WO2015178667A1 - Use of nkx6.3 for prediction of cancer occurrence and early diagnosis of cancer and as therapeutic agent for cancer - Google Patents

Abstract

The present invention relates to a use of NKX6.3 for prediction of cancer occurrence and early diagnosis of cancer and as a therapeutic agent for cancer and, more specifically, to a use of NKX6.3 for prediction of cancer occurrence and early diagnosis of cancer on the basis of the expression level of NKX6.3 protein, the expression level of mRNA and the number of DNA copies, and for prevention and treatment of cancer by administrating an NKX6.3 protein or gene. The NKX6.3 according to the present invention can be used as a diagnostic composition for early diagnosis of cancer because the activity or expression thereof is inhibited in a cancer cell. Also, NKX6.3 exerts the functions of inhibiting the survival and proliferation of cancer cells and inducing apoptosis when the expression thereof is induced, and thus can be usefully used as a composition for anti-cancer treatment.

Description

Use of NKX6.3 as a predictor, early diagnosis and treatment of cancer

FIELD OF THE INVENTION The present invention relates to the use of NKX6.3 as a predictor, early diagnosis and treatment of cancer, and more particularly to early diagnosis of cancer through the amount of protein expression, mRNA expression, and DNA copy number of NKX6.3. And the use of NKX6.3 to treat cancer by administering a NKX6.3 protein or gene.

The gastric epithelium forms tubular branched gastric glands, which have a complex structure containing a variety of cells. In general, gastric epithelial cells have very fast cell turnover rate and induce epithelial regeneration every 3 to 5 days, but epithelial cells of gastric gland maintain homeostasis through apoptosis despite fast turnover rate. Pluripotent stem cells are located in the neck or isthmus of the gastric gland, and stem cells migrate above or below the gastric gland after mitosis and differentiate into various types of cells. Recent studies have shown that inflammation of the gastric mucosa is mainly caused by infection with Helicobacter pylori and is involved in the development of gastric cancer through various stages of atrophic gastritis, metaplasia to intestinal epithelium or dysplasia. The double intestinal metaplasia is known as the precancerous lesion of gastric cancer because gastric mucosal epithelial cells differentiate into intestinal mucosal epithelium. Although many studies have been conducted and progressed to conquer gastric cancer, the exact molecular mechanisms for gastric cancer development and progression are still unknown, and gastric cancer is one of the most malignant tumors with the highest incidence and mortality in the world. do.

Many homeodomain transcription factors play an important role in the differentiation and development of cells. Among these homeobox genes, NKX family members are known to play an important role in the development and differentiation process, such as determining the fate of cells in the central nervous system, gastrointestinal tract and pancreas. Among these, NKX6.3, the third member of NKX6, a subfamily of the NKX gene, is mainly expressed in the lower / base region of epithelial cells of the gastric distal end in mouse experiments. In humans, NKX6.3 is located on chromosome 8p11.21 and encodes 266 amino acid proteins. NKX6.3 is expressed in mobile cells that differentiate up and down after mitosis of gastric units, and NKX6 in gastric cancer. .3 Heterozygosity loss of the chromosome 8p11 region in which the gene is present is frequently observed. The present inventors have found that inactivation of the NKX6.3 gene may lead to abnormal differentiation of gastric mucosal epithelial cells and imbalance of homeostasis. It was predicted that this may be a cause of adult gastritis and cancer.

In order to confirm whether the NKX6.3 gene is related to the development of gastric cancer, the present inventors conducted genetic, epigenetic and expression analysis of NKX6.3 gene using 55 gastric cancer samples, and used AGS and MNK1 gastric cancer cell lines. Functional analysis was conducted. As a result, the expression of NKX6.3 was significantly decreased in gastric cancer tissues compared to normal gastric mucosal tissues, and we confirmed that NKX6.3 acts as a tumor suppressor gene that regulates the differentiation, cell proliferation and apoptosis of gastric mucosal epithelial cells. The invention has been completed.

An object of the present invention is to provide an anticancer composition comprising a polynucleotide encoding the NKX6.3 (NK6 homeobox3) protein or NKX6.3 protein.

Another object of the present invention is to predict the occurrence of cancer by confirming the expression level of NKX6.3 protein, the expression level of mRNA encoding NKX6.3 protein or the DNA copy number of NKX6.3 gene in a biological sample collected from a subject, It provides the information necessary for diagnosis or prognostic analysis.

It is still another object of the present invention to provide an anticancer screening method comprising the step of treating an anticancer agent to a cell and measuring the effect on the activity of NKX6.3 protein or an increase in intracellular expression level.

In order to achieve the above object, the present invention provides an anticancer composition comprising a polynucleotide encoding the NKX6.3 (NK6 homeobox3) protein or NKX6.3 protein.

In one embodiment of the present invention, the NKX6.3 protein may be composed of the amino acid sequence of SEQ ID NO: 1.

In one embodiment of the present invention, the polynucleotide encoding the NKX6.3 protein may be composed of the nucleotide sequence of SEQ ID NO: 2.

In one embodiment of the present invention, the polynucleotide may be contained in an expression vector.

In one embodiment of the invention, the cancer is breast cancer (breast cancer), liver cancer (liver cancer), bladder cancer, brain cancer (brain cancer), cervical cancer (cervical cancer), colorectal cancer (colorectal cancer), Esophageal cancer, gallbladder cancer, head and neck cancer, kidney cancer, lung cancer (small and / or non-small cell) )), Melanoma, ovarian cancer, ovary (germ cell cancer), prostate cancer, pancreatic cancer, penile cancer, skin cancer ( skin cancer, soft-tissue sarcoma, squamous cell carcinomas, stomach cancer, testicular cancer, thyroid cancer and uterine cancer One or more cancers selected.

In addition, the present invention is to predict the occurrence of cancer by confirming the expression amount of NKX6.3 protein, the expression amount of mRNA encoding NKX6.3 protein or the DNA copy number of NKX6.3 gene in a biological sample collected from a subject, cancer It provides a method for providing information necessary for diagnosis or prognostic analysis.

In one embodiment of the present invention, the biological sample for predicting cancer occurrence may be noncancerous (precancerous) tissue, and the biological sample for cancer diagnosis or prognostic analysis may be cancer tissue.

In one embodiment of the present invention, the noncancerous (precancerous) tissue has a reduced amount of expression of NKX6.3 protein compared to normal tissue at its periphery; The amount of expression of mRNA encoding NKX6.3 protein is decreased; Or when the DNA copy number of the NKX6.3 gene decreases; The risk of cancer is high.

