KR20140067602A - The biomarkers for identification of exposure to deiodinase inhibiting disruptors and the identification method of deiodinase inhibiting disruptors exposure using the same biomarkers - Google Patents

Abstract

The present invention relates to a biomarker for identifying specific exposure to deiodinase inhibiting disruptors which are main targets causing thyroid hormone disruption, and to an identification method using the same and, specifically, to a biomarker in which gene expression is increased or decreased specifically by the deiodinase inhibiting disruptors, and to a method for identifying specific exposure to the deiodinase inhibiting disruptors using the same. The biomarker of the present invention can be suitably used as a biomarker which can screen the deiodinase inhibiting disruptors using response genes selected through DNA microarray chips and as a method for identifying exposure to the deiodinase inhibiting disruptors.

Description

Technical Field [0001] The present invention relates to a biomarker for detecting exposure to an inhibitor of dehydroiodination enzyme activity and a method for identifying the biomarker using the biomarker,

The present invention relates to a biomarker for confirming exposure to a chemical substance that inhibits deiodination enzyme activity and a method for identifying the biomarker. More particularly, the present invention relates to a biomarker for inhibiting deiodination enzyme activity, And to a method for identifying the specific exposure of a de-iodinating enzyme activity inhibiting chemical using the biomarker.

Thyroid peroxidase, sodium iodide symporter, TH transport protein transthyretin, TH receptor, etc., cause the disorder of thyroid hormone (Schmutzler C. et al., Endocrinology, 148 (6): 2835-2844, 2007). In addition, deiodinase (Deiodinase) is used as a target to study the action of thyroid hormone in the target tissue by deiodination enzyme.

Thyroid hormones (T3 and T4) are derivatives of iodine bound to tyrosine, T3 and T4 are active, and free T3 is inactive. About 20% of iodine in the blood is actively ingested and removed by the thyroid gland. Iodine is released into the blood by organiation, coupling (T3, T4), proteolytic degradation, and deiodination. The thyroid hormone thus produced is metabolized in each tissue through deiodination, deamination, decarboxylation, and conformation. Most of the thyroid hormones secreted from the thyroid are T4 type, which is converted to T3 by deiodination in tissues such as liver and kidney, acting on thyroid hormone receptors and acting on various tissues such as nerve cells. Therefore, the deiodination of the thyroid hormone by the deiodination enzyme is important, and evaluation of the action of the deiodination enzyme on the target tissue by the action of the thyroid hormone by the chemical exposure is an important target in the evaluation of the human body effect.

85% of the T3 in blood is a form in which T4 is converted to T3 by 5'-de-iodination. 30% of T4 is 5'-de-iodinated to T3 by type 1 deiodinase (DIO1) or type 2 deiodinase (DIO2), and 40% of T4 is dehydroiodinated Type 3 deiodinase (DIO3). These de-iodination enzymes have recently been reported to be involved in the tissue specificity of thyroid hormones. Especially, the specific expression and activity level in brain tissue plays an important role during the development of thyroid hormone. These studies have been conducted using Xenopus, and studies have been conducted on how brain function in neonatal brain development through the level of thyroid hormone expression is examined.

The activity of the de-iodination enzyme is 2,3,7,8-tetrachlorodibenzo-p-dioxin, propylthiouracil, beta-estradiol, , 3-methylcholanthrene, amiodarone, arochlor 1254, cadmium chloride, dimethoate, fenvalerate, iopanoic acid, iopanoic acid, methoxychlor, and 4-methylbenzylidene-camphor (see Table 1). In other words, since exposure to various chemical substances has an effect on the inhibition of deiodination enzyme activity, it affects each tissue of the human body, and a method for evaluating exposure to these chemicals is required.

To the de-iodination enzyme  Affecting Chemicals chemical substance De-iodidase activity references 2,3,7,8-tetrachlorodibenzo dioxin D1 ↓, D2 ↓ Boas et al., 2006; Ahmed, 2011 Propylthiouracil D1 ↓ Mol et al., 1997; Howdeshell, 2002; Miller et al., 2009; Liu et al., 2011 Beta estradiol D1 ↓ Schmutzler et al., 2004; 2007b 3-Methylcholanthrene D1 ↓ Howdeshell, 2002 Amiodarone D1 ↓, D2 ↓ Howdeshell, 2002; Tedelind et al., 2006; Rosene et al., 2010 Aroclor 1254 D1 ↓ Howdeshell, 2002; Coimbra et al., 2005 Cadmium chloride D1 ↓ Ghosh et al., 1992; Howdeshell, 2002 Dimethoate D1 ↓ Maiti & Kar, 1997; Howdeshell, 2002 Fenvalerate D1 ↓ Maiti et al., 1995; Howdeshell 2002 Iopanoic acid D1 ↓, D2 ↓, D3 ↓ Cardoso et al., 2002; Howdeshell, 2002; Colantuoni et al., 2005 Methoxychlor D1 ↓ Howdeshell, 2002; Boas et al., 2006 4-methylbenzylidene camphor D1 ↓ Schmutzler et al., 2004; 2007b

D1: de-iodination enzyme type 1;

D2: dehydroiodidase type 2; And

D3: Dehydroiodination enzyme type 3.

