JP5725490B2 - Identification of genes that determine various effects of retinoic acid receptor ligands, including antitumor and carcinogenic effects - Google Patents

Description

  The present invention relates to an expression control agent for controlling the expression of a specific gene containing a retinoid, or a method for screening a gene whose expression is controlled by a retinoid.

  The number of liver cancer cases in the world is ranked fifth in comparison with other malignant tumors, and the number of deaths is third (Non-patent Document 1). Known primary malignant tumors in the liver include hepatocellular carcinoma (HCC), cholangiocarcinoma, and mixed forms thereof. Currently commonly used HCC treatments include surgical excision, hepatic artery embolization, ethanol infusion therapy, radiofrequency ablation therapy, and microwave coagulation therapy, but the results are never satisfactory. The current situation is difficult. In addition, HCC occurs from liver cirrhosis, and its recurrence rate is very high. For this reason, research and development of new treatments for liver cancer, in which higher therapeutic effects reduce the number of deaths from liver cancer and improve prognosis, are essential.

  Retinoid is a general term for compounds including retinol, retinal, retinoic acid (RA), and derivatives thereof. All-trans-RA (ATRA) and 9-cis-RA are known as biologically active RAs, and they function through nuclear receptors, which are ligand-induced transcription factors. (Non-Patent Document 2). There are two types of receptors for RA, retinoic acid receptor (RAR) and retinoid X receptor (RXR), and it is known that each has three isotypes (α, γ, β). . In the presence of RA, these receptors regulate the expression of various genes by binding to an RA response element (RARE) present in the promoter region of the target gene (Non-patent Document 3).

  In addition, in order to elucidate the physiological functions of RA in vivo, knockout mice of these receptors were created, but they showed growth retardation, early lethality, malformations, abnormal reproductive functions, etc. Analysis of RA function after occurrence was difficult (Non-Patent Document 4). Saitou et al., On the other hand, show that a mutant (RAR-E) in which RARα 303rd glycine is substituted with one amino acid for glutamate acts as a dominant negative for all subtypes of endogenous RARα, β, and γ. Reported (Non-Patent Document 5). That is, by expressing the RAR-E gene in a tissue-specific manner, it became possible to avoid the problem of embryonic lethality and examine the physiological function of RA in a tissue-specific manner. Therefore, the inventors of the present application previously produced a RAR-E transgenic mouse (RAR-E Tg) that expresses the RAR-E gene under the control of an albumin enhancer / promoter, and analyzed the function of RA in the liver. As a result, it has been clarified that suppression of liver-specific RA signal causes HCC to occur frequently through fat deposition, iron overdeposition, and increased oxidative stress (Non-patent Document 6).

  In past studies, acyclic retinoids significantly reduced the recurrence of HCC after surgical resection or ethanol injection therapy (Non-patent Document 7), and retinoids in the liver (Non-patent Document 8) and head and neck (non-patented). Reference 9), skin (non-patent document 10), lung (non-patent document 11), breast (non-patent document 12), acting on tumors such as hematopoietic cells (non-patent document 13), etc. Has been reported. These results strongly suggest that the RA signal acts suppressively on HCC. Actually, functional analysis of hepatic RA signal has been conducted so far to elucidate the mechanism of HCC generation based on such hypotheses (Non-Patent Document 14).

  There is a cDNA microarray as a tool for comprehensive gene expression analysis, and several RA-responsive genes have actually been identified using this analysis technique (Non-patent Document 15).

Parkin DM, Bray F, Ferlay J, Posani P. Estimating the world cancer burden: globocan 2000. Int. J. Cancer 2001; 94: 153-156. Chambon P. A decade of molecular biology of retinoic acid receptors. FASEB J 1996; 10: 940-954. Bastien J, Egly CR. Nuclear retinoid receptors and the transcription of retinoid-target genes. Gene 2004; 328: 1-16. Ross SA, McCaffery PJ, Drager UC, De Luca LM.Retinoids in embryonal development.Physiol Rev. 2000; 80 (3): 1021-54. Saitou M, Narumiya S, Kakizuka A. Alteration of a single amino acid residue in retinoic acid receptor causes dominant-negative phenotype. J Biol Chem 1994; 269: 19191-7. Tsuchiya H, Akechi Y, Ikeda R, Nishio R, Sakabe T, Terabayashi K, Matsumi Y, Ashla AA, Hoshikawa Y, Kurimasa A, Suzuki T, Ishibashi N, Yanagida S, Shiota G. Suppressive effects of retinoids on iron-induced oxidative stress in the liver. Gastroenterology. 2009; 136 (1): 341-350. Muto Y, Moriwaki H, Ninomiya M, Adachi S, Saito A, Takasaki KT, Tanaka T, Tsurumi K, Okuno M, Tomita E, Nakamura T, Kojima T. Prevention of second primary tumors by an acyclic retinoid, polyrenoic acid, in patiens with hepatocellular carcinoma. N Engl J Med 1996; 334: 1561-1567. Nakamura N, Shidoji Y, Yamada Y, Hatakeyama H, Moriwaki H, Muto Y. Induction of apoptosis by acyclic retinoid in the human hepatoma-derived cell line, HuH-7. Biochem Biophys Res Commun 1995; 207: 382-388. Lotan R. Retinoids in cancer chemoprevention. FASEB J 1996; 10: 1031-1039. Cheepala SB, Syed Z, Trutschl M, Cvek U, Clifford JL.Retinoids and skin: microarrays shed new light on chemopreventive action of all-trans retinoic acid. Mol Carcinog. 2007; 46: 643-649. Bogos K, Renyi-Vamos F, Kovacs G, Tovari J, Dome B. Role of retinoic receptors in lung carcinogenesis.J Exp Clin Cancer Res. 2008; 27:18. Leslie J. Donato, Jean H. Shu, Noa Noy.Suppression of Mammary Carcinoma Cell Growth by Retinoic Acid: the Cell Cycle Control Gene Btg2 Is a Direct Target for Retinoic Acid Receptor Signaling.Cancer Res 2007; 67: 609-615. Sarah J Freemantle, Michael J Spinella, Ethan Dmitrovsky.Retinoids in therapy and chemoprevention: promise meets resistance.Onco 2003; 22: 7305-7315. Shimizu M, Takai K, Moriwaki H. Strategy and mechanism for the prevention of hepatocellular carcinoma: phosphorylated retinoid X receptor alpha is a critical target for hepatocellular carcinoma chemoprevention. Cancer Sci. 2009; 100 (3): 369-74. Mamoon A, Ventura-Holman T, Maher JF, Subauste JS. Retinoic acid responsive genes in the murine hepatocyte cell line AML 12. Gene. 2008; 31; 408 (1-2): 95-103.

However, the prior art described in the above literature has room for improvement in the following points.
First, the in vivo mechanism of retinoids has been gradually elucidated in the above-mentioned literature, but there are still many unclear points at the molecular level about the mechanism by which retinoids are involved in disease or healing. Yes. For this reason, it has been difficult to develop a new treatment method using a biological mechanism related to retinoid.

  Secondly, attempts have been made to identify RA-responsive genes using cDNA microarrays. However, cDNA microarrays have problems such as difficulty in confirming reproducibility, complexity of operation, and limitations on detection limits. Furthermore, only known genes can be analyzed by this method, which is unsuitable for searching for unknown new genes genome-wide. Therefore, it is considered that there are still many RA target genes that have been overlooked in the conventional microarray analysis.

  This invention is made | formed in view of the said situation, and it aims at providing the expression control agent which controls the expression of a specific gene containing a retinoid. Another object of the present invention is to provide an efficient or highly accurate screening method for genes whose expression is controlled by retinoids.

  According to the present invention, a retinoid-containing expression control agent that controls the expression of a specific gene, comprising colony stimulating factor 3 receptor (granulosite), thioredoxin interacting protein, OTU domain containing 7B, Tudor domain Containing 5, Kinesin family member 21B, Odo-Skip Dried 1 (Drosophila), Protocadherin beta 4, NHS-like, Cytochrome P450, Family 26, Subfamily A, Polypeptide 1, Tyrosine hydroxylase, Olfactomedin -Like 1, Frizzled homolog 4 (Drosophila), Ataxin 2, RAS protein activator like 1 (GAP1 like), Isocytolate dehydrogenase 3 (NAD +) alpha, zinc finger Rotin 710, selenophosphate synthetase 2, carboxypeptidase D, lectin rich repeat containing 37, member A2, G protein-coupled receptor 108, FXYD domain containing ion transport regulator 3, and keratin associated protein 19-6 An expression control agent that controls the expression of one or more genes selected from the group consisting of:

  This expression control agent contains a retinoid that has been demonstrated to control the expression of the above-described gene in Examples described later. Therefore, if this expression control agent is used, the expression of the gene can be controlled according to various purposes such as treatment of diseases related to the gene.