In one embodiment of the invention, the expression of NKX6.3 protein, Sox2 protein, and Muc5ac protein is lost in the noncancerous (precancerous) tissue, especially in the stomach, compared to normal tissues in the periphery, and Cdx2 and Muc2 If the expression of the intestinal metaplasia is in progress, it can be determined that the cancer is high.

In one embodiment of the present invention, the cancer tissue is reduced in the amount of expression of NKX6.3 protein compared to the normal tissue of the periphery thereof; The amount of expression of mRNA encoding NKX6.3 protein is decreased; Or when the DNA copy number of the NKX6.3 gene decreases; The cancer may be diagnosed or determined to be highly malignant.

In one embodiment of the invention, the cancer is breast cancer (breast cancer), liver cancer (liver cancer), bladder cancer, brain cancer (brain cancer), cervical cancer (cervical cancer), colorectal cancer (colorectal cancer), Esophageal cancer, gallbladder cancer, head and neck cancer, kidney cancer, lung cancer (small and / or non-small cell) )), Melanoma, ovarian cancer, ovary (germ cell cancer), prostate cancer, pancreatic cancer, penile cancer, skin cancer ( skin cancer, soft-tissue sarcoma, squamous cell carcinomas, stomach cancer, testicular cancer, thyroid cancer and uterine cancer One or more cancers selected.

The present invention also provides an anticancer screening method comprising the step of treating the anticancer drug candidates to cells to determine the effect on the activity of NKX6.3 protein or an increase in intracellular expression level.

In one embodiment of the present invention, when the candidate increases the activity of the NKX6.3 protein or increases the expression level of the NKX6.3 gene in the cell, it may be added to determine the anticancer agent having anticancer activity Can be.

In one embodiment of the present invention, the activity or expression level of the NKX6.3 protein is immunoprecipitation (coimmunoprecipitation), radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, polymerase chain Reaction (PCR), Western blotting (Western Blotting) and flow cytometry (FACS) may be performed by any one method selected from the group consisting of.

NKX6.3 according to the present invention can be utilized as a diagnostic composition for early diagnosis of cancer because the activity or expression is inhibited in cancer cells, induces the expression of NKX6.3 to inhibit the survival of cancer cells and apoptosis It can be usefully used as an anti-cancer composition to exert a function of inducing and inhibiting intestinal metaplasia, which is a precancerous lesion, by inducing differentiation into gastric mucosal cells.

1 shows expression of NKX6.3 in gastric cancer cell lines and gastric cancer tissues. A) AGS, MKN1, MKN28 and MKN45 gastric cancer cell lines do not show NKX6.3 protein expression, but AGS cell lines transiently transfected with NKX6.3. Or NKX6.3 protein expression was confirmed in stably transfected AGS ^N6.3 and MNK1 ^N6.3 cell lines, B) expression of NKX6.3 protein was lost or decreased in most gastric cancer tissues, and C) Both methylated and unmethylated DNA of the NKX6.3 gene promoter region was observed in the non-cancerous gastric mucosal tissue corresponding to each example. (U, upper stomach; M, middle stomach, L, lower stomach; N, normal; T , gastric cancer; M, methylated PCR product; U, unmethylated PCR product)

FIG. 2 shows NKX6.3 DNA copy number and NKX6.3 mRNA expression patterns in non-cancerous gastric mucosa and gastric cancer tissues. A) SYBR of NKX6.3 DNA levels in 55 gastric cancer and corresponding gastric mucosal tissues. After real time-QPCR analysis using green and normalized with GAPDH, B) NKX6.3 mRNA expression was normalized with GAPDH after QRT-PCR with SYBR green and C & D) 35 normal gastric mucosal tissues And quantitative correlation between NKX6.3 DNA copy number and mRNA expression in gastric cancer tissue samples (P <0.0001).

Figure 3 shows the expression of NKX6.3 in atrophic gastritis and intestinal metaplasia of gastric mucosal tissues. A) Real-time RT-PCR analysis of mRNA expression changes in 55 gastric mucosal tissues, B) Monocyte infiltration, atrophicity Scatter plot analysis of NKX6.3 mRNA expression in gastritis and intestinal metaplasia, C) ROC curve analysis using NKX6.3 to distinguish gastric mucosal tissue from atrophic gastritis or intestinal metaplasia D) Cdx2 in mononuclear cell infiltration, atrophic gastritis and intestinal metaplasia Scattering analysis of mRNA expression E) ROC curve analysis using Cdx2 to distinguish gastric mucosal tissue from atrophic gastritis or intestinal metaplasia.

Figure 4 confirms that NKX6.3 is an expression regulator of gastric differentiation marker Sox2 and intestinal differentiation marker Cdx2, A) NKX6.3 is a transcription factor by binding to the 3 ~ 4 kb promoter region in the Sox2 gene. B) NKX6.3 increased the binding capacity as a transcription factor at the 5 kb promoter region of Cdx2 gene, C) NKX6.3 induced Sox2 gene expression in a time-dependent manner, and D) inhibited the expression of Cdx2 gene. The result is.

FIG. 5 shows that normal gastric gland in human gastric mucosal tissues expresses NKX6.3, Sox2 and Muc5ac, but gastric gland with intestinal metaplasia has lost their expression and expressed intestinal differentiation markers Cdx2 and Muc2. The cell line study results were verified in human gastric mucosal tissue.

To Figure 6 confirm the effect of NKX6.3 on cell survival and cell ^{death, A) AGS mock, MKN1 mock} , AGS NKX6.3, ^NKX6.3 MNK1 was confirmed in the cell line to cell survival by MTT assay, B) AGS ^mock, MKN1 ^mock, AGS ^NKX6.3, was confirmed in the cell proliferation inhibitory ability MNK1 ^NKX6.3 cell line as ^{BrdU incorporation assay, C) AGS NKX6.3} , has confirmed that the colony formation inhibitory ability at MNK1 ^NKX6.3 cell lines, D) AGS ^Inhibition of cell cycle progression was confirmed in ^Nb6.3 and MNK1 ^Nb6.3 cell lines.

Figure 7 confirmed the apoptosis induction ability of NKX6.3 in AGS ^N6 and MNK1 ^N6,3 cell lines by annexin V-binding assay.