Thyroid hormone activity and thyroid hormone receptor binding and gene expression changes in thyroid hormone disorders have been reported, but they are mostly limited to mouse studies. Although there is a possibility that human thyroid and other tissues may be impaired by the inhibition of the activity of the deiodination enzyme as mentioned above, there is not enough human risk assessment data, and the search method of exposure is GC-MS (Gas Chromatography- Mass Spectrometer) or HPLC (High Performance Liquid Chromatography). When using the above-described GC-MS or HPLC method, quantitative analysis is possible, but appropriate conditions for analysis are required and expensive equipment is required. Therefore, a quick and easy screening method, for example, a real-time reverse transcription polymerase chain reaction (RT-PCR) using a primer or a DNA microarray, It is an important task to identify and analyze molecular markers that can detect toxic effects and gene expression in the human body and to take appropriate measures and manage exposure to chemical substances that inhibit deiodination enzyme activity.

The National Center for Biotechnology Information (NCBI) has completed a series of genome sequencing projects for 6 species of mammals and 292 species of microorganisms. Based on the huge amount of data thus obtained, - Genome-wide expression studies are being conducted. That is, DNA microarray analysis is generally performed to analyze the expression of thousands of genes in a single experiment (Schena, M., et al., Proc. Natl. Acad. 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. 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. et al., J. Am. Acad. Dermatol., 51: 681-692, 2004). The oligonucleotide microarray is prepared by direct synthesis on a chip using photolithography in Affymetrix. Agillent and the like produce the oligonucleotide by immobilizing the synthesized oligonucleotide. (Sellheyer, K. et al., J. Am. Acad. Dermatol. 51: 681-692, 2004).

For the analysis of gene expression, RNA is obtained from a sample such as a tissue or a cell, and a hybridization reaction is performed with an oligonucleotide in the DNA microarray. The obtained RNA is labeled with fluorescence or isotope and converted into cDNA. Oligo microarray is mainly labeled with two different fluorescence (for example, Cy3 or Cy5) in the control group and the experimental group, and simultaneously hybridization reaction is performed on the same chip After performing the optical scanning, the image is scanned to obtain the intensity of the fluorescence and the result is analyzed. As a result, the expression of the gene is determined by the ratio of the two fluorescence intensities (Somasundaram, K. et al., Genomics Proteomics I: 1-10, 2002).

Recently, advanced technology using DNA microarray technology has been combined with toxicogenomics research, and it has been widely used in high throughput for drug candidates, prospective drug candidates, as well as representative environmental pollutants and specific tissues and cell lines The expression pattern of the expressed genes can be analyzed and quantitative analysis becomes possible. Thus, by analyzing the frequency of expression of a specific gene in a specific cell, it is possible to identify a gene related to harmful action of environmental pollutants, drug action and side effect, And to search for substances that cause side effects.

Thus, the present inventors have used a oligo microarray in which 62,000 human genes have been integrated to determine a gene expression profile by a de-iodination enzyme activity inhibiting chemical, such as a neuroblastoma cell line derived from human knockdown a biomarker capable of specifically detecting only a substance inhibiting de-iodination enzyme activity by observing and analyzing in a neuroblastoma SH-SY5Y cell line and locating specifically overexpressed or underexpressed genes only by a de-iodidation enzyme inhibiting chemical And a method for confirming the exposure using the same were established and the present invention was completed.

An object of the present invention is to provide a biomarker which is specifically overexpressed or under-expressed by exposure to a deiodinase activity inhibiting chemical and a method of confirming the specific exposure of a chemical inhibitor of deiodination enzyme activity using the biomarker .

In order to achieve the above object, the present invention provides a biomarker for specifically detecting exposure to a dehydroiodidizing enzyme activity inhibiting chemical substance that specifically causes an expression change by exposure to a deiodinase activity inhibiting chemical substance.

Also, the present invention provides a DNA microarray chip for confirming specific exposure of a nucleic acid sequence of any one or more genes selected from the group consisting of the following genes or complementary strand molecules integrated therein:

Gene registration number (Genebank) NM_016569 (T-box 3) and NM_002213 (integrin, beta 5).

Further, according to the present invention,

1) separating each RNA from a somatic cell isolated from a test subject suspected of being exposed to a chemical substance inhibiting the activity of the deiodo-oxidizing enzyme and a somatic cell isolated from a normal individual as a control;

2) labeling the experimental group and the control group with different fluorescent substances while synthesizing the RNA of the experimental group and the control group of step 1) with cDNA;

3) hybridizing the cDNA labeled with different fluorescent substances of step 2) with the DNA microarray chip of claim 1;

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

5) determining the degree of expression of the gene integrated in the DNA microarray chip of claim 1 by comparing the data analyzed in step 4) with that of the control group to provide a method of confirming exposure to the dehydroiodinating enzyme activity inhibiting chemical do.

Further, according to the present invention,

1) separating each RNA from a somatic cell isolated from a test subject suspected of being exposed to a chemical substance inhibiting the activity of the deiodo-oxidizing enzyme and a somatic cell isolated from a normal individual as a control;

2) Performing Real-time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using primer pairs complementary to the following genes in the experimental group and the control group of step 1) and capable of amplifying the following genes:

Gene registration numbers NM_016569 (T-box 3) and NM_002213 (integrin, beta 5); And

3) comparing the gene product of step 2) with that of the control, and confirming the expression level of the gene product of step 2).

In addition, the present invention provides a kit for confirming exposure to an activity inhibiting chemical of a de-iodination enzyme comprising a microarray chip of the present invention.

In addition, the present invention provides a kit for confirming exposure to a dehydroiodinating enzyme activity inhibiting chemical comprising a pair of primers complementary to each of the following genes and capable of amplifying each of the following genes:

Gene registration numbers NM_016569 (T-box 3) and NM_002213 (integrin, beta 5).