  Moreover, according to this invention, the reagent containing the said expression control agent is provided.

  This reagent contains an expression control agent containing a retinoid that has been demonstrated to control the expression of the above-described gene in Examples described later. Therefore, it can be used as a reagent for various studies related to the expression of the above genes.

  Further, according to the present invention, there is provided a screening method for a gene whose expression is controlled by a retinoid, the base of RGKTCANNNNNRGKTCA (R = A / G, K = G / T, N = A / G / C / T) upstream A step of selecting a gene having a sequence by in silico analysis, a step of examining retinoid-dependent binding of RAR or RXR to the nucleotide sequence of RGKTCANNNNNRGKTCA located upstream of the selected gene, and RAR or RXR is a retinoid And a step of analyzing a retinoid-dependent change in the expression level of the gene located downstream of the base sequence of RGKTCANNNNNRGKTCA exhibiting dependent binding.

  This screening method has been demonstrated to enable efficient or high-precision screening of genes whose expression is controlled by retinoids in the examples described later. Therefore, if this screening method is used, a novel gene whose expression is controlled by a retinoid can be selected efficiently or with high accuracy.

  According to the present invention, an expression control agent containing retinoid and controlling the expression of a specific gene can be obtained. Alternatively, an efficient or highly accurate screening method for genes whose expression is controlled by retinoids can be obtained.

FIG. 1 is a diagram showing typical retinoid types and molecular structures. FIG. 2 shows a typical RAR and RXR mechanism. FIG. 3 shows the results of investigating the effects of ATRA treatment on various cell proliferations. FIG. 4 shows the results of investigating ChIP-PCR time variation due to ATRA treatment. FIG. 5A shows the results of primary screening of TRRA (TRRA1-80) by ChIP assay. FIG. 5B shows the results of primary screening of TRRA (TRRA81-160) by ChIP assay. FIG. 5C shows the result of primary screening of TRRA (TRRA161-201) by ChIP assay. FIG. 6A shows the results of investigating the time variation of CYP26A1 expression by ATRA treatment by RT-PCR. FIG. 6B is a result of investigating time variation of TRRA13 expression by RT-PCR by ATRA treatment. FIG. 6C shows the results of investigating the time variation of TRRA52 expression by RT-PCR by ATRA treatment. FIG. 6D shows the results of investigating the time variation of TRRA111 expression by RT-PCR by ATRA treatment. FIG. 6E shows the results of investigating the time variation of TRRA121 expression by RT-PCR by ATRA treatment. FIG. 7A shows the result of secondary screening of TRRA (TRRA4-116) by RT-PCR. FIG. 7B shows the result of secondary screening of TRRA (TRRA117-197) by RT-PCR. FIG. 7C is a diagram summarizing the results of secondary screening of TRRA by RT-PCR.

<Explanation of terms>
The meaning of various terms in this specification will be described as follows.

(1) Retinoid
Retinoid is a general term for compounds including retinol, retinal, retinoic acid (RA), and derivatives thereof. All-trans-RA (ATRA) and 9-cis-RA are known as biologically active RAs, and they function through nuclear receptors, which are ligand-induced transcription factors. Has been. Retinoids include the molecules of FIG.

(2) Retinoic acid receptor and retinoid X receptor Retinoic acid receptor (RAR) and retinoid X receptor (RXR) are RA receptors, each of which has three isotypes (α , Γ, β) are known. In general, in the presence of RA, these receptors regulate the expression of various genes by binding to an RA response element (RARE, retinoic acid response element) present in the promoter region of the target gene. For example, a reaction mechanism as shown in FIG. 2 has been estimated.

Hereinafter, embodiments of the present invention will be described in detail. For similar contents,
In order to avoid repeated complications, description will be omitted as appropriate.

<Embodiment 1: Expression control agent containing retinoid>
The present embodiment is an expression control agent that controls the expression of a specific gene containing a retinoid, such as colony stimulating factor 3 receptor (granulocyte), thioredoxin interacting protein ( thioredoxin interacting protein), OTU domain containing 7B, tudor domain containing 5, kinesin family member 21B, Odo-Skip Dried 1 (Drosofia) (Odd-skipped related 1 (Drosophila)), protocadherin beta 4 (NH), like NHS-like, cytochrome P450, family 26, subfamily A, polypeptide 1 (cytochrome P450, family 26, subfamily A, polypeptide 1), tyrosine hydride Tyrosine hydroxylase, olfactomedin-like 1, frizzled homolog 4 (Drosophila), ataxin 2 and RAS protein activator-like 1 GAP1 like) (RAS protein activator like 1 (GAP1 like)), isocytorate dehydrogenase 3 (NAD +) alpha (isocitrate dehydrogenase 3 (NAD +) alpha), zinc finger protein 710 (zinc finger protein 710), selenophosphate synthetase 2 (selenophosphate synthetase 2), carboxypeptidase D, lectin rich repeat containing 37, member A2 (leucine rich repeat containing 37, member A2), G protein-coupled receptor 108 , FXYD domain containing ion transpo An expression control agent that controls the expression of one or more genes selected from the group consisting of a regulator 3 (FXYD domain containing ion transport regulator 3) and a keratin associated protein 19-6 (keratin associated protein 19-6) It is. A retinoid has the characteristic which controls the expression of said gene, as demonstrated by the Example mentioned later. Therefore, the expression control agent containing a retinoid can be suitably used for controlling the expression of the gene according to various purposes such as treatment of a disease associated with the gene.

  Details of the nucleotide sequence and the like related to the above gene can be confirmed in GenBank, which is a database of National Center for Biotechnology Information (NCBI). For example, colony stimulating factor 3 receptor (granulosite) (Uzumaki et al., Proc. Natl. Acad. Sci. USA 86 (23): 9323-6.) Is a gene encoding cytokines, and thioredoxin interacting protein (Yu et al., PLoS One. 2009 Dec 22; 4 (12): e8397.) Is considered to be a gene encoding a protein involved in redox in vivo.

  OTU domain containing 7B has been reported as a gene that indirectly suppresses NF-κB. In recent years, suppression of NF-κB has been reported to suppress tumor formation and enhance the effects of antitumor drugs in breast cancer, stomach cancer, HBV-infected HCC, oral squamous cell carcinoma, and the like.

  Protocadherin beta 4 is a membrane protein. In recent years, there have been reports that some of the genes belonging to the same family as protocadherin beta 4 have a cancer suppressing action.

  Frizzled homolog 4 (Drosophila) is a gene presumed to be related to the wnt / β-catenin signal. It was found in [Yanagitani et al., Hepatology. 2004 Aug; 40 (2): 366-75.] That the expression of β-catenin in RAR-E transgenic mice was significantly increased in the liver compared to wild type mice. Have been described.

  RAS protein activator-like 1 (GAP1-like) has been reported as a gene that suppresses the RAS signaling pathway. So far, the action of RAS as an oncogene has been well studied.

  Thus, all of the above genes are considered to have an important role in life activities, and thus can be a drug discovery target for new drugs. Therefore, treatment based on a novel mechanism of action becomes possible by controlling these expressions with the above-mentioned expression control agent. In addition, since the expression of the above gene is regulated by retinoid, it is considered that the gene is involved in a disease involving retinoid and a healing mechanism. In particular, OTU domain containing 7B, protocadherin beta 4, frizzled homolog 4 (Drosophila), and RAS protein activator-like 1 (GAP1 like) have been strongly suggested to be associated with disease It is a drug discovery target.

  The above expression control agents are colony stimulating factor 3 receptor (granulosite), OTU domain containing 7B, kinesin family member 21B, odo-skip recited 1 (Drosophila), protocadherin beta 4, NHS-like, cytochrome P450 , Family 26, Subfamily A, Polypeptide 1, Tyrosine hydroxylase, Ataxin 2, RAS protein activator-like 1 (GAP1 like), Isocytolate dehydrogenase 3 (NAD +) alpha, Zinc finger protein 710, Selenophosphate synthetase 2, carboxypeptidase D, lectin rich repeat containing 37, member A2, G protein-coupled receptor 108, and FXYD domain containing ion trans Inducing the expression of one or more genes selected from the group consisting of over preparative regulator 3, it may be an expression control agent. This is because the retinoid contained in the expression control agent has a characteristic of inducing the expression of the gene as demonstrated in Examples described later. In this case, the expression control agent containing a retinoid can be suitably used for inducing expression of the gene according to various purposes such as treatment of a disease associated with the gene.

