KR102029198B1 - Gastric cancer stem cell markers and uses thereof - Google Patents

Abstract

The present invention relates to a gastric cancer stem cell marker and its use, and specifically comprises SOX2 (sex determining region Y-box 2), E-cadherin (E-cadherin) and ERBB3 (Erb-B2 Receptor Tyrosine Kinase 3). Gastric cancer stem cell composition comprising an agent for measuring the expression level of one or more genes selected from the group, kit comprising the composition, a method for providing information for distinguishing gastric cancer stem cells, and less than / long form gastric cancer therapeutic agent It relates to a screening method of.
SOX2 and E-cadherin genes are subtypes; And since the ERBB3 gene can be used as an enteric gastric cancer stem cell marker, the composition of the present invention for measuring the expression level of the gene can not only distinguish between the enteric / less than gastric cancer cells, the therapeutic agent according to the pathological characteristics of gastric cancer It can be useful for development.

Description

Gastric cancer stem cell markers and uses thereof}

The present invention relates to a gastric cancer stem cell marker and its use, and specifically comprises SOX2 (sex determining region Y-box 2), E-cadherin (E-cadherin) and ERBB3 (Erb-B2 Receptor Tyrosine Kinase 3). Gastric cancer stem cell composition comprising an agent for measuring the expression level of one or more genes selected from the group, kit for gastric cancer stem cell discrimination comprising the composition, method for providing information for gastric cancer stem cell discrimination, and less It relates to a method of screening for a type / long-term gastric cancer therapeutic agent.

Stomach cancer is the fifth most common cancer in the world and the third most common cancer. In the treatment of gastric cancer, chemotherapy using surgical methods, cytotoxic compounds, and targeted therapies is being performed. However, after surgery or treatment, about 40 to 60% of all patients with gastric cancer lose their lives due to recurrence. Cancer stem cells have been suggested as resistance to gastric cancer treatment or as a cause of relapse (Cancer Res, 2003 Sep, 15). 63 (18): 5821-8.).

Cancer stem cells have the ability of self-replicating and, under certain conditions, differentiate into cells with different characteristics. As studies have been published that targeting and treating such cancer stem cells can be an effective cancer treatment, many studies have been conducted to characterize and isolate their characteristics. For example, a method of using cancer stem cells to form tumors (Stem Cells. 2009 May; 27 (5): 1006-20.), Or isolating cancer stem cells using cell surface markers has been used. However, some cell surface markers, such as identified CD44 and CD90, cannot be utilized as gastric cancer stem cell markers (J Cell Physiol. 2012 Jun; 227 (6): 2686-93.), And tumorigenic capacity. The method using the microenvironment in which the tumor grows differently depending on the mouse, there was a disadvantage that a lot of research costs.

Accordingly, researches for separating stem cells and confirming their characteristics through the spear forming ability of cancer stem cells have been continued. In this regard, methods for promoting differentiation and cultivating tonsil-derived stem cells in the form of sustained spheroids (Korean Patent Publication) 10-2015-0073919), adult stem cell aggregates for transplantation in the form of cell spheroids and a method of manufacturing the same (Korean Patent No. 10-1017710) have been developed.

On the other hand, cancer is subdivided by genetic characteristics, histopathological characteristics, etc., and treatment is applied differently according to each subtype. For the classification of gastric cancer, the Lauren method, which is classified into Diffuse type or Intestinal type according to histopathological characteristics, is widely used (J Surg Oncol. 2005; 90: 144-33.) . Recently, the opinion of cancer stem cell may be different according to the subtype of cancer, and the variation pattern of gene is also suggested (Nature. 2014; 513: 202-9). For example, RhoA gene is long Studies have been reported to be activated in subtype gastric cancer cell lines rather than gastric cancer cell lines, which are involved in anticancer drug resistance (Clin Cancer Res. 2016 Feb 15; 22 (4): 971-83.). However, although targeting and treating cancer stem cells is an effective method, only cancer gas cell markers are currently being studied, and cancer stem cell specific marker genes have not been reported.

Under these backgrounds, the present inventors have made diligent efforts to develop methods for effectively diagnosing and treating gastric cancer according to the type. As a result, the present inventors have formed and isolated spear cells exhibiting gastric cancer stem cell characteristics of subtype / long form, and are specific to the cells. As a result of identifying the gene to be expressed as a target, the present invention was completed by confirming that the gene can be used as a specific marker for a small-type / long-term gastric cancer stem cell.

One object of the present invention is to provide a protein comprising at least one gene selected from the group consisting of sex determining region Y-box 2 (SOX2), E-cadherin and Erb-B2 Receptor Tyrosine Kinase 3 (ERBB3). It is to provide a composition for distinguishing gastric cancer stem cells, including an agent for measuring the expression level.

Another object of the present invention is to provide a kit for distinguishing gastric cancer stem cells, comprising the composition.

Another object of the present invention is to (a) use the composition to measure the expression level of one or more genes selected from the group consisting of SOX2, E-cadherin and ERBB3 from biological samples isolated from gastric cancer patients Making; And (b) comparing the expression level measured in step (a) with that of the control sample, to provide a method for providing information for gastric cancer stem cell differentiation.

Another object of the present invention comprises the steps of: (a) measuring the expression level of SOX2 or E-cadherin gene in subtype gastric cancer stem cells treated with the candidate substance of the subtype gastric cancer; And (b) comparing the expression level of step (a) with that of the sample not treated with the candidate.

Another object of the present invention comprises the steps of (a) measuring the expression level of the ERBB3 gene in enteric gastric cancer stem cells treated with a candidate for enteric gastric cancer treatment; And (b) comparing the expression level of step (a) with that of the sample not treated with the candidate.

In order to achieve the above object, one embodiment of the present invention is from the group consisting of sex determining region Y-box 2 (SOX2), E-cadherin and Erb-B2 Receptor Tyrosine Kinase 3 (ERBB3) It provides a composition for distinguishing gastric cancer stem cells, comprising an agent for measuring the expression level of one or more genes selected.

In the present invention, the spear cells exhibiting the characteristics of the subtyped / intestinal gastric cancer stem cells were formed and separated, respectively, in the case of the subtype gastric cancer stem cells, the SOX2 and E-cadherin genes, and the enteric gastric cancer stem cells. It was confirmed that the ERBB3 gene is specifically expressed, and the gene was confirmed to be utilized as a specific gene marker for each of the subtype / intestinal gastric cancer stem cells, thereby completing the present invention.

As used herein, the term "sex determining region Y-box 2" refers to a gene encoding sex determining region Y-box 2 and is also referred to as SOX2. The SOX2 plays an essential role in the maintenance of self-replicating, multipotent, etc. of undifferentiated embryonic stem cells, and plays an important role in the development of mammals. A type of Sox family that is a transcription factor, the protein family of genes share a conserved DNA binding domain known as a high-mobility group (HMG) box domain. The base sequence and amino acid sequence of SOX2 can be obtained from a known database such as GenBank of NCBI (eg GenBank Accession NM_003106, NP_003097.1, etc.). Specifically, the SOX2 may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

The term "E-cadherin" of the present invention refers to a gene encoding E-cadherin, which is a kind of cadherin, and may also be named CDH1 (Cadherin 1). Cadherins (calcium-dependent adhesion) are a type-1 transmembrane proteins that form cell-adherent junctions that allow cells to bind to each other in tissues. Mediate. In vertebrates, there are more than 100 kinds of cadrerin, and in addition to the E-cadherin mainly found in epithelial tissue, N-cadherin mainly found in neurons, P-cadherin mainly found in placenta, and T1- Kadherin, T2-cadherin, H-cadherin, M-cadherin and the like. The base sequence and amino acid sequence of the E-cadherin can be obtained from a known database such as GenBank of NCBI (eg GenBank Accession NM_004360, NM_001317184, NP_004351.1, NP_001304113.1, etc.). Specifically, the E-cadherin may be composed of the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

As used herein, the term “ERBB3 (Erb-B2 Receptor Tyrosine Kinase 3)” refers to a gene encoding Erb-B2 Receptor Tyrosine Kinase 3, and may also be referred to as human epidermal growth factor receptor 3 (HER3). ERBB3 is a type of epidermal growth factor receptor (EGFR / ERBB) family of receptor tyrosine kinase that can form homodimers with ERBB3 and can also form heterodimers with proteins belonging to other ERBB families. Evidence that ERBB3 is overexpressed in cancer cells has not been shown, but constitutive activation or mutation of ERBB3 has been reported to cause cancer (Zhang K, et al., 1996, J. Biol. Chem., 271 (7). ): 3884-90.). The base sequence and amino acid sequence of ERBB3 can be obtained from a known database such as GenBank of NCBI (eg GenBank Accession NM_001982, NM_001005915, NP_001005915.1, NP_001973.2, etc.). Specifically, the ERBB3 may be composed of the nucleotide sequence of SEQ ID NO: 3, but is not limited thereto.

