KR20200049383A - Composition for inhibiting stemness of stem cell comprising material for inhibiting methylation of OCT4 - Google Patents

Abstract

The present invention relates to a composition for inhibiting stemness of stem cells, including an OCT4 methylation-inhibiting material. The OCT4 methylation-inhibiting material inhibits methylation of the 179^th arginine in the amino acid sequence of mouse OCT4, 186^th arginine in the amino acid sequence of human OCT4, the 215^th lysine in the amino acid sequence of mouse OCT4, or the 222^th lysine in the amino acid sequence of human OCT4, and thus can effectively reduce stemness of various stem cells. Therefore, the composition can be used effectively for inhibiting cancer proliferation, cancer recurrence, cancer metastasis, generation of resistance against anti-cancer agents in cancer, or the like. In addition, since the composition can reduce stemness in general stem cells, it is expected that the composition can reduce the time and can increase the efficiency in the differentiation of embryotic stem cells to specific cells.

Description

Composition for inhibiting stemness of stem cell comprising material for inhibiting methylation of OCT4}

The present invention relates to a composition for inhibiting stem cellity of stem cells containing a substance that inhibits methylation of OCT4.

Most of the anti-cancer drugs that have been actively developed in recent years and used in anti-cancer treatment are drugs targeting rapidly proliferating cancer cells. In the case of anti-cancer treatment using these drugs, it is initially shown that cancer cells are effectively killed and cancer is cured, but in the end, cancer stem cells remaining in the body are not removed and cancer recurrence and / or metastasis are actively In the end, interest in cancer stem cells has recently increased because problems that indicate resistance to existing anti-cancer therapies often occur.

Cancer stem cells are cancer cells with unlimited regenerative capacity similar to normal stem cells. They proliferate slowly and differently from normal cancer cells. They are cancer cells with autogenous regeneration or differentiation ability that are unique to stem cells. It is known to have other mechanisms. Cancer stem cells show drug and radiation resistance in a specific subpopulation that exhibits the potential to differentiate into various cancer cell groups and self-renewal, which causes cancer recurrence as well as cancer initiation. Since it causes metastasis, preventing cancer stem cell proliferation is important in cancer treatment.

On the other hand, in the case of stem cell therapeutics that have been actively researched recently, it is difficult to control the proliferation, survival, etc. of stem cells, and thus side effects are caused. A typical side effect may be caused by the undifferentiated stem cells remaining in the body and becoming darkened after treatment is completed.

Therefore, if there is a method that can regulate the stem cell properties of stem cells, including cancer stem cells, it can be safely applied to various stem cell applications, and effectively suppress cancer recurrence, metastasis, and resistance by increasing the therapeutic effect of cancer. It is expected to be possible.

On the other hand, octamer-binding transcription factor 4 (OCT4), also known as POU5F1 (POU domain, class 5, transcription factor 1), is a transcription factor expressed in conventional embryonic stem cells and differentiation of cells It plays a role in preventing and disappears when natural differentiation of cells begins, and is known as a marker specific for embryonic stem cells that are starch-potentiating ability.

However, as conventionally known in the treatment of cancer, OCT4 is not only used as a marker for confirming stem cellity, but further, by regulating methylation of a specific amino acid sequence among the amino acid sequences of OCT4, stem cells are directly regulated by OCT4 itself. The relationship between methylation of OCT4 and stem cell regulation, such as regulating sex, is not yet known.

Domestic Publication Patent 10-2014-0143989

The present invention was devised to solve the problems in the prior art as described above, and includes a substance that inhibits the function of OCT4 by inhibiting the methylation of OCT4 as an active ingredient, stem cell properties of various stem cells such as cancer stem cells. An object thereof is to provide a composition that can be suppressed.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description. There will be.

To achieve the above object, the present invention is the 179th arginine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4), the 186th arginine in the amino acid sequence of human OCT4, the 215th lysine in the amino acid sequence of mouse OCT4, Or it provides a composition for inhibiting stem cell stem cell, comprising a substance that inhibits the methylation of the 222th lysine in the amino acid sequence of human OCT4.

As an embodiment of the present invention, the amino acid sequence of the mouse OCT4 may be represented by the amino acid sequence of SEQ ID NO: 1.

As another embodiment of the present invention, the amino acid sequence of human OCT4 may be represented by the amino acid sequence of SEQ ID NO: 2.

As another embodiment of the present invention, the material may be any one selected from the group consisting of peptides, proteins, nucleotides, and compounds.

As another embodiment of the present invention, the stem cells may be any one selected from the group consisting of embryonic stem cells, germ cells, and cancer stem cells.

As another embodiment of the present invention, the composition can inhibit the proliferation, self-renewal, colonization, or survival of stem cells.

In addition, the present invention is from the amino acid sequence of mouse OCT4 (octamer-binding transcription factor 4) 179th arginine, human OCT4 amino acid sequence of 186th arginine, mouse OCT4 amino acid sequence of 215th lysine, or human OCT4 amino acid sequence Provided is a cell therapeutic agent comprising a substance that inhibits methylation of the 222nd lysine and stem cells for cell therapy as an active ingredient.

As an embodiment of the present invention, the cell therapeutic agent can inhibit metastasis, survival, or cancer metastasis of therapeutic stem cells.

In addition, in the present invention, in vitro , the 179th arginine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4), the 186th arginine in the amino acid sequence of human OCT4, the 215th lysine in the amino acid sequence of mouse OCT4, or human OCT4 Characterized in that it comprises the step of inhibiting the methylation of the 222th lysine in the amino acid sequence of, provides a method for suppressing stem cellity through OCT4 methylation function control.