The biomarker for confirming exposure to a chemical substance that inhibits the activity of the deiodinase of the present invention and the identification method using the same are characterized in that the reaction genes selected through a DNA microarray are used as biomarkers to activate the deiodination enzyme As a tool to identify toxic mechanisms caused by chemicals that inhibit the activity of de-iodination enzymes.

1 is a diagram showing the degree of expression of three kinds of deiodination enzyme genes in a deiodinase knockdown human neuron-derived cell line:
D1: deiodination enzyme type 1 (type 1 deiodinase, DIO1),
D2: deiodinase type 2 (type 2 deiodinase, DIO2), and
D3: deiodination enzyme type 3 (type 3 deiodinase, DIO3).
FIG. 2 is a graph showing changes in thyroid hormone (T3) in human neuronal cell line derived from de-iodinated enzyme knockdown:
NC: negative control, and
DKD: DKD cell line knockdown with de-iodination enzyme.
FIG. 3 is a diagram showing the expression pattern of the TBX3 gene when various chemicals inhibiting the activity of deiodination enzyme using real-time quantitative PCR are exposed to human neuron-derived cell lines:
propylthiouracil: propylthiouracil,
2,3,7,8-tetrachlorodibenzo-p-dioxin: 2,3,7,8-tetrachlorodibenzodioxin,
beta-estradiol: beta-estradiol,
3-methylcholanthrene: 3-methylcholanthrene,
amiodarone: amiodarone,
arochlor 1254: Aroclark 1254,
cadmium chloride: cadmium chloride,
dimethoate: dimethoate,
fenvalerate: fenvalerate,
iopanoic acid:
methoxychlor: methoxychlor, and
4-methylbenzylidene-camphor: 4-methylbenzylidene camphor.
FIG. 4 is a diagram showing the expression pattern of the ITGB5 gene when various chemical substances inhibiting de-iodination enzyme activity are exposed to a human neuron-derived cell line using real-time quantitative PCR:
propylthiouracil: propylthiouracil,
2,3,7,8-tetrachlorodibenzo-p-dioxin: 2,3,7,8-tetrachlorodibenzodioxin,
beta-estradiol: beta-estradiol,
3-methylcholanthrene: 3-methylcholanthrene,
amiodarone: amiodarone,
arochlor 1254: Aroclark 1254,
cadmium chloride: cadmium chloride,
dimethoate: dimethoate,
fenvalerate: fenvalerate,
iopanoic acid:
methoxychlor: methoxychlor, and
4-methylbenzylidene-camphor: 4-methylbenzylidene camphor.

Hereinafter, the present invention will be described in detail.

The present invention provides a biomarker for confirming the exposure of a dehydroiodidizing enzyme activity inhibiting chemical substance that specifically induces an expression change by exposure to a dehydroiodinating enzyme activity inhibiting chemical.

The biomarker is a gene whose expression is increased or decreased by 1.5 times or more. The biomarker is composed of two kinds of genes specifically expressed by a chemical substance inhibiting enzyme activity inhibiting activity.

Preferably, the biomarker exhibiting a specific expression change according to the degree of exposure of the de-iodination enzyme activity inhibiting chemical is selected from the group consisting of:

Gene registration number (Genebank) NM_016569 (T-box 3) and NM_002213 (integrin, beta 5).

In a specific embodiment of the present invention, the inventors of the present invention conducted a study to identify a biomarker for confirming the specific inhibition of chemical activity of de-iodination enzyme activity, using SH-SY5Y cell line derived from human neuron using siRNA of de-iodination enzyme An enzyme knockdown human neuron-derived cell line was constructed to confirm the low expression of each of three kinds of deiodination enzyme genes, and it was confirmed that the production of T3 thyroid hormone was decreased (see FIGS. 1 and 2). In order to discover a chemical-specific biomarker inhibiting de-iodination enzyme activity, a gene expression profile was secured from a cell line derived from de-iodination enzyme knockdown human neuron based on the above-mentioned experiment. MRNA was isolated from the cell line derived from de-iodinated enzyme knockdown human neurons and gene expression patterns were analyzed using an 8 × 60 k human genome whole genome microarray (Agilent, USA). When the Cy5 / Cy3 ratio was 1.5 times or more, it was classified as an overexpressed gene. When the Cy5 / Cy3 ratio was 0.67 or less, it was classified as a reduced expression gene. As a result of the analysis, it was confirmed that 1.858% (1,152 of 62,000 genes) and 1.845% (1,144 of 62,000 genes) of the genes whose Cy5 / Cy3 ratio was increased more than 1.5 times were observed. In the above, gene expression number NM_016569 (T-box 3) was overexpressed 1.5 times or more and gene expression number NM_002213 (integrin, beta 5) was 1.5 times or more low expression in the cell line derived from de-iodinated enzyme knockdown human neuron ).

When the previously reported chemical substances are exposed to normal human neuron-derived cell lines at a concentration that does not exhibit cytotoxicity (see Table 5) in relation to the inhibition of deiodination enzyme activity, the two types of genes The genes were observed to be over-expressed and underexpressed, respectively (see FIGS. 3 and 4). This gene has not been reported to be related to toxicity in human neuron-derived cell lines.

