KR101274256B1 - A NOVEL USE OF Rani AS AN APOPTOSIS REGULATOR - Google Patents

Abstract

The present invention relates to a novel use of Rani as an apoptosis modulator. According to the present invention, Rani can inhibit apoptosis by inhibiting the activity of protease caspase (Caspase) essential for cell death. The Rani gene or protein encoded by the present invention, or a substance that modulates its activity, such as an antibody, siRNA, chemicals, etc., can be usefully used for the treatment of diseases related to apoptosis.

Rani, apoptosis, siRNA

Description

A new application of RANE as apoptosis regulator {A NOVEL USE OF Rani AS AN APOPTOSIS REGULATOR}

Figure 1 schematically shows the isolation of cell lines resistant to apoptosis induced by p53 and the cloning method of apoptosis inhibitor genes therefrom.

2 shows homology at the protein level between the Rani protein C terminus and HECT regions derived from various proteins. Figure 2a is a comparison of the protein containing the HECT region at the Rani and amino acid sequence level, Figure 2b is expressed as a percentage of homology in the HECT region. * Indicates a cysteine residue important for thioester bond formation.

Figure 3 shows the activity of Rani protein as E3 ubiquitin ligase.

4 is Rani protein shows inhibitory effect on apoptosis induced by p53, TNFα and STS (strausporine).

5 shows that Rani protein inhibits the activity of protease caspase 9 (FIG. 5A) and caspase 3 (FIG. 5B), which is a protease activated thereby, in an apoptosis cascade.

6, Rani expression in various organ-derived cancers and normal tissues is shown.

Figure 7 shows that apoptosis of cancer cells is promoted by siRNA (small interfering RNA) that inhibits the expression of the Rani gene.

8 shows that the sensitivity of cancer cells to anticancer agents is increased by siRNA (small interfering RNA) that inhibits the expression of the Rani gene.

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The present invention relates to a novel use of Rani that can inhibit cell death.

Apoptosis refers to the destruction or suicide of cells in eukaryotic cells (Kerr et. al ., 1972) is a basic intracellular process for maintaining homeostasis of an individual, including regulating normal development of the animal and removing unnecessary or abnormal cells. Apoptosis occurs in response to various external and internal stimuli. With the progression of apoptosis, in the cell, cellular destruction, cell membrane blistering, cytoskeletal changes, cell contraction, chromosome condensation and DNA fragmentation, etc. The same characteristic change occurs (Kerr et al., 1972).

This apoptosis is a physiologically important phenomenon, which is precisely regulated by complex mechanisms in vivo, and induction of inappropriate cell death, ie, inhibition or promotion of apoptosis due to abnormal regulation, is associated with many diseases.

In particular, excessive apoptosis can lead to the destruction of certain cells and thereby mediated loss of function in vivo, such as central nervous system diseases such as a number of acute and chronic degenerative planetary diseases (eg, Alzheimer's disease, Parkinson's disease, Cerebral ischemia / stroke) (Mochizuki et al., 1996; Smale et al., 1995; Thomas et al., 1995; Robertson, et al., 2000), cardiovascular disease (Yue et al., 1999), and Graves' disease And autoimmune diseases such as type 2 diabetes (O'Reilly et al., 1999; Magge, 1998).

On the other hand, a representative disease associated with abnormal suppression of apoptosis is cancer, which is known to be closely related to the development of cancer due to the accumulation of tumor cells that are not properly removed due to the underactivity of apoptosis (Vogelstein et al., 2000). Vousden et al., 2002).

Therefore, the identification of factors and regulatory mechanisms that regulate apoptosis can be useful in developing treatment / diagnostic agents for the above-mentioned apoptosis-related diseases by understanding the mechanism of occurrence of various diseases.

In this regard, a number of apoptosis regulators are known which act at each stage leading to apoptosis.

Pathways leading to apoptosis can be broadly classified into those that pass through the apoptosis receptor and those that do not. The former is apoptosis induced by the binding of a specific ligand to its receptor, and the receptors involved are Fas, tumor necrosis factor receptor 1 (TNFR), and TRAIL (TNF-related apoptosis-inducing ligand). The latter is apoptosis induced by stress, and stresses that can cause cell death include ultraviolet rays, heat shock, gamma irradiation, and hypoxia. Apoptosis caused by this stimulus is completed via factors acting in the downstream stages, a typical caspase protease. They are a group of proteins that act sequentially in the signaling pathway leading to cell death and are involved in cell death by activating a series of proteins by cleaving specific amino acid (aspartate) sites.