In addition, the present invention is from the amino acid sequence of mouse OCT4 (octamer-binding transcription factor 4) 179th arginine, human OCT4 amino acid sequence of 186th arginine, mouse OCT4 amino acid sequence of 215th lysine, or human OCT4 amino acid sequence Provided is a pharmaceutical composition for preventing or treating cancer, comprising a substance that inhibits methylation of the 222nd lysine.

As an embodiment of the present invention, the pharmaceutical composition may further include an anti-cancer agent.

As another embodiment of the present invention, the pharmaceutical composition may inhibit cancer proliferation, survival, metastasis, relapse, or anticancer drug resistance.

In addition, the present invention provides a method for screening a stem cell inhibitory substance of stem cells, comprising the following steps:

(a) processing a candidate substance in cells expressing mouse octamer-binding transcription factor 4 (OCT4) or human OCT4;

(b) Confirmation of inhibition of methylation of the 179th arginine in the amino acid sequence of the mouse OCT4, the 186th arginine in the amino acid sequence of the human OCT4, the 215th lysine in the amino acid sequence of the mouse OCT4, or the 222th lysine in the amino acid sequence of human OCT4 To do; And

(c) when methylation of arginine or lysine is inhibited in step (b), selecting the candidate substance as a stem cell inhibitory substance of stem cells.

In addition, the present invention provides a method for providing information for diagnosing whether stem cells are inhibited by stem cells, comprising the following steps:

(a) Mouse octamer-binding transcription factor 4 (OCT4) or human OCT4 from cells expressing mouse OCT4 in the amino acid sequence of 179th arginine, human OCT4 amino acid sequence of 186th arginine, mouse OCT4 amino acid sequence of 215th lysine , Or determining whether to inhibit the methylation of the 222nd lysine in the amino acid sequence of human OCT4; And

(b) When the methylation of arginine or lysine is inhibited in step (a), diagnosing that stem cellity of stem cells is suppressed.

In addition, the present invention provides a method for preventing or treating cancer comprising administering the pharmaceutical composition to an individual.

In addition, the present invention provides a cancer prevention or treatment use of the pharmaceutical composition.

A substance that inhibits the methylation of OCT4 according to the present invention is 179th arginine in the amino acid sequence of mouse OCT4, 186th arginine in amino acid sequence of human OCT4, 215th lysine in amino acid sequence of mouse OCT4, or 222 in the amino acid sequence of human OCT4 By inhibiting the methylation of th lysine, it is possible to effectively reduce the stem cell properties of various stem cells. Therefore, it can be effectively used for suppressing the proliferation of cancer, suppressing recurrence of cancer, suppressing metastasis of cancer, and suppressing the occurrence of anticancer drug resistance of cancer, and can also reduce stem cellity in normal stem cells, so as specific cells of embryonic stem cells. In the differentiation of, time can be shortened and efficiency can be increased. In addition, when the OCT4 methylation inhibitory substance of the present invention is used for cell therapy using embryonic stem cells, undifferentiated embryonic stem cells remaining after treatment can be completely removed, and the side effects of cancer development can be effectively suppressed. It is expected that the stability of cell therapy using stem cells can be significantly increased.

1 is a FLAG-OCT4 FLAG-OCT4 separated by immunoprecipitation using an anti-FLAG antibody in transgenic embryonic stem cells expressing FLAG-OCT4 according to an embodiment of the present invention shows the results confirmed using SDS-PAGE It is a drawing.
2 is a view showing the results of confirming the separated FLAG-OCT4 protein according to an embodiment of the present invention using a mass spectrometer.
3 is a view showing the results of confirming the nucleotide sequence of a vector mutated to methylate the methylation position of the mouse OCT4 (179th arginine and 215th lysine) according to an embodiment of the present invention.
4 is a view showing the results confirming that the mutant OCT4 was expressed in an amount similar to that of the wild type (WT) in the transformed cell line according to an embodiment of the present invention.
5 is a view showing the results of confirming the effect of the non-methylation of OCT4 on the stem cell self-renewal ability in the transformed cell line according to one embodiment of the present invention as a cluster formation of embryonic stem cells using alkaline phosphatase staining to be.

The present invention is the 179th arginine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4), the 186th arginine in the amino acid sequence of human OCT4, the 215th lysine in the amino acid sequence of mouse OCT4, or the 222nd in the amino acid sequence of human OCT4. Provided is a composition for inhibiting stem cell stem cells, comprising a substance that inhibits methylation of lysine.

In the present invention, the amino acid sequence of the mouse OCT4 can be represented by the amino acid sequence of SEQ ID NO: 1 (NCBI Reference Sequence: NP_038661.2), and the amino acid sequence of the human OCT4 is the amino acid sequence of SEQ ID NO: 2 (NCBI Reference Sequence) : NP_002692.2).

In the present invention, the amino acid sequence of SEQ ID NO: 1 may be encoded by the base sequence of SEQ ID NO: 3 (NCBI Reference Sequence: NM_013633.3).

In the present invention, the amino acid sequence of SEQ ID NO: 2 may be encoded by the base sequence of SEQ ID NO: 4 (NCBI Reference Sequence: NM_002701.5).

In the present invention, the substance may be any one selected from the group consisting of peptides, proteins, nucleotides, compounds, but 179th arginine of mouse OCT4, 186th arginine of human OCT4, 215th lysine of mouse OCT4, or human OCT4 If it is a substance capable of inhibiting the methylation of the 222nd lysine of, it is not limited thereto.

