KR20200031480A - Biomarker and screening method for carcinogens using thereof in colon cancer stem cells - Google Patents

Abstract

The present invention relates to a gene biomarker for the search for genotoxic carcinogens, a composition for the search for the genotoxic carcinogens comprising an agent which measures the expression level of the gene biomarker, a kit for the search for genotoxic carcinogens containing the composition, and a method of screening for the genotoxic carcinogen using the kit. By measuring the expression level of the gene biomarker of the present invention in colorectal cancer stem cells treated with a test substance, it is possible to determine the carcinogenic potential of a specific substance with improved accuracy, low cost, and high efficiency compared to existing methods, thereby being used in the safety evaluation during the development of a new drug.

Description

Biomarker and screening method for carcinogens using thereof in colon cancer stem cells}

The present invention relates to a biomarker for retrieving carcinogens using colon cancer stem cells and a method for retrieving carcinogens.

　 Despite many efforts to overcome cancer, cancer remains a major life-threatening disease, and the incidence and mortality rates continue to rise. There are many causes of cancer, and there is substantial evidence that environmental chemicals and genetic factors play an important role in carcinogenesis. Accordingly, various approaches have been developed to detect the carcinogenicity of environmental pollutants and to study the exact mechanisms in carcinogenesis. Carcinogenicity has been measured using long-term rodent bioassays for the past several decades, but this method is time consuming and expensive, and the accuracy of detecting carcinogenicity to humans is only 60% despite the fact that many animals have to be killed. There was. Therefore, as an alternative to animal experiments, a gene expression profiling method using a microarray has been applied to various fields including toxicity detection and prediction in vivo (non-patent document 001). However, microarray analysis is a limited solution in that it requires expensive specialized equipment and bioinformatics approaches for data analysis.

In the past research, many researchers analyzed the specific surface protein of stem cells through the labeling method, and found some of the cells with excellent tumorigenicity by comparing the tumor ability in the selected cells by finding the stem cell marker in cancer tissue. They were found and they were called cancer stem cells (Reya, T. et al., (2001) Stem cells, cancer, and cancer stem cells, Nature 414,105-111; Collins, A. et al (2005) Prospective identification of tumorigenic prostate cancer stem cells, Cancer Res 65,10946-10951). Through this study, the hypothesis was suggested that only some cancer cells have the ability to form cancer, and it began to be studied as cancer stem cells in a similar concept to stem cells. Cancer stem cells maintain the ability to self-replicate and produce cancer stem cells of the same type as their own, while also producing several different types of cancer cells. Unlike cancer stem cells, these differentiated cells do not have the ability to self-replicate and cannot produce cancer on their own. Therefore, it can be said that in normal cancer tissues, a small number of cancer stem cells capable of self-replicating, self-producing cancer, and general cancer cells that cannot self-reproducing and self-producing cancer are mixed to generate cancer.

The discovery of these cancer stem cells is important in the clinical field. Through the study of cancer stem cell markers, it is possible to recognize cancer cells that must be removed in order to treat cancer among various cancer cells. It will help research treatment methods that kill cancer stem cells that could not be treated with treatment methods. Removing cancer stem cells is expected to bring about a breakthrough in cancer treatment by removing the roots that make up the cancer cells. Cancer stem cell markers have been found in several cancers, and cancer stem cells can be isolated from cancer tissues using various experimental methods. It is expected that separating cancer stem cells from various cancers and finding a way to kill cancer stem cells using the characteristics of cancer stem cells is expected to help cancer treatment.

In this way, research on markers of cancer stem cells and studies on characteristics of cancer stem cells are being conducted all over the world, and studies on various types of cancer are being conducted. Among them, the present invention has been conducted for colorectal cancer stem cells, and this colorectal cancer is the second most malignant tumor with high incidence and mortality in the West, and the incidence and mortality increase gradually as the dietary habits have been westernized in Korea. According to statistics, it ranks third among all malignant tumors. It is known that the mechanism by which malignant tumors occur in the large intestine is caused by over-proliferation by mutation in normal stem cells involved in mucosal formation in the crypt located below the large intestine mucosa. In particular, recent cancer stem cell research has made significant progress in the development of biomarkers and causes of tumor formation in colon cancer epithelial cells.