Thus, various factors acting in this series of steps may be targets for the regulation of apoptosis, for example, Walczak et al. (Walczak et al., 1999) suggest that apoptosis-promoting factors are induced through cell receptors. The use of TRAIL protein as a cancer therapy is disclosed.

Fulda et al. (Fulda et al., 2000) suggest the use of Fas-L as a target of cytoreceptor-induced apoptosis regulation.

WO03 / 076647 discloses a JADE gene, a protein and a method for screening apoptosis-modulating substances using the same that function as apoptosis and cell cycle regulators in a downstream stage of cell death.

Korean Patent Application Publication No. 2001-113088 discloses CIA proteins, genes and their use to interact with specific DNases activated by caspases to regulate apoptosis.

Korean Patent Application Publication No. 2002-40521 discloses an anticancer agent comprising microlactone using apoptosis.

Furthermore, a representative protein associated with induction of apoptosis is p53, and it has been found that p53 is involved in the removal of these cells as mediators of apoptosis in cells showing abnormal cell growth by oncogenic genes such as Myc (Vogelstein). et al ., 2000; Vousden et al ., 2002). p53 is a representative tumor suppressor protein involved in the repair of damaged DNA and cell cycle regulation. It acts as a transcriptional regulator with high affinity for the target sequence in the promoter of a specific gene to regulate the expression of the target gene at the transcriptional level. Plays an important role in preventing the progression of cancer by inhibiting the division of cells or selectively destroying abnormal cells with damaged DNA or abnormal division (Oda et al., 2000).

However, in response to various stimuli, the regulation of apoptosis for the accurate understanding of the mechanism of apoptosis induced by complex steps interacting with each other and the development of effective therapeutic / diagnostic agents specific and specific to the specific disease based thereon There is a need to develop additional factors that are involved.

Accordingly, the present inventors have identified a new factor capable of regulating the activity of apoptosis induced by cell receptors or various external stresses, and thus resistance to p53-induced apoptosis using cells lacking p53. The present invention was completed by cloning a gene to give a gene and confirming its novel activity as an apoptosis inhibitor.

The present invention is to provide a new use of Rani protein, apoptosis regulator of the gene encoding the same, specifically, apoptosis regulator and apoptosis-related disease treatment comprising a Rani gene, protein or a substance that modulates its activity as an active ingredient. .

In order to achieve the above object, the present invention provides a cell death regulator comprising a Rani protein, a gene encoding the same, or a functional equivalent thereof as an active ingredient.

The present invention also provides a therapeutic or therapeutic adjuvant of cancer comprising a Rani gene or protein expression inhibitor as an active ingredient.

The present invention also provides a pharmaceutical composition useful for the treatment or prevention of a disease associated with the under activity of apoptosis, including Rani gene or protein expression inhibitor as an active ingredient. .

Hereinafter, the present invention will be described in detail.

The present inventors screened a mouse-derived cDNA library using a Saos-2 cell line, and isolated about 2.2 kb of cDNA that confers strong resistance to p53-induced apoptosis. Abbreviation).

Results of homology analysis using a computer using a program called BLASTN or BLASTX (Altschul et al 1990; http://www.ncbi.nlm.nih.gov.) Developed based on BLAST (Karlin et al., 1993) Rani has been identified as a sequence constituting part of GenBank AK077015 (mouse) and AB002315 (human), but its function is unknown at present. However, as a result of homology analysis using the above program, the C-terminal region of Rani showed very low similarity with the HECT (Homology to E6-AP Carboxy Terminus) region.

The HECT (Homology to E6-AP Carboxy Terminus) region is a conserved region consisting of about 350 amino acid residues, which is primarily an E3 ubiquitin ligase involved in the ubiquitination of proteins, such as E6-AP (Huibregtse et al., 1995), Nedd4 (Harvey and Kumar, 1999), SMURF2 (Lin et al., 2000), and PUP1 (Arendt and Hochstrasser, 1997) and the like (Huibregtse et al., 1995). In mammals, E3 ubiquitin ligase has been found to cover about 20 HECT regions to date, but their role varies greatly depending on the intracellular function involved in ubiquitination (You and Pickart, 2001). Therefore, even a protein containing an HECT region requires many experiments that are not obvious to those skilled in the art before elucidating its specific function in the cell. In particular, as Rani of the present invention, E6-AP, hNedd4, hSMURF2, This is even the case when both the HECT region and homology of hPUP1 and yRSP5 are very low, less than 35% (FIG. 2).