The present inventors have investigated the expression of NKX6.3 protein in gastric cancer tissues, taking into consideration that cancer may occur when abnormalities in cell differentiation occur. As expected, the expression of NKX6.3 is lost in gastric cancer tissues. It was confirmed that the decrease significantly.

That is, expression of NKX6.3 was not confirmed in gastric cancer cell lines such as AGS, MKN1, MKN28 and MKN45, and NKX6 was expressed in 33 (94.3%) of 35 gastric cancer tissues expressing NKX6.3 in noncancerous mucosal tissues. The expression of 3 was found to be significantly reduced or lost compared to normal tissue (see FIGS. 1A and 1B).

To determine whether this decreased expression of NKX6.3 is caused by epigenetic changes in somatic cells of the NKX6.3 gene, mutations in the NKX6.3 gene were identified in gastric cancer tissues. However, no somatic mutation of the NKX6.3 gene that was specific in gastric cancer tissue was identified (data not shown).

Thus, the present inventors analyzed the methylation to determine whether the expression of NKX6.3 is changed in gastric cancer tissue by the methylation change of the NKX6.3 gene promoter region, but methylation of the NKX6.3 gene in gastric cancer tissue and normal mucosal tissue It was confirmed that there is no significant difference (see FIG. 1C), and it was concluded that somatic mutation and promoter hypermethylation of NXK6.3 did not significantly affect the development of gastric cancer during gastric cancer development.

Since the present inventors further analyzed the DNA copy number and mRNA transcription level by real-time QPCR and real-time RT-PCR to further determine the mechanism by which NKX6.3 expression is reduced in gastric cancer, compared to non-cancerous gastric mucosal tissues. DNA copy number and mRNA transcription expression in gastric cancer tissues were found to be reduced in 18 (32.7%) and 34 (61.8%) of 55 samples, respectively (see FIGS. 2A and 2B). On the other hand, the present inventors compared the relationship between NKX6.3 DNA copy number and mRNA transcript expression, and found that there is a significant correlation between DNA copy number and mRNA transcript in both non-cancerous mucosal tissue and gastric cancer tissue. (P <0.0001) (see FIG. 2D). From the above results, the present inventors determined that the DNA copy number and mRNA transcription level of the reduced NKX6.3 gene in gastric cancer were the main cause of the decreased expression of the NKX6.3 protein.

NKX6.3 is a novel transcription factor and is known to play a role in the differentiation of gastric mucosal epithelial cells. The present inventors have focused on the fact that the intestinal metaplasia, which is one of the abnormalities in the cell differentiation process, is a precancerous lesion of gastric cancer, and inhibiting the intestinal metaplasia can prevent cancer, and thus the amount of NKX6.3 expression in tissues showing atrophic gastritis and enteroplastic lesions. The changes were examined. In all tissues showing atrophic gastritis and enteroplastic lesions, the expression of NKX6.3 was significantly decreased, and the expression of Cdx2 was increased (see FIG. 3).

In addition, the effect of NKX6.3 on protein expression related to cell differentiation was further investigated. NKX6.3 was evaluated for the expression of Sox2 gene, which induces differentiation into gastric mucosa, and Cdx2, which induces differentiation into intestinal mucosa. It was confirmed that the inhibition of intestinal metaplasia by adjusting.

That is, NKX6.3 induces the expression of gastric differentiation marker Sox2 as a transcription factor that binds to the promoter regions of Sox2 and Cdx2 genes, while inhibiting the expression of the intestinal differentiation marker Cdx2 (see FIG. 4).

 In addition, normal gastric gland expressed NKX6.3, Sox2 and Muc5ac in human gastric mucosa, but gastric gland with enteroplasia lost their expression and expressed Cdx2 and Muc2 markers ( See FIG. 5).

In addition, the present inventors observed the cell survival by constructing the AGS ^N6.3 and MNK1 ^N6.3 cell lines stably expressing NKX6.3 to determine how NKX6.3 affects the growth and death of gastric cancer cells. Expression of NKX6.3 was found to inhibit time-dependent survival, cell proliferation and colony formation of these gastric cancer cell lines (see FIGS. 6A, 5B, 5C & 5D). In addition, it was found that AGS ^NKX6.3 and MNK1 ^NKX6.3 cell lines induce apoptosis in these gastric cancer cell lines in a time-dependent manner (see FIG. 7). From these results, the present inventors found that NKX6.3 functions as a tumor suppressor that induces normal differentiation of gastric cancer cells, inhibits cell proliferation and controls apoptosis.

Therefore, the present invention can provide an anticancer composition comprising a NKX6.3 protein or a polynucleotide encoding the NKX6.3 protein.

Preferably, the NKX6.3 protein may be one having an amino acid sequence represented by SEQ ID NO: 1, the polynucleotide encoding the NKX6.3 protein may be one having a nucleotide sequence described in SEQ ID NO: 2.

In addition, the NKX6.3 protein according to the present invention may preferably be a functional equivalent to the polypeptide having the amino acid sequence of SEQ ID NO: 1. The term 'functional equivalent' refers to a sequence homology of at least 70%, preferably 70%, more preferably 80% or more with the amino acid sequence represented by SEQ ID NO: 1 as a result of the addition, substitution, or deletion of amino acids. It refers to a polypeptide that exhibits substantially homogeneous activity with NKX6.3 of the present invention. Here, "substantially homogeneous activity" means the activity of NKX6.3 described above. Such functional equivalents may include, for example, amino acid sequence variants in which some of the amino acids of the amino acid sequence of NKX6.3 according to the invention have been substituted, deleted or added. Substitution of amino acids may preferably be conservative substitutions, examples of conservative substitutions of amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of amino acids may preferably be located at portions not directly involved in the activity of NKX6.3 of the present invention. The functional equivalent may also include polypeptide derivatives in which some chemical structures of the polypeptide are modified while maintaining the basic backbone of NKX6.3 and its physiological activity. For example, fusion proteins made by fusion with other proteins while maintaining structural changes and physiological activities for altering the stability, storage, volatility or solubility of the polypeptide of the present invention may be included.

In addition, the polynucleotide encoding the NKX6.3 protein may be introduced into an expression vector, such as a plasmid or viral vector, by a known method, followed by transduction or transfection of the expression vector by various methods known in the art. transfection) can be introduced into the target cell in the phenotype.