As used herein, the term "stomach cancer stem cell" means a cancer stem cell derived from gastric cancer. Cancer stem cells have the same characteristics as normal stem cells, and generate tumors through self-renewal and differentiation ability. It also generates new tumors that distinguish them from other tumor populations, leading to cancer recurrence and metastasis.

For the purposes of the present invention, the gastric cancer stem cells may be diffuse type gastric cancer stem cells or intestinal type gastric cancer stem cells.

As used herein, the term “diffuse type gastric cancer stem cell” refers to a cancer stem cell derived from gastric cancer divided into subtypes according to a Lauren classification method based on pathological characteristics. For the purposes of the present invention, the diffuse gastric cancer stem cells may refer to sphere cells derived from the diffuse gastric cancer cell line. At this time, the term "less than gastric cancer" refers to gastric cancer that exhibits a characteristic that individual cancer cells infiltrate the stomach wall in a state in which a cancer cell has low adhesion and no clear mass is formed.

As used herein, the term "intestinal type gastric cancer stem cell" refers to a cancer stem cell derived from gastric cancer divided into intestines according to a pathological characteristic based Lauren classification method. For the purpose of the present invention, the enteric gastric cancer stem cells may refer to spear cells derived from the enteric gastric cancer cell line. At this time, the term, "stomach gastric cancer" refers to gastric cancer showing the characteristics that the cancer cells are clustered in one place to grow into a lump.

As used herein, the term "agent for measuring the expression level of a gene" is an agent for measuring the expression level of an mRNA or a protein of the gene, and specifically binds to or recognizes an mRNA or a protein of the gene or the mRNA. Or an agent capable of amplifying a protein. Specifically, primers or probes that specifically bind to the mRNA; Or an antibody or probe that specifically binds to the protein.

The agent may be labeled directly or indirectly for measuring the expression level of the gene. Specifically, the label may include ligands, beads, radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent materials, chemiluminescent materials, magnetic particles, hapten and dyes, but are not limited thereto. It doesn't work. As a specific example, the ligand includes biotin, avidin, streptoavidin, and the like, and the enzyme includes luciferase, peroxidase, beta galactosidase, and the like, and the fluorescent substance includes fluorescein, coumarin, rhodamine, Phycoerythrins and sulforodamic acid chlorides (Texas red) and the like. Most of the known labels can be used as such detectable labels, and those skilled in the art will be able to select a suitable label for the purpose of the present invention.

As used herein, the term "primer" refers to a base sequence having a short free 3 'hydroxyl group, capable of forming complementary templates and base pairs and for template strand copying. A short sequence that functions as a starting point. In the present invention, the primers used for mRNA amplification of the genes can be prepared by appropriate conditions (e.g., four different nucleoside triphosphates and polymerizers such as DNA, RNA polymerase or reverse transcriptase) and appropriate temperature in appropriate buffers. It can be a single-stranded oligonucleotide that can serve as a starting point for template-directed DNA synthesis under the appropriate length of the primer, depending on the intended use. The primer sequence need not be completely complementary to the polynucleotide of the mRNA of the gene or its complementary polynucleotide, and may be used if it is sufficiently complementary to hybridize.

Specifically, the primer specifically binding to the gene is a pair of primers for measuring SOX2 of SEQ ID NOs: 4 and 5, a pair of primers for measuring E-cadherin of SEQ ID NOs: 6 and 7, and a primer for measuring ERBB3 of SEQ ID NOs: 8 and 9 It may be one or more selected from the group consisting of a pair, but is not limited thereto.

The base sequence used in the present invention is to be construed to include a sequence showing substantial identity with the sequence described in the sequence list, in consideration of the biologically equivalent variation. The term 'substantial identity' means that if the alignment of the sequence of the present invention with any other sequence is maximally matched, the aligned sequence is analyzed using algorithms commonly used in the art. By 60% homology, more specifically 70% homology, even more specifically 80% homology, most specifically 90% homology.

Therefore, a base sequence having high homology with the nucleotide sequence represented by SEQ ID NOs: 1 to 9, for example, having a homology of 70% or more, specifically 80% or more, more specifically 90% or more. Base sequences should also be construed as being included in the scope of the present invention.

As used herein, the term “antibody” refers to a protein molecule that acts as a receptor for an antigen that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen. It includes both clone antibodies, whole antibodies and antibody fragments. The whole antibody is a structure having two full length light chains and two full length heavy chains, and each light chain is linked by a heavy chain and disulfide bond. The total antibody includes IgA, IgD, IgE, IgM and IgG, and IgG is a subtype, including IgG1, IgG2, IgG3 and IgG4.

As used herein, the term "probe" refers to a labeled nucleic acid fragment or peptide capable of specific binding with an mRNA or protein. Specific examples thereof include oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, oligonucleotide peptide probes, and polypeptide probes. It can be produced as.

In one specific embodiment of the present invention, a subtype of SNU638 or SNU484; Or spear cells having cancer stem cell characteristics in the enteric gastric cancer cell line of MKN28 or MKN74 (FIG. 1), and confirm that the spear cells exhibit cancer stem cell characteristics by confirming that they have excellent tumorigenicity and anticancer drug resistance. 2 and 3. In addition, in the spear cells having the characteristics of the cancer stem cells, the gene expression was increased compared to the normal gastric cancer cells, the expression of SOX2 and E-cadherin gene is increased in the gastric spear cancer cells of the gastric, Stomach gastric spear In cells, the expression of the ERBB3 gene was confirmed to be increased (FIGS. 4 and 5).

This suggests that the SOX2 and E-cadherin genes can be used as markers for diffuse gastric cancer stem cells, and the ERBB3 gene can be used as markers for enteric gastric cancer stem cells.

Another aspect provides a kit for distinguishing gastric cancer stem cells, comprising the composition.

At this time, the definition of "stomach cancer stem cells" is as described above.

Kit of the present invention, by using the composition for distinguishing gastric cancer stem cells, can distinguish gastric cancer stem cells by measuring the expression level of the gene, that is, the expression level of mRNA or protein. Specifically, the kit may be an RT-PCR kit, a DNA chip kit, or a protein chip kit, but is not limited thereto.

As a specific example, the kit may be a kit including essential elements necessary for performing RT-PCR. For example, the RT-PCR kit can be used in addition to the respective primers specific for the gene, in addition to test tubes or other appropriate containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), didioxynucleotides (ddNTPs) Enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also include primer pairs specific for the gene used as a quantitative control.

As another example, the kit of the present invention may be a DNA chip kit for distinguishing gastric cancer stem cells including essential elements necessary to perform a DNA chip. As used herein, the term "DNA chip" refers to one DNA microarray capable of identifying each base of hundreds of thousands of DNA at a time. The DNA chip kit is a substrate in which a nucleic acid species is attached in a gridded array to a glass surface, which is generally not larger than a flat solid support plate, typically a microscope slide. Multiple hybridization reactions occur between nucleic acids on a chip and complementary nucleic acids contained in a solution treated on a chip surface, thereby enabling mass parallel analysis.

As another example, the kit may be a protein chip kit for distinguishing gastric cancer stem cells including essential elements necessary for performing a protein chip. The protein chip may be one or more antibodies to the protein of the gene is arranged at a predetermined position on the substrate is fixed at a high density. In a method of analyzing a sample using a protein chip, gastric cancer is isolated by separating the protein from the sample, hybridizing the separated protein with the protein chip to form an antigen-antibody complex, and reading the same to confirm the presence or expression level of the protein. Stem cells can be distinguished.

Another embodiment comprises (a) measuring the expression level of one or more genes selected from the group consisting of SOX2, E-cadherin and ERBB3 from biological samples isolated from gastric cancer patients using the composition; And (b) comparing the expression level measured in step (a) with that of the control sample, providing a method for providing information for gastric cancer stem cell differentiation.

At this time, the definition of "SOX2", "E-cadherin", "ERBB3" and "gastric cancer stem cells" are as described above.

As used herein, the term "sample" refers to a direct subject which is isolated from gastric cancer patients and the expression level of the SOX2, E-cadherin or ERBB3 gene is measured, and may be, for example, cancer stem cells derived from gastric cancer. As long as the expression level can be measured, it is not limited thereto.

As used herein, the term "control sample" refers to a direct subject which is isolated from a gastric cancer patient and whose expression level of the SOX2, E-cadherin or ERBB3 gene is measured, and specifically, a general gastric cancer cell or tissue which does not include gastric cancer stem cells. And so on. For the purposes of the present invention, the control sample may be isolated from gastric cancer patients, or may be cells derived from known gastric cancer cell lines such as MKN45, SNU484, SNU601, SNU668, MKN28, MKN74, SNU216, SNU719, AGS, etc. The expression level of the gene can be measured and is not limited thereto unless it includes gastric cancer stem cells.