The present inventors continuously tried to develop a simple and cost-effective alternative carcinogenic screening assay for microarray-based assays, and then increased or decreased markers after exposing carcinogens to human colon cancer stem cells using quantitative PCR (qPCR) technique. The present invention was completed by identifying a gene and establishing a carcinogenic screening assay using the same.

 M.R. Fielden, A. Adai, R.T. Dunn, 2nd, A. Olaharski, G. Searfoss, J. Sina, J. Aubrecht, E. Boitier, P. Nioi, S. Auerbach, D. Jacobson-Kram, N. Raghavan, Y. Yang, A. Kincaid, J. Sherlock, SJ Chen, B. Car, C.W.G. Predictive Safety Testing Consortium, Development and evaluation of a genomic signature for the prediction and mechanistic assessment of nongenotoxic hepatocarcinogens in the rat, Toxicological sciences: an official journal of the Society of Toxicology, 124 (2011) 54-74.

The present inventors have made diligent research efforts to discover biomarkers capable of searching for carcinogens using cancer stem cells. As a result, the present invention was completed by exposing a carcinogen to human colon cancer stem cells, identifying a marker gene that increases or decreases, and establishing a carcinogenic screening assay using the marker gene.

Accordingly, an object of the present invention is to provide a biomarker for detecting genotoxic carcinogens.

Another object of the present invention is to provide a kit for detecting carcinogens.

Another object of the present invention is to provide a method for screening for carcinogens.

According to an aspect of the present invention for achieving the above object, the present invention provides a biomarker for detecting a genotoxic carcinogen comprising one or more genes selected from the group consisting of GOLGA7B, S100A7A, KRTAP4-6 and MS4A4E.

According to another aspect of the present invention, the present invention provides a kit for detecting a genotoxic carcinogen comprising an agent that measures the expression level of the biomarker mRNA or protein thereof.

According to another aspect of the present invention, the present invention provides a method for screening for carcinogens comprising the following steps.

(i) treating the test substance with cancer stem cells;

(ii) measuring the expression level of the biomarker gene according to claim 1 in cancer stem cells treated with the test substance; And

(iii) comparing the gene expression level of step (ii) with the gene expression level of the control group not treated with the test substance.

Since the biomarker of the present invention exhibits very good indicator efficiency for carcinogens, it is possible to quickly and accurately determine whether a test substance is a carcinogen, and in particular, whether a test substance is a carcinogen having genetic toxicity. have.

In addition, according to the carcinogen screening method of the present invention, since it can screen carcinogens at a higher efficiency and at a lower cost than the method using a conventional microarray, it can be widely used for stability evaluation during a new drug development process.

1A is a graph confirming that the AOM, ENU, MNZ, NKK, and DMAB compounds are treated on HCT116 colon cancer stem cells, and microarrays show distinct patterns in hierarchical clustering.
FIG. 1B is a result of performing a principle component analysis for exploring changes in gene expression caused by chemicals using five data sets.
Figure 1c is a result showing the number of genes showing more than 2 fold expression variation between groups in the five GC groups.
2 shows GeneMANIA and GIANT analysis methods, which are methods for analyzing the candidate candidate carcinogenicity biomarker network.

Hereinafter, the present invention will be described in more detail.

In the present invention, it was intended to discover factors other than the known cancer stem cell surface markers and use them in the carcinogenicity prediction and evaluation system. As a result, it includes one or more genes selected from the group consisting of GOLGA7B, S100A7A, KRTAP4-6 and MS4A4E. It was confirmed that the biomarker for retrieval of genotoxic carcinogens has an increased or decreased expression level by treatment with carcinogens and thus has an index efficiency for carcinogen detection.

In the present invention, the selection process of genes involved in the search for genotoxic carcinogens is as follows. First, after treating five cancer-causing chemicals in cancer stem cells, microarray analysis was performed to derive 16 genes related to carcinogens. Subsequently, 25 genotoxic and non-genotoxic carcinogens were additionally treated, and then qPCR was performed to confirm the predictability of carcinogens of the selected genes and 4 GC biomarkers associated with genotoxic carcinogens (GOLGA7B, S100A7A, KRTAP4) -6 and MS4A4E) were finally selected.