  The expression regulator is one selected from the group consisting of thioredoxin interacting protein, Tudor domain containment 5, olfactedin-like 1, frizzled homolog 4 (Drosophila), and keratin associated protein 19-6. It may be an expression control agent that suppresses the expression of the above genes. This is because the retinoid contained in the expression control agent has a feature of suppressing the expression of the gene as demonstrated in Examples described later. In this case, the expression control agent containing a retinoid can be suitably used to suppress the expression of the gene according to various purposes such as treatment of a disease associated with the gene.

  Further, the retinoid contained in the expression regulator may be all-trans retinoic acid (ATRA) or a derivative thereof, and ATRA is particularly preferable. This is because ATRA has the characteristic of controlling the expression of the gene as demonstrated in the examples described later.

  The gene whose expression is controlled by the expression control agent includes a mutant gene of the gene. Here, the mutant type includes those resulting from differences in DNA sequences between individuals. Further, the base sequence of the mutant gene may be a base sequence in which one or several bases are deleted, substituted or added in the wild-type base sequence of the above gene derived from human. The “one or several” is preferably 30 or less, more preferably 20 or less, more preferably 15 or less, more preferably 10 or less, more preferably 5 or less. Preferably, it is 4 or less, more preferably 3 or less, more preferably 2 or less, and still more preferably 1. This is because the gene encoded by the base sequence in which one or several bases are deleted, substituted or added has fewer base deletions, substitutions or additions than the base sequence of the wild-type gene. This is because it has characteristics close to those of the gene encoding the base sequence of the wild-type gene.

  In addition, the base sequence of the mutant gene may be a base sequence having 80% or more homology with the wild-type base sequence of the human-derived gene. Here, “80% or more” is preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and most preferably 98% or more. This is because the higher the homology of the above-described base sequence having “80% or more” homology with the base sequence of the wild-type gene, the closer to the gene encoding the base sequence of the wild-type gene. It is because it is doing.

  The base sequence of the mutant gene is a base sequence of a nucleic acid that hybridizes under stringent conditions to a nucleic acid consisting of a base sequence complementary to the wild-type base sequence of the human-derived gene. There may be.

  Further, the origin of the gene is not limited as long as the base sequence of RGKTCANNNNNRGKTCA is present upstream of the gene, for example, human, mouse, rat, rabbit, pig, sheep, cow, horse, cat, dog, monkey, Or it may be a chimpanzee. Preferred are mouse, rat, monkey, chimpanzee and human, and particularly preferred is human. This is because humans can be used to treat human diseases and develop therapeutic drugs. In addition, mice, rats, monkeys, and chimpanzees are widely used as model animals for research all over the world, and many characteristics have been clarified. Therefore, by controlling the expression of the above genes in these organisms, therapeutic drugs can be obtained. Especially useful information for the development of

  Moreover, other embodiment of this invention is a reagent containing the said expression control agent. This reagent is a research reagent, an additive for maintaining the function and survival rate of cells or tissues in regenerative medicine, or an additive for assisting animal growth in livestock production through the above-mentioned gene expression control action. Can be used as an agent.

  As used herein, “inducing expression” includes significantly increasing the expression level of a specific gene in a test sample as compared to a control sample. In addition, “suppressing expression” includes significantly reducing the expression level of a specific gene in a test sample as compared to a control sample. The significance is evaluated, for example, by using a Student's t-test to evaluate the statistical significance of the expression level before and after changing the expression of a specific gene in vitro or in vivo. It can be considered statistically significant when <0.05.

  As used herein, “cancer” includes a disease caused by normal cells undergoing mutation and continuing to grow. Malignant cancer cells arise from all organs and tissues throughout the body, and when cancer cells grow, they become a mass of cancer tissues and invade and destroy surrounding normal tissues. Cancer is lung cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, kidney cancer, adrenal cancer, biliary tract cancer, breast cancer, colon cancer, small intestine cancer, cervical cancer, endometrial cancer, ovarian cancer, bladder cancer, prostate cancer, Ureteral cancer, renal pelvic cancer, ureteral cancer, penile cancer, testicular cancer, brain tumor, cancer of central nervous system, cancer of peripheral nervous system, head and neck cancer (oral cancer, pharyngeal cancer, laryngeal cancer, nasal cavity / sinus cancer, Salivary gland cancer, thyroid cancer, etc.), skin cancer, melanoma, thyroid cancer, salivary gland cancer, blood cancer, malignant lymphoma, carcinoma or sarcoma.

  As used herein, “liver cancer” includes diseases caused by continued proliferation of liver cells. Hepatocytes capable of causing liver cancer include cells of blood vessels such as bile ducts, portal veins, dendritic cells or hepatocytes. Also included are primary liver cancer and metastatic liver cancer. Many primary liver cancers are known to be hepatocellular carcinoma (HCC).

As used herein, “bond” means a connection between substances. The linkage may be either covalent or non-covalent, and includes, for example, ionic bonds, hydrogen bonds, hydrophobic interactions, or hydrophilic interactions.

  As used herein, “homology” is a ratio of the number of identical amino acids in an amino acid sequence between two or more amino acids calculated according to a method known in the art. Before calculating the ratio, the amino acid sequences of the amino acid sequences to be compared are aligned, and a gap is introduced into a part of the amino acid sequence if necessary to maximize the same ratio. Nor do any conservative substitutions be considered identical. Further, it means the ratio of the same number of amino acids to all amino acid residues including overlapping amino acids in an optimally aligned state. Alignment methods, percentage calculation methods, and related computer programs are well known in the art and use common sequence analysis programs (eg GENETYX, GeneChip Sequence Analysis, etc.) Can be measured. In addition, “homology” is obtained by calculating the ratio of the same base in two or more DNA strands or two or more RNA strands according to a method known in the art as described above. is there.

  In this specification, “stringent conditions” means, for example, (1) low ionic strength and high temperature for washing, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.005 at 50 ° C. Using 1% sodium dodecyl sulfate, (2) denaturing agents such as formamide during hybridization, eg 50% (vol / vol) formamide and 0.1% bovine serum albumin / 0.1% ficoll / 42 ° C. With 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5) and 750 mM sodium chloride, 75 mM sodium citrate, or (3) 50% formamide, 5 × SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (PH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate at 0 ° C. at 42 ° C. Conditions may include stringent washing with 0.1 × SSC containing EDTA at 55 ° C. followed by washing in 2 × SSC (sodium chloride / sodium citrate) and 55 ° C. formamide. Examples of moderately stringent conditions are 20% formamide, 5 × SSC, 50 mM sodium phosphate (pH 7.6), 5 × Denhart solution, 10% dextran sulfate, and 20 mg / ml denatured sheared salmon sperm DNA. Conditions such as overnight incubation at 37 ° C. in a solution containing, followed by washing of the filter at 37-50 ° C. in 1 × SSC. The stringency of the hybridization reaction can be easily determined by those skilled in the art and generally depends on the probe length, the washing temperature, and the salt concentration. In general, longer probes require higher temperatures for proper annealing, and shorter probes require lower temperatures. In general, stringency is inversely proportional to salt concentration.

  As used herein, “hybridization” as applied to a polynucleotide refers to the property of allowing pairing between nucleotides by hydrogen bonding between nucleotide bases. Base pairs can occur in Watson-Crick base pairs, Hoogsteen base pairs, or any other sequence specific form.

In this specification, “derivative” means that when an organic compound is considered as a matrix, the structure and properties of the matrix are greatly increased by introduction of a functional group, oxidation, reduction, substitution of atoms or addition of atoms. It means a compound that has been modified to the extent that it does not change. The modification may be performed as an actual chemical reaction, but it may be performed on a desk. The derivative is preferably a pharmacologically acceptable derivative. In the present specification, the “pharmacologically acceptable derivative” is not limited as long as it has a desired effect, and is a pharmacologically acceptable salt, solvate, isomer, or compound of the present invention. Includes forms of prodrugs and the like (eg esters). This pharmacologically acceptable derivative also includes any other compound that, when administered to a patient, is provided (directly or indirectly) a compound according to the invention or an active metabolite thereof or residue thereof. Such pharmacologically acceptable derivatives can be obtained by those skilled in the art without undue experimentation. For example, [Burger's Medicinal Chemistry And Drug Discovery, 5 th Edition, Vol 1: Principles and Practice] can refer to. A pharmacologically acceptable salt or solvate of the compound according to the present invention is preferred. The desired effect includes an effect substantially equivalent to that of the compound according to the present invention.