In the present invention, the step (a) of measuring the expression level of the gene, that is, the mRNA or protein level of the gene, can be used by any method known to those skilled in the art. Specific examples include PCR, ligase chain reaction (LCR), transcription amplification, autonomous sequence replication, nucleic acid based sequence amplification (NASBA), western blot, co-immunoprecipitation assay (Co- Immunoprecipitation assay, ELISA (Enzyme Linked Immunosorbent Assay), real-time RT-PCR, electrophoresis, tissue immunostaining (Fluorescence activated cell sorter) and FACS (Fluorescence activated cell sorter) may be used, but is not limited thereto.

At this time, when the expression level of SOX2 or E-cadherin measured in step (a) is increased than that of the control sample, it is determined that the sample contains less gastric cancer stem cells; When the expression level of ERBB3 measured in step (a) is higher than that of the control sample, it is determined that the sample contains enteric gastric cancer stem cells; Or when the expression levels of SOX2, E-cadherin and ERBB3 measured in step (a) are equal to or lower than those of the control sample, it is determined that the sample does not contain subtype and enteric gastric cancer stem cells. Can be.

In a specific embodiment of the present invention, compared to the normal gastric cancer cells, the SOX2 and E-cadherin genes are increased in the expression of diffuse gastric spear cells, the ERBB3 gene is confirmed to increase the expression in gastric gastric spear cells 4 and 5.

This suggests that gastric cancer stem cells can be distinguished according to types of genes with increased expression levels, and that gastric cancer cells and gastric cancer stem cells can be distinguished.

Another embodiment comprises the steps of: (a) measuring the expression level of SOX2 or E-cadherin gene in the subtype gastric cancer stem cells treated with the candidate substance of the subtype gastric cancer; And (b) comparing the expression level of step (a) with that of the sample not treated with the candidate.

At this time, the definition of "SOX2", "E-cadherin" and "less than gastric cancer" is as described above.

In the present invention, the diffuse gastric stem cells may be isolated by using the composition for distinguishing gastric cancer stem cells of the present invention.

As used herein, the term "candidate substance for less than gastric cancer treatment agent" is a substance that is expected to be able to treat diffuse gastric cancer, and any substance that is expected to improve or improve diffuse gastric cancer can be used without limitation. It is possible and includes all therapeutically predictable substances such as compounds, genes or proteins.

In the present invention, the step (a) of measuring the expression level of the gene, that is, the mRNA or protein level of the gene, can be used by any method known to those skilled in the art. Specific examples include PCR, ligase chain reaction (LCR), transcription amplification, autonomous sequence replication, nucleic acid based sequence amplification (NASBA), western blot, co-immunoprecipitation assay (Co- Immunoprecipitation assay, ELISA (Enzyme Linked Immunosorbent Assay), real-time RT-PCR, electrophoresis, tissue immunostaining (Fluorescence activated cell sorter) and FACS (Fluorescence activated cell sorter) may be used, but is not limited thereto.

In addition, the present invention, when the expression level of the SOX2 or E-cadherin measured in step (a) is reduced than that of the sample not treated with the candidate, judging the candidate as a less gastric cancer treatment ( c) may further comprise a step.

Another embodiment comprises the steps of (a) measuring the expression level of the ERBB3 gene in enteric gastric cancer stem cells treated with a candidate for enteric gastric cancer treatment; And (b) comparing the expression level of step (a) with that of the sample not treated with the candidate.

At this time, the definition of the "ERBB3" and "janggyeong stomach cancer" is as described above.

In the present invention, the enteric gastric cancer stem cells may be isolated using the composition for distinguishing gastric cancer stem cells of the present invention.

As used herein, the term "cancer candidate for treating gastric cancer" is a substance that is expected to be able to treat gastric cancer, and any substance that is expected to improve or improve intestinal gastric cancer can be used without limitation. It includes all therapeutically predictable substances such as compounds, genes or proteins.

In the present invention, the step (a) of measuring the expression level of the gene, that is, the mRNA or protein level of the gene, can be used by any method known to those skilled in the art. Specific examples include PCR, ligase chain reaction (LCR), transcription amplification, autonomous sequence replication, nucleic acid based sequence amplification (NASBA), western blot, co-immunoprecipitation assay (Co- Immunoprecipitation assay, ELISA (Enzyme Linked Immunosorbent Assay), real-time RT-PCR, electrophoresis, tissue immunostaining (Fluorescence activated cell sorter) and FACS (Fluorescence activated cell sorter) may be used, but is not limited thereto.

In addition, the present invention further comprises the step (c) of judging the candidate as an enteric gastric cancer when the expression level of ERBB3 measured in step (a) is lower than that of the sample not treated with the candidate. It may include.

SOX2 and E-cadherin genes are subtypes; And since the ERBB3 gene can be used as an enteric gastric cancer stem cell marker, the composition of the present invention for measuring the expression level of the gene can not only distinguish between the enteric / less than gastric cancer cells, the therapeutic agent according to the pathological characteristics of gastric cancer It can be useful for development.

Figure 1 relates to a method of forming a spear cell and the appearance of the formed cells from each gastric cancer cell line, a is a schematic diagram showing a method of forming a spear cell, b is a schematic diagram showing a method of forming a spear cell, b is a diffuse (diffuse) type) image showing a spheroid or non-spheroid cell formed from SNU638, a gastric cancer cell line, c is a graph and image showing the number of spear cells and the number of surviving spear cells according to the formation stage, and d is an image showing the shape of the spear cells formed along the gastric cancer cell line. MKN45, SNU484, SNU601 and SNU668 are diffuse type gastric cancer cell lines; MKN28, MKN74, SNU216, SNU719 and AGS refer to an intestinal type gastric cancer cell line.
Figure 2 shows the tumor-forming ability of the spear cells formed from the MKN28 cell line according to an embodiment of the present invention, a shows the size of tumors formed in normal gastric cancer cells (parents) and spheroid cells (spheroid) It is a graph. b is a table showing the degree of tumor formation according to the number of injected cancer cells, and is described in the form of '(number of mice forming tumors) / (number of mice tested)'. c is an image showing the appearance of the formed tumor.
Figure 3 shows the resistance of the spear cells to the anticancer agent formed from each gastric cancer cell line according to an embodiment of the present invention, a is a normal gastric cancer cells (parents; parents) and spheroid cells (spheroid) cells when the anticancer agent treatment FACS analysis showing the degree of death, b is a graph quantifying the result of the a. c is a Western blot image analyzing the expression of proteins related to apoptosis / cell survival in normal gastric cancer cells and spear cells when treated with anticancer drugs. At this time, DMSO (dimethyl sulfoxide), 5FU (Fluorouracil), CDDP (Cisplatin) and PTX (Paclitaxel) were used as a control.
Figure 4 shows the results of analyzing the expression amount of the OCT4, SOX2 and NANOG genes in spear cells, and normal parent cells formed from each gastric cancer cell line according to an embodiment of the present invention. a is a graph analyzing the mRNA expression amount of the gene, which is expressed as the expression amount in the spear cells compared to the expression amount in the parent cell. b is a Western blot image analyzing the protein expression amount of the gene, P means parent cells, S means spear cells. At this time, β-actin was used as a standard gene of expression amount.
Figure 5 is a protein of the E-cadherin, phosphorylated (Phospho) -ERBB3 (Y1289) and ERBB3 genes are expressed in the sphere cells formed from each gastric cancer cell line, and normal parent cells according to an embodiment of the present invention Western blot image of the amount of expression analyzed. P means parent cell, S means spear cell. At this time, β-actin was used as a standard gene of expression amount.
Figure 6 relates to the sphere-forming ability of the subtype gastric cancer cell line (SNU638 or SNU484) suppressed expression of the SOX2 or E-cadherin gene produced according to one embodiment of the present invention. a is a Western blot image showing the amount of SOX2 or E-cadherin protein expressed in SNU638 or SNU484, b is a image showing spear cells formed from SNU638 or SNU484, c is a graph showing the number of spear cells formed, and d is a graph showing the diameter of the formed sphere cells. At this time, Dox or Doxy means doxycycline (Doxycycline), β-actin was used as a standard gene of the expression amount.
Figure 7 relates to the sphere-forming ability of the enteric gastric cancer cell line (MKN28 or MKN74) suppressed the expression of the ERBB3 gene produced according to one embodiment of the present invention. a is a Western blot image showing the amount of protein of ERBB3 expressed in the MKN28 or MKN74, b is an image showing the sphere cells formed from the MKN28 or MKN74, c is a graph showing the number of sphere cells formed, and d is a sphere cell formed Graph showing the diameter of the In this case, Dox or Doxy means doxycycline, and β-actin was used as a standard gene for the expression amount.
Figure 8 relates to the tumorigenic capacity of the enteric gastric cancer cell line (MKN28) suppressed the expression of the ERBB3 gene produced according to one embodiment of the present invention. a and b are graphs and images comparing the size of tumors formed when the expression of the ERBB3 gene is normal (Doxy-) or inhibited (Doxy +). In this case, Doxy means doxycycline.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example  One. Spear and cultivation of (sphere) cells

In order to discover gastric cancer stem cell specific markers, spear cells having gastric cancer stem cell characteristics were first isolated and cultured by the method according to Examples 1-1 to 1-3.