According to the carcinogen screening method of the present invention, the test substance is treated with cells containing one or more biomarker genes selected from the group consisting of GOLGA7B, S100A7A, KRTAP4-6 and MS4A4E.

In general, the cells that can be used in the present invention may be cancer stem cells, colorectal cancer stem cells, and cells transformed with one or more genes selected from the group consisting of GOLGA7B, S100A7A, KRTAP4-6 and MS4A4E. It includes.

The test substance determined to be a carcinogen by the present invention is not particularly limited, and includes a single compound or a mixture of compounds. Test substances can be obtained from libraries of synthetic or natural compounds. Methods for obtaining libraries of these compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories ( USA) and MycoSearch (USA). In an embodiment of the present invention, AOM, ENU, MNZ, NKK, and DMAB were used in the gene selection process related to the initial carcinogen, and the screening of carcinogens using the selected gene markers and the end of the marker for predicting genotoxic carcinogens In the selection process, 30 types of chemicals shown in [Table 1] were used, including the chemicals.

In the carcinogen screening method of the present invention, a test substance is contacted with a cell, and then the expression level of one or more genes selected from the group consisting of GOLGA7B, S100A7A, KRTAP4-6 and MS4A4E is measured in the cell. If the expression of the gene in the cell is measured to be increased or decreased, it is determined as a carcinogen.

"Increased or decreased expression of a gene in a cell" means that the expression level is increased or decreased compared to the expression level of the gene in untreated cells. The expression level is the expression level measured by methods such as RT-PCR, qPCR, etc. that are commonly performed in the industry.

According to an embodiment of the present invention, when the result of comparison with the level of gene expression in the untreated cells corresponds to any one or more of the following (a) to (f), the test substance is determined as a genotoxic carcinogen .

(a) down regulation of GOLGA7B expression levels;

(b) down regulation of S100A7A expression level;

(c) down regulation of MS4A4E expression level;

(d) down-regulation of KRTAP4-6 expression levels;

More specifically, when the expression level of the gene is 2 times or more or 2 times or less compared to the expression level of the gene in untreated cells, it is determined as a significant increase in expression or a decrease in expression, and the test substance is determined as a carcinogen. .

When carrying out the present invention based on genetic analysis, primers or probes that specifically bind one or more gene sequences selected from the group consisting of GOLGA7B, S100A7A, KRTAP4-6 and MS4A4E are used.

When using a primer, a gene amplification reaction is performed to investigate the expression level of one or more genes selected from the group consisting of GOLGA7B, S100A7A, KRTAP4-6, and MS4A4E. Since the present invention analyzes the expression level of one or more genes selected from the group consisting of GOLGA7B, S100A7A, KRTAP4-6, and MS4A4E, the amount of gene expression is determined by examining the mRNA level of the gene in the cell.

Therefore, in the present invention, in principle, mRNA in a sample is used as a template, and a gene amplification reaction is performed using primers that bind to mRNA or cDNA.

To obtain mRNA, total RNA is isolated from the sample. Isolation of total RNA can be performed according to conventional methods known in the art (Sambrook, J. et al., Molecular Cloning.A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Tesniere , C. et al., Plant Mol. Biol. Rep., 9: 242 (1991); Ausubel, FM et al., Current Protocols in Molecular Biology, John Willey & Sons (1987); and Chomczynski, P. et al ., Anal. Biochem. 162: 156 (1987)). For example, total RNA in cells can be easily separated using Trizol.

Then, cDNA is synthesized from the isolated mRNA, and the cDNA is amplified. Since the total RNA of the present invention is isolated from a human sample, it has a poly-A tail at the end of mRNA, and cDNA can be easily synthesized using oligo dT primers and reverse transcriptases using these sequence characteristics. Next, the synthesized cDNA is amplified through a gene amplification reaction.