Plasmid expression vectors are FDA's approved gene delivery methods for human use and deliver plasmid DNA directly to human cells (Nabel, EG et al., Science, 249: 1285-1288, 1990). Unlike viral vectors, there is an advantage that can be purified homogeneously. As the plasmid expression vector that can be used in the present invention, mammalian expression plasmids known in the art can be used, and in one embodiment of the present invention, the NKX6.3 gene is inserted using a pcDNA3.1 (Invitrogen) plasmid. A recombinant expression vector pcDNA3.1-NKX6.3 having a cleavage map of 12 was prepared.

Plasmid expression vectors comprising nucleic acids according to the present invention are methods known in the art, such as, but not limited to, transient transfection, microinjection, transduction, Cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection Can be introduced into tumor cells by electroporation, gene guns and other known methods for introducing DNA into cells (Wu. Et al., J. Bio. Chem., 267: 963-967, 1992; Wu. Et al., Bio. Chem., 263: 14621-14624, 1988). Preferably, transient transfection methods can be used.

In addition, the vector capable of expressing NKX6.3 may be administered to cells, tissues or the body by known methods, for example, topically, parenteral, oral, nasal, intravenous, intramuscular, subcutaneous. Or by other suitable means. In particular, the vector can be injected directly into a target cancer or tumor cell in an effective amount for treating tumor cells of the target tissue. In particular, in the case of cancer or tumors in the body cavity such as the eye, gastrointestinal tract, urogenital tract, lungs and bronchial system, the pharmaceutical composition of the present invention may be used as a hollow organ affected by cancer or a tumor. It can be injected directly using a needle, catheter or other type of transport tube within.

In addition, the anticancer composition according to the present invention can be used as a pharmaceutical composition that can prevent and treat cancer, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and that, when administered to a human, typically does not cause allergic or similar reactions, such as gastrointestinal disorders, dizziness, and the like. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like, and parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols, and the like. And the like may further comprise stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to those described in the following documents (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). The pharmaceutical composition according to the present invention may be formulated in a suitable form according to methods known in the art together with the pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral dosage forms according to known methods, and isotonic aqueous solution or suspension is preferable as an injectable formulation as a typical parenteral dosage form. Injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills and capsules.

Pharmaceutical compositions formulated in such a manner can be administered in a effective amount via several routes, including oral, transdermal, subcutaneous, intravenous or intramuscular. As used herein, the term 'effective amount' refers to an amount exhibiting a prophylactic or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may be appropriately selected depending on the route of administration, subject to administration, age, gender weight, individual difference and disease state. Preferably, the pharmaceutical composition of the present invention may vary the content of the active ingredient according to the degree of disease, preferably 1 μg to 10000 μg mg / kg body weight / day, more preferably 10 to 1000 μg / It may be administered repeatedly several times a day at an effective dose of kg / day body weight. In addition, the anticancer composition of the present invention may be administered in parallel with a known compound having the effect of preventing, improving or treating cancer.

In addition, the present invention provides an amount of expression of NKX6.3 protein, an expression amount of mRNA encoding NKX6.3 protein or NKX6 protein in a biological sample taken from a subject to provide information necessary for cancer prevention, diagnosis or prognostic analysis. The method of diagnosing cancer can be provided by checking the DNA copy number of the 3 gene.

The term "prevention" in the context of the present invention encompasses the prevention of cancer development in precancerous lesions of cancer. Preferably, prevention in the present invention means the possibility of fundamentally inhibiting the occurrence of cancer.

As used herein, the term “diagnosis” refers to determining the susceptibility of an object to a particular disease or condition, determining whether an object currently has a particular disease or condition, a particular disease or condition Determining the prognosis (eg, identifying a metastatic or metastatic cancer state, determining the stage of the cancer, or determining the responsiveness of the cancer to treatment), or therametrics (eg, treatment) Monitoring the state of the object to provide information about its efficacy.

The term "prognosis" in the context of the present invention includes the prediction in terms of the process of disease progression, in particular, the degree of disease remission, disease regeneration, tumor recurrence, metastasis and death. Preferably, the prognosis in the present invention means the possibility that the disease of a cancer patient will be cured. On the other hand, the high malignancy of cancer in the present invention includes a state of high invasion and metastasis of cancer and a high risk of recurrence.

According to a preferred embodiment of the present invention, the present invention can be carried out by immunoassay (ie, antigen-antibody reaction). In this case, the antibody or aptamer specifically binds to the cancer marker of the present invention described above.

The antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Antibodies may be commonly used in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) Or phage antibody library methods (Clackson et al, Nature, 352: 624-628 (1991) and Marks et al, J. Mol. Biol., 222: 58, 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing immortalized cell lines with antibody-producing lymphocytes, and the techniques required for this process are well known to those skilled in the art and can be readily implemented. Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting antisera from the animal, and then isolating the antibody from the antisera using known affinity techniques.

When the method of the present invention is carried out using antibodies or aptamers, the present invention can be used to diagnose cancer by performing according to conventional immunoassay methods. Such immunoassays can be performed according to various quantitative or qualitative immunoassay protocols developed in the prior art. The immunoassay format may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbant assay (ELISA), capture-ELISA, inhibition or hardwood analysis, sandwich analysis, flow cytometry, immunoassay. Including but not limited to fluorescent staining and immunoaffinity purification. The immunoassay or method of immunostaining is described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Sprin Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, when the method of the present invention is carried out in accordance with radioimmunoassay methods, antibodies labeled with radioisotopes (eg, C14, I125, P32 and S35) can be used to detect marker molecules of the present invention. have.

When the method of the present invention is carried out in an ELISA method, certain embodiments of the present invention comprise the steps of: (1) coating an unknown cell sample lysate to be analyzed on the surface of a solid substrate; (2) reacting the cell lysate with an antibody to a marker as a primary antibody; (3) reacting the resultant of step (2) with a secondary antibody to which an enzyme is bound; And (4) measuring the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates. Enzymes bound to the secondary antibody include, but are not limited to, enzymes catalyzing color reaction, fluorescence, luminescence or infrared reaction, for example, alkaline phosphatase, β-galactosidase, hose Radish peroxidase, luciferase and cytochrome P450. When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as a substrate Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis) when chromogenic reaction substrates such as -B1-phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase is used -N-methylacridinium nitrate), lesoruppin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), TMB (tetramethylbenzidine), ABTS (2,2-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS ( substrates such as phenzaine methosulfate) Can be.