  In the present specification, the “pharmacologically acceptable salt” is not particularly limited. For example, it is formed with an anion salt formed with any acidic (eg, carboxyl) group, or formed with any basic (eg, amino) group. Cationic salt. Salts include inorganic and organic salts, including those described in [Berge, Bighley and Monkhouse, J. Pharm. Sci., 1977, 66, 1-19]. As used herein, a “solvate” is a compound formed by a solute and a solvent. As for the solvate, for example, [J. Honig et al., The Van Nostrand Chemist's Dictionary P650 (1953)] can be referred to. If the solvent is water, the solvate formed is a hydrate. This solvent is preferably one that does not interfere with the biological activity of the solute. Examples of such preferred solvents include, without limitation, water, ethanol, and acetic acid. The most preferred solvent is water. The compound according to the present invention or a salt thereof absorbs moisture when exposed to air or recrystallizes, and in some cases has moisture absorption water or can be a hydrate. As used herein, “isomer” includes molecules having the same molecular formula but different structures. Includes enantiomers (enantiomers), geometric (cis / trans) isomers, or isomers having one or more asymmetric centers that are not mirror images of one another (diastereomers). As used herein, a “prodrug” is a compound that is a precursor, and undergoes a chemical change by metabolic processes or various chemical reactions when the compound is administered to a subject, and the compound according to the present invention or a compound thereof Including compounds that yield salts or solvates thereof. Regarding prodrugs, for example, [T. Higuchi and V. Stella, “Pro-Drugs as Novel Delivery Systems”, A.C.S. Symposium Series, Volume 14] can be referred to.

  In the present specification, the “derivative” includes, for example, a compound having a 90% or higher similarity (tanimoto coefficient) of Compound Structure Search of PubChem as long as it is a compound having a desired effect. The lower limit value of the tanimoto coefficient to be included in the derivative is not particularly limited, but is preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more. Yes, more preferably 99% or more. The greater the degree of similarity (tanimoto coefficient), the higher the possibility that it is a derivative whose structure, physiological activity, etc. approximate to the parent body. The formula for calculating the tanimoto coefficient is as follows, and is the similarity coefficient between the authoritative compounds most frequently used in the field of chemoinformatics. The tanimoto coefficient calculation formula can also be referred from the PubChem website.

Tanimoto = AB / (A + B-AB)
Where:
Tanimoto is the Tanimoto score, a fraction between 0 and 1.
AB is the count of bits set after bit-wise & of fingerprints A and B
A is the count of bits set in fingerprint A
B is the count of bits set in fingerprint B

  Examples of the compound having a similarity (tanimoto coefficient) of 95% or more with respect to ATRA include, for example, PubChem CID (Compound ID) of 5538, 444795, 449171, 4136524, 5282379, 5268825, 5496917, 6419708, 6439661, 6439749, 6660993 , 6913131,6913136,6913160,9796370,9839397,9861147,9972326,9972327,9995220,10017822,10017935,10040620,10041353,10063649,10086397,10086398,10149682,10267048,10286439,10335106,10357701,10380944,10525032,10470200 , 10518761,10566385,10638113,10881132,11738545,12358676,12358678,18458354,18637768,19609228,21590819,23275881,25145416,44725022,9860303,9929074,10015486,10125803,10266931,10286753,10314319,10474100,10712359,11141 , 18696002,18696006,21651187,22239079,25141345,44393163,44579060,167095,5355027,10087786,10193246,10215224,10358907,10426543,11266097,15125882,18977383,19063167,19360964,19609253,2 0830941, 2226220, 23002673, 23181726, 23208908, 25011742, 4314230, 44579056, 44579100, 9830767, 10636975, or 11000660, or one or more compounds selected from the group consisting of 11000660.

<Embodiment 2: Retinoid-responsive gene screening method>
Another embodiment of the present invention is a method for screening a gene whose expression is controlled by a retinoid, wherein RGKTCANNNNNRGKTCA upstream (R = A / G, K = G / T, N = A / G / C / T) A step of selecting a gene having the nucleotide sequence of RAR or RXR with respect to the nucleotide sequence of RGKTCANNNNNRGKTCA located upstream of the selected gene, RAR or RXR Analyzing the change in the retinoid-dependent expression level of the above gene located downstream of the base sequence of RGKTCANNNNNRGKTCA, which showed retinoid-dependent binding. According to this screening method, as demonstrated in Examples described later, genes whose expression is controlled by retinoids can be screened efficiently or with high accuracy.

  Here, the upstream in the above in silico analysis may be within 5 kb upstream. This is because, as demonstrated in Examples described later, by examining within 5 kb upstream, genes whose expression is controlled by retinoids can be screened efficiently or with high accuracy.

  The test for retinoid-dependent binding of RAR or RXR may be a test using ChiP (Chromatin immunoprecipitation) analysis. This is because, as demonstrated in the examples described later, by examining by ChiP analysis, genes whose expression is controlled by retinoids can be screened efficiently or with high accuracy. ChiP analysis is a method for investigating the interaction between a nucleic acid and a protein, or the binding site of a protein on the nucleic acid, using an antibody against the protein.

  The retinoid-dependent change in expression level may be analyzed using RT-PCR. This is because, as demonstrated in the examples described later, by performing analysis by RT-PCR, genes whose expression is controlled by retinoids can be screened efficiently or with high accuracy.

  In addition, in the above screening method, the retinoid-dependent binding of RAR or RXR to the base sequence of RGKTCANNNNNRGKTCA located upstream of the gene selected by in silico analysis in the above-described examination of retinoid-dependent binding of RAR or RXR. Is negative, and when a sequence overlapping with the base sequence around the base sequence of RGKTCANNNNNRGKTCA exists on the genome, retinoid-dependent expression of the gene selected by the above in silico analysis The screening method may further include a step of analyzing the change in the amount. This is because, as demonstrated in the examples described later, when a screening method including this step is used, genes whose expression is controlled by retinoids can be screened efficiently or with high accuracy. .

  In this specification, “in silico analysis” means analysis performed using a computer. In fields such as molecular biology, it is generally handled with a concept different from in vitro and in vivo, which actually handle cells and various biomolecules.

  In the present specification, “retinoid-dependent binding property” represents a property of increasing or decreasing the binding property due to the presence of a retinoid. For example, it is known that RAR or RXR binds to RA in the presence of RA and then binds to the target gene RARE.

  In the present specification, “change in expression level dependent on retinoid” refers to the property that the expression level of a gene increases or decreases due to the presence of a retinoid. For example, RAR or RXR is known to bind to the target gene RARE in the presence of RA and subsequently increase or decrease the expression of the target gene.

  RT-PCR (Reverse Transcription Polymerase Chain Reaction) is a method in which reverse transcription is performed using an RNA chain as a template, and PCR is performed on the generated cDNA. For example, a commercially available kit such as Titan One-Tube RT-PCR Kit (Roche) can be used. Total RNA can be extracted from cells using the guanidine thiocyanate method, commercially available reagents or kits. Cells can be purchased from Sanko Junyaku Co., Ltd. or Takara Bio Inc. Real-time PCR is a method for monitoring nucleic acids amplified by PCR in real time.

  As mentioned above, although embodiment of this invention was described, these are illustrations of this invention and various structures other than the above are also employable.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these.

<Used cells>
Hepatocellular carcinoma cell lines Huh7, HepG2, breast cancer cell line MCF-7

<Reagent used>
・ DMEM
4.75 g of Dulbecco's Modified Eagle's Medium powder (day water) was dissolved in ultrapure water to make a total volume of 500 mL and sterilized by high-pressure steam. Then 10 mL 10% NaHCO3 (Wako), 5 mL 100 × glucose (NACALAI TESQUE) (final concentration 3500 mg / L), 10 mL 50 × L-glutamine (NACALAI TESQUE) (final concentration 584 mg / L), non- Activated fetal bovine serum (FBS) (final concentration 10% or 1%: JRH Biosciences) was added.

・ PBS (-)
NaCl, Na2HPO4 · 12H2O, KCl (above, NACALAI TESQUE), and KH2PO4 (Wako) were added to ultrapure water at final concentrations of 137 mM, 8.10 mM, 2.68 mM, and 1.47 mM, respectively, and then autoclaved.

・ ATRA
ATRA powder (SIGMA) was dissolved in DMSO (SIGMA) to a concentration of 40 mM and stored at −30 ° C.

・ Protease inhibitor cocktail (PIC)
Protease inhibitor cocktail tablets (Roche Diagnostics) were added to 2 mL of ultrapure water, made 25 × PIC, stored at −30 ° C., and thawed appropriately.

・ Low Salt Immune Complex Wash Buffer
Prepare with ultra pure water so that 0.1% SDS, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl (above, NACALAI TESQUE), 1% Triton X-100 (Wako), and 4 ° C Saved with.