The term "amplification reaction" described herein refers to a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the industry, which include polymerase chain reaction (hereinafter referred to as PCR), reverse transcriptase-polymerase chain reaction (hereinafter referred to as RT-PCR), and ligase chain reaction (LCR). , Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA), self-sustained sequence replication, target Selective amplification of target polynucleotide sequences, consensus sequence primed polymerase chain reaction (CP-PCR), randomly primed polymerase chain reaction (AP) -PCR), nucleic acid sequence based amplification (NASBA), strand displacement amplification and ring-mediated constant temperature amplification (lo op mediated isothermal amplification (LAMP).

The primer used in the amplification reaction in the present invention is an oligonucleotide having a sequence complementary to the cDNA sequence of one or more genes selected from the group consisting of GOLGA7B, S100A7A, KRTAP4-6 and MS4A4E. The term “primer” herein is a single-strand capable of acting as a starting point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in a suitable buffer at a suitable temperature. Means oligonucleotide. The suitable length of the primer varies depending on various factors, such as temperature and the use of the primer, but is typically 15-30 nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template.

One or more cDNA selected from the group consisting of GOLGA7B, S100A7A, KRTAP4-6 and MS4A4E thus amplified is analyzed by a suitable method to investigate the expression level of the gene. For example, gel electrophoresis is performed on the result of the amplification reaction described above, and a resultant band is observed and analyzed to investigate the expression level of the biomarker gene.

Through these amplification reactions, when the expression of one or more genes selected from the group consisting of GOLGA7B, S100A7A, KRTAP4-6, and MS4A4E in cells is higher or lower than that of untreated cells, the test material is determined as a carcinogen.

Carcinogens can be determined by hybridization-based analysis using a probe that hybridizes to a biomarker gene or cDNA.

The term “probe” herein is a single chain nucleic acid molecule and includes a sequence complementary to a target nucleic acid sequence. According to one embodiment of the present invention, the probe of the present invention can be modified within the scope of not impairing the advantages of the probe of the present invention, that is, improving hybridization specificity. These modified labels can provide a signal to detect whether or not hybridization, which can be linked to an oligonucleotide. Suitable labels include fluorophores (e.g. fluorescein, phycoerythrin, rhodamine, lisamine, and Cy3 and Cy5 (Pharmacia)), chromophores, chemiluminescent groups, magnetic particles, and radioisotopes Elements (P32 and S35), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, substrates for enzymes, heavy metals (eg gold) and antibodies, streptavidin, biotin , Hapten having a specific binding partner such as digoxigenin and chelating group, but is not limited thereto.

According to one embodiment of the present invention, the probe hybridized to the cDNA of the biomarker is immobilized on an insoluble carrier (eg, a nitrocellulose or nylon filter, glass plate, silicon and fluorocarbon support), thereby producing a microarray. . In microarrays, the probe is used as a hybridizable array element. Immobilization with an insoluble carrier is carried out by a chemical bonding method or a covalent bonding method such as UV. According to one specific embodiment of the present invention, the hybridization array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bonded by UV on a polylysine coated surface. In addition, the hybridization array element can be attached to a carrier through a linker (eg, ethylene glycol oligomer and diamine).

The cDNA of EBP50 can be labeled in place of the probe to perform hybridization reaction-based analysis. When using a probe, the probe is hybridized with a cDNA molecule.

After the hybridization reaction, a hybridization signal coming out through the hybridization reaction is detected. The hybridization signal can be carried out in various ways depending on the type of the label bound to the probe, for example. For example, when the probe is labeled with an enzyme, hybridization may be confirmed by reacting the substrate of the enzyme with the result of the hybridization reaction. Combinations of enzymes / substrates that can be used include peroxidase (eg horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, and lucigenin (bis-N-methylacridinium) Nitrate), resorphin benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazin), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine (TMB), ABTS (2 , 2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine; Alkaline phosphatase and bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF substrates; Glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate). When the probe is labeled with gold particles, it can be detected by silver staining using silver nitrate.

By analyzing the intensity of the hybridization signal by the hybridization process described above, it can be determined whether the test substance is a carcinogen. That is, when a signal for one or more cDNA selected from the group consisting of GOLGA7B, S100A7A, KRTAP4-6, and MS4A4E in cells treated with the test substance is stronger or weaker than the untreated cell, the test substance is determined as a carcinogen.