When the method of the invention is carried out in a capture-ELISA mode, certain embodiments of the invention comprise the steps of: (1) coating the surface of a solid substrate with an antibody against the marker of the invention as a capturing antibody; (2) reacting the capture antibody with the sample; (3) reacting the resultant of step (2) with a detecting antibody which has a label generating a signal and which specifically reacts with NKX6.3 protein; And (4) measuring the signal resulting from the label.

The detection antibody carries a label which generates a detectable signal. The label may include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P450), radioactive substances (eg C14, I125, P32 and S35) , Fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent, and fluorescence resonance energy transfer (FRET). Various labels and labeling methods are described in Ed. Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

Measurement of the final enzyme activity or signal in the ELISA method and the capture-ELISA method can be carried out according to various methods known in the art. Detection of such signals allows for qualitative or quantitative analysis of the markers of the invention. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used.

According to another variant of the present invention, an aptamer that specifically binds to the marker of the present invention may be used instead of the antibody. Aptamers are oligonucleic acid or peptide molecules, the general contents of which are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95 (24): 142727 (1998).

Cancer can be diagnosed by analyzing the final signal intensity by the above-described immunoassay process. That is, if the protein of the marker of the present invention is low in the biological sample and the signal is significantly weaker than the normal biological sample (eg, normal gastric tissue, blood, plasma or serum), the cancer may be diagnosed.

In addition, the present invention can diagnose cancer by confirming the expression amount of mRNA encoding the NKX6.3 protein or the DNA copy number of the NKX6.3 gene.

The amount of mRNA expression or DNA copy number of the NKX6.3 gene can be confirmed by PCR amplification, and can be confirmed by microarray.

The term "amplification" described in the specification means a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the art, which include polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822), ligase chain reaction (LCR) ( 17, 18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA, WO 88/10315), self-maintaining sequence replication (self sustained sequence replication, WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction ( CP-PCR), US Pat. No. 4,437,975), optional print Arbitrarily primed polymerase chain reaction (AP-PCR), US Pat. Nos. 5,413,909 and 5,861,245, Nucleic acid sequence based amplification (NASBA), US Pat. No. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification (21, 22) and loopmediated isothermal amplification; LAMP) 23, but is not limited thereto. Other amplification methods that can be used are described in US Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and US Pat. No. 09 / 854,317.

PCR is the best known nucleic acid amplification method, and many modifications and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (inverse polymerase) chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information on PCR, see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.

The probe or primer used for diagnosis in the present invention has a sequence complementary to the NKX6.3 nucleotide sequence. As used herein, the term "complementary" means having complementarity sufficient to selectively hybridize to the above-described nucleotide sequence under certain specific hybridization or annealing conditions. Thus, the term complementary has a meaning different from that of the term perfectly complementary, and the primers or probes of the present invention may be capable of selectively hybridizing to the above-described nucleotide sequence, so long as one or more mismatch bases are used. May have a sequence.

As used herein, the term “primer” refers to a single that can act as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in a suitable buffer at a suitable temperature. -Refers to stranded oligonucleotides. Suitable lengths of primers are typically 15-30 nucleotides, although varying with various factors, such as temperature and the use of the primer. Short primer molecules generally require lower temperatures to form hybrid complexes that are sufficiently stable with the template.

The sequence of the primer does not need to have a sequence that is completely complementary to some sequences of the template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with the template to perform the primer-specific function. Therefore, the primer in the present invention does not need to have a sequence that is perfectly complementary to the above-described nucleotide sequence as a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing to the gene sequence and acting as a primer. The design of such primers can be easily carried out by those skilled in the art with reference to the above-described nucleotide sequence, for example, by using a program for primer design (eg, PRIMER 3 program).

As used herein, the term “probe” refers to a linear oligomer of natural or modified monomers or linkages, includes deoxyribonucleotides and ribonucleotides, and can specifically hybridize to a target nucleotide sequence, naturally Present or artificially synthesized. Probes of the invention are preferably single chain and oligo deoxyribonucleotides.

The label of the probe can provide a signal that allows detection of hybridization, which can be linked to oligonucleotides. Suitable labels include fluorophores (eg fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia), chromophores, chemilumines, magnetic particles, 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 And hapten with specific binding partners such as digoxigenin and chelating groups. Labeling is carried out in a variety of ways conventionally practiced in the art, such as nick translation methods, random priming methods (Multiprime DNA labeling systems booklet, "Amersham" (1989)) and chination methods (Maxam & Gilbert, Methods). in Enzymology, 65: 499 (1986)). Labels provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetric, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, nanocrystals.

The nucleic acid sample to be analyzed may be prepared using mRNA obtained from various biological samples, and preferably, may be prepared using mRNA obtained from stomach tissue cells. The hybridization reaction-based assay may be performed by labeling the cDNA to be analyzed instead of the probe.

On the other hand, NKX6.3 of the present invention has the activity of inhibiting the proliferation of cancer cells and promote cell death, the present invention can provide an anticancer screening method using the gene.

In other words, the anticancer drug screening method according to the present invention may include the step of measuring the effect on the activity of NKX6.3 protein or increase in the expression level of cells by treating the anticancer agent candidate cells. In addition, when the candidate substance increases the activity of the NKX6.3 protein or increases the expression level of the NKX6.3 gene in the cell, it may further comprise the step of judging it as an anticancer agent having anticancer activity.

The increase in the activity or expression level of the NKX6.3 protein in the cell means that the expression of the NKX6.3 gene is increased or the degradation of the NKX6.3 protein is inhibited, thereby increasing the concentration of the NKX6.3 protein. The NKX6.3 gene expression includes the transcription and translation of the NKX6.3 gene into protein.

In addition, the activity or intracellular expression level of NKX6.3 protein may be measured by immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, polymerase chain reaction (PCR), and Western blotting. (Western Blotting) and flow cytometry (FACS) can be performed by any one method selected from the group consisting of.

In addition, the screening method targeting NKX6.3 of the present invention can apply high throughput screening (HTS). HTS is a method of testing multiple candidates in parallel and screening simultaneously or nearly simultaneously for the biological activity of the multiple candidates. In certain embodiments, cell lines can be cultured in 96-well microtiter plates or 192-well microtiter plates, treated with a number of candidates therein and then the level of expression of NKX6.3 can be measured by immunochemical methods. The wells typically require assay volumes ranging from 50 μl to 500 μl and, in addition to plates, a number of instruments, instruments, pipettes, robots, robots, etc., to suit 96-well formats for a wide range of homogeneous and heterogeneous assays. Plate washers and plate readers are commercially available.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only intended to illustrate 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 to these examples.