The term "OCT4 (octamer-binding transcription factor 4)" in the present invention is expressed in pluripotent embryonic stem cells and germ cells as one of the family of POU transcription factors. Members of the POU transcription factor family have conserved DNA binding domains, or POU domains, which were first identified in the transcription factors Pit-1, Oct-1, Oct-2 and Unc-86. OCT4 activates transcription through an octamer motif located adjacent to or at a distance from the transcription initiation site.

OCT4 mRNA is commonly found in totipotent and pluripotent stem cells of long-acting pregastrulation embryos, including oocytes, early-split-stage embryos, and inner cell mass of spore cysts. do. In addition, the expression of the gene is down-regulated in the differentiation process, suggesting that OCT4 plays an important role in mammalian development.

In addition, knocking out the OCT4 gene in the mouse causes initial lethality due to deficiency in the formation of endocytosis, which represents an important action of OCT4 in self-renewal of embryonic stem cells. In human development, the expression of OCT4 is confirmed at least until the blastocyst stage, and OCT4 is involved in regulating gene expression.

In the present invention, the term "stem cell (stem cell)" refers to the broad concept of the ability to differentiate into various types of tissue cells, ie, undifferentiated cells with stem cellity (stemness). These stem cells are largely divided into embryonic stem cells, adult stem cells, adult stem cells, gamete, cancer stem cells, etc., which can be prepared using embryos. Stem cells refer to the stage of cell lumps before forming a specific organ within 14 days after fertilization, and recently, embryonic stem cells are also produced from normal cells through dedifferentiation. Therefore, the cells that can differentiate into all cells and tissues constituting the body are not limited thereto. Adult stem cells are extracted from umbilical cord blood, bone marrow, and blood, and mean primitive cells immediately before being differentiated into cells of specific organs such as bone, liver, and blood. Germ cells are cells that transmit genetic information to the next generation through reproduction, and humans have sperm and eggs, but are not limited thereto.

In addition, the stem cells can self-replicate in the process of forming a cluster of cells by forming a clone, thereby maintaining one new stem cell in the cluster, and have the ability to form one or more characteristic cell types through differentiation. Cells.

In the present invention, the term "stemness" refers to the unique ability of stem cells, as well as pluripotency, which can differentiate into all kinds of cells constituting organisms such as nerves, blood, and cartilage. In addition, it includes both multipotency and unipotency, and includes both self-renewal and self-renewal. In addition, it means that it has the ability to form a cluster through self-replication or self-regeneration ability. At this time, the stem cellity may include the ability to differentiate embryonic stem cells obtained from embryos generated from fertilized eggs into various types of cells, and although functionally different, they are retained in each body tissue of an adult body. The ability to differentiate adult stem cells into different types of cells may also be included.

Cancer stem cells refer to cancer cells in a comprehensive sense having stem cell-specific self-renewal or differentiation ability, which is a characteristic of stem cells. Cancer stem cells generally proliferate at a slower rate than normal cancer cells under normal tumor growth conditions (referring to a state in which there is sufficient nutrients (glucose) required for cell growth and no growth of the tumor microenvironment, and no cell stress). Maintaining a dormant state may have resistance to anti-cancer agents. For example, the expression of transcription regulators such as PGC-1α is controlled unlike normal tumor cells, so that the function of major metabolic regulators is normal cancer cells. It may be different compared to. It refers to cells with invasion and metastasis that have acquired resistance to apoptosis in a nutritional deficiency state through the regulation of these different metabolic regulation capabilities and the cell signaling system mechanically linked thereto. However, it is not limited to cells that can differentiate into normal cancer cells.

In the present invention, the stem cells may be any one selected from the group consisting of embryonic stem cells, germ cells, and cancer stem cells, but the type of stem cells in which OCT4 protein is expressed is not limited thereto.

In the present invention, the composition can inhibit stem cell proliferation, stem cell proliferation, self-renewal, colony ability, survival, etc., thereby controlling stem cells in all fields using stem cells. It can be used in any way.

In addition, the present invention is from the amino acid sequence of mouse OCT4 (octamer-binding transcription factor 4) 179th arginine, human OCT4 amino acid sequence of 186th arginine, mouse OCT4 amino acid sequence of 215th lysine, or human OCT4 amino acid sequence Provided is a cell therapeutic agent comprising a substance that inhibits methylation of the 222nd lysine and stem cells for cell therapy as an active ingredient.

In the present invention, the term "cell therapy" refers to the proliferation or selection of autologous, allogenic, xenogenic cells in vitro to restore the function of cells and tissues, or changes the biological properties of cells by other methods. Drugs used for the purpose of treatment, diagnosis, and prevention through a series of methods such as prescribing.

In the present invention, the cell therapy may control the stem cells by inhibiting the metastasis and survival of the therapeutic stem cells, or may suppress the occurrence of cancer due to the cancer of the remaining therapeutic stem cells after treatment. If the effect is induced by suppressing the function is not limited to this.

In addition, in the present invention, in vitro , the 179th arginine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4), the 186th arginine in the amino acid sequence of human OCT4, the 215th lysine in the amino acid sequence of mouse OCT4, or human OCT4 Characterized in that it comprises the step of inhibiting the methylation of the 222th lysine in the amino acid sequence of, provides a method for suppressing stem cellity through OCT4 methylation function control.