If the kit for detecting a carcinogen of the present invention is applied to a PCR amplification process, the kit of the present invention is optionally a reagent required for RT-PCR amplification, such as buffer, reverse transcriptase, DNA polymerase, DNA polymerase joiner and dNTPs.

The kit of the present invention can be manufactured in a number of separate packaging or compartments containing the reagent components described above.

As demonstrated in the examples below, the biomarker of the present invention exhibits very good indicator efficiency against carcinogens.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

1. Materials and Methods

1-1. Cell culture and test substance

The HCT116 human colon cancer cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). HCT116 cells were cultured in DMEM medium (Hyclone, Logan, UT, USA) supplemented with 10% (v / v) fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured in a humidifier incubator with 5% CO ₂ at 37 ° C. For cancer stem cells, 1000 cells / mL were inoculated onto a cryogenic attachment plate (Corning, Inc., Corning, NY, USA) and cultured as described above. The test materials used are listed in Table 1 below. Candidate concentrations were selected based on in vivo data to identify the highest concentrations that did not show cytotoxicity in vitro .

[Table 1]

1-2. Cell viability test

Cell viability was quantified using a cell count kit-8 (CCK-8) assay (Promega®, Madison, WI, USA). Cells were seeded at 5000 cells / well in 96-well culture plates containing 100% μL DMEM medium supplemented with 10% fetal bovine serum, 100 μg / mL, penicillin and 0.25 μg / mL, streptomycin, and cultured overnight. After 20 hours of culture, various concentrations of chemicals were added to the wells, followed by an additional 72 hours of culture. Cell viability was analyzed by performing CCK-8 analysis according to the manufacturer's instructions. Optical density was measured at 450 nm using a microplate reader (Apollo LB 9110; Berthold Technologies GmbH, Bad Wildbad, Germany).

1-3. Spherical formation

HCT116 colon cancer cells were counted and plated on a low adherence plate (Corning) at a constant density of 2000 viable cells per mL. DMEM / F12 medium containing 1% fetal bovine serum supplemented with cells supplemented with 20 ng / mL epidermal growth factor (Sigma), 10 ng / mL basic fibroblast growth factor (Sigma) and 10 ng / mL basic fibroblast growth factor. (Invitrogen, Carlsbad, CA, USA), 1% N2 (Stemcell Technologies, Inc., 밴쿠버 Canada Vancouver) and 5% B27 (Gibco, Grand Island, NY, USA) for 7 days.

1-4. Microarray

Microarray analysis was performed to identify genes regulated by AOM, ENU, MNZ, NKK and DMAB in cancer stem cells. Total RNA was extracted using the Ribospin Kit (GeneAll, Seoul, Korea) according to the manufacturer's instructions. RNA quality was measured with an RNA 6000 nanochip (Agilent Technologies, Santa Clara, CA, USA), and RNA amount was measured with an ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). 300 ng of each RNA sample was used as input in Affymetrix according to the manufacturer's instructions. Total RNA of each sample was reverse transcribed with cDNA. cDNA was fragmented, terminally labeled with a terminal transferase reaction, and linked to biotinylated dideoxy nucleotides. The fragmented and terminally labeled cDNA was hybridized to the Gene Chip® Human Gene 1.0 ST array according to the manufacturer's instructions. After 16 hours of hybridization, the chips were stained and washed using Gene chip Fluidics Station 450 (Affymetrix), and scanned with Gene chip Array scanner 3000 7G (Affymetrix). The Affymetrix Gene Chip® Human Gene 1.0 ST Array was used to analyze the transcription profile of HCT116 cancer stem cells treated with 6 samples, untreated HCT116 cancer stem cells and carcinogens. The array of Affymetrix covers 28,869 human genes and includes a 764,885 25-mer oligonucleotide probe. Affymetrix analysis was performed using steps such as image acquisition, data extraction, normalization, differentially expressed gene selection and functional grouping. A robust multi-array average was used for normalization. A web-based tool, the Database for Annotation, Visualization, and Integrated Discovery (DAVID), was used to identify the biological function of the differentially expressed gene. Next, these genes were classified according to the gene function in the Gene Ontology and KEGG Pathway database ( http://david.abcc.ncifcrf.gov/ home.jsp ).