Example  1-1. Culture of Common Gastric Cancer Cells

SNU638 and SNU484 were used as the diffuse type gastric cancer cell lines, and MKN28 and MKN74 were used as the intestinal type gastric cancer cell lines. The cell lines were all purchased from the Bank of Korea Cell Line, and general gastric cancer cells were cultured using RPMI1640 medium containing 10% FBS, 1% antibiotic, 25 mM HEPES, 25 mM NaHCO ₃ . All cell cultures were run at 37 ° C. in a wet incubator containing 5% carbon dioxide.

Example  1-2. Spear  Formation and Culture of Cells

In order to form spear cells with gastric cancer stem cell characteristics, a spheroid film (Spheroid Film, Incyto) was used.

Specifically, spear cells were formed according to the method shown in FIG. First, each gastric cancer cell line (NU638, SNU484, MKN28, MKN74) was dispensed in 5 x 10 ³ numbers on a spheroid film fixed to a 60 mm Petri dish. After 30 minutes of cell dispensing, the medium was replaced to remove cells that did not enter each microwell, and microscopic confirmation of the entry of one cell into each microwell was followed by incubation for 2 weeks with medium replacement once every 4 days. Thereby forming spear cells.

In this case, RPMI1640 medium containing recombinant human EGF 20ng / mL, recombinant human FGF 10ng / mL, 1X B27 supplement, 1% antibiotic, 10mM HEPES was used for culturing the spear cells.

Example  1-3. Spear  Isolation of Cells

As spear cells, cells were closely adhered to form a clear boundary, and spherical cells were selected to have a diameter of 100 μm. At this time, the adhesion ability between the cells was excluded to form a grape cluster shape.

Thereafter, the spear cells isolated by the above method were passaged to obtain a large number of spear cells. Cells separated into individual cells by treatment with 0.05% trypsin-EDTA for 5 minutes were incubated again in a new spheroid film for 2 weeks in the same manner. In addition, the number of wells in which the spheres were formed was counted to confirm the change in the sphere formation capacity.

As a result, as shown in b and d of FIG. 1, spherical spear cells could be formed from SNU638 and SNU484, which are diffuse gastric cancer cells, and MKN28 and MKN74, which are enteric gastric cancer cell lines, as shown in c of FIG. 1. It was confirmed that a higher number of surviving spear cells can be obtained at subculture.

Example  2. Spear  Determining the ability of tumor cells to form

One characteristic of cancer stem cells is their ability to replicate. Thus, in order to confirm that the spear cells isolated in Example 1 have the characteristics of cancer stem cells, the tumor formation ability of the spear cells was confirmed.

Specifically, 5 week old male immunosuppressed mice were used. Parent cells (general gastric cancer cells) and spear cells of the MKN28 cell line were prepared in 100 uL PBS with 5 x 10 ⁵ cells each and injected into the left and right flanks of mice to form tumors. Euthanasia was performed to relieve pain when the tumor was larger than 1,000 mm ³ or necrosis occurred. Experimental groups were assigned to four.

As a result, as can be seen in Figure 2a, as compared to the parent cells, it was confirmed that the tumor formation ability in the isolated sphere cells. In addition, as shown in b and c of FIG. 2, when 5 x 10 ⁵ cancer cells were injected, tumors were formed in all 4 mice injected with the parent cells and the spear cells. It was confirmed that the size is superior to the tumor derived from the parent cell. Through the above results, it was found that the spear cells exhibit the characteristics of stem cells with high tumorigenic capacity.

Example  3. Spear  Confirmation of anticancer drug resistance of cells

One of the characteristics of cancer stem cells is resistance to cancer and relapse. Thus, in order to confirm whether the spear cells isolated in Example 1 have the characteristics of cancer stem cells, the anticancer drug resistance of the spear cells was confirmed.

Specifically, 200 μg / mL fluorouracil, 1 μg / mL paclitaxel, 50 μg / mL cisplatin were used as anticancer agents, and DMSO was used as a control. Each material was treated with parent cells (general gastric cancer cells) and spear cells, and killed cells were measured 24 hours later using an Annexin V-FITC apoptosis detection kit. Cells were analyzed by FACS calibur, and early apoptotic and necrotic cells were distinguished by Annexin V-FITC and PI staining.

As a result, as shown in a and b of Figure 3, compared to the parent cells, the isolated spear cells were confirmed that the number of cells killed by the anticancer agent is relatively reduced. Through the results, it was found that the spear cells exhibit the characteristics of stem cells with high anticancer drug resistance.

In addition, changes in protein expression associated with apoptosis were analyzed using Western blot method. 70-80% grown cells were lysed using PRO-PREP lysis buffer, and SDS-PAGE was performed using 30 μg of protein by measuring the concentration of total lysate through BCA assay. The protein was then transferred to PVDF membrane and blocked for 1 hour with PBS-T solution containing 5% skim milk powder. The primary antibody was reacted overnight at 4 ° C, and the secondary antibody conjugated with HRP (horseradish phosphatase) was reacted at room temperature for 1 hour. Protein expression bands were identified with ECL solution and observed through ChemiDoc system. At this time, the primary antibody was PARP, Caspase3, Bcl-xL, Bcl-2.

As a result, as shown in c of Figure 3, compared to the parent cells, the isolated spear cells, the expression of Cleaved PARP, Cleaved Caspase 3, which is a protein related to cell death during anticancer treatment, did not change, but the protein related to cell survival The expression of Bcl-xL was confirmed to increase. Through the results, it was found that the spear cells exhibit the characteristics of stem cells with high anticancer drug resistance.

Example  4. according to the type of stomach cancer Spear  Cellular Marker  Genetic Identification

Example  4-1. Self-replicating Regulator Expression amount  Confirm

To identify marker genes of spear cells, ie, gastric cancer stem cells, according to gastric cancer types, first, mRNA and protein levels of OCT4, SOX2, and NANOG, which are known as self-replicating regulators in stem cells, were measured by subcutaneous gastric cancer cell lines SNU638, SNU484; And MKN28 and MKN74 cells, which are enteric gastric cancer cell lines.

Specifically, total RNA was isolated by TRIzol method. 1 μg of total RNA was quantified by Nanodrop and cDNA was synthesized using M-MLV reverse transcriptase. Amplification of cDNA was performed using CFX, iQ SYBR Green Supermix. MRNA expression level of each gene amplified by real-time PCR was corrected by the mRNA value of GAPDH. At this time, the primers used are summarized in Table 1 below.

gene division Sequence (5 '→ 3') OCT4
Forward direction SEQ ID NO: 4 GCA GCG ACT ATG CAC AAC GA Reverse SEQ ID NO: 5 CCA GAG TGG TGA CGG AGA CA SOX2
Forward direction SEQ ID NO: 6 CAT CAC CCA CAG CAA ATG ACA Reverse SEQ ID NO: 7 GCT CCT ACC GTA CCA CTA GAA CTT NANOG
Forward direction SEQ ID NO: 8 AAT ACC TCA GCC TCC AGC AGA TG Reverse SEQ ID NO: 9 TGC GTC ACA CCA TTG CTA TTC TTC GAPDH
Forward direction SEQ ID NO: 10 CAG CCT CAA GAT CAT CAG CAA TG Reverse SEQ ID NO: 11 TCA TGA GTC CTT CCA CGA TAC CA

As a result, as can be seen in Figure 4a showing the amount of expression in the spear cells relative to the amount of gene expression in the parent cells, the spear cells of SNU638 and SNU484, the diffuse gastric cancer cell lines, compared to the parent cells (general gastric cancer cells), It was confirmed that the mRNA expression of SOX2 and NANOG genes increased significantly. However, in MKN28 and MKN74, the enteric gastric cancer cell lines, it was confirmed that the mRNA expression level of the genes in the spear cell was not changed compared with the parent cell (general gastric cancer cell).

In addition, Western blot was performed in the same manner as in Example 3 to analyze the protein expression level of the OCT4, SOX2, NANOG gene in the cells.

As a result, as can be seen in Figure 4b, it was confirmed that the spear cells (S) of the diffuse gastric cancer cell lines SNU638 and SNU484 significantly increased the amount of protein expression of the SOX2 gene compared to the parent cell (P). However, in the gastric cancer cell lines MKN28 and MKN74, it was confirmed that the amount of protein expression of the genes in spear cells was unchanged or decreased compared with the parent cells.

Through the above results, it was found that the SOX2 gene can be used as a marker of diffuse gastric cancer stem cells.

