim 5, wherein said somatic cells of step 1) are mouse lung cells or lung tissue.
7. The method for identifying exposure to PM2.5 of claim 6, wherein said lung tissue is BALB/C mouse lung tissue.
8. The method for identifying exposure to PM2.5 as recited in claim 5, wherein said fluorescent substance of step 2) is selected from the group consisting of:
cy3, Cy5, poly L-lysine-fluorescein isothiocyanate, rhodamine-B-isothiocyanate and rhodamine.
9. Use of cDNA of the following micrornas in the preparation of microarray chips for identifying exposure to PM 2.5:
miRNA Mm-miR-1943-3p, the microrna accession number of Mm-miR-1943-3p in miRbase being MIMAT0017342, the sequence of said Mm-miR-1943-3p consisting of seq.id no: 1, the method is shown in the specification,
wherein the cDNA of the miRNA Mm-miR-1943-3p is integrated on the microarray chip.
10. Use according to claim 9, wherein the PM2.5 is a water-soluble extract of PM2.5 or an organic-soluble extract of PM 2.5.
11. Use of cDNA of the following micrornas in the preparation of a kit for identifying exposure to PM 2.5:
miRNA Mm-miR-1943-3p, the microrna accession number of Mm-miR-1943-3p in miRbase being MIMAT0017342, the sequence of said Mm-miR-1943-3p consisting of seq.id no: 1, the method is shown in the specification,
the kit comprises a microarray chip, wherein the cDNA of the miRNA Mm-miR-1943-3p is integrated on the microarray chip.
12. The use of claim 11, wherein the kit further comprises mouse lung cells or tissues.
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