1-5. Gene network using compound-mediated signaling

Data sets were collected from GeneMANIA, including co-expression, physical interactions, pathways and gene interactions. A data set was generated excerpting five carcinogens from the GeneMANIA database. GIANT (Genome-scale Integrated Analysis of gene Networks in Tissues) was used for tissue-specific pathway analysis. This data set is available at http://giant.princeton.edu/ .

1-6. Reverse transcription polymerase chain reaction

Cells were lysed in 1 mL easy-BLUE Total RNA Extraction Kit (iNtRON Biotechnology, Sungnam, Korea) and RNA was isolated according to the manufacturer's instructions. Oligo (dT) -primer RNA (5 μg) was reverse transcribed using M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA, USA). Quantitative reverse transcription polymerase chain reaction (QRT-using Rotor-Gene 6000 series software 1.7 (Qiagen, Hilden, Germany), SensiFAST ^TM SYBR NO-ROX Kit (BIOLINE, London, UK) and primer set of [Table 2] PCR).

[Table 2]

Relative gene expression differences were calculated using a critical cycle (CT) value normalized to the GAPDH gene as an internal control in the same sample.

1-7. Statistical analysis

Statistical significance was evaluated by permutation t-test (p value <0.05) because the distribution of qPCR does not follow a normal distribution and has very large outliers. Since extremely large outliers are distributed differently among the 3 replicated data, permutation t-test was first performed on each data separately. In addition, two data sets were constructed by taking the median or the averaging average of three replicated data. Appropriate genotoxic carcinogen (GCs) biomarker candidates were selected based on criteria that met the p-value (p <0.05) of the permutation t-test between the GC and NC qPCR results from the five data sets.

2. Results

2-1. Identification of gene expression changes by compounds in colorectal cancer stem cells and selection of genes related to prediction of carcinogenicity

The purpose of this study was to identify commonalities in gene expression changes induced by GCs with similar behavior patterns. The concentration was selected based on 1/10 of the concentration used in mice to determine the optimal concentration of chemicals, including GCs and NCs, for colon cancer cells. Mice concentrations are reported in the test data in a summary form in a chemo carcinogenic research information system that can be retrieved from the NLM TOXNET system. The largest amount of chemical was selected for CCK cell viability analysis using the HCT116 colon cancer cell line (data not shown). The effect of this concentration on HCT116 colorectal cancer stem cells was analyzed if the cell viability was not reduced by 90% due to the maximum exposure of the chemical. Next, a microarray was performed to identify candidate GC biomarkers using GC. For the evaluation of cancer stem cell carcinogenicity using biomarkers, colon cancer stem cells (HCT116) were treated with AOM, ENU, MNZ, NKK and DMAB 'compounds and cultured for 7 days, followed by purification of RNA to perform microarray.

As a result, it can be seen that at least 1,663 genes in each group and at most 10,927 genes were changed compared to the control group, and a distinct pattern was seen in hierarchical clustering (FIG. 1A). Principle component analysis was performed to explore changes in gene expression caused by chemicals using five data sets (Figure 1B). NKK had the greatest impact, followed by DMAB. Next, the number of significant deregulated probe sets in the 5 GC groups was determined (FIG. 1C). Genes that change identically between groups of genes were selected, but the selection criteria were increased or decreased by 2 times or more, and genes that changed in the same manner were selected in all treatment groups (Table 3).

[Table 3]

Sixteen genes were selected, except for a set of "unknown" probes that were not revealed by gene symbols and functions. Among the selected marker genes, PGA5, SCT, MS4A4E, KRTAP4-6, RNPS1 and PLCXD1 were confirmed to be up-regulated in the gene treatment group compared to the control group without treatment with chemicals, S100A7A, ENO1, GOLGA7B, H3F3C, ZNF844, CDKL4, HIST1H2BH, RPS12, C6orf47 and CPA4 genes were confirmed to be down-regulated. The identified GC biomarker candidates are involved in calcium ion binding, DNA binding, cyclin-dependent kinase activity, and nucleotide binding.