Example  4-2. Expression of major signaling factors

In order to identify marker genes of spear cells according to the types of gastric cancer, ie, gastric cancer stem cells, protein expression levels of signaling factors that have a major influence on the development of cancer were measured in the subtype gastric cancer cell lines SNU638 and SNU484; And MKN28 and MKN74 cells, which are enteric gastric cancer cell lines.

Specifically, Western blot was performed in the same manner as in Example 3 to investigate the expression levels of E-cadherin, phosphorylated (Phospho) -ERBB3 (Y1289), and ERBB3 in the cells. At this time, E-cadherin is involved in the epithelial-to-mesenchymal transition (EMT) process important for cancer development (Clin Cancer Res. 2004 Jun 15; 10 (12 Pt 1): 4125-33.), ERBB3 and phosphorylation -ERBB3 is involved in ERBB3 signaling, which is important for the proliferation of cancer cells (Acta Biochim Biophys Sin (2014) 46 (3): 190-198).

As a result, as can be seen in Figure 5, the spear cells (S) of the diffuse gastric cancer cell lines SNU638, SNU484 induce protein expression of the E-cadherin gene compared to the parent cells (general gastric cancer cells, P), It was confirmed that the amount increased significantly. In addition, it was confirmed that the spear cells of the enteric gastric cancer cell lines MKN28 and MKN74 induce protein expression of the ERBB3 gene and significantly increase the amount of the ERBB3 gene.

Through the above results, it can be seen that E-cadherin gene can be used as a marker of diffuse gastric cancer stem cells, ERBB3 gene can be used as a marker of enteric gastric cancer stem cells.

Example 5 Spear Formation Ability of Gastric Cancer Stem Cells with Inhibited Marker Gene Expression

Example 5-1. Construction method of vector which suppresses expression of marker gene

In order to produce gastric cancer stem cells in which the expression of SOX2, E-cadherin or ERBB3 was suppressed as a marker gene, a recombinant vector capable of inhibiting the expression of the gene was constructed and used.

First, a Tet-pLKO-puro-Scrambled (# 47541) vector and a Tet-pLKO-puro-SOX2 (# 47540) vector were used to prepare a control group or a SOX2 expression suppressing group, respectively.

Meanwhile, pLKO-Tet-On (# 21915) vectors were used for cloning to construct vectors targeting E-cadherin and ERBB3. AgeI / EcoRI restriction enzymes were treated to remove the stuffer portion and ERBB3 shRNA oligomers were introduced at that location. At this time, the oligomers used are listed in Table 2 below.

Kinds Top Bottom ERBB3
shRNA oligomer 5'-CCGGCCAGAGCTTCAAGACTGTTTACTCGAGTAAACAGTCTTGAAGCTCTGGTTTTT-3 '
(SEQ ID NO: 12) 5'-AATTAAAAACCAGAGCTTCAAGACTGTTTACTCGAGTAAACAGTCTTGAAGCTCTGG-3 '
(SEQ ID NO: 13) E-Cardherin
shRNA oligomer 5'-CCGGGCAGAAATTATTGGGCTCTTTCTCGAGAAAGAGCCCAATAATTTCTGCTTTTT-3 '
(SEQ ID NO: 14) 5'-AATTAAAAAGCAGAAATTATTGGGCTCTTTCTCGAGAAAGAGCCCAATAATTTCTGC-3 '
(SEQ ID NO: 15)

Example 5-2. Production of virus containing vector and infection with target cell

In order to prepare a cell line of gastric cancer stem cells whose expression of SOX2, E-cadherin or ERBB3 was suppressed, a virus comprising the vector constructed in Example 5-1 was prepared. Each vector was transfected with HEK-293T cell line using lipofectamin with pMD2.G (# 12259) envelope plasmid and psPAX2 (# 12260) packaging plasmid. Obtained. Afterwards, the produced virus was infected with the subtype gastric cancer stem cell lines SNU638 and SNU484, the enteric gastric cancer stem cells MKN28 and MKN74, and selected with a culture medium to which 1 μg / mL of puromycin was added. Lastly, in order to induce shRNA expression in selected cells, gastric cancer stem cells with suppressed expression of each gene were prepared by culturing for 72 hours in medium containing 500 ng / mL of doxycycline or not.

Example 5-3. Spear Formation Capacity of Gastric Cancer Stem Cells with Inhibition of Marker Gene Expression

Spear formation ability of gastric cancer stem cells whose expression of SOX2, E-cadherin or ERBB3 was suppressed was confirmed.

As a result, as can be seen in Figure 6, when the expression of SOX2 and E-cadherin is suppressed in SNU638 and SNU484 Diffuse gastric cancer stem cell lines, it was confirmed that the sphere formation ability of the diffuse gastric cancer stem cells.

Specifically, as can be seen in Figure 6a, when the shRNA of each gene is expressed by the treatment of toxin cyclin, it was confirmed that the expression of each gene is reduced. In addition, as can be seen from b to d of Figure 6, it was confirmed that when the expression of each gene is reduced by shRNA, the size (diameter) and the number of spheres is significantly reduced.

As a result, as shown in Figure 7, when the expression of ERBB3 is suppressed in the enteric gastric cancer stem cell lines MKN28 and MKN74, it was confirmed that the sphere-forming ability of the enteric gastric cancer stem cells.

Specifically, as shown in Figure 7a, when the shRNA of each gene is expressed by the treatment of toxin cyclin, it was confirmed that the expression of each gene is reduced. In addition, as can be seen from b to d of FIG. 7, it was confirmed that the size (diameter) and the number of spheres decreased significantly when the expression of each gene was reduced by shRNA.

In particular, as shown in Figure 8, when the expression of ERBB3 in enteric gastric cancer stem cells was confirmed that the tumor formation ability is also reduced.

Through the above results, expression of the marker gene SOX2 or E-cadherin is suppressed in diffuse gastric cancer stem cells, and expression of the marker gene ERBB3 is inhibited in intestinal gastric cancer stem cells. It was found that the spear forming ability or the tumor forming ability decreased. Accordingly, it can be seen that the genes can be used as specific marker genes for the subtype or enteric gastric cancer stem cells, respectively.