<Example 1>

1-1. Obtaining Gastric Cancer Samples

A total of 55 frozen gastric cancer tissues and non-cancerous mucosal tissues around the cancer were obtained from Hwasun Hospital, Chonnam National University, Korea National Biobank, which is supported by the Ministry of Health and Welfare. Informed consent was provided in accordance with the Declaration of Helsinki, whereby written consent was obtained from all patients, and all experiments were approved by the Institutional Audit Committee of the Catholic University Medical College (MC15SISI0015). None of the patients participating in the trial exhibited familial cancer.

1-2. Cell Culture and Transfection of NKX6.3

AGS, MKN1, MKN28 and MNK45 gastric cancer cell lines were incubated at 37% in 5% CO ₂ using RPMI-1640 medium with 10% FBS inactivated by heat. After cloning the entire NKX6.3-cDNA into the pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) expression vector, Lipofectamine Plus transfection reagent (Invitrogen) was applied to AGS and MKN1 cell lines in total 5 mg of the expression plasmid in 60 mm dishes. Was temporarily transfected according to the manufacturer's method.

In addition, AGS and MKN1 cell lines AGS ^NKX6.3 and MNK1 ^NKX6.3 cell lines stably expressing human NKX6.3 were ^prepared, and stable NKX6.3 protein expression was confirmed by Western blot.

1-3. Statistical analysis

Student's t-test was used to analyze the effect of NKX6.3 on cell survival. Data are expressed as mean ± standard deviation after at least three independent experiments. Linear regression correlation and Chi-square tests were used to analyze the correlation with mRNA and protein expression according to the DNA copy number change of NKX6.3. p <0.05 was interpreted as statistically significant.

<Example 2>

Reduction of NKX6.3 Protein Expression in Gastric Cancer Cell Lines and Gastric Cancer Tissues

Expression of NKX6.3 protein was examined by Western blotting on gastric cancer tissues collected from the 35 patients, and the expression level of NKX6.3 in non-cancerous-gastrointestinal mucosa tissues corresponding to each example was also examined. Gastric cancer cell lines such as AGS, MKN1, MKN28, and MNK45 were also examined for expression levels of NKX6.3. Cells were lysed for western blotting, electrophoresed on 20% polyacrylamide gels, then transferred to Hybond-PVDF membranes (Bio-Rad, Hercules, Calif., USA) and anti-NKX6.3 monoclones. Reaction with antibody (Abcam, Cambridge, UK). Protein bands were detected with ECM Western Blotting Detection Reagent (Bid-Rad).

As a result, the expression of NKX6.3 protein was not observed in AGS, MNK1, MKN28 and MKN45 gastric cancer cell lines, and it was confirmed that NKX6.3 was completely inactivated in these gastric cancer cell lines (see FIG. 1A). In 35 samples of non-cancerous gastric mucosa tissue including the upper [cardia], middle [fundus, corpus], and lower [antrum and pylorus] Expression of the NKX6.3 protein was confirmed, and in 33 (94.3%) of these gastric cancer tissues, the expression was found to be lost or decreased (see FIG. 1B).

<Example 3>

Mutation Analysis of NKX6.3 in Gastric Cancer Tissues

Sequence analysis was performed to determine whether the reduced expression of NKX6.3 of Example 2 is associated with mutation of the gene. To this end, genomic DNA from each tumor cell and corresponding non-cancerous gastric mucosa cells was amplified with six sets of primers covering the entire coding region of the NKX6.3 gene. Specific primers for detecting mutations of NKX6.3 include GenBank accession No. Designed according to the genome sequence of NC_000008.11, specific primer sequences are shown in Table 1 below.

 Each polymerase chain reaction (PCR) procedure was performed under standard conditions. In brief, the reaction mixture is denatured at 94 ° C. for 12 minutes, followed by denaturation at 94 ° C. for 40 seconds, annealing at 56-60 ° C. for 40 seconds and extension at 72 ° C. for 40 seconds, for 35 cycles. I was. The final extension step at 72 ° C. was carried out for 5 minutes. Sequencing of PCR products was performed using a cycle sequencing kit (Perkin Elmer, Foster City, CA, USA) according to the manufacturer's protocol.

As a result, mutation of NKX6.3 was not confirmed in gastric cancer cells. In order to ensure reliability, PCR and sequencing were performed twice, respectively, but the results were the same.

<Example 4>

Analysis of Methylation Status of NKX6.3 in Gastric Cancer Tissues

The reduced expression of NKX6.3 of Example 2 was determined by methylation of the promoter region of the NXK6.3 gene. For this purpose, the methylation status of the promoter region of the NKX6.3 gene was measured by methylation specific PCR (MSP) after sodium hydrogen sulfite treatment on DNA. 5 μl of disulfate modified DNA was MSP using two sets of NKX6.3 primers for analyzing methylated or unmethylated NKX6.3. Primers used at this time are shown in Table 1 above. PCR was performed in a total volume of 30 μl using 5 μl template DNA, 0.5 μM of each primer, 0.2 μM of each dNTP, 1.5 mM MgCl ₂ , 0.4 unit of Ampli Taq gold polymerase (Perkin-Elmer), and 3 μl 10 × buffer. . The reaction solution was initially denatured at 95 ° C. for 1 minute. Amplification was carried out for 40 seconds for 30 seconds at 95 ° C, 30 seconds at 59 ° C, and 30 seconds at 72 ° C, and further extended for 5 minutes at 72 ° C. PCR products were directly electrophoresed on 2% agarose gels, stained with EtBr and confirmed by UV illumination.

As a result, methylation and unmethylation of the NKX6.3 gene was shown in all normal and gastric cancer tissues (see FIG. 1C), so that specific methylation of NKX6.3 did not affect NKX6.3 expression in gastric cancer tissues. Could confirm that it does.

Example 5

DNA copy number and mRNA expression patterns of NKX6.3 in gastric cancer tissues

The aim of this study was to determine whether the decrease in the expression level of NKX6.3 is related to the change of DNA copy number and mRNA expression of NKX6.3. To this end, genomic DNA and mRNA were extracted and quantified from gastric cancer tissues and corresponding non-cancerous gastric mucosal tissues, and then real-time SYBR Green QPCR was performed using the Stratagene Mx 3000P QPCR system. Specific primers for confirming NKX6.3 DNA copy number are described in Genbank accession No. It was designed using the genomic sequence of NC_000008.11. All samples were normalized by PCR amplification with GAPDH specific primers. Primers for SYBR Green analysis were designed based on gene specific gene-specific non-homologous DNA sequences, primer sequences as described in Table 1 above.