Therefore, the use of the gene registration number NM_016569 (T-box 3) and NM_002213 (integrin, beta 5) of the present invention as a biomarker is used to monitor the chemical substances that inhibit the activity of the deiodination enzyme and determine the risk and the activity of the deiodination enzyme Can be usefully used as a tool to identify toxic mechanisms caused by chemicals that inhibit it.

The present invention also provides a DNA microarray chip for confirming specific exposure of a nucleic acid sequence of any one or more genes selected from the group consisting of the following genes or complementary strand molecules integrated therein:

Gene registration number (Genebank) NM_016569 (T-box 3) and NM_002213 (integrin, beta 5).

The DNA microarray chip for the propionaldehyde 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. A micropipetting method using a piezo electric method or a pin-type spotter for immobilizing the searched biomarker as a probe DNA molecule on a substrate of a DNA chip, But the present invention is not limited thereto. In a preferred embodiment of the present invention, a micro array, which is a pin-shaped spotter, is used. The substrate of the DNA microarray chip is preferably coated with one activator selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde, But is not limited thereto. The substrate may be selected from the group consisting of a slide glass, a plastic, a metal, a silicon, a nylon film, and a nitrocellulose membrane, but the present invention is not limited thereto. In a preferred embodiment of the present invention, Coated slide glass was used.

In the DNA microarray chip for confirming the specific exposure of the dehydroiodinating enzyme activity inhibiting chemical of the present invention, the activity inhibiting chemical of the deiodinating enzyme is 2,3,7,8-tetrachlorodibenzodioxin (2,3, Propylthiouracil, beta-estradiol, 3-methylcholanthrene, amiodarone, arochlor 1254, and the like. Cadmium chloride, dimethoate, fenvalerate, iopanoic acid, methoxychlor, and 4-methylbenzylidene-camphor. ), But the present invention is not limited thereto.

In addition, the present invention provides a method for confirming the specific exposure of a dehydroiodinating enzyme activity inhibiting chemical using the biomarker of the present invention.

The present invention provides a method for identifying a specific iodination enzyme activity inhibiting chemical substance including the following steps:

1) separating each RNA from a somatic cell isolated from a test subject suspected of being exposed to a chemical substance inhibiting the activity of the deiodo-oxidizing enzyme and a somatic cell isolated from a normal individual as a control;

2) labeling the experimental group and the control group with different fluorescent substances while synthesizing the RNA of the experimental group and the control group of step 1) with cDNA;

3) hybridizing the cDNA labeled with different fluorescent substances of step 2) with the DNA microarray chip of the present invention;

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

5) comparing the degree of expression of the gene integrated in the DNA microarray chip of claim 1 with the control group in the analyzed data of step 4).

In the above exposure confirmation method, the somatic cell of step 1) is preferably a SH-SY5Y cell line, which is a human neuron-derived cell, but is not limited thereto.

In the above-described method of confirming the exposure, the fluorescent material of step 3) is selected from the group consisting of Cy3, Cy5, poly-L-lysine-fluorescein isothiocyanate (RITC), rhodamine-B-isothiocyanate (RITC), and rhodamine But it is not limited thereto, and fluorescent materials known to those skilled in the art are all usable.

In the above exposure confirmation method, the hybridization in step 3) is preferably performed at 40 ° C to 50 ° C for 10 to 20 hours, but is not limited thereto.

In the above exposure confirmation method, the DNA microarray chip of step 4) can be used if it is a microarray chip having the gene registration number NM_016569 (T-box 3) or NM_002213 (integrin, beta 5) of the present invention among the human genome (Whole Human Genome Oligo Microarray, Agilent, USA), but the present invention is not limited thereto, and it is most preferable to use the DNA microarray chip manufactured by the present inventor.

In the above exposure confirmation method, it is preferable that the hybridization microarray chip is washed and stained after the hybridization, and then the image processing is performed and then the statistical analysis is performed. In addition, dyeing and washing were performed using a Fluidics Station 450, a scan using a GeneChip scanner 3000  3000), and image processing using an Affymetrix GeneChip Operating System (GCOS), and all procedures are performed according to the protocol of the Affymetrix company. The statistical analysis is preferably performed using GenPlex software version 2.3, but is not limited thereto, and analysis software known to those skilled in the art may be used.

In the above exposure confirmation method, the activity inhibiting chemicals of the deiodinating enzyme are 2,3,7,8-tetrachlorodibenzodioxin, propylthiouracil, betaestradiol, 3-methylcolanthrene, amiodarone, Aroclor 1254 , Cadmium chloride, dimethoate, fenvalerate, iopanoic acid, methoxychlor and 4-methylbenzylidene camphor. However, the present invention is not limited thereto.

Further, according to the present invention,

1) separating each RNA from a somatic cell isolated from a test subject suspected of being exposed to a chemical substance inhibiting the activity of the deiodo-oxidizing enzyme and a somatic cell isolated from a normal individual as a control;

2) Performing Real-time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using primer pairs complementary to the following genes in the experimental group and the control group of step 1) and capable of amplifying the following genes:

Gene registration numbers NM_016569 (T-box 3) and NM_002213 (integrin, beta 5); And

3) comparing the gene product of step 2) with that of the control, and confirming the expression level of the gene product of step 2).

In the above exposure confirmation method, it is preferable that the primer pair of step 2) is a pair of forward and reverse primers having a length of 18 to 30 m, capable of amplifying the gene of step 2), and is composed of primer pairs 1 to 2 More preferably, it is selected from the group consisting of:

Primer pair 1: a forward primer represented by SEQ ID NO: 9 and a reverse primer represented by SEQ ID NO: 10, and

Primer pair 2: the forward primer described in SEQ ID NO: 11 and the reverse primer set forth in SEQ ID NO: 12.