<110> Korea University Research and Business Foundation <120> Gastric cancer stem cell markers and uses approximately <130> KPA161352-KR-P1 <150> KR 10-2017-0002076 <151> 2017-01-05 <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 2450 <212> DNA <213> Homo sapiens <400> 1 aagggggaaa gtagtttgct gcctctttaa gactaggact gagagaaaga agaggagaga 60 gaaagaaagg gagagaagtt tgagccccag gcttaagcct ttccaaaaaa taataataac 120 aatcatcggc ggcggcagga tcggccagag gaggagggaa gcgctttttt tgatcctgat 180 tccagtttgc ctctctcttt ttttccccca aattattctt cgcctgattt tcctcgcgga 240 gccctgcgct cccgacaccc ccgcccgcct cccctcctcc tctccccccg cccgcgggcc 300 ccccaaagtc ccggccgggc cgagggtcgg cggccgccgg cgggccgggc ccgcgcacag 360 cgcccgcatg tacaacatga tggagacgga gctgaagccg ccgggcccgc agcaaacttc 420 ggggggcggc ggcggcaact ccaccgcggc ggcggccggc ggcaaccaga aaaacagccc 480 ggaccgcgtc aagcggccca tgaatgcctt catggtgtgg tcccgcgggc agcggcgcaa 540 gatggcccag gagaacccca agatgcacaa ctcggagatc agcaagcgcc tgggcgccga 600 gtggaaactt ttgtcggaga cggagaagcg gccgttcatc gacgaggcta agcggctgcg 660 agcgctgcac atgaaggagc acccggatta taaataccgg ccccggcgga aaaccaagac 720 gctcatgaag aaggataagt acacgctgcc cggcgggctg ctggcccccg gcggcaatag 780 catggcgagc ggggtcgggg tgggcgccgg cctgggcgcg ggcgtgaacc agcgcatgga 840 cagttacgcg cacatgaacg gctggagcaa cggcagctac agcatgatgc aggaccagct 900 gggctacccg cagcacccgg gcctcaatgc gcacggcgca gcgcagatgc agcccatgca 960 ccgctacgac gtgagcgccc tgcagtacaa ctccatgacc agctcgcaga cctacatgaa 1020 cggctcgccc acctacagca tgtcctactc gcagcagggc acccctggca tggctcttgg 1080 ctccatgggt tcggtggtca agtccgaggc cagctccagc ccccctgtgg ttacctcttc 1140 ctcccactcc agggcgccct gccaggccgg ggacctccgg gacatgatca gcatgtatct 1200 ccccggcgcc gaggtgccgg aacccgccgc ccccagcaga cttcacatgt cccagcacta 1260 ccagagcggc ccggtgcccg gcacggccat taacggcaca ctgcccctct cacacatgtg 1320 agggccggac agcgaactgg aggggggaga aattttcaaa gaaaaacgag ggaaatggga 1380 ggggtgcaaa agaggagagt aagaaacagc atggagaaaa cccggtacgc tcaaaaagaa 1440 aaaggaaaaa aaaaaatccc atcacccaca gcaaatgaca gctgcaaaag agaacaccaa 1500 tcccatccac actcacgcaa aaaccgcgat gccgacaaga aaacttttat gagagagatc 1560 ctggacttct ttttggggga ctatttttgt acagagaaaa cctggggagg gtggggaggg 1620 cgggggaatg gaccttgtat agatctggag gaaagaaagc tacgaaaaac tttttaaaag 1680 ttctagtggt acggtaggag ctttgcagga agtttgcaaa agtctttacc aataatattt 1740 agagctagtc tccaagcgac gaaaaaaatg ttttaatatt tgcaagcaac ttttgtacag 1800 tatttatcga gataaacatg gcaatcaaaa tgtccattgt ttataagctg agaatttgcc 1860 aatatttttc aaggagaggc ttcttgctga attttgattc tgcagctgaa atttaggaca 1920 gttgcaaacg tgaaaagaag aaaattattc aaatttggac attttaattg tttaaaaatt 1980 gtacaaaagg aaaaaattag aataagtact ggcgaaccat ctctgtggtc ttgtttaaaa 2040 agggcaaaag ttttagactg tactaaattt tataacttac tgttaaaagc aaaaatggcc 2100 atgcaggttg acaccgttgg taatttataa tagcttttgt tcgatcccaa ctttccattt 2160 tgttcagata aaaaaaacca tgaaattact gtgtttgaaa tattttctta tggtttgtaa 2220 tatttctgta aatttattgt gatattttaa ggttttcccc cctttatttt ccgtagttgt 2280 attttaaaag attcggctct gtattatttg aatcagtctg ccgagaatcc atgtatatat 2340 ttgaactaat atcatcctta taacaggtac attttcaact taagttttta ctccattatg 2400 cacagtttga gataaataaa tttttgaaat atggacactg aaaaaaaaaa 2450 <210> 2 <211> 4845 <212> DNA <213> Homo sapiens <400> 2 tcagtggcgt cggaactgca aagcacctgt gagcttgcgg aagtcagttc agactccagc 60 ccgctccagc ccggcccgac ccgaccgcac ccggcgcctg ccctcgctcg gcgtccccgg 120 ccagccatgg gcccttggag ccgcagcctc tcggcgctgc tgctgctgct gcaggtctcc 180 tcttggctct gccaggagcc ggagccctgc caccctggct ttgacgccga gagctacacg 240 ttcacggtgc cccggcgcca cctggagaga ggccgcgtcc tgggcagagt gaattttgaa 300 gattgcaccg gtcgacaaag gacagcctat ttttccctcg acacccgatt caaagtgggc 360 acagatggtg tgattacagt caaaaggcct ctacggtttc ataacccaca gatccatttc 420 ttggtctacg cctgggactc cacctacaga aagttttcca ccaaagtcac gctgaataca 480 gtggggcacc accaccgccc cccgccccat caggcctccg tttctggaat ccaagcagaa 540 ttgctcacat ttcccaactc ctctcctggc ctcagaagac agaagagaga ctgggttatt 600 cctcccatca gctgcccaga aaatgaaaaa ggcccatttc ctaaaaacct ggttcagatc 660 aaatccaaca aagacaaaga aggcaaggtt ttctacagca tcactggcca aggagctgac 720 acaccccctg ttggtgtctt tattattgaa agagaaacag gatggctgaa ggtgacagag 780 cctctggata gagaacgcat tgccacatac actctcttct ctcacgctgt gtcatccaac 840 gggaatgcag ttgaggatcc aatggagatt ttgatcacgg taaccgatca gaatgacaac 900 aagcccgaat tcacccagga ggtctttaag gggtctgtca tggaaggtgc tcttccagga 960 acctctgtga tggaggtcac agccacagac gcggacgatg atgtgaacac ctacaatgcc 1020 gccatcgctt acaccatcct cagccaagat cctgagctcc ctgacaaaaa tatgttcacc 1080 attaacagga acacaggagt catcagtgtg gtcaccactg ggctggaccg agagagtttc 1140 cctacgtata ccctggtggt tcaagctgct gaccttcaag gtgaggggtt aagcacaaca 1200 gcaacagctg tgatcacagt cactgacacc aacgataatc ctccgatctt caatcccacc 1260 acgtacaagg gtcaggtgcc tgagaacgag gctaacgtcg taatcaccac actgaaagtg 1320 actgatgctg atgcccccaa taccccagcg tgggaggctg tatacaccat attgaatgat 1380 gatggtggac aatttgtcgt caccacaaat ccagtgaaca acgatggcat tttgaaaaca 1440 gcaaagggct tggattttga ggccaagcag cagtacattc tacacgtagc agtgacgaat 1500 gtggtacctt ttgaggtctc tctcaccacc tccacagcca ccgtcaccgt ggatgtgctg 1560 gatgtgaatg aagcccccat ctttgtgcct cctgaaaaga gagtggaagt gtccgaggac 1620 tttggcgtgg gccaggaaat cacatcctac actgcccagg agccagacac atttatggaa 1680 cagaaaataa catatcggat ttggagagac actgccaact ggctggagat taatccggac 1740 actggtgcca tttccactcg ggctgagctg gacagggagg attttgagca cgtgaagaac 1800 agcacgtaca cagccctaat catagctaca gacaatggtt ctccagttgc tactggaaca 1860 gggacacttc tgctgatcct gtctgatgtg aatgacaacg cccccatacc agaacctcga 1920 actatattct tctgtgagag gaatccaaag cctcaggtca taaacatcat tgatgcagac 1980 cttcctccca atacatctcc cttcacagca gaactaacac acggggcgag tgccaactgg 2040 accattcagt acaacgaccc aacccaagaa tctatcattt tgaagccaaa gatggcctta 2100 gaggtgggtg actacaaaat caatctcaag ctcatggata accagaataa agaccaagtg 2160 accaccttag aggtcagcgt gtgtgactgt gaaggggccg ctggcgtctg taggaaggca 2220 cagcctgtcg aagcaggatt gcaaattcct gccattctgg ggattcttgg aggaattctt 2280 gctttgctaa ttctgattct gctgctcttg ctgtttcttc ggaggagagc ggtggtcaaa 2340 gagcccttac tgcccccaga ggatgacacc cgggacaacg tttattacta tgatgaagaa 2400 ggaggcggag aagaggacca ggactttgac ttgagccagc tgcacagggg cctggacgct 2460 cggcctgaag tgactcgtaa cgacgttgca ccaaccctca tgagtgtccc ccggtatctt 2520 ccccgccctg ccaatcccga tgaaattgga aattttattg atgaaaatct gaaagcggct 2580 gatactgacc ccacagcccc gccttatgat tctctgctcg tgtttgacta tgaaggaagc 2640 ggttccgaag ctgctagtct gagctccctg aactcctcag agtcagacaa agaccaggac 2700 tatgactact tgaacgaatg gggcaatcgc ttcaagaagc tggctgacat gtacggaggc 2760 ggcgaggacg actaggggac tcgagagagg cgggccccag acccatgtgc tgggaaatgc 2820 agaaatcacg ttgctggtgg tttttcagct cccttccctt gagatgagtt tctggggaaa 2880 aaaaagagac tggttagtga tgcagttagt atagctttat actctctcca ctttatagct 2940 ctaataagtt tgtgttagaa aagtttcgac ttatttctta aagctttttt ttttttccca 3000 tcactcttta catggtggtg atgtccaaaa gatacccaaa ttttaatatt ccagaagaac 3060 aactttagca tcagaaggtt cacccagcac cttgcagatt ttcttaagga attttgtctc 3120 acttttaaaa agaaggggag aagtcagcta ctctagttct gttgttttgt gtatataatt 3180 ttttaaaaaa aatttgtgtg cttctgctca ttactacact ggtgtgtccc tctgcctttt 3240 tttttttttt aagacagggt ctcattctat cggccaggct ggagtgcagt ggtgcaatca 3300 cagctcactg cagccttgtc ctcccaggct caagctatcc ttgcacctca gcctcccaag 3360 tagctgggac cacaggcatg caccactacg catgactaat tttttaaata tttgagacgg 3420 ggtctccctg tgttacccag gctggtctca aactcctggg ctcaagtgat cctcccatct 3480 tggcctccca gagtattggg attacagaca tgagccactg cacctgccca gctccccaac 3540 tccctgccat tttttaagag acagtttcgc tccatcgccc aggcctggga tgcagtgatg 3600 tgatcatagc tcactgtaac ctcaaactct ggggctcaag cagttctccc accagcctcc 3660 tttttatttt tttgtacaga tggggtcttg ctatgttgcc caagctggtc ttaaactcct 3720 ggcctcaagc aatccttctg ccttggcccc ccaaagtgct gggattgtgg gcatgagctg 3780 ctgtgcccag cctccatgtt ttaatatcaa ctctcactcc tgaattcagt tgctttgccc 3840 aagataggag ttctctgatg cagaaattat tgggctcttt tagggtaaga agtttgtgtc 3900 tttgtctggc cacatcttga ctaggtattg tctactctga agacctttaa tggcttccct 3960 ctttcatctc ctgagtatgt aacttgcaat gggcagctat ccagtgactt gttctgagta 4020 agtgtgttca ttaatgttta tttagctctg aagcaagagt gatatactcc aggacttaga 4080 atagtgccta aagtgctgca gccaaagaca gagcggaact atgaaaagtg ggcttggaga 4140 tggcaggaga gcttgtcatt gagcctggca atttagcaaa ctgatgctga ggatgattga 4200 ggtgggtcta cctcatctct gaaaattctg gaaggaatgg aggagtctca acatgtgttt 4260 ctgacacaag atccgtggtt tgtactcaaa gcccagaatc cccaagtgcc tgcttttgat 4320 gatgtctaca gaaaatgctg gctgagctga acacatttgc ccaattccag gtgtgcacag 4380 aaaaccgaga atattcaaaa ttccaaattt ttttcttagg agcaagaaga aaatgtggcc 4440 ctaaaggggg ttagttgagg ggtagggggt agtgaggatc ttgatttgga tctcttttta 4500 tttaaatgtg aatttcaact tttgacaatc aaagaaaaga cttttgttga aatagcttta 4560 ctgtttctca agtgttttgg agaaaaaaat