In addition, the present invention is from the amino acid sequence of mouse OCT4 (octamer-binding transcription factor 4) 179th arginine, human OCT4 amino acid sequence of 186th arginine, mouse OCT4 amino acid sequence of 215th lysine, or human OCT4 amino acid sequence Provided is a pharmaceutical composition for preventing or treating cancer, comprising a substance that inhibits methylation of the 222nd lysine.

In addition, the present invention provides a method for preventing or treating cancer comprising administering the pharmaceutical composition to an individual.

In addition, the present invention provides a cancer prevention or treatment use of the pharmaceutical composition.

The term "cancer" in the present invention is an aggressive property in which cells divide and grow, ignoring normal growth limits, an invasive property that penetrates surrounding tissue, and metastatic that spreads to other parts of the body. It is a generic term for diseases caused by cells with characteristics.

The main cause of cancer death is cancer metastasis and treatment resistance, and the fact that the root cause is stem cell cancer cells has been proved in recent years. Therefore, it is expected that cancer-related death can be substantially reduced through an anticancer strategy that reduces the stem cellity of cancer cells.

The path for cancer cells to acquire stem cellity can be broadly summarized into two types, one in which normal stem cells become cancerous, and the other in cases where cancer cells acquire stem cellity through dedifferentiation. . In cancer patients, in particular, the key factors of embryonic stem cells play an important role in the maintenance of stem cells of cancer cells, and as malignant cancer progresses, the expression of embryonic stem cell-related gene groups rather than normal tissue stem cell-related genes is reported to increase. Became.

The term "methylation" in the present invention largely includes DNA methylation and histone methylation. DNA methylation is the attachment of a methyl group to the 5th carbon of the cytosine base of the nucletotide 5'-CpG-3 ', where DNA methyltransferase functions as an enzyme. On the other hand, histone methylation is to attach a methyl group to the amino acid residues of arginine (R) and lysine (K) of histone H3 and histone H4. Histone methylation functions as a transcription inhibition or transcriptional activation function depending on the number of residues of arginine or lysine. Arginine residues can be methylated to monomethylarginine or dimethylarginine by Peptidylarginine methyltransferase (PRMT), of which 2 methyl groups are bound to one nitrogen atom by two methyl groups Can form an asymmetric structure (Asymmetric) or a symmetric structure (Symmetric) in which methyl groups are respectively bonded to two nitrogen atoms. Up to three methyl groups can be attached to a lysine residue by lysine methyltransferase.

In one embodiment of the present invention, as a result of analyzing the protein by separating and purifying the OCT4 protein from mouse embryonic stem cells to confirm the new methylation position of OCT4, the analysis was performed using a mass spectrometer. It was confirmed that new methylation occurs at the 186th arginine position of human OCT4) and the 215th lysine position of mouse OCT4 (222th lysine position of human OCT4) (see Example 1).

In another embodiment of the present invention, as a result of confirming the association between OCT4 methylation and OCT4 activity, it was confirmed that the number of embryonic stem cells in which colonies are formed decreases when the OCT4 methylation-inhibiting mutant cell lines (R179K and K215R) are compared with the wild type. When methylation of mouse 179th arginine (186th arginine of human OCT4) or mouse 215th lysine of OCT4 (222th lysine of human OCT4) was inhibited, it was confirmed that stem cellity was reduced (see Example 2), and the mouse A substance that inhibits the methylation of the 179th arginine in the amino acid sequence of OCT4, the 186th arginine in the amino acid sequence of human OCT4, the 215th lysine in the amino acid sequence of mouse OCT4, or the 222th lysine in the amino acid sequence of human OCT4 functions for OCT4. Inhibits, and this causes normal stem cells to lose and differentiate stem cells, and cancer cells to stem cells. Loss makes long-term survival impossible, and prevents recurrence after chemotherapy.

In the present invention, the cancer is preferably made of thyroid cancer, cervical cancer, brain cancer, lung cancer, ovarian cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, rectal cancer, colon cancer, blood cancer, etc. It may be, more preferably gastric cancer, colon cancer, lung cancer, liver cancer, prostate cancer, thyroid cancer, breast cancer, cervical cancer, ovarian cancer, etc., but is not limited to cancer associated with OCT4.

In the present invention, the term "prevention" means any action that suppresses or delays the onset of diseases such as cancer by administration of the composition according to the present invention.

In the present invention, the term "treatment" means any action in which symptoms of diseases such as cancer are improved or advantageously changed by administration of the composition according to the present invention.

The term "administration" in the present invention means to provide the composition of the present invention to a subject in any suitable way.

The term "individual" in the present invention means a subject to which the composition of the present invention can be administered in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, dog, cat , Horses, and cows.

The term "pharmaceutical composition" in the present invention means that it is manufactured for the purpose of preventing or treating cancer, and can be used by being formulated in various forms according to a conventional method. For example, powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc. can be formulated into oral dosage forms, creams, gels, patches, sprays, ointments, warnings, lotions, linen agents, pasta agents, or It can be formulated and used in the form of external preparations for skin, suppositories and sterile injectable solutions such as cataplasma.

The pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. At this time, carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose ) Or by mixing lactose, gelatin, and the like. In addition, lubricants such as magnesium styrene talc are used in addition to simple excipients. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, sweeteners, flavoring agents, fragrances, and preservatives, can be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally, or topically) according to a desired method, and the dosage may be adjusted according to the patient's condition, weight, and disease. It depends on the degree, drug type, route of administration, and time, but can be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "a pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, activity of the drug, Sensitivity to the drug, time of administration, route of administration and rate of discharge, duration of treatment, factors including co-drugs, and other factors well known in the medical field can be determined. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art. The administration may be administered once a day, or may be divided into several times.