In order to confirm mRNA expression, cDNA was synthesized according to the manufacturer's method using a reverse transcription kit manufactured by Roche Molecular System (Roche, Mannheim, Germany). For QPCR, 50ng of cDNA was amplified with the Stratagene Mx 3000P QPCR system using Fullvelocity SYBR Green QPCR Master Mix (Stratagene, La Jolla, Calif., USA) and 20 pmol / μl forward and reverse primers.

However, due to the low Ct value, real-time RT PCR was performed for 45 cycles. In order to secure the reliability of mRNA extraction and reverse transcription, all samples were normalized by PCR amplification with GAPDH specific primers. Standard curves were used to quantify the relative amount of gene expression, which provides subunit normalized expression values that can be used for direct comparison of the relative amounts of target DNA and mRNA in other samples. All samples were tested in duplicate and the average value was used. The DNA copy number and mRNA expression were defined as decreased when the mean test (cancer) / reference (mucosa) ratio was 0.5 times or less.

As a result, the DNA copy number of NKX6.3 was found to be reduced in 18 gastric cancer DNAs compared to the gastric mucosal DNA in the periphery thereof (see FIG. 2A). The NKX6.3 gene transcript was expressed in non-cancerous gastric mucosal tissue corresponding to all examples (see FIG. 2B). Loss or decrease in the expression of mRNA transcripts was observed in 34 (61.8%) of the 55 gastric cancer tissues analyzed. In both non-cancerous gastric mucosal and gastric cancer tissues, there was a significant correlation between DNA copy number and mRNA transcript expression of NKX6.3 (P <0.0001) (see Figures 2C and 2D).

<Example 6>

Effect of NKX6.3 Expression on Cdx2 Expression and Intestinal Metaplasia

The mRNA expression level of NKX6.3 was examined in 55 stomach tissues by real-time RT-PCR analysis. The average value of mRNA expression in gastric mucosa without infection with H. pyroli., Atrophic gastritis and enteroplasia was used as a control. Changes in mRNA expression in each case were normalized to the mean value of the gastric mucosa of the control group.

As a result, mRNA expression of NKX6.3 was decreased in 28 tissues (50.9%). A decrease in NKX6.3 mRNA expression was observed in 19 (95.0%) of 20 gastric tissue samples with enteroplasia (see Figure 3A).

In the scatter plot analysis, NKX6.3 mRNA expression was closely related to mononuclear cell infiltration (Student t-test, P <0.0001), atrophic gastritis (P = 0.0274) and enteroplasia (P <0.0001). There was no correlation with infiltration (P = 0.3550) (see FIG. 3B).

To distinguish atrophic gastritis or intestinal metaplasia from human gastric mucosal tissue, ROC curve analysis was performed using NKX6.3. As a result, the expression level of NKX6.3 was 0.6892 (95% confidence interval [CI], 05463-0.8321; P = 0.02386) and 0.9414 (95% CI, 08847-0.9981; P) in atrophic gastritis and enteroplastic tissue, respectively. <0.0001) (see FIG. 3C).

Scatter plot analysis showed that Cdx2 mRNA expression was closely related to mononuclear cell infiltration (Student t-test, P = 0.0036), atrophy (P = 0.0038), and enteroplasia (P <0.0001).

ROC curve analysis was performed using Cdx2 to distinguish atrophic gastritis or enteroplasia from human gastric mucosal tissue. As a result, the expression level of Cdx 2 was 0.7613 (95% confidence interval [CI], 0.6250-0.8976; P = 0.001812) and 0.9971 (95% CI, 0.9895-1.005; P <) in atrophic gastritis and enteroplastic tissue, respectively. 0.0001) (see FIG. 3E).

In conclusion, the expression of NKX6.3 is decreased and the expression of Cdx2 is increased in atrophic gastritis or intestinal metaplasia stages before cancer development, atrophic gastritis, a lesion before developing cancer Increasing the expression of NKX6.3 or reducing the expression of Cdx2 in the intestinal metaplasia phase was able to prevent the occurrence of cancer.

<Example 7>

Effect of NKX6.3 Expression on Differentiation of Gastric Mucosal Epithelial Cells

In order to confirm the function of NKX6.3 in gastric epithelial cells, NKX6.3 was stably expressed in AGS and MKN1 cell lines. Production and MNK1 AGS cell line, AGS stably expressing the ^NKX6.3 and NKX6.3 cell lines MNK1 ^NKX6.3 sepojueul To this end, and were produced AGS ^mock and MNK1 ^mock cell line stably expressing the control group for this is NKX6.3 Western blotting was confirmed.

In order to analyze the effect of NKX6.3 on cell differentiation, the present inventors investigated whether NKX6.3 acts as a transcription factor of Sox2, a gastric differentiation marker, and Cdx2, an intestinal differentiation marker, to control expression by Chromatin immunoprecipitation-PCR. It was.

As a result, it was confirmed that NKX6.3 induces expression of gastric differentiation marker Sox2 and inhibits the expression of intestinal differentiation marker Cdx2 as a transcription factor that binds to promoter regions of Sox2 and Cdx2 genes (see FIG. 4). In addition, normal gastric gland in human gastric mucosal tissues expresses NKX6.3, Sox2 and Muc5ac, but gastric gland with intestinal metaplasia is lost in their expression and intestinal differentiation markers Cdx2 and Muc2 are expressed. The results of the study were verified (see FIG. 5).

<Example 8>

Analysis of Cell Proliferation Inhibition Effect of Gastric Cancer Cell Line by NKX6.3

In order to confirm the function of NKX6.3 in gastric epithelial cells, it was possible to stably express NKX6.3 in AGS and MKN1 cell lines. Production and MNK1 AGS cell line, AGS stably expressing the ^NKX6.3 and NKX6.3 cell lines MNK1 ^NKX6.3 sepojueul To this end, and to prepare a ^mock AGS and MNK1 ^mock cell line, the control for this. Stable expression of NKX6.3 in AGS ^{NB6.3 NKX6.3} and MNK1 ^NB6.3 cell lines was confirmed by Western blotting.