caaccctgca atcacttttt ggaattgtct 4620 tgatttttcg gcagttcaag ctatatcgaa tatagttctg tgtagagaat gtcactgtag 4680 ttttgagtgt atacatgtgt gggtgctgat aattgtgtat tttctttggg ggtggaaaag 4740 gaaaacaatt caagctgaga aaagtattct caaagatgca tttttataaa ttttattaaa 4800 caattttgtt aaaccattaa aaaaaaaaaa aaaaaaaaaa aaaaa 4845 <210> 3 <211> 5765 <212> DNA <213> Homo sapiens <400> 3 actccagcct cgcgcgggag ggggcgcggc cgtgactcac ccccttccct ctgcgttcct 60 ccctccctct ctctctctct ctcacacaca cacacccctc ccctgccatc cctccccgga 120 ctccggctcc ggctccgatt gcaatttgca acctccgctg ccgtcgccgc agcagccacc 180 aattcgccag cggttcaggt ggctcttgcc tcgatgtcct agcctagggg cccccgggcc 240 ggacttggct gggctccctt caccctctgc ggagtcatga gggcgaacga cgctctgcag 300 gtgctgggct tgcttttcag cctggcccgg ggctccgagg tgggcaactc tcaggcagtg 360 tgtcctggga ctctgaatgg cctgagtgtg accggcgatg ctgagaacca ataccagaca 420 ctgtacaagc tctacgagag gtgtgaggtg gtgatgggga accttgagat tgtgctcacg 480 ggacacaatg ccgacctctc cttcctgcag tggattcgag aagtgacagg ctatgtcctc 540 gtggccatga atgaattctc tactctacca ttgcccaacc tccgcgtggt gcgagggacc 600 caggtctacg atgggaagtt tgccatcttc gtcatgttga actataacac caactccagc 660 cacgctctgc gccagctccg cttgactcag ctcaccgaga ttctgtcagg gggtgtttat 720 attgagaaga acgataagct ttgtcacatg gacacaattg actggaggga catcgtgagg 780 gaccgagatg ctgagatagt ggtgaaggac aatggcagaa gctgtccccc ctgtcatgag 840 gtttgcaagg ggcgatgctg gggtcctgga tcagaagact gccagacatt gaccaagacc 900 atctgtgctc ctcagtgtaa tggtcactgc tttgggccca accccaacca gtgctgccat 960 gatgagtgtg ccgggggctg ctcaggccct caggacacag actgctttgc ctgccggcac 1020 ttcaatgaca gtggagcctg tgtacctcgc tgtccacagc ctcttgtcta caacaagcta 1080 actttccagc tggaacccaa tccccacacc aagtatcagt atggaggagt ttgtgtagcc 1140 agctgtcccc ataactttgt ggtggatcaa acatcctgtg tcagggcctg tcctcctgac 1200 aagatggaag tagataaaaa tgggctcaag atgtgtgagc cttgtggggg actatgtccc 1260 aaagcctgtg agggaacagg ctctgggagc cgcttccaga ctgtggactc gagcaacatt 1320 gatggatttg tgaactgcac caagatcctg ggcaacctgg actttctgat caccggcctc 1380 aatggagacc cctggcacaa gatccctgcc ctggacccag agaagctcaa tgtcttccgg 1440 acagtacggg agatcacagg ttacctgaac atccagtcct ggccgcccca catgcacaac 1500 ttcagtgttt tttccaattt gacaaccatt ggaggcagaa gcctctacaa ccggggcttc 1560 tcattgttga tcatgaagaa cttgaatgtc acatctctgg gcttccgatc cctgaaggaa 1620 attagtgctg ggcgtatcta tataagtgcc aataggcagc tctgctacca ccactctttg 1680 aactggacca aggtgcttcg ggggcctacg gaagagcgac tagacatcaa gcataatcgg 1740 ccgcgcagag actgcgtggc agagggcaaa gtgtgtgacc cactgtgctc ctctggggga 1800 tgctggggcc caggccctgg tcagtgcttg tcctgtcgaa attatagccg aggaggtgtc 1860 tgtgtgaccc actgcaactt tctgaatggg gagcctcgag aatttgccca tgaggccgaa 1920 tgcttctcct gccacccgga atgccaaccc atggagggca ctgccacatg caatggctcg 1980 ggctctgata cttgtgctca atgtgcccat tttcgagatg ggccccactg tgtgagcagc 2040 tgcccccatg gagtcctagg tgccaagggc ccaatctaca agtacccaga tgttcagaat 2100 gaatgtcggc cctgccatga gaactgcacc caggggtgta aaggaccaga gcttcaagac 2160 tgtttaggac aaacactggt gctgatcggc aaaacccatc tgacaatggc tttgacagtg 2220 atagcaggat tggtagtgat tttcatgatg ctgggcggca cttttctcta ctggcgtggg 2280 cgccggattc agaataaaag ggctatgagg cgatacttgg aacggggtga gagcatagag 2340 cctctggacc ccagtgagaa ggctaacaaa gtcttggcca gaatcttcaa agagacagag 2400 ctaaggaagc ttaaagtgct tggctcgggt gtctttggaa ctgtgcacaa aggagtgtgg 2460 atccctgagg gtgaatcaat caagattcca gtctgcatta aagtcattga ggacaagagt 2520 ggacggcaga gttttcaagc tgtgacagat catatgctgg ccattggcag cctggaccat 2580 gcccacattg taaggctgct gggactatgc ccagggtcat ctctgcagct tgtcactcaa 2640 tatttgcctc tgggttctct gctggatcat gtgagacaac accggggggc actggggcca 2700 cagctgctgc tcaactgggg agtacaaatt gccaagggaa tgtactacct tgaggaacat 2760 ggtatggtgc atagaaacct ggctgcccga aacgtgctac tcaagtcacc cagtcaggtt 2820 caggtggcag attttggtgt ggctgacctg ctgcctcctg atgataagca gctgctatac 2880 agtgaggcca agactccaat taagtggatg gcccttgaga gtatccactt tgggaaatac 2940 acacaccaga gtgatgtctg gagctatggt gtgacagttt gggagttgat gaccttcggg 3000 gcagagccct atgcagggct acgattggct gaagtaccag acctgctaga gaagggggag 3060 cggttggcac agccccagat ctgcacaatt gatgtctaca tggtgatggt caagtgttgg 3120 atgattgatg agaacattcg cccaaccttt aaagaactag ccaatgagtt caccaggatg 3180 gcccgagacc caccacggta tctggtcata aagagagaga gtgggcctgg aatagcccct 3240 gggccagagc cccatggtct gacaaacaag aagctagagg aagtagagct ggagccagaa 3300 ctagacctag acctagactt ggaagcagag gaggacaacc tggcaaccac cacactgggc 3360 tccgccctca gcctaccagt tggaacactt aatcggccac gtgggagcca gagcctttta 3420 agtccatcat ctggatacat gcccatgaac cagggtaatc ttggggagtc ttgccaggag 3480 tctgcagttt ctgggagcag tgaacggtgc ccccgtccag tctctctaca cccaatgcca 3540 cggggatgcc tggcatcaga gtcatcagag gggcatgtaa caggctctga ggctgagctc 3600 caggagaaag tgtcaatgtg taggagccgg agcaggagcc ggagcccacg gccacgcgga 3660 gatagcgcct accattccca gcgccacagt ctgctgactc ctgttacccc actctcccca 3720 cccgggttag aggaagagga tgtcaacggt tatgtcatgc cagatacaca cctcaaaggt 3780 actccctcct cccgggaagg caccctttct tcagtgggtc tcagttctgt cctgggtact 3840 gaagaagaag atgaagatga ggagtatgaa tacatgaacc ggaggagaag gcacagtcca 3900 cctcatcccc ctaggccaag ttcccttgag gagctgggtt atgagtacat ggatgtgggg 3960 tcagacctca gtgcctctct gggcagcaca cagagttgcc cactccaccc tgtacccatc 4020 atgcccactg caggcacaac tccagatgaa gactatgaat atatgaatcg gcaacgagat 4080 ggaggtggtc ctgggggtga ttatgcagcc atgggggcct gcccagcatc tgagcaaggg 4140 tatgaagaga tgagagcttt tcaggggcct ggacatcagg ccccccatgt ccattatgcc 4200 cgcctaaaaa ctctacgtag cttagaggct acagactctg cctttgataa ccctgattac 4260 tggcatagca ggcttttccc caaggctaat gcccagagaa cgtaactcct gctccctgtg 4320 gcactcaggg agcatttaat ggcagctagt gcctttagag ggtaccgtct tctccctatt 4380 ccctctctct cccaggtccc agcccctttt ccccagtccc agacaattcc attcaatctt 4440 tggaggcttt taaacatttt gacacaaaat tcttatggta tgtagccagc tgtgcacttt 4500 cttctctttc ccaaccccag gaaaggtttt ccttattttg tgtgctttcc cagtcccatt 4560 cctcagcttc ttcacaggca ctcctggaga tatgaaggat tactctccat atcccttcct 4620 ctcaggctct tgactacttg gaactaggct cttatgtgtg cctttgtttc ccatcagact 4680 gtcaagaaga ggaaagggag gaaacctagc agaggaaagt gtaattttgg tttatgactc 4740 ttaaccccct agaaagacag aagcttaaaa tctgtgaaga aagaggttag gagtagatat 4800 tgattactat cataattcag cacttaacta tgagccaggc atcatactaa acttcaccta 4860 cattatctca cttagtcctt tatcatcctt aaaacaattc tgtgacatac atattatctc 4920 attttacaca aagggaagtc gggcatggtg gctcatgcct gtaatctcag cactttggga 4980 ggctgaggca gaaggattac ctgaggcaag gagtttgaga ccagcttagc caacatagta 5040 agacccccat ctctttaaaa aaaaaaaaaa aaaaaaaaaa aaaactttag aactgggtgc 5100 agtggctcat gcctgtaatc ccagccagca ctttgggagg ctgagatggg aagatcactt 5160 gagcccagaa ttagagataa gcctatggaa acatagcaag acactgtctc tacaggggaa 5220 aaaaaaaaaa gaaactgagc cttaaagaga tgaaataaat taagcagtag atccaggatg 5280 caaaatcctc ccaattcctg tgcatgtgct cttattgtaa ggtgccaaga aaaactgatt 5340 taagttacag cccttgttta aggggcactg tttcttgttt ttgcactgaa tcaagtctaa 5400 ccccaacagc cacatcctcc tatacctaga catctcatct caggaagtgg tggtgggggt 5460 agtcagaagg aaaaataact ggacatcttt gtgtaaacca taatccacat gtgccgtaaa 5520 tgatcttcac tccttatccg agggcaaatt cacaaggatc cccaagatcc acttttagaa 5580 gccattctca tccagcagtg agaagcttcc aggtaggaca gaaaaaagat ccagcttcag 5640 ctgcacacct ctgtcccctt ggatggggaa ctaagggaaa acgtctgttg tatcactgaa 5700 gttttttgtt ttgtttttat acgtgtctga ataaaaatgc caaagttttt tttcagcaaa 5760 aaaaa 5765 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for OCT4 <400> 4 gcagcgacta tgcacaacga 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for OCT4 <400> 5 ccagagtggt gacggagaca 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for SOX2 <400> 6 catcacccac agcaaatgac a 21 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for SOX2 <400> 7 gctcctaccg taccactaga actt 24 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for NANOG <400> 8 aatacctcag cctccagcag atg 23 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for NANOG <400> 9 tgcgtcacac cattgctatt cttc 24 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for GAPDH <400> 10 cagcctcaag atcatcagca atg 23 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for GAPDH <400> 11 tcatgagtcc ttccacgata cca 23 <210> 12 <211> 57 <212> DNA <213> Artificial Sequence <220> 223 ERBB3 shRNA oligomer (top) <400> 12 ccggccagag cttcaagact gtttactcga gtaaacagtc ttgaagctct ggttttt 57 <210> 13 <211> 57 <212> DNA <213> Artificial Sequence <220> 223 ERBB3 shRNA oligomer (bottom) <400> 13 aattaaaaac cagagcttca agactgttta ctcgagtaaa cagtcttgaa gctctgg 57 <210> 14 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> E-cadherin shRNA oligomer (top) <400> 14 ccgggcagaa attattgggc tctttctcga gaaagagccc aataatttct gcttttt 57 <210> 15 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> E-cadherin shRNA oligomer (bottom) <400> 15 aattaaaaag cagaaattat tgggctcttt ctcgagaaag agcccaataa tttctgc 57