In the present invention, the pharmaceutical composition may further include an anti-cancer agent, and the anti-cancer agent may be doxorubicin, cisplatin, gemcitabine, oxaliplatin, 5-FU, etc., but is not limited to the compound used as an anti-cancer agent. Does not.

In the present invention, the pharmaceutical composition inhibits the stem cellularity of cancer stem cells, through which it is possible to suppress the proliferation, survival, metastasis, recurrence, or the development of anticancer drug resistance, but the stem cellularity of cancer stem cells If the effect can be caused by suppressing, it is not limited thereto.

In addition, the present invention provides a method for screening a stem cell inhibitory substance of stem cells, comprising the following steps:

(a) processing a candidate substance to cells expressing mouse OCT4 or human OCT4;

(b) Confirmation of inhibition of methylation of the 179th arginine in the amino acid sequence of the mouse OCT4, the 186th arginine in the amino acid sequence of the human OCT4, the 215th lysine in the amino acid sequence of the mouse OCT4, or the 222th lysine in the amino acid sequence of human OCT4 To do; And

(c) when methylation of arginine or lysine is inhibited in step (b), selecting the candidate substance as a stem cell inhibitory substance of stem cells.

In the present invention, the candidate material may be a nucleotide, DNA, RNA, amino acid, aptamer, protein, compound, natural product, natural extract, and the like, and is not limited to any material that can be used for stem cell suppression of stem cells.

In addition, the present invention provides a method for providing information for diagnosing whether stem cells are inhibited by stem cells, comprising the following steps:

(a) From a cell expressing mouse OCT4 or human OCT4, in the amino acid sequence of mouse OCT4, the 179th arginine, the amino acid sequence of human OCT4, the 186th arginine, the mouse OCT4 amino acid sequence of 215th lysine, or the human OCT4 amino acid sequence. Determining whether to inhibit methylation of the 222nd lysine; And

(b) When the methylation of arginine or lysine is inhibited in step (a), diagnosing that stem cellity of stem cells is suppressed.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example : OCT4  Protein methylation OCT4  Check active association

Example  1. From mouse embryonic stem cells OCT4  Methylation identification

In order to confirm the new methylation position of octamer-binding transcription factor 4 (OCT4), analysis was performed using a mass spectrometer after separation and purification of OCT4 protein from mouse embryonic stem cells.

First, in order to identify the OCT4 protein, a mouse embryonic stem cell line in which the FLAG fusion OCT4 protein is expressed in the same amount as the endogenous OCT4 was prepared in place of the endogenous OCT4. Then, only the OCT4 protein was identified using immunoprecipitation using the anti-FLAG protein antibody in the cell line, and the results are shown in FIG. 1.

Thereafter, as a result of analysis using a mass spectrometer, as shown in FIG. 2, the 179th arginine position of mouse OCT4 (the 186th arginine position of human OCT4) and the 215th lysine position of mouse OCT4 (human OCT4) It was confirmed that a new methylation occurs at the 222th lysine position)

Example  2. OCT4  With methylation OCT4  Check active association

The association between methylation of OCT4 and OCT4 activity can be confirmed by a method of evaluating embryonic stem cell maintenance. To accomplish this, first, as shown in FIG. 3, R179K and K215R, methylation inhibitory mutants that substituted OCT4 179th arginine with lysine or 215th lysine with arginine, were prepared through site-directed mutatgenesis. Thereafter, OCT4 inherent in the embryonic stem cells was removed, and a new cell line expressing OCT4 normal or mutants was prepared. For cell production, a retrovirus expressing OCT4 wild type and puromycine resistance genes in the pMSCV-Flag-puro vector, or OCT4 and puromycin resistance genes substituted with R179A, K215A, which are methylation-inhibiting types of OCT4 Was treated with doxycycline and transfected into the mouse embryonic stem cell line ZHBTc4, which can inhibit the expression of endogenous OCT4. Then, the medium was treated with puromycin to isolate the normally transfected cell line, and the isolated cell line was treated with doxycycline so that the expression of endogenous OCT4 was inhibited and the substituted exogenous OCT4 protein was expressed. Subsequently, in order to confirm that the same dose of retrovirus was transfected, the mouse was infected with NIH3T3, a mouse fibroblast line that does not express OCT4. Protein expression was confirmed by Western blotting with OCT4 antibody (SantaCruz), and ACTB antibody (Abcam) was used as a control.

As a result, as shown in FIG. 4, it was confirmed that OCT4 substituted with methylation-inhibiting types R179A and K215A in the NIH3T3 cell line was expressed in an amount similar to that of the wild type (WT).

In addition, prior to treatment with doxycycline, the cell line was washed with a phosphate buffered saline (PBS), the cell population was separated into single cells using TE (Trypsin-EDTA), and then the final cell lysate was collected. Cell culture medium was added so that the volume was 1 ml. Thereafter, 100 ul of cell lysate, a volume of 1/10 per well, was dispensed into a 6-well plate and treated with doxycycline and puromycin, and cultured for 5 to 7 days at 37 ° C and 5% CO ₂ conditions. The cultured cells were stained using an alkaline phosphatase assay kit (Medsource Ozone Biomedicals), and observed under a microscope.

As a result, as shown in FIG. 5, it was confirmed that the number of embryonic stem cells in which the colonies are formed is reduced when compared to the wild-type cell line as a control group in the case of the OCT4 methylation-inhibiting mutant cell line.