2-2. Screening of carcinogens using selected markers and final selection of markers for predicting genotoxic carcinogens

To test the carcinogenic predictive power of the candidate genes of [Table 1], a total of 30 positive and negative substances, including 25 additional drugs, were processed to observe whether the genes were changed by qPCR. QPCR analysis was performed using 30 chemicals including GCs and NCs shown in Table 1 above. To confirm the correctness of the 16 species, HCT116 colon cancer stem cells were treated with 30 chemicals for 7 days, and then mRNA was extracted, cDNA was synthesized, and qPCR was performed, and the results are shown in [Table 4].

[Table 4]

Based on the qPCR results, it is statistically accessed through T-test analysis, and finally GOLGA7B, S100A7A, KRTAP4-6 and MS4A4E Four genes were optimized (Table 5). Statistical significance was evaluated by the t-test because the distribution of qPCR does not follow a normal distribution and has very large outliers (p value <0.05). Since the extremely large outliers were distributed in three different ways, a permutation t-test was first performed separately on each data. In addition, two data sets were constructed by taking the median or the averaging mean of three replicated data. Appropriate GC biomarker candidates were selected from five data sets. Determined by criteria that follow the p-value (p <0.05) of the permutation t-test between the qPCR results of GC and NC.

 [Table 5]

As a result, carcinogenicity and non-carcinogenicity were measured using four genes GOLGA7B, S100A7A, KRTAP4-6, and MS4A4E among the genes having significant results (p <0.05). Substances that have non-carcinogenic properties when 30 substances are treated on HCT116 cancer stem cells and the expression values of the genes are within the cut-off range, and when the genes having such expression values are 50% or more in 4 genes ( -), On the contrary, when the expression value of genes does not fall within the cut-off range, and when the gene having the expression value is 50% or more, it is designated as a carcinogen (+). The results of confirming the carcinogenic and non-carcinogenic predictive power using 30 test substances are shown in [Table 6].

[Table 6]

In [Table 6], + is a symbol used when having a value outside the cut-off range, and-is a symbol used when having a value included in the cut-off range.

Then, GeneMANIA and GIANT analysis were conducted to determine the relationship between the identified candidates. The network consists of 24 genes, including 4 identified genes and 20 additional genes identified by GeneMANIA (A in Figure 2). Physical interaction (67.64%), co-expression (13.5%), predicted (6.35%), pathway (4.35%), co-localization (6.17%), genetic interaction (1.4%) and shared protein (0.59%) ) Domain literature. In the present invention, the mechanism of action of carcinogen-treated colon cancer stem cells was evaluated and an alternative carcinogenicity test method was developed. Of the 16 genes predicted by microarray analysis based on data treated with 18 GCs and 5 NCs using qPCR analysis, 4 predicted genes (GOLGA7B, S100A7A, KRTAP4-6 and MS4A4E) were selected. Candidates identified in this regard can be used as GC biomarkers. They clearly distinguished GCs and NCs from the apparent cellular signaling pathway. The present invention also presents a unique approach to measuring colon carcinogenesis using new GC biomarkers. These measurable biomarkers can be used to detect carcinogenesis, and alternative carcinogenicity screening tests using PCR and colon cancer stem cells can be useful as additional carcinogenicity testing strategies.