・ High Salt Immune Complex Wash Buffer
Prepare with ultrapure water so that 0.1% SDS, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl (above, NACALAI TESQUE), 1% Triton X-100 (Wako), and 4 ° C Saved with.

・ LiCl Immune Complex Wash Buffer
0.1% SDS, 1 mM EDTA, 20 mM Tris-HCl (pH 8.1), 250 mM LiCl (above, NACALAI TESQUE), 1% deoxycholic acid (sodium salt) (Wako), 1% IGEPAL-CA360 (MP Biomedicals) The sample was prepared with ultrapure water and stored at 4 ° C.

・ TE Buffer
It was prepared with ultrapure water to 10 mM Tris-HCl (pH 8.1) and 1 mM EDTA (above, NACALAI TESQUE) and stored at 4 ° C.

・ Elution Buffer
The product was prepared with ultrapure water so as to be 10 mM DTT (Wako), 1% SDS, 0.1 M NaHCO3 (NACALAI TESQUE).

・ Phenol / Chloroform
TE saturated Phenol and Chloroform (both NACALAI TESQUE) were mixed 1: 1 and stored at 4 ° C.

・ 70% Ethanol
35 mL 100% ethanol (NACALAI TESQUE) and 15 mL ultrapure water were mixed and stored at room temperature.

・ 1a + 1b
10 μL LightCycler FastStart Enzyme (1a) (Roche Diagnostics) was added to LightCycler FastStart Reaction Mix SYBR GreenI, 10XConc. (1b) (Roche Diagnostics) and stored at −30 ° C.

・ Acidic Phenol / Chloroform
Citrate buffer saturated Phenol and Chloroform (both NACALAI TESQUE) were mixed 1: 1 and stored at 4 ° C.

<Example 1: Selection of transcription region having retinoic acid responsive element upstream>
Table 1 shows the database used for in silico analysis.

  An rnaCluster sequence file was created using the location information of each rnaCluster on the chromosome and the human genome sequence (rnaClusterSeq). Furthermore, the base sequence from the most upstream (5 'terminal base) of each rnaCluster to 5 kbp upstream was cut out to create a 5 kbp sequence file upstream of rnaCluster (upstream5KSeq). As consensus RARE, DR5 in which two half-sites (5'-RGKTCA-3 '(R = A / G, K = G / T)) are connected in tandem with an arbitrary 5-base spacer sequence is applied (RGKTCANNNNNRGKTCA (R = A / G, K = G / T, N = A / G / C / T)), whether or not this DR5 exists within 5 kbp upstream of rnaCluster was searched. A transcription region having DR5 within 5 kbp upstream of rnaCluster was designated as Target RNA of Retinoic Acid (TRRA). Moreover, the gene corresponding to each TRRA was confirmed using the Genome browser of UCSC.

  As described above, in order to identify an unknown new RA target gene, in silico analysis was performed focusing on the fact that RA target gene generally has RARE upstream thereof. As a result, a total of 201 TRRAs with RAREs within 5 kbp upstream were found. Furthermore, as a result of confirming which genes are supported using UCSC Genome browser, 111 TRRAs corresponded to RefSeq Genes (TRRA-1, 3, 4, 5, 8, 9, 12 , 13, 14, 16, 17, 19, 20, 26, 30, 31, 32, 33, 36, 37, 38, 44, 46, 49, 50, 52, 55-58, 63, 67-71, 74 75, 81-86, 89, 91, 92, 94-96, 99-102, 104, 106, 109-113, 116, 117, 120-122, 129, 132, 137, 138, 140, 141, 143 -145, 147-149, 152-154, 156, 159-161, 163, 166, 167, 171, 175, 176, 178-185, 186-188, 190-192, 195-200). On the other hand, the remaining 90 were not annotated as RefSeq Genes on the Genome browser. However, rnaCluster was originally a transcriptional region determined from spliced EST and mRNA, suggesting the possibility of the existence of an unknown gene. In addition,

<Example 2: Effect of ATRA treatment on cell proliferation>
(2-1) Cell culture
Huh7, MCF-7, and HepG2 were cultured in 10% FBS-added DMEM using 10-cm dish (FALCON) at 5% CO2, 37 ° C, saturated water vapor. In a state of 70-90% confluence, wash once with PBS (-), add 2 mL PBS (-) and 300 μL of 0.25% Trypsin / 1 mM EDTA Solution (NACALAI TESQUE) to detach the cells. It was collected. After centrifuging at 1000 rpm for 3 minutes and removing the supernatant, the suspension was resuspended in 10% FBS DMEM, and 1 dish was divided into 4 dishes and subcultured.

(2-2) WST assay
Cells (Huh7, MCF-7, HepG2) were collected, resuspended in 10% FBS DMEM to 5000 cells / 50 μL, and seeded in 50 μL each well of a 96-well plate (FALCON). After overnight culture, the medium was changed to 1% FBS DMEM. 24 hours later, the solution was diluted with 1% FBS DMEM so that the final concentration of ATRA was 0, 1, 5, 10 μM, and incubated at 37 ° C. 1, 2 or 3 days after drug treatment, add TetraColor ONE (Biochemical Biobusiness) diluted to 1% FBS DMEM to a final concentration of 5%, and incubate for 2 hours in a 37 ° C CO2 incubator. Absorbance at 450 nm was measured using a 96-well plate Micro Plate Reader (MRP-A4i, TOSOH). A wavelength of 630 nm was measured as a control wavelength and corrected.

  As described above, in order to determine the cell line to be used for screening the RA responsiveness of TRRA, 0, 1, and 0 for the liver cancer cell line (Huh7, HepG2) and the breast cancer cell line (MCF-7), respectively. After culturing in the presence of 5, 10 μM ATRA, WST assay was performed. In Huh7, almost no change in proliferation due to ATRA treatment was observed (FIG. 3 (a)). However, MCF-7 showed growth inhibition by ATRA treatment (FIG. 3 (b)), and HepG2 showed growth enhancement (FIG. 3 (c)). The effect on proliferation was dependent on the dose of ATRA. By comparing these cell lines, we think that TRRA with antitumor effect can be selected efficiently. In future analysis, these three cell lines will be used to screen TRRA with 5 μM ATRA treatment. Decided to do.

  In addition, the influence on the cell proliferation by the ATRA treatment of FIG. 3 was investigated by the following procedure. Each cancer cell line was seeded at a density of 5000 cells / well in a 96-well plate, and after overnight culture, the medium was replaced with 1% FBS DMEM. After 24 hours, ATRA treatment was performed at each concentration, and WST assay was performed 1, 2, and 3 days later. The absorbance of Day 0 cells was taken as 100%, and the absorbance of each day was graphed (*, P <0.05, **, P <0.01 (t test), n = 3).

<Example 3: Primary screening by ChIP method chromatin immunoprecipitation method (ChIP method)>
10 ⁶ cells were seeded in a 10-cm dish, cultured overnight, and then the medium was changed to 1% FBS DMEM. After 24 hours, the cells were treated with 1% FBS DMEM supplemented with 5 μM ATRA or DMSO. In addition, one cell was sowed and the cell was counted. 270 μL 37% formaldehyde was added and placed at 37 ° C. for 10 minutes to fix. Thereafter, experiments were conducted on ice. The plate was washed twice with 5 mL of cold PBS (−) and scraped with 1 mL of cold PBS (−) (including PIC). The supernatant was removed by centrifugation (700 × g, 4 ° C., 3 minutes). 10 ⁶ cells / 500 μL SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1), PIC) was resuspended and placed on ice for 10 minutes. Using Bioruptor (Cosmo Bio), 30 seconds (250W) sonication was applied 10 times. The supernatant was collected by centrifugation (13 krpm, 4 ° C., 10 minutes). Using a low adsorption tube (BioScience), add 100 μL of supernatant to 900 μL of dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl, PIC) and anti-RARα (sc-551) (Santa cruz), anti-RARβ (sc-552) (Santa cruz), anti-RAR (sc-773) (Santa cruz) To each μg, 25 μL Magnetic beads (Activemotif) was added and rotated at 4 ° C. overnight. Further, 10 μL was collected as input and stored at −30 ° C.

  Next, the following buffer was added, and washing was performed by repeatedly tumbling 50 times. Washing was performed twice with 1400 μL Low Salt Immune Complex Wash Buffer, twice with 1400 μL High Salt Immune Complex Wash Buffer, twice with 1400 μL LiCl Immune Complex Wash Buffer, and twice with 1400 μL TE Buffer. Thereafter, experiments were performed at room temperature. 125 μL Elution Buffer was added and rotated for 15 minutes, and the supernatant was collected. This was done once more. Here, the stored Input was taken out, 240 μL Elution Buffer was added, and the same operation as the ChIP sample was performed thereafter. 10 μL of 5 M NaCl was added and incubated at 65 ° C. overnight to defix.