Therefore, through the above results, it was confirmed that when the methylation of the 179th arginine (human 186th arginine) of mouse OCT4 or the 215th lysine (human 222th lysine) of mouse OCT4 is inhibited, stem cellity of stem cells is suppressed.

The above description of the present invention is for illustration only, and those skilled in the art to which the present invention pertains can understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and not restrictive.

<110> National Cancer Center <120> Composition for inhibiting stemness of stem cell comprising          material for inhibiting methylation of OCT4 <130> MP18-212 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 352 <212> PRT <213> Mus musculus <220> <221> PEPTIDE <222> (1) .. (352) <223> POU domain, class 5, transcription factor 1 isoform 1 <400> 1 Met Ala Gly His Leu Ala Ser Asp Phe Ala Phe Ser Pro Pro Pro Gly   1 5 10 15 Gly Gly Asp Gly Ser Ala Gly Leu Glu Pro Gly Trp Val Asp Pro Arg              20 25 30 Thr Trp Leu Ser Phe Gln Gly Pro Pro Gly Gly Pro Gly Ile Gly Pro          35 40 45 Gly Ser Glu Val Leu Gly Ile Ser Pro Cys Pro Pro Ala Tyr Glu Phe      50 55 60 Cys Gly Gly Met Ala Tyr Cys Gly Pro Gln Val Gly Leu Gly Leu Val  65 70 75 80 Pro Gln Val Gly Val Glu Thr Leu Gln Pro Glu Gly Gln Ala Gly Ala                  85 90 95 Arg Val Glu Ser Asn Ser Glu Gly Thr Ser Ser Glu Pro Cys Ala Asp             100 105 110 Arg Pro Asn Ala Val Lys Leu Glu Lys Val Glu Pro Thr Pro Glu Glu         115 120 125 Ser Gln Asp Met Lys Ala Leu Gln Lys Glu Leu Glu Gln Phe Ala Lys     130 135 140 Leu Leu Lys Gln Lys Arg Ile Thr Leu Gly Tyr Thr Gln Ala Asp Val 145 150 155 160 Gly Leu Thr Leu Gly Val Leu Phe Gly Lys Val Phe Ser Gln Thr Thr                 165 170 175 Ile Cys Arg Phe Glu Ala Leu Gln Leu Ser Leu Lys Asn Met Cys Lys             180 185 190 Leu Arg Pro Leu Leu Glu Lys Trp Val Glu Glu Ala Asp Asn Asn Glu         195 200 205 Asn Leu Gln Glu Ile Cys Lys Ser Glu Thr Leu Val Gln Ala Arg Lys     210 215 220 Arg Lys Arg Thr Ser Ile Glu Asn Arg Val Arg Trp Ser Leu Glu Thr 225 230 235 240 Met Phe Leu Lys Cys Pro Lys Pro Ser Leu Gln Gln Ile Thr His Ile                 245 250 255 Ala Asn Gln Leu Gly Leu Glu Lys Asp Val Val Arg Val Trp Phe Cys             260 265 270 Asn Arg Arg Gln Lys Gly Lys Arg Ser Ser Ile Glu Tyr Ser Gln Arg         275 280 285 Glu Glu Tyr Glu Ala Thr Gly Thr Pro Phe Pro Gly Gly Ala Val Ser     290 295 300 Phe Pro Leu Pro Pro Gly Pro His Phe Gly Thr Pro Gly Tyr Gly Ser 305 310 315 320 Pro His Phe Thr Thr Leu Tyr Ser Val Pro Phe Pro Glu Gly Glu Ala                 325 330 335 Phe Pro Ser Val Pro Val Thr Ala Leu Gly Ser Pro Met His Ser Asn             340 345 350 <210> 2 <211> 360 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1) .. (360) <223> POU domain, class 5, transcription factor 1 isoform 1 <400> 2 Met Ala Gly His Leu Ala Ser Asp Phe Ala Phe Ser Pro Pro Pro Gly   1 5 10 15 Gly Gly Gly Asp Gly Pro Gly Gly Pro Glu Pro Gly Trp Val Asp Pro              20 25 30 Arg Thr Trp Leu Ser Phe Gln Gly Pro Pro Gly Gly Pro Gly Ile Gly          35 40 45 Pro Gly Val Gly Pro Gly Ser Glu Val Trp Gly Ile Pro Pro Cys Pro      50 55 60 Pro Pro Tyr Glu Phe Cys Gly Gly Met Ala Tyr Cys Gly Pro Gln Val  65 70 75 80 Gly Val Gly Leu Val Pro Gln Gly Gly Leu Glu Thr Ser Gln Pro Glu                  85 90 95 Gly Glu Ala Gly Val Gly Val Glu Ser Asn Ser Asp Gly Ala Ser Pro             100 105 110 Glu Pro Cys Thr Val Thr Pro Gly Ala Val Lys Leu Glu Lys Glu Lys         115 120 125 Leu Glu Gln Asn Pro Glu Glu Ser Gln Asp Ile Lys Ala Leu Gln Lys     130 135 140 Glu Leu Glu Gln Phe Ala Lys Leu Leu Lys Gln Lys Arg Ile Thr Leu 145 150 155 160 Gly Tyr Thr Gln Ala Asp Val Gly Leu Thr Leu Gly Val Leu Phe Gly                 165 170 175 Lys Val Phe Ser Gln Thr Thr Ile Cys Arg Phe Glu Ala Leu Gln Leu             180 185 190 Ser Phe Lys Asn Met Cys Lys Leu Arg Pro Leu Leu Gln Lys Trp Val         195 200 205 Glu Glu Ala Asp Asn Asn Glu Asn Leu Gln Glu Ile Cys Lys Ala Glu     210 215 220 Thr Leu Val Gln Ala Arg Lys Arg Lys Arg Thr Ser Ile Glu Asn Arg 225 230 235 240 Val Arg Gly Asn Leu Glu Asn Leu Phe Leu Gln Cys Pro Lys Pro Thr                 245 250 255 