In the above exposure confirmation method, the activity inhibiting chemicals of the deiodinating enzyme are 2,3,7,8-tetrachlorodibenzodioxin, propylthiouracil, betaestradiol, 3-methylcolanthrene, amiodarone, Aroclor 1254 , Cadmium chloride, dimethoate, fenvalerate, iopanoic acid, methoxychlor and 4-methylbenzylidene camphor. However, the present invention is not limited thereto.

In addition, the present invention provides a kit for confirming exposure to an activity inhibiting chemical of a de-iodination enzyme comprising a microarray chip of the present invention.

In the above-mentioned kit, it is preferable, but not limited to, include human somatic cells. Although the human somatic cell is preferably SH-SY5Y, it is not limited thereto, and any cell derived from human nerve-derived cells and tissues can be used.

The kit may further comprise a fluorescent material, wherein the fluorescent material is a streptavidin-like phosphatease conjugate, a chemifluorescent agent, and a chemiluminescent agent. However, the present invention is not limited thereto. In a preferred embodiment of the present invention, Cy3 and Cy5 are used.

The reaction reagent may further comprise a buffer solution used for hybridization, a reverse transcriptase for synthesizing cDNA from RNA, cNTPs and rNTP (pre-mixed or separate feed type), a chemical of a fluorescent dye An indicator reagent such as an inducer, and a washing buffer solution. However, the present invention is not limited thereto, and any reaction reagent necessary for the hybridization reaction of a DNA microarray chip known to a person skilled in the art may be included.

There is provided a kit for confirming exposure to a chemical substance which inhibits the activity of a deiodinating enzyme comprising a pair of primers complementary to each of the following genes and capable of amplifying each of the following genes:

Gene registration numbers NM_016569 (T-box 3) and NM_002213 (integrin, beta 5).

The primer pair of the confirmation kit may be selected from the group consisting of the following primer pairs 1 to 2, but it is not limited thereto, and the amplification product of the biomarker gene may be 15 to 50 mPa A pair of forward and reverse primers of length, preferably 15 to 30, and more preferably 18 to 25, are all usable:

Primer pair 1: a forward primer represented by SEQ ID NO: 9 and a reverse primer represented by SEQ ID NO: 10, and

Primer pair 2: the forward primer described in SEQ ID NO: 11 and the reverse primer set forth in SEQ ID NO: 12.

In the kit, the activity inhibiting chemicals of the deiodinating enzyme are 2,3,7,8-tetrachlorodibenzodioxin, propylthiouracil, beta estradiol, 3-methylcolanthrene, amiodarone, Aroclor 1254, cadmium chloride , Dimethoate, fenvalerate, iopanoic acid, methoxychlor, and 4-methylbenzylidene camphor. However, the present invention is not limited thereto.

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> De-iodination enzyme ( Deiodinase ) Knockdown ( knockdown ) Human neuron-derived cell shareholder building

<1-1> Cell culture

The SH-SY5Y cell line, a human neuron-derived cell line, was cultured in MEM medium (50% F-12 Nutrient Mixture, Gibco) supplemented with 2% penicillin / streptomycin and 10% FBS (Gibco, USA) At 37 ° C and 5% CO ₂ .

<1-2> Construction of three de-iodinated enzyme knockdown SH-SY5Y cell lines using siRNA

The culture medium was MEM medium containing 10% CD-FBS. NC siRNA (Bioneer) was used as a negative control siRNA to compare the knockdown efficiency of the target gene in the siRNA experiment under the same conditions as the target siRNA experiment.

Specifically, SH-SY5Y cells were cultured in a 6-well plate at 50 × 10 ⁴ cells / ml at 37 ° C and 5% CO ₂ for 48 hours and then cultured in DharmaFECT 1 (Dharmacon, Thermo Fisher) (Type 2 deiodinase) siRNA (siRNA No. 1041596; Bioneer, Korea) in a concentration of 0.4 μM and treated with 50 nM of DIO1 (siRNA No. 1041596; Bioneer, 100 nM &lt; / RTI &gt; After 24 hours, total RNA isolation using trizol (Invitrogen life technologies, USA) was performed. Total RNA was isolated from the treated cells using a trizol reagent according to the manufacturer's procedure and purified using an RNeasy mini kit (Qiagen, USA). Genomic DNA was removed during RNA purification using an RNase-free DNase set (Qiagen, USA). The amount of each total RNA sample was measured with a spectrophotometer, and the concentration was confirmed by Experion (Automated Electrophoresis station, Bio-Rad, USA).

Quantitative real-time RT-PCR was performed using My IQ real-time PCR (Bio-Rad, USA) to investigate and quantify the degree of expression of the three genes of the de-iodination enzymes.

Specifically, cDNA was synthesized by performing reverse transcription using oligo dT primer and superscript kit (Omniscipt kit, Qiagen, Co., USA). PCR products were stained with SYBR Green I staining (Bio-rad, USA). CyberGreen I staining binds to double-stranded DNA. As the amount of double-stranded DNA produced during PCR increases, the fluorescence intensity increases. First, primers for the target gene used for PCR and the primer for endogenous control (RPLPO) were added to a cyber green master mix and subjected to PCR, and then subjected to a primer optimization process Respectively. The synthesized cDNA sample and each primer (Table 2) were mixed and PCR was performed after adding Cyber Green Mastermix and analyzed using quantitative software.