  0.5 μL Protease K (20 mg / ml) (NACALAI TESQUE), 5 μL 0.5 M EDTA (pH 8.0), 10 μL 1 M Tris-HCl (pH 6.8) were added, and the mixture was incubated at 45 ° C. for 1 hour. 270 μL Phenol / Chloroform was added, vortexed, and centrifuged (15 krpm, 5 minutes). Collect the supernatant, add 270 μL 100% isopropanol (NACALAI TESQUE), 13.5 μL 4 M NaCl, 1 μL glycogen (20 mg / ml) (Roche Diagnostics), and centrifuge (15 krpm, 4 ° C., 15 minutes) did. It was rinsed with 70% ethanol, air-dried, dissolved in 50 μL ultrapure water, and analyzed by real-time PCR (ChIP-PCR). 5 μL DNA, 0.5 μL 10 μM primer (F), 0.5 μL 10 μM primer (R), 0.8 μl 25 mM MgCl2 (Roche Diagnostics), 2.7 μL ultrapure water, 0.5 μL 1a + 1b into capillary (Roche Diagnostics) And use LightCycler 1.5 (Roche Diagnostics) for 10 minutes at 95 ° C, followed by 5 seconds at optimal temperature (annealing), 10 seconds at 72 ° C (extension), 0 seconds at 72 ° C (fluorescence measurement) The test was repeated 55 times at 95 ° C. for 10 seconds (thermal denaturation). Analyzed with LightCycler Software Version 3.5 (Roche Diagnostics) and corrected with Input value. In addition, in the TRRA screening, the value of the negative control was further corrected. The primers are shown in Table 2.

  As described above, in order to examine whether or not RAR binds to RRA of TRRA obtained by in silico analysis in an ATRA-dependent manner, primary screening was performed by ChIP-PCR. First, in order to determine the ATRA treatment time in screening, the time variation of ChIP-PCR of RARβ and CYP26A1, which are typical retinoid target genes, was examined. At this time, in addition to primers (RARβ_DR5, CYP26A1_DR5) that amplify the region containing RARE (DR5) as primers for PCR, primers (RARβ_DR5_ds4kbp, CYP26A1_DR5_ds4kbp) that amplify the region 4 kbp downstream from each DR5 are used. A negative control was used. As a result, in the samples subjected to ATRA treatment for 3 hours, the value of CYP26A1_DR5 was significantly increased with respect to the negative control in all cell lines. Therefore, the ATRA treatment time of the primary screening was 3 hours (FIG. 4 (a ), (B), (c)).

The time variation of ChIP-PCR due to ATRA treatment in FIG. 4 was investigated by the following procedure. 10 ⁶ cells were seeded in a 10 cm dish, cultured overnight, and then replaced with 1% FBS DMEM. 24 hours later, the cells were treated with 1% FBS DMEM supplemented with 5 μM ATRA or DMSO, and ChIP-PCR was performed 1.5, 3, and 6 hours later. It was corrected by the input collected before immunoprecipitation (*, P <0.05, **, P <0.01 (t test), n = 3).

  On the other hand, regarding RARβ_DR5, there was no difference in MCF-7 subjected to ATRA treatment. This was consistent with the fact that RARβ was hardly expressed in MCF-7 (data not shown). Next, ChIP-PCR of TRRA was performed (Tables 3 to 9 and FIGS. 5A to 5C).

  As in the initial study, CYP26A1_DR5_ds4kbp was applied as a negative control primer, and relative occupancy [=% input (target region) /% input (CYP26A1_DR5_ds4kbp)] was obtained. TRRA with a relative occupancy of 1.5 times or more was determined in the next secondary screening I decided to consider it. In addition, regarding the nucleotide sequence around the target DR5, there was an overlapping region on the genome, so TRRA for which specific ChIP-PCR could not be performed was also examined in the secondary screening. As a result, 126 of 201 TRRAs were examined in the next secondary screening.

<Example 4: Secondary screening of TRRA by RT-PCR>
(4-1) Total RNA extraction The cell line was cultured in a 3.5-cm dish, DMEM was removed, 1 mL TRIzol (invitrogen) was added, and the mixture was shaken for 5 minutes at room temperature. After collecting in a 1.5-mL tube and adding 200 μL chloroform, the mixture was vigorously mixed for 30 seconds and then centrifuged (15 krpm, 4 ° C., 15 minutes). Thereafter, a dedicated RNA instrument was used. 500 μL of the supernatant was collected, 500 μL isopropanol was added, the mixture was stirred with vortex, and then centrifuged (15 krpm, 4 ° C., 20 minutes). The supernatant was removed, 300 μL 70% ethanol was added, and the mixture was centrifuged (15 krpm, 4 ° C., 5 minutes). The supernatant was removed, and RNA was dissolved in 7 μL ultrapure water. 1 μL 10 × DNase buffer (Nippon gene), 1 μL 2 mg / ml BSA (Wako) and 1 μL DNase (1 U / μL) (Nippon gene) were added and incubated at 37 ° C. overnight. 278 μL ultrapure water, 12 μL 5 M NaCl, 300 μL Acidic Phenol / Chloroform was added, vortexed, and centrifuged (15 krpm, 4 ° C., 5 minutes). 300 μL of the supernatant was collected, 700 μL of 100% ethanol was added, and centrifuged (15 krpm, 4 ° C., 15 minutes). It was rinsed with 70% ethanol, lightly air-dried, and dissolved in 10 μL ultrapure water.

(4-2) RT reaction (SuperScript First-Strand Synthesis for RT-PCR Kit (invitrogen))
The total volume was adjusted to 12 μL with 5 μg RNA, 3 μL random hexamers (50 ng / μL), 1 μL 10 mM dNTPs, and ultrapure water. Using a Thermal Cycler (TaKaRa Bio), annealing was carried out by placing at 65 ° C. for 5 minutes, once removed, and placed on ice for 2 minutes. Then, add 4 μL 5 × FS buffer (invitrogen), 1 μL ultrapure water, 2 μL 0.1 M DTT, 1 μL 50 U / μl SuperScriptII, and again with Thermal Cycler for 10 minutes at 25 ° C and 2 more at 42 ° C. The reaction was stopped by extension for a period of time, followed by 10 minutes at 70 ° C. 380 μL of ultrapure water was added to dilute, and stored at −30 ° C.

(4-3) RT reaction (NCode VILO miRNA cDNA Synthesis Kit (invitrogen))
The total volume was adjusted to 20 μL with 1 μg RNA, 4 μL 5 × reaction Mix, 2 μL 10 × SuperScript Enzyme Mix, and ultrapure water. Using Thermal Cycler (TaKaRa Bio), the reaction was stopped at 37 ° C. for 60 minutes, followed by 5 minutes at 95 ° C. to stop the reaction. 180 μL of ultrapure water was added to dilute, and stored at −30 ° C.

(4-4) cDNA real-time PCR
Place 5 μL cDNA, 0.5 μL 10 μM primer (F), 0.5 μL 10 μM primer (R), 0.8 μL 25 mM MgCl ₂ 2.7 μL ultrapure water, 0.5 μL 1a + 1b into the capillary, and use LightCycler 1.5. First at 95 ° C for 10 minutes, then at optimal temperature for 5 seconds (annealing), 72 ° C for 7 seconds (extension), 72 ° C for 0 second (fluorescence measurement), 95 ° C for 1 second (thermal denaturation) Was performed 55 times. Analyzed with LightCycler Software Version 3.5 and corrected with β-actin value. Primers are shown in Table 10.

(4-5) Statistical analysis At least three independent samples were analyzed, and a significant difference was determined by t-test.

  As described above, RT-PCR was performed in order to examine whether TRRA selected by the primary screening of ChIP-PCR shows an expression change by ATRA treatment. First, in order to determine the ATRA treatment time, the time variation of CYP26A1 expression in the presence of ATRA was examined by RT-PCR (FIG. 6A (a), (b), (c)).

The time variation of RT-PCR by ATRA treatment in FIG. 6 was investigated by the following procedure. 1.5 × 10 ⁵ cells were seeded in a 3.5 cm dish, and after overnight culture, the medium was replaced with 1% FBS DMEM. After 24 hours, 5 μM ATRA treatment was performed, and after 0, 1, 3, 6, 9, 12, 18, and 24 hours, total RNA was collected and subjected to RT reaction. Quantification was performed by real-time PCR and corrected with β-actin values (*, P <0.05, **, P <0.01 (t test), n = 3).