Leu Gln Gln Ile Ser His Ile Ala Gln Gln Leu Gly Leu Glu Lys Asp             260 265 270 Val Val Arg Val Trp Phe Cys Asn Arg Arg Gln Lys Gly Lys Arg Ser         275 280 285 Ser Ser Asp Tyr Ala Gln Arg Glu Asp Phe Glu Ala Ala Gly Ser Pro     290 295 300 Phe Ser Gly Gly Pro Val Ser Phe Pro Leu Ala Pro Gly Pro His Phe 305 310 315 320 Gly Thr Pro Gly Tyr Gly Ser Pro His Phe Thr Ala Leu Tyr Ser Ser                 325 330 335 Val Pro Phe Pro Glu Gly Glu Ala Phe Pro Pro Val Ser Val Thr Thr             340 345 350 Leu Gly Ser Pro Met His Ser Asn         355 360 <210> 3 <211> 1353 <212> RNA <213> Mus musculus <220> <221> gene <222> (1) .. (1353) <223> POU domain, class 5, transcription factor 1 (Pou5f1), transcript          variant 1, mRNA <400> 3 gaggtgaaac cgtccctagg tgagccgtct ttccaccagg cccccggctc ggggtgccca 60 ccttccccat ggctggacac ctggcttcag acttcgcctt ctcaccccca ccaggtgggg 120 gtgatgggtc agcagggctg gagccgggct gggtggatcc tcgaacctgg ctaagcttcc 180 aagggcctcc aggtgggcct ggaatcggac caggctcaga ggtattgggg atctccccat 240 gtccgcccgc atacgagttc tgcggaggga tggcatactg tggacctcag gttggactgg 300 gcctagtccc ccaagttggc gtggagactt tgcagcctga gggccaggca ggagcacgag 360 tggaaagcaa ctcagaggga acctcctctg agccctgtgc cgaccgcccc aatgccgtga 420 agttggagaa ggtggaacca actcccgagg agtcccagga catgaaagcc ctgcagaagg 480 agctagaaca gtttgccaag ctgctgaagc agaagaggat caccttgggg tacacccagg 540 ccgacgtggg gctcaccctg ggcgttctct ttggaaaggt gttcagccag accaccatct 600 gtcgcttcga ggccttgcag ctcagcctta agaacatgtg taagctgcgg cccctgctgg 660 agaagtgggt ggaggaagcc gacaacaatg agaaccttca ggagatatgc aaatcggaga 720 ccctggtgca ggcccggaag agaaagcgaa ctagcattga gaaccgtgtg aggtggagtc 780 tggagaccat gtttctgaag tgcccgaagc cctccctaca gcagatcact cacatcgcca 840 atcagcttgg gctagagaag gatgtggttc gagtatggtt ctgtaaccgg cgccagaagg 900 gcaaaagatc aagtattgag tattcccaac gagaagagta tgaggctaca gggacacctt 960 tcccaggggg ggctgtatcc tttcctctgc ccccaggtcc ccactttggc accccaggct 1020 atggaagccc ccacttcacc acactctact cagtcccttt tcctgagggc gaggcctttc 1080 cctctgttcc cgtcactgct ctgggctctc ccatgcattc aaactgaggc accagccctc 1140 cctggggatg ctgtgagcca aggcaaggga ggtagacaag agaacctgga gctttggggt 1200 taaattcttt tactgaggag ggattaaaag cacaacaggg gtggggggtg ggatggggaa 1260 agaagctcag tgatgctgtt gatcaggagc ctggcctgtc tgtcactcat cattttgttc 1320 ttaaataaag actgggacac acagtagata gct 1353 <210> 4 <211> 1430 <212> RNA <213> Homo sapiens <220> <221> gene <222> (1) .. (1430) <223> POU class 5 homeobox 1 (POU5F1), transcript variant 1, mRNA <400> 4 agagaggggt tgagtagtcc cttcgcaagc cctcatttca ccaggccccc ggcttggggc 60 gccttccttc cccatggcgg gacacctggc ttcggatttc gccttctcgc cccctccagg 120 tggtggaggt gatgggccag gggggccgga gccgggctgg gttgatcctc ggacctggct 180 aagcttccaa ggccctcctg gagggccagg aatcgggccg ggggttgggc caggctctga 240 ggtgtggggg attcccccat gccccccgcc gtatgagttc tgtgggggga tggcgtactg 300 tgggccccag gttggagtgg ggctagtgcc ccaaggcggc ttggagacct ctcagcctga 360 gggcgaagca ggagtcgggg tggagagcaa ctccgatggg gcctccccgg agccctgcac 420 cgtcacccct ggtgccgtga agctggagaa ggagaagctg gagcaaaacc cggaggagtc 480 ccaggacatc aaagctctgc agaaagaact cgagcaattt gccaagctcc tgaagcagaa 540 gaggatcacc ctgggatata cacaggccga tgtggggctc accctggggg ttctatttgg 600 gaaggtattc agccaaacga ccatctgccg ctttgaggct ctgcagctta gcttcaagaa 660 catgtgtaag ctgcggccct tgctgcagaa gtgggtggag gaagctgaca acaatgaaaa 720 tcttcaggag atatgcaaag cagaaaccct cgtgcaggcc cgaaagagaa agcgaaccag 780 tatcgagaac cgagtgagag gcaacctgga gaatttgttc ctgcagtgcc cgaaacccac 840 actgcagcag atcagccaca tcgcccagca gcttgggctc gagaaggatg tggtccgagt 900 gtggttctgt aaccggcgcc agaagggcaa gcgatcaagc agcgactatg cacaacgaga 960 ggattttgag gctgctgggt ctcctttctc agggggacca gtgtcctttc ctctggcccc 1020 agggccccat tttggtaccc caggctatgg gagccctcac ttcactgcac tgtactcctc 1080 ggtccctttc cctgaggggg aagcctttcc ccctgtctcc gtcaccactc tgggctctcc 1140 catgcattca aactgaggtg cctgcccttc taggaatggg ggacaggggg aggggaggag 1200 ctagggaaag aaaacctgga gtttgtgcca gggtttttgg gattaagttc ttcattcact 1260 aaggaaggaa ttgggaacac aaagggtggg ggcaggggag tttggggcaa ctggttggag 1320 ggaaggtgaa gttcaatgat gctcttgatt ttaatcccac atcatgtatc acttttttct 1380 taaataaaga agcctgggac acagtagata gacacactta aaaaaaaaaa 1430