Claims (20)

Formulations that measure the expression level of one or more genes selected from the group consisting of sex determining region Y-box 2 (SOX2), E-cadherin and Erb-B2 Receptor Tyrosine Kinase 3 (ERBB3) A composition for distinguishing a diffuse type gastric cancer stem cell or an intestinal type gastric cancer stem cell comprising.
The composition of claim 1, wherein the SOX2 consists of the nucleotide sequence of SEQ ID NO: 1.
According to claim 1, The E-cadherin is composed of the nucleotide sequence of SEQ ID NO: 2.
The composition of claim 1, wherein the ERBB3 consists of the nucleotide sequence of SEQ ID NO: 3.
delete The composition of claim 1, wherein the expression level of the gene is its mRNA or protein level.
The composition of claim 6, wherein the mRNA level of the gene is measured using primers or probes that specifically bind to the gene.
8. The primer according to claim 7, wherein the primer consists of a pair of primers for measuring SOX2 of SEQ ID NOs: 4 and 5, a pair of primers for measuring E-cadherin of SEQ ID NOs: 6 and 7, and a pair of primers for measuring ERBB3 of SEQ ID NOs: 8 and 9 The composition is one or more selected from the group.
The composition of claim 6, wherein the level of the protein is measured using an antibody or probe specific for the protein.
Claim 1 to 4 and 6 to claim 9, comprising a composition of any one of the type (diffuse type) gastric cancer cells or intestinal type gastric cancer stem cell discriminating kit.
The kit of claim 10, wherein the kit is an RT-PCR kit, a DNA chip kit or a protein chip kit.
(a) Sex determining region Y-box 2 (SOX2), E-cadherin, from a biological sample isolated from a gastric cancer patient, using the composition of any one of claims 1-4 and 6-9. Measuring the expression level of at least one gene selected from the group consisting of (E-cadherin) and ERBB3 (Erb-B2 Receptor Tyrosine Kinase 3); And
(b) a method of providing information for distinguishing diffuse type gastric cancer stem cells or intestinal type gastric cancer stem cells, comprising comparing the expression level measured in step (a) to that of the control sample; .
The method of claim 12, wherein the biological sample is cancer stem cells derived from gastric cancer.
The method of claim 12, wherein when the expression level of SOX2 or E-cadherin measured in step (a) is higher than that of the control sample, it is determined that the sample contains diffuse gastric cancer stem cells. , Way.
The method of claim 12, wherein when the expression level of ERBB3 measured in step (a) is increased than that of the control sample, it is determined that the sample contains enteric gastric cancer stem cells.
The method of claim 12, wherein when the expression level of SOX2, E-cadherin and ERBB3 measured in step (a) is equal to or lower than that of the control sample, the sample contains subtype and enteric gastric cancer stem cells. The method of judging that there is not.
(A) measuring the expression level of sex determining region Y-box 2 (SOX2) or E-cadherin gene in subtype gastric cancer stem cells treated with the candidate substance of the subtype gastric cancer treatment; And
(b) comparing the expression level of step (a) with that of the sample not treated with the candidate substance.
18. The method according to claim 17, wherein when the expression level of SOX2 or E-cadherin measured in step (a) is lower than that of the sample not treated with the candidate, the candidate is judged to be a gastric cancer treatment. (c) further comprising the step.
(A) measuring the expression level of the ERBB3 (Erb-B2 Receptor Tyrosine Kinase 3) gene in the enteric gastric cancer stem cells treated with candidates for enteric gastric cancer treatment; And
(b) comparing the expression level of step (a) with that of the sample that has not been treated with the candidate.
20. The method of claim 19, wherein if the expression level of ERBB3 measured in step (a) is lower than that of the sample that has not been treated with the candidate, adding the step (c) to determine the candidate as an enteric gastric cancer treatment That, including.

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