As a result, it was confirmed by real-time RT-PCR that gene expression of DIO3 (type 3 deiodinase) not treated with siRNA as well as DIO1 and DIO2 treated with siRNA decreased as shown in Fig. 1 (Fig. 1).

DIO1 , DIO2 , DIO3 primer  order Gene name PCR primer sequence (5 '- &gt; 3') SEQ ID NO: DIO1 F AAGCAACACTTGAGCTGTTCAGCC One R TGCAGGGAATGTGGTCTCCAAAGT 2 DIO2 F ATTAGGGACGAGTACGCCAGCTTT 3 R CATTCAATTCGGAGCTGCTGCCTT 4 DIO3 F GAGGGTGTAAACATCCAACGGACA 5 R TCCCAAGCGTGACTTGGTTTGA 6 RPLPO F TGCAGCTGATCAAGACTGGAGACA 7 R TCCAGGAAGCGAGAATGCAGAGTT 8

< Example  2> De-iodination enzyme In knockdown  Thyroid hormone T3 ) Biosynthesis Inhibition Measure

T3 expression level was measured by luminescence immunoassay for inhibition of thyroid hormone (T3) biosynthesis in DKD cell line knockdown of deiodination enzyme.

Specifically, a luminescence immunoassay kit (Cat No. 175-300; Monobind Inc., Lake Forest, CA, USA) capable of measuring total T3 in the cell lysate and cell supernatant of the cell line was used . Cells obtained by the method of Example <1-2> were washed with PBS and then lysed in a lysis buffer (20 mM Tris, pH 8.0) supplemented with a protease inhibitor (Roche applied science, Mannheim, Germany) , 137 mM NaCl, 2 mM EDTA, 10% (v / v) NP-40] was added to the cell lysate at 4 ° C for 20 minutes to obtain a cell lysate. The obtained cell lysate was incubated at 12,000 rpm for 10 minutes The supernatant was collected by centrifugation and used for luminescence immunoassay. The amount of protein in the supernatant was quantitated using BCA assay kit (Pierce, USA).

As a result, a decrease in the level of thyroid hormone (T3) was observed in the DKD cell line knockdown the deiodination enzyme as shown in Fig. 2 (Fig. 2).

< Example  3> Microarray  Experiment

<3-1> Labeled cDNA  Produce

For the oligomicroarray analysis, cDNA was prepared using total RNA of the de-iodinated enzyme knockdown cell line obtained by the method of Example <1-2>.

Specifically, 30 μg of the obtained total RNA was mixed with 2 μg (1 μg / μl) of an oligo (dT) primer, reacted at 65 ° C. for 10 minutes, and immediately subjected to annealing in ice. Reagents were mixed as shown in Table 3 for the reverse transcription of the annealed RNA.

Reverse transcription  Reagent composition for reaction Configuration Volume ([mu] l) 5x first strand buffer 6 dNTPs 0.6 0.1 M DDT 3 SuperScript II enzyme 3 Cy-3 or Cy-5 dUTP 2

Total RNA isolated from the control SH-SY5Y cell line was labeled with Cy3-dUTP (green), and RNA isolated from the de-iodination enzyme knockdown SH-SY5Y cell line was labeled with Cy5-dUTP (red). The two samples were mixed and purified using a Microcon YM-30 column (Millipore, USA).

<3-2> Hybridization ( hybridzation ) reaction

Hybridization and washing were performed according to the instructions of Zinocheck (Korea). Hybridization was carried out in a 62 [deg.] C oven for 12 hours. As a DNA microarray, a 44 k human whole genome oligo microarray (Agilent, USA) was used. After washing (2 × SSC / 0.1% SDS for 2 minutes, 1 × SSC for 3 minutes and 0.2 × SSC for 2 minutes), the slides were centrifuged for 3 minutes at 800 rpm.

<3-3> Fluorescence image acquisition

Hybridization images on the slides were scanned on a Jeunpix 4000B (Genepix 4000B, Axon Instruments, USA), and chips that washed unbound genes were obtained fluorescence images using a laser fluorescence scanner. At this time, the green fluorescence image shows the activity level of the control gene and the red fluorescence image shows the gene specific expression only in the experimental group, and the yellow fluorescence image means the green and red complementary colors, and the expression of the two groups is not significantly different. Scanned images were analyzed with GenePix 4.1 software (Axon Instruments, USA) to obtain gene expression rates. From the data obtained by the above method, marker genes for cell lines derived from human neuron derived from de-iodinated enzyme knockdown were selected (Table 4).

As a result, as shown in Table 4, among the approximately 62,000 genes present on the oligonucleotide, the gene whose cell ratio of Cy5 / Cy3 was 1.5 times or more increased specifically in the neuron-derived cell line derived from deiodination enzyme knockdown was 1.858% (1,152 out of 62,000 genes), and 1.845% (1,144 out of 62,000 genes) were found to have decreased expression. Among these genes, genes encoding specifically expressed thiodeoxygenase knockdown were identified (Table 4).