  In Huh7 and HepG2, a significant increase in expression was observed after 1 hour of ATRA treatment, and continued to increase until 24 hours later. On the other hand, MCF-7 also showed a significant increase in expression after 1 hour, but reached a peak after 6 hours of treatment and maintained a relatively high expression level until 24 hours later. Next, according to the result of this initial examination, the TRRA expression level at 6 hours after ATRA treatment was analyzed by RT-PCR (Tables 11 to 14, FIGS. 7A to 7C).

  As a result, there were 27 TRRAs that showed expression changes by ATRA treatment in at least one cell line (Table 15).

  In addition, regarding TRRA for which antitumor effects were expected from past reports, the temporal variation of expression in the presence of ATRA was also examined (FIGS. 6B (d) to 6E (n)). TRRA13 showed a significant increase in expression in Huh7 and MCF-7 from 3 hours after ATRA treatment and was maintained until 24 hours (FIGS. 6B (d) and (e)). On the other hand, in HepG2, a significant increase in expression was observed 9 hours after ATRA treatment, and was maintained until 24 hours later (FIG. 6B (f)). The expression of TRRA52 was increased in Huh7 and MCF-7 by ATRA treatment, and the expression was more strongly induced in MCF-7 (FIGS. 6C (g) and (h)). However, it was not induced at all in HepG2 (FIG. 6C (i)). The expression of TRRA111 was decreased in Huh7 and MCF-7 by ATRA treatment, and the decrease in expression was maintained in MCF-7 until 24 hours later (FIGS. 6D (j) and (k)). On the other hand, in HepG2, a significant increase in expression was observed 24 hours after ATRA treatment (FIG. 6D (l)). TRRA121 showed a transient increase in expression due to ATRA in MCF-7 (FIG. 6E (m)). On the other hand, expression was not observed in Huh7 (data not shown). Moreover, regarding HepG2, expression was observed, but induction by ATRA treatment was not observed (FIG. 6E (n)).

<Consideration of results>
As a result of examining the influence of ATRA on cell proliferation, growth suppression was not observed at a concentration of 10 μM or less in the HCC cell line (FIGS. 3A and 3C). In previous reports, 0.1-20 μM ATRA treatment did not cause apoptosis in Huh7, whereas apoptosis in Hep3B and HepG2 caused apoptosis by high concentrations of 100 μM and 166 μM, respectively (Muto et al., N Engl J Med. 1996 Jun 13; 334 (24): 1561-7.). However, since the induction of retinoid target gene expression was sufficiently induced by 5 μM ATRA treatment (FIGS. 6 (a), (b), (c)), apoptosis caused by excessive ATRA concentration of 5 μM or more is There is a possibility that it works independently from the regulation of gene expression by ATRA via RARE. Therefore, the WST assay was not performed in this study at an ATRA concentration of 10 μM or more, and as a result, it was considered that growth inhibition by ATRA treatment was not observed in the HCC cell line. On the other hand, MCF-7 (Elstner et al., Proc Natl Acad Sci US A. 1998 Jul 21; 95 (15): 8806-11.), Which has been reported to suppress growth by ATRA even at low concentrations, In the above examination, cell growth was actually significantly suppressed with 5 μM ATRA (FIG. 3B). Based on these results, it was decided to use the HCC cell lines Huh7 and HepG2 and the breast cancer cell line MCF-7 for screening for TRRA showing ATRA responsiveness. Thus, by finding MCF-7-specific, or Huh7, HepG2-specific TRRA, it is possible to more efficiently identify genes that can explain the difference in proliferation ability in the presence of ATRA.

  In this case, 201 TRRAs were analyzed in ChIP-PCR in the primary screening. As a result, we were able to narrow down the TRRA to 126, about half. Furthermore, in consideration of the possibility of including false positives, secondary screening was performed in which RNA in ATRA-treated cell lines was measured by RT-PCR. In the secondary screening by RT-PCR, first, the temporal variation of CYP26A1 expression was examined. As a result, 6 hours when sufficient expression induction was confirmed in the three cell lines was determined as the ATRA treatment time in the secondary screening. Subsequently, RT-PCR for 126 TRRAs selected in the primary screening was performed (Tables 11 to 14). As a result, it was shown that the expression of 27 TRRAs was significantly regulated by ATRA.

  The 27 TRRAs are likely to have an important role in various diseases such as HCC involving retinoids. For example, TRRA13, 52, 111, and 121 are expected to have an antitumor effect from changes in expression in each cancer cell line treated with ATRA and reports of antitumor effects in past literature. Therefore, regarding these TRRAs, time-variation analysis by RT-PCR was also subsequently performed (FIGS. 6B (d) to 6E (n)).

  TRRA13 has been reported as a gene that indirectly suppresses NF-κB. In recent years, suppression of NF-κB has been reported to suppress tumor formation and enhance the effects of antitumor drugs in breast cancer, stomach cancer, HBV-infected HCC, oral squamous cell carcinoma, and the like. It has also been reported that acyclic retinoids suppress NF-κB and further suppress adult T-cell leukemia. These reports suggest that TRRA13 may play a part in the antitumor effect of retinoids.

  TRRA52 is a membrane protein. In recent years, there have been reports that some of the genes belonging to the same family as TRRA52 have a cancer suppressing action. Furthermore, in the examination by RT-PCR, since it was strongly induced in MCF-7 (FIG. 6C (h)), it is considered that TRRA52 may also be involved in the antitumor effect by retinoid.

  TRRA111 is a gene presumed to be involved in the wnt / β-catenin signal. Previously, the expression of RAR-E Tg produced in our laboratory in the liver of β-catenin was greatly enhanced compared to wild type mice. Furthermore, in MCF-7, the expression of TRRA111 was strongly suppressed by ATRA (FIG. 6D (k)). The above suggests that TRRA111 may play a part of the antitumor effect of RA signal.

  TRRA121 has been reported as a gene that suppresses the RAS signaling pathway. So far, the action of RAS as an oncogene has been well studied. In addition, in the expression analysis of TRRA121 in this study, it was induced only by MCF-7 (FIG. 6E (m)). Therefore, it is suggested that TRRA121 may also be a gene useful for cancer suppression.

  As described above, it was shown that the expression of 27 TRRAs is significantly regulated by ATRA. Of the 27 TRRAs, 4 TRRAs are likely to define the antitumor effects of RA signals, 5 are unknown gene regions, and the remaining 18 are mostly related to ATRA responsiveness. The gene has not been reported yet.

  It was shown that the series of methods used in this example is effective as a method for searching for a novel ATRA-responsive gene. Furthermore, it was found that ATRA can control the expression of the above 27 genes that can be drug discovery targets for various diseases. In addition, TRRA found in this example is considered to be an important gene that is a drug discovery target for various diseases.

In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

Claims (4)