Claims (15)

Methylation of 179th arginine in amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4), 186th arginine in amino acid sequence of human OCT4, 215th lysine in amino acid sequence of mouse OCT4, or 222th lysine in amino acid sequence of human OCT4 Containing a substance that inhibits, stem cell composition for inhibiting stem cells.
According to claim 1,
The amino acid sequence of the mouse OCT4 is characterized in that represented by the amino acid sequence of SEQ ID NO: 1, stem cell stem cell composition for inhibiting.
According to claim 1,
The amino acid sequence of the human OCT4 is characterized in that represented by the amino acid sequence of SEQ ID NO: 2, stem cell stem cell composition for inhibiting.
According to claim 1,
The material is any one selected from the group consisting of peptides, proteins, nucleotides, and compounds, the composition for inhibiting stem cell stem cells.
According to claim 1,
The stem cell is characterized in that any one selected from the group consisting of embryonic stem cells, germ cells, and cancer stem cells, stem cell stem cell composition for inhibiting.
According to claim 1,
The composition is characterized in that to inhibit the proliferation, self-renewal, colony, or survival of stem cells, stem cell composition for inhibiting stem cells.
Methylation of 179th arginine in amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4), 186th arginine in amino acid sequence of human OCT4, 215th lysine in amino acid sequence of mouse OCT4, or 222th lysine in amino acid sequence of human OCT4 Cell therapy comprising a substance for inhibiting and stem cells for cell therapy as an active ingredient.
The method of claim 7,
The material is a cell therapeutic agent, characterized in that any one selected from the group consisting of peptides, proteins, nucleotides, and compounds.
The method of claim 8,
The cell therapy is characterized in that to inhibit the metastasis, survival, or cancer of the stem cells for treatment, cell therapy.
In vitro , the 179th arginine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4), the 186th arginine in the amino acid sequence of human OCT4, the 215th lysine in the amino acid sequence of mouse OCT4, or 222 in the amino acid sequence of human OCT4 The method comprising the step of inhibiting the methylation of the lysine, stem cell suppression method through OCT4 methylation function control.
Methylation of 179th arginine in amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4), 186th arginine in amino acid sequence of human OCT4, 215th lysine in amino acid sequence of mouse OCT4, or 222th lysine in amino acid sequence of human OCT4 A pharmaceutical composition for preventing or treating cancer, comprising a substance that inhibits the cancer.
The method of claim 11,
The pharmaceutical composition is characterized in that it further comprises an anti-cancer agent, pharmaceutical composition.
The method of claim 11,
The pharmaceutical composition is characterized in that to inhibit the proliferation, survival, metastasis, relapse or anticancer drug resistance of cancer, pharmaceutical composition.
A method for screening a stem cell inhibitory substance of a stem cell, comprising the following steps:
(a) processing a candidate substance in cells expressing mouse octamer-binding transcription factor 4 (OCT4) or human OCT4;
(b) Confirmation of inhibition of methylation of the 179th arginine in the amino acid sequence of the mouse OCT4, the 186th arginine in the amino acid sequence of the human OCT4, the 215th lysine in the amino acid sequence of the mouse OCT4, or the 222th lysine in the amino acid sequence of human OCT4 To do; And
(c) when methylation of arginine or lysine is inhibited in step (b), selecting the candidate substance as a stem cell inhibitory substance of stem cells.
A method of providing information for diagnosing whether stem cells are inhibited by stem cells, including the following steps:
(a) Mouse octamer-binding transcription factor 4 (OCT4) or human OCT4 from cells expressing mouse OCT4 in the amino acid sequence of 179th arginine, human OCT4 amino acid sequence of 186th arginine, mouse OCT4 amino acid sequence of 215th lysine , Or determining whether to inhibit the methylation of the 222nd lysine in the amino acid sequence of human OCT4; And
(b) When the methylation of arginine or lysine is inhibited in step (a), diagnosing that stem cellity of stem cells is suppressed.

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