De-iodination enzyme  Genes whose expression is altered by active inhibitory chemicals Gene registration number Genetic Gene name Oligonucleotide array value Normal SH-SY5Y cell line De-iodination enzyme knockdown SH-SY5Y cell line NW_016569 TBX3 T-box 3 1.00 3.20 NW_002213 ITGB5 integrin, beta 5 1.00 0.32

< Example  4> De-iodination enzyme  Treatment of Active Inhibiting Chemicals

<4-1> De-iodination enzyme  Active inhibitory chemicals

The present inventors have found that 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin), propylthio But are not limited to, propylthiouracil, beta-estradiol, 3-methylcholanthrene, amiodarone, arochlor 1254, cadmium chloride, dimethoate, At least one selected from the group consisting of fenvalerate, iopanoic acid, methoxychlor and 4-methylbenzylidene-camphor was used, This was dissolved in DMSO and the vehicle concentration was used below 0.1% in all experiments.

<4-2> De-iodination enzyme  Determination of concentration of active inhibitory chemicals

MTT experiments were carried out by the method of Mossman et al. (J. Immunol. Methods, 65, 55-63, 1983) using the SH-SY5Y cell line cultured by the method of Example <1-1>.

Specifically, cells were cultured in 24-well plates at 25 x 10 ⁴ cells / well in MEM medium (Gibro-BRL, USA) in the presence of the above 2,3,7,8-tetrachlorodi Benzodioxane, propylthiouracil, beta estradiol, 3-methylcholanthrene, amiodarone, Aroclor 1254, cadmium chloride, dimethoate, fenvalerate, iopanoic acid, methoxychlor, 4-methylbenzylidene camphor After 48 hours, the mixture was mixed with 5 mg / ml of 3- (4,5-dimethylthiazol-2,5-diphenyltetra zolium bromide), and the mixture was incubated at 37 ° C. for 3 hours. The formazan crystal was dissolved in 500 μl of DMSO, transferred to a 96-well plate and aliquoted and the OD value was measured at an absorbance of 540 nm.

In the SH-SY5Y cell line, 2,3,7,8-tetrachlorodibenzo dioxin, propylthiouracil, beta estradiol, 3-methylcolanthrene, amiodarone, Aroclor 1254, cadmium chloride, dimethoate, As a result of examining the cytotoxicity of iopanoic acid, methoxychlor and 4-methylbenzylidene camphor, cytotoxicity-free concentrations were observed (Table 5), and the concentrations were determined, Real-time RT-PCR experiments were performed on two genes of NM_016569 (T-box 3) and NM_002213 (integrin, beta 5).

chemical substance Concentration (uM) Propylthiouracil 40 Iodoacetic acid One Methylmercury One Methimazole 400 2,3,7,8-tetrachlorodibenzo dioxin 0.01 Beta estradiol 10 3-Methylcholanthrene 25 Aminotriazole 500 Amiodarone 10 Aroclor 1254 100 Cadmium chloride 25 Dimethoate 25 Fenvalerate 40 Octylmethoxycinnamate (Octylmethoxycinnamate) 50 Iopanoic acid 90 Methoxychlor 50 4-methylbenzylidene camphor 60

<4-3> Real time RT -PCR ( Real - time reverse transcriptase 중합체  chain reaction ) Quantity

(Gene registration number NM_016569 (T-box 3) and NM_002213 (integrin, beta 5)) related to the inhibition of the deiodination enzyme activity determined by Example <3-3> Real-time RT-PCR was carried out in the same manner as in the method described in [2], and the primers for the above two genes are shown in Table 6.

primer  order Gene registration number Gene name PCR primer sequence (5 '- &gt; 3') SEQ ID NO: NM_016569
T-box 3
sense TCAGAAACGTCGGTTGCATTGAGC 9 Antisense AATCCGCACTGAGGGAGATGTCTT 10 NM_002213
integrin, beta 5
sense TGTGAGTGCGACAACTTCTCCTGT 11 Antisense TAACCTGCATGGCACTTGCATTCC 12

As a result, gene expression number NM_016569 (T-box 3) increased gene expression and gene expression number NM_002213 (integrin, beta 5) decreased gene expression on a 1.5-fold basis as shown in FIG. 3 and FIG. 4 (Figs. 3 and 4).

<110> Korea Institute of Science and Technology <120> The biomarkers for identification of exposure to deiodinase          inhibiting disruptors and the identification method of deiodinase          inhibiting disruptors exposure using the same biomarkers <130> 12P-11-21 <160> 12 <170> Kopatentin 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DIO1_F <400> 1 aagcaacact tgagctgttc agcc 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DIO1_R <400> 2 tgcagggaat gtggtctcca aagt 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DIO2_F <400> 3 attagggacg agtacgccag cttt 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DIO2_R <400> 4 cattcaattc ggagctgctg cctt 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DIO3_F <400> 5 gagggtgtaa acatccaacg gaca 24 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> DIO3_R <400> 6 tcccaagcgt gacttggttt ga 22 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RPLPO_F <400> 7 tgcagctgat caagactgga gaca 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RPLPO_R <400> 8 tccaggaagc gagaatgcag agtt 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> T-box 3_sense <400> 9 tcagaaacgt cggttgcatt gagc 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> T-box 3_antisense <400> 10 aatccgcact gagggagatg tctt 24 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> integrin, beta 5_ense <400> 11 tgtgagtgcg acaacttctc ctgt 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> integrin, beta 5_antisense <400> 12 taacctgcat ggcacttgca ttcc 24

Claims (16)