<110> Industry-Academic Cooperation Foundation, Konkuk University and Ministry of Food and Drug Safety <120> Biomarker and screening method for carcinogens using thereof in          colon cancer stem cells <130> 1063476 <160> 34 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of S100A7A <400> 1 ttcacaaata caccggacgt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of S100A7A <400> 2 cgaggtaatg tatgcccttt 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of EN01 <400> 3 ttcacaaata caccggacgt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of EN01 <400> 4 gcaggcgcaa tagttttatt 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of GOLGA7B <400> 5 ttatccagag actactacag c 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of GOLGA7B <400> 6 ttttaggcaa ctcccagaag 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of PGA5 <400> 7 gacttcctga agaagcacaa 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of PGA5 <400> 8 catatccagg tagttctcca 20 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of SCT <400> 9 gacgcagaga acagcatg 18 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of SCT <400> 10 cagctggttc tgaaaccat 19 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of MS4A4E <400> 11 ttctgattgc cttgatgagc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of MS4A4E <400> 12 taaggataca tcactgaccc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of H3F3C <400> 13 caagcagact gctcgtaaat 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of H3F3C <400> 14 ggtcgacttc tgataacgac 20 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of KRTAP4-6 <400> 15 cctcttgctg tgaatccag 19 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of KRTAP4-6 <400> 16 gtggaaatga cacaggttgg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of ZNF844 <400> 17 cagagaagtg atgcaggaaa 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of ZNF844 <400> 18 aaagtagttg agagagaggg 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of CDKL4 <400> 19 cagaaacaaa acctctggac 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of CDKL4 <400> 20 ctcgatgagg ttcacaagat 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of RNPS1 <400> 21 tgaaaaggag aggaaaaggc 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of RNPS1 <400> 22 cacgggcatg tcaatcattt 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of HIST1H2BH <400> 23 gaagaaggat ggcaagaagc 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of HIST1H2BH <400> 24 ggaattcatg atccccatgg 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of RPS12 <400> 25 tctttgtgtg cttgcatcca 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of RPS12 <400> 26 ttacaaaggc ctacccattc 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of C6orf47 <400> 27 catcccaaga ctaaggactc 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of C6orf47 <400> 28 ccacttgagg gaatccattc 20 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of CPA4 <400> 29 gggaccaagt ttgaggatt 19 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of CPA4 <400> 30 ggagggagat ttccagaaat 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of PLCXD1 <400> 31 gacacactca cggaaatctc 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of PLCXD1 <400> 32 atgttcttga tacaggcgac 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of GAPDH <400> 33 tgggctacac tgagcaccag 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of GAPDH <400> 34 cgaggtaatg tatgcccttt 20

Claims (10)

Biomarker for detecting genotoxic carcinogens containing one or more genes selected from the group consisting of GOLGA7B, S100A7A, KRTAP4-6 and MS4A4E.
The biomarker of claim 1, wherein the biomarker is one or more genes selected from colon cancer stem cell genes.
A composition for retrieving genotoxic carcinogens comprising an agent for measuring the mRNA of the biomarker of claim 1.
The composition of claim 3, wherein the agent that measures the expression level of the mRNA comprises a primer pair, probe or antisense nucleotide that specifically binds the biomarker.
A kit for detecting a genotoxic carcinogen comprising the composition of claim 3 or 4.
The kit of claim 5, wherein the kit is an RT-PCR kit, a competitive RT-PCR kit, a real-time RT-PCR kit or a DNA chip kit.
A method of screening for a genotoxic carcinogen comprising the following steps:
(i) treating the test substance with cancer stem cells;
(ii) measuring the expression level of one or more marker genes selected from the group consisting of GOLGA7B, S100A7A, KRTAP4-6 and MS4A4E in cancer stem cells treated with the test substance; And
(iii) comparing the gene expression level of step (ii) with the gene expression level of the control group not treated with the test substance.
The method of claim 7, wherein measuring the gene expression level comprises measuring the mRNA level of the biomarker through RT-PCR.
The screening method according to claim 7, further comprising determining a test substance as a genotoxic carcinogen when the comparison result of step (iii) corresponds to any one or more of the following (a) to (f). Way.
(a) down regulation of GOLGA7B expression levels;
(b) down regulation of S100A7A expression level;
(c) down regulation of MS4A4E expression level;
(d) Down-regulation of KRTAP4-6 expression levels.
The method of claim 7, wherein the carcinogen is AOM, MNZ, NKK, ENU, DMAB, 2,4-Diaminotoluene, Furan, QN, 4-NQO, DMBA, MMC, BaP, 2-NP, SDMH, O-Nitroanisole, DSS, PMA, PTX, 1-Chloro, 1-nitro, 2,5-TDS, MC, TAA, EE2, ACTD any one or more chemicals selected from the group consisting of, screening method.

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