As a result, as can be seen in Figure 1, the AGS ^NB6.3 and MNK1 ^NB6.3 cell lines were found to stably express NKX6.3 protein as compared to the control group.

The present inventors have NKX6.3 is to analyze the effect on cell viability, MTT [3- (4,5 dimethylthiazol- 2-yl -2,5-diphenyltetrazoliumbromide] by a method AGS ^{mock, mock} MNK1, AGS ^NKX6.3 Cell viability was analyzed at 24, 48 and 72 hours in ^MNK1 and NNK6.3 cell lines, and absorbance was measured using a spectrophotometer at 540 nm, and cell viability was observed relative to mock (empty vector + lipofectamine). .

As a result, it was found that ^NKX6.3 significantly inhibited the survival of the gastric cancer cells over time in the AGS ^Nv6.3 and MNK1 ^Nv6.3 cell lines compared to the control group (see FIG. 6A). In addition, as a result of examining the effect on cell proliferation by BrdU incorporation method, NKX6.3 significantly inhibited the proliferation of gastric cancer cells over time and colony formation compared to the control group (see FIGS. 6B and 5C). In addition, NKX6.3 inhibited cell cycle progression in AGS ^N6.3 and MNK1 ^N6.3 cell lines (see FIG. 6D).

Example 9

Apoptosis Analysis of Gastric Cancer Cell Lines by NKX6.3

To determine if cell death was induced in NKX6.3, the cells were stained by FITC-labeled annexin V staining in the AGS ^NB6.3 and MNK1 ^NB6.3 cell lines stably expressing ^NKX6.3. Cell death was analyzed. To this end, Annexin V-binding assay was performed at 24, 48 and 72 hours after incubation in AGS ^N6.3 and MNK1 ^N6.3 cell lines according to the manufacturer's method. To this end, the AGS ^NB6.3 and MNK1 ^NB6.3 cell lines were washed twice with cold PBS, resuspended using 100 μL binding buffer, and then 100 μL of the supernatant was mixed with 400 μL of blocking solution, 5 μl (1 μg / ml) of annexin V-FITC and 5 μl of PI (2 μg / ml) were added to the mixed solution and reacted in the dark for 15 minutes. Cells were then analyzed by flow cytometry (FACS, BD Biosciences, San Jose, Calif., USA) and only fluorescence-positive cells without PI staining were considered dead cells.

As a result, the number of apoptotic cells was increased over time in the AGS ^N6.3 and MNK1 ^N6.3 cell lines expressing ^NKX6.3 (see FIG. 7).

Through the above results, it can be seen that NKX6.3 is specifically reduced in gastric cancer cells, and when NKX6.3 is introduced into such gastric cancer cell line, the gastric cancer cell line specifically inhibits cell proliferation and induces cell death. As can be seen, it was found that NKX6.3 can be treated with gastric cancer treatment.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (14)

An anticancer composition comprising a NKX6.3 (NK6 homeobox3) protein or a polynucleotide encoding a NKX6.3 protein. The method of claim 1, The NKX6.3 protein is an anticancer composition, characterized in that consisting of the amino acid sequence of SEQ ID NO: 1. The method of claim 1, The polynucleotide encoding the NKX6.3 protein is an anticancer composition, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2. The method of claim 1, The polynucleotide composition for anticancer, characterized in that contained in the expression vector. The method of claim 1, The cancer may include breast cancer, liver cancer, bladder cancer, brain cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, head and neck cancer, kidney cancer, lung cancer (small and / or non-small cell), melanoma, Ovarian cancer, ovary (germ cell cancer), prostate cancer, pancreatic cancer, penile cancer, skin cancer, soft tissue sarcoma anticancer, characterized in that it is at least one cancer selected from the group consisting of tissue sarcoma, squamous cell carcinomas, stomach cancer, testicular cancer, thyroid cancer and uterine cancer Composition. The amount of expression of NKX6.3 protein, the amount of mRNA encoding NKX6.3 protein, or the number of DNA copies of NKX6.3 gene in a biological sample collected from a subject was checked to predict cancer occurrence, cancer diagnosis, or prognostic analysis. How to give you the information you need. The method of claim 6, And wherein said biological sample for predicting cancer occurrence is noncancerous (precancerous) tissue and said biological sample for cancer diagnosis or prognostic analysis is cancer tissue. The method of claim 7, wherein The noncancerous (precancerous) tissue has reduced expression of NKX6.3 protein compared to normal tissue at its periphery; The amount of expression of mRNA encoding NKX6.3 protein is decreased; Or when the DNA copy number of the NKX6.3 gene decreases; A method characterized by determining that the risk of cancer is high. The method of claim 8, In the gastric mucosa, the noncancerous (precancerous) tissue loses the expression of NKX6.3 protein, Sox2 protein and Muc5ac protein compared to the normal tissues of its periphery, and enteroplasia is progressing when the expression of Cdx2 and Muc2 is increased. A method characterized by the high incidence of cancer. The method of claim 7, wherein The cancer tissue has a decreased expression level of NKX6.3 protein compared to normal tissue in its periphery; The amount of expression of mRNA encoding NKX6.3 protein is decreased; Or when the DNA copy number of the NKX6.3 gene decreases; Diagnosing cancer or determining the cancer malignancy is high. The method of claim 6, The cancer may include breast cancer, liver cancer, bladder cancer, brain cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, head and neck cancer, kidney cancer, lung cancer (small and / or non-small cell), melanoma, Ovarian cancer, ovary (germ cell cancer), prostate cancer, pancreatic cancer, penile cancer, skin cancer, soft tissue sarcoma tissue sarcoma, squamous cell carcinomas, stomach cancer, testicular cancer, testicular cancer, thyroid cancer, and uterine cancer. . A method for screening an anticancer agent comprising treating the cell with an anticancer agent and measuring the effect on the activity of NKX6.3 protein or an increase in the level of expression in the cell. The method of claim 12, When the candidate substance increases the activity of the NKX6.3 protein or increases the expression level of the NKX6.3 gene in the cell, the anticancer drug screening method, characterized in that it further comprises the step of determining the anticancer agent having anticancer activity. The method of claim 12, The activity or intracellular expression level of the NKX6.3 protein was measured by immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, polymerase chain reaction (PCR), and Western blotting ( Western Blotting) and flow cytometry (FACS) of the anticancer drug screening method, characterized in that carried out by any one method selected from the group consisting of.

Images