A nucleic acid sequence of any one or more genes selected from the following group or a DNA microarray chip for confirming the specific inhibition of a de-iodination enzyme activity-inhibiting chemical substance in which the complementary strand molecule is integrated:
Gene registration number (Genebank) NM_016569 (T-box 3) and NM_002213 (integrin, beta 5).
The method of claim 1, wherein the activity inhibiting chemical of the deiodination enzyme is selected from the group consisting of 2,3,7,8-tetrachlorodibenzo-p-dioxin, propylthiouracil, Beta-estradiol, 3-methylcholanthrene, amiodarone, arochlor 1254, cadmium chloride, dimethoate, fenvalerate, Wherein the enzyme is at least one selected from the group consisting of fenvalerate, iopanoic acid, methoxychlor and 4-methylbenzylidene-camphor. A DNA microarray chip for identifying specific inhibitors of inhibitory chemicals.
1) separating each RNA from a somatic cell isolated from a test subject suspected of being exposed to a chemical substance inhibiting the activity of the deiodo-oxidizing enzyme and a somatic cell isolated from a normal subject as a control;
2) labeling the experimental group and the control group with different fluorescent substances while synthesizing the RNA of the experimental group and the control group of step 1) with cDNA;
3) hybridizing the cDNA labeled with different fluorescent substances of step 2) with the DNA microarray chip of claim 1;
4) analyzing the reacted DNA microarray chip of step 3); And
5) determining the degree of expression of the gene integrated in the DNA microarray chip of claim 1 by comparing the data analyzed in step 4) with that of the control group; and confirming exposure to the dehydroiodinating enzyme activity inhibiting chemical.
4. The method according to claim 3, wherein the somatic cells of step 1) are derived from human neurons or tissues.
5. The method according to claim 4, wherein the human neuron is SH-SY5Y.
The method according to claim 3, wherein the fluorescent material of step 3) is selected from the group consisting of Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate rhodamine-B-isothiocyanate (RITC), and rhodamine. The method according to claim 1, wherein the enzyme is selected from the group consisting of rhodamine-B-isothiocyanate (RITC) and rhodamine.
The method of claim 3, wherein the activity inhibiting chemical of the deiodinating enzyme is selected from the group consisting of 2,3,7,8-tetrachlorodibenzodioxine, propylthiouracil, beta estradiol, 3-methylcolanthrene, amiodarone, A method for confirming exposure to a dehydroiodinating enzyme activity inhibiting chemical, characterized in that it is at least one selected from the group consisting of chloride, dimethoate, fenvalerate, iopanoic acid, methoxychlor and 4-methylbenzylidene camphor .
1) separating each RNA from a somatic cell isolated from a test subject suspected of being exposed to a chemical substance inhibiting the activity of the deiodo-oxidizing enzyme and a somatic cell isolated from a normal individual as a control;
2) Performing Real-time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using primer pairs complementary to the following genes in the experimental group and the control group of step 1) and capable of amplifying the following genes:
Gene registration numbers NM_016569 (T-box 3) and NM_002213 (integrin, beta 5); And
3) comparing the gene product of step 2) with that of the control group to confirm the degree of expression, and then confirming whether or not the activity inhibitory chemical of the deiodination enzyme is exposed.
9. The method according to claim 8, wherein the primer pair of step 2) is a pair of forward and reverse primers of 18 to 30 primers capable of amplifying the gene of step 2). How to check if you are exposed.
9. The method according to claim 8, wherein the primer pair of step 2) comprises the following primer pairs 1 to 2: Method for confirming exposure to an activity inhibiting chemical of a de-iodinating enzyme:
Primer pair 1: a forward primer represented by SEQ ID NO: 9 and a reverse primer represented by SEQ ID NO: 10, and
Primer pair 2: the forward primer described in SEQ ID NO: 11 and the reverse primer set forth in SEQ ID NO: 12.
9. The method of claim 8, wherein the activity inhibiting chemical of the deiodinating enzyme is selected from the group consisting of 2,3,7,8-tetrachlorodibenzodioxin, propylthiouracil, beta estradiol, 3-methylcolanthrene, amiodarone, Wherein the inhibitor is at least one selected from the group consisting of chloride, dimethoate, fenvalerate, iopanoic acid, methoxychlor and 4-methylbenzylidene camphor. Way.
A kit for confirming exposure to an activity inhibiting chemical of a de-iodination enzyme comprising the microarray chip of claim 1.
A kit for confirming exposure to a chemical substance that inhibits the activity of a deiodinating enzyme comprising a pair of primers complementary to each of the following genes and capable of amplifying each of the following genes:
Gene registration numbers NM_016569 (T-box 3) and NM_002213 (integrin, beta 5).
16. The method according to claim 15, wherein the primer pair is a pair of forward and reverse primers having a length of 18 to 30 m designed to have an amplification product of 100 to 300 bp. Kits.
16. The kit according to claim 15, wherein the pair of primers comprises the following primer pairs 1 to 2: a kit for confirming exposure to an activity inhibiting chemical of a de-iodinating enzyme:
Primer pair 1: a forward primer represented by SEQ ID NO: 9 and a reverse primer represented by SEQ ID NO: 10, and
Primer pair 2: the forward primer described in SEQ ID NO: 11 and the reverse primer set forth in SEQ ID NO: 12.
14. The method according to claim 12 or 13, wherein the activity inhibiting chemical of the deiodinating enzyme is selected from the group consisting of 2,3,7,8-tetrachlorodibenzodioxine, propylthiouracil, beta- estradiol, 3-methylolanthrene, Wherein the inhibitor is at least one selected from the group consisting of Clark 1254, cadmium chloride, dimethoate, fenvalerate, iopanoic acid, methoxychlor and 4-methylbenzylidene camphor. Kits for checking exposure.

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