An expression-enhancing agent that increases the expression of a specific gene containing retinoid (except for a preventive or therapeutic agent for liver cancer),
The retinoid is retinol, retinal, retinoic acid or derivatives thereof, retinyl ester, all-trans retinol, 14-hydroxy-retro retinol, β-carotene, all-trans retinal, all-trans retinoic acid, and 9-cis retinoic acid One or more compounds selected from the group consisting of:
The derivative is
3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid
(2E, 4E, 6E, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4E, 6Z, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
3- [2- (2,6,6-trimethylcyclohexen-1-yl) ethenyl] hept-2-enoic acid,
(2Z, 4E, 6E, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2Z, 4Z, 6E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6-trienoic acid,
(2E, 4Z, 6E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6-trienoic acid,
(2Z, 4E, 6Z, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2Z, 4E, 6Z, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4E, 6E, 8E) -9- (2-ethyl-6,6-dimethylcyclohexen-1-yl) -3,7-dimethylnona-2,4,6,8-tetraenoic acid,
(2E, 4Z, 6E, 8Z) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2Z, 4E, 6Z, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) -5,6-ditrithionona-2,4,6,8-tetraenoic Acid,
(2Z, 4E, 6Z, 8E) -3,7-Dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) -4,5-ditrithionona-2,4,6,8-tetraenoic Acid,
(2Z, 4E, 6Z, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) -5-trithionona-2,4,6,8-tetraenoic acid,
(2E, 4Z, 6E, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4E, 6Z, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) -5-trithionona-2,4,6,8-tetraenoic acid,
(2E, 4Z, 6Z, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4Z, 6E, 8E) -3,7-Dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) -4,5-ditrithionona-2,4,6,8-tetraenoic Acid,
(2Z, 4Z, 6E, 8E) -3,7-Dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) -4,5-ditrithionona-2,4,6,8-tetraenoic Acid,
(2E, 4Z, 6Z, 8E) -3,7-Dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) -4,5-ditrithionona-2,4,6,8-tetraenoic Acid,
(2E, 4E, 6Z, 8E) -9- (3,4-dideuterio-2,6,6-trimethylcyclohexen-1-yl) -3,7-dimethylnona-2,4,6,8-tetraenoic Acid,
(2Z, 4E, 6E, 8E) -3,7-Dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) -4,5-ditrithionona-2,4,6,8-tetraenoic Acid,
(2E, 4E, 6Z, 8E) -9- (4,5-dideuterio-2,6,6-trimethylcyclohexen-1-yl) -3,7-dimethylnona-2,4,6,8-tetraenoic Acid,
(2E, 4E, 6E, 8E) -3-ethyl-7-methyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4E, 6Z, 8E) -9- [2,6-Dimethyl-6- (trideuteriomethyl) cyclohexen-1-yl] -3,7-dimethylnona-2,4,6,8-tetraeno Ic Acid,
(2E, 4E, 6Z, 8E) -3,7-dimethyl-9- (2,6,6-trimethyl-3,4-ditrithiocyclohexen-1-yl) nona-2,4,6,8-tetra Enoic Acid,
(2Z, 4Z, 6Z, 8E) -3,7-Dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) -4,5-ditrithionona-2,4,6,8-tetraenoic Acid,
(2E, 4E, 6Z, 8E) -3,6,7-trimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4E, 6Z, 8E) -3,7-Dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) -4,5-ditrithionona-2,4,6,8-tetraenoic Acid,
(2E, 4E, 6Z, 8E) -7-ethyl-3-methyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4E, 6E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6-trienoic acid,
(2E, 4E, 6Z, 8E) -3,7-Dimethyl-9- (2,6,6-trimethyl-4,5-ditrithiocyclohexen-1-yl) nona-2,4,6,8-tetra Enoic Acid,
(2E, 4E, 6Z, 8E) -3-ethyl-7-methyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4E, 6Z, 8E) -4,5-Dideuterio-3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraeno Ic Acid,
(2E, 4Z, 6Z, 8E) -4,5-Dideuterio-3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraeno Ic Acid,
(2E, 4E, 8E) -3-Methyl-7-methylidene-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,8-trienoic acid,
(2E, 4E, 6Z, 8E) -7-Methyl-9- (2,6,6-trimethylcyclohexen-1-yl) -3- (tritrithiomethyl) nona-2,4,6,8-tetraeno Ic Acid,
(2E, 4E, 6Z, 8E) -7-Methyl-3- (trideuteriomethyl) -9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetra Enoic Acid,
(2E, 4E, 6E, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2Z, 4Z, 6E, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4E, 6E, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) deca-2,4,6,8-tetraenoic acid,
(2E, 4E, 6E, 8E) -9- [3,3-dideuterio-6,6-dimethyl-2- (trideuteriomethyl) cyclohexen-1-yl] -3,7-dimethylnona-2,4, 6,8-tetraenoic acid,
(2E, 4E, 6E, 8Z) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2Z, 4E, 8E) -3-Methyl-7-methylidene-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,8-trienoic acid,
(2Z, 4Z, 6E, 8Z) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2Z, 4E, 6Z, 8Z) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4E, 6E, 8E) -9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4E, 6E, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid; zinc,
(2E, 4E, 6E) -5-methyl-7- (2,6,6-trimethylcyclohexen-1-yl) hepta-2,4,6-trienoic acid,
(2E, 4E, 6Z) -3-methyl-7-[(E) -2- (2,6,6-trimethylcyclohexen-1-yl) ethenyl] deca-2,4,6-trienoic acid,
(2E, 4E, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,8-trienoic acid,
(2E, 4E, 6Z, 8E) -7-tert-Butyl-3-methyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid ,
(2E, 4E, 6E, 8E, 10E) -5,9-Dimethyl-11- (2,6,6-trimethylcyclohexen-1-yl) undeca-2,4,6,8,10-pentaenoic acid ,
(Z) -3-[(E) -2- (2,6,6-trimethylcyclohexen-1-yl) ethenyl] hept-2-enoic acid,
(2E, 4E, 6E, 8E, 10E, 12E) -3,7,11-trimethyl-13- (2,6,6-trimethylcyclohexen-1-yl) trideca-2,4,6,8,10, 12-hexaenoic acid,
(2E, 4E, 6Z) -3-methyl-7-[(E) -2- (2,6,6-trimethylcyclohexen-1-yl) ethenyl] undeca-2,4,6-trienoic acid,
(2E, 4E, 6E, 8E) -4,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4E, 6E, 8E) -7-methyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4E, 6E, 8E) -9- (2-butyl-6,6-dimethylcyclohexen-1-yl) -3,7-dimethylnona-2,4,6,8-tetraenoic acid,
(2Z, 4E, 8E) -7-methylidene-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,8-trienoic acid,
(2E, 4E, 6Z, 8E) -9- (2-butyl-6,6-dimethylcyclohexen-1-yl) -3,7-dimethylnona-2,4,6,8-tetraenoic acid,
3-methyl-5- (2,6,6-trimethylcyclohexen-1-yl) penta-2,4-dienoic acid,
(2E, 4E) -3-methyl-5- (2,6,6-trimethylcyclohexen-1-yl) penta-2,4-dienoic acid,
(Z, 4E) -3-Methyl-4- [3-[(E) -2- (2,6,6-trimethylcyclohexen-1-yl) ethenyl] cyclohex-2-en-1-ylidene] but- 2-Enoic acid,
(2E, 4E) -3-Methyl-6- [1-[(E) -2- (2,6,6-trimethylcyclohexen-1-yl) ethenyl] cyclopropyl] hexa-2,4-dienoic Acid,
(2E, 4E, 6Z, 8E) -3-Methyl-7-propan-2-yl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraeno Ic Acid,
(Z, 4E) -3-Methyl-4-[(4E) -3-methyl-4-[(2,6,6-trimethylcyclohexen-1-yl) methylidene] cyclohexa-2,5-diene-1- Iriden] But-2-Enoic Acid,
(E, 4E) -3-Methyl-4- [3-[(E) -2- (2,6,6-trimethylcyclohexen-1-yl) ethenyl] cyclohex-2-en-1-ylidene] but- 2-Enoic acid,
(2Z, 4E, 8E) -3-methyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,8-triene-6-inoic acid,
(2E, 4E, 6E, 8E) -2,3,7-trimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4E, 6E, 8E) -3,7-dimethyl-9- (2,5,6,6-tetramethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4E) -3-Methyl-5- [2-[(E) -2- (2,6,6-trimethylcyclohexen-1-yl) ethenyl] cyclopenten-1-yl] penta-2,4- Jenoic acid,
(2E, 4E, 6Z, 8E) -3,7-dimethyl-9- (2,5,6,6-tetramethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 4E, 6E, 8E) -2,3-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid,
(2E, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,8-dienoic acid,
(2Z, 5E) -7-methyl-3-[(E) -2- (2,6,6-trimethylcyclohexen-1-yl) ethenyl] nona-2,5-dienoic acid,
(2E, 4E, 6E, 8E) -9- [6,6-Dimethyl-2- (2-methylpropyl) cyclohexen-1-yl] -3,7-dimethylnona-2,4,6,8-tetraeno Ic Acid,
(2E, 4E, 6Z, 8E) -9- [6,6-Dimethyl-2- (2-methylpropyl) cyclohexen-1-yl] -3,7-dimethylnona-2,4,6,8-tetraeno Ic Acid,
(2Z, 4E, 6Z, 8E) -9- (6,6-dimethylcyclohexen-1-yl) -3,7-dimethylnona-2,4,6,8-tetraenoic acid,
(2E, 4E, 6E, 8E) -9- (6,6-dimethylcyclohexen-1-yl) -3,7-dimethylnona-2,4,6,8-tetraenoic acid, and
(2E, 4E, 6Z, 8E) -9- (6,6-dimethylcyclohexen-1-yl) -3,7-dimethylnona-2,4,6,8-tetraenoic acid,
One or more compounds selected from the group consisting of:
An expression enhancer that increases the expression of colony stimulating factor 3 receptor (granulosite).   The retinoid is selected from the group consisting of retinol, retinal, retinoic acid, retinyl ester, all-trans retinol, 14-hydroxy-retro retinol, β-carotene, all-trans retinal, all-trans retinoic acid, and 9-cis retinoic acid. 2. The expression enhancer according to claim 1, which is one or more compounds. The expression enhancer according to claim 1 or 2,
An expression enhancer, wherein the gene comprises a mutant gene.   A reagent comprising the expression enhancer according to any one of claims 1 to 3.