Thus, the present inventors found that Rani can effectively inhibit cell death as an E3 ubiquitin ligase containing a HECT region.

Specifically, it was found in the present invention that the Rani protein has an E3 ubiquitin ligase activity through in vitro experiments (FIG. 3), and overexpression of the Rani gene is p53, TNF-alpha and staurosporine (STS) through in vivo experiments. It was found that the cell death caused by and the like can be significantly suppressed (about 50% or more, Figure 4). Furthermore, the expression of Rani in normal and cancerous tissues of cancer patients showed that overexpression of Rani in 48%, 46%, 33%, and 42% of various cancers, such as gastric cancer, colorectal cancer, liver cancer, and lung cancer, respectively. This was observed (FIG. 6). In addition, inhibition of endogenous Rani protein expression by siRNA (Small Interfering RNA) induced induction of cell death as well as sensitivity to anticancer agents (FIGS. 7 and 8). These results demonstrate the new use of the Rani gene of the present invention as a novel apoptosis regulator.

The present invention provides an apoptosis regulator comprising a Rani protein, a gene encoding the same, or a functional equivalent thereof as an active ingredient.

The Rani protein, the gene sequence encoding it, can be used from a variety of mammals, but is preferably of human origin and they are purified from natural sequence sources that are present in all cell types of human and non-human mammal species. It includes all, chemically synthesized, produced by DNA recombination techniques, or prepared by a combination of these and / or other methods.

Furthermore, the Rani protein of the present invention, and the gene encoding the same, include its "functionally equivalent variants". Functionally equivalent variants for the purposes of the present invention refer to compounds that exhibit biological activity comparable to the naturally occurring sequences found. In consideration of the object of the present invention, a functionally equivalent variant means that the cell death can be inhibited. Specifically, functionally equivalent variants include GenBank Accession numbers AB002315 (human) (SEQ ID NO: 1) and AK077015 (mouse) (SEQ ID NO: 2) for genes, and BAA20775 (mouse) and BAC36566 (human) sequences for proteins. It is derived from.

In addition, such functionally equivalent variants include nucleotides constituting the gene sequence or sequence variants in which the amino acid sequence constituting the protein sequence is substituted, deleted, added, and / or inserted as long as it has the activity of inhibiting apoptosis.

Such protein sequence variations are well known to those skilled in the art, for example, site-directed mutagenesis, Kramer, W. & Fritz, HJ. Oligonucleotide-directed construction of mutagenesis via gapped duplux DNA. Enzymology, 154, 350-367, 1987) and the like. Polynucleotide sequence variations also occur naturally.

Such variations in sequence may or may not involve amino acid sequence variations that make up the protein. For example, there are degeneracy mutations in which protein sequence variations do not involve amino acid variations in polypeptides, and such degeneracy mutants are also included in the genes of the invention.

Genes encoding variants that are functionally equivalent to the Rani protein are known in the art, such as hybridization techniques (Southern, EM, Journal of Molecular Biology, 98, 503, 1975) or PCR methods (Saiki et al., Science). 230: 1350-1354, 1985; Saiki et al., Science, 239: 487-491, 1988). For example, those skilled in the art will be able to isolate a gene having high homology with the Rani gene by designing a primer that can hybridize specifically to the Rani gene from the above-mentioned Rani protein sequence.

The protein encoded by this isolated gene has high homology with the native Rani protein at the amino acid level. High homology refers to the identity of at least 50%, in particular at least 70%, more particularly at least 90% (eg, at least 95%) of sequences throughout an amino acid sequence.

The homology of amino acid sequences or nucleotide sequences is based on BLAST (Proc. Natl. Acad. Sci. USA, 90, 5873-5877, 1993), a program called BLASTN or BLASTX (Altschul et al, J. Mol. Biol. , 215, 403-410, 1990) and specific methods are known on the following website (http://www.ncbi.nlm.nih.gov.).

In addition, the Rani protein, a gene encoding it, also includes fragments having biological activity equivalent to its native sequence. As used herein, the term “fragment” refers to a sequence corresponding to a portion of a gene or protein, in the case of genes comprising physical, endonuclease, or chemical cleavage, and in the case of proteins, cleaved with protease. Or chemically cleaved ones. Furthermore, in the case of proteins, variants for altering the safety, shelf life, solubility, etc. of the Rani protein, or variants for altering interaction with other proteins that interact with are also included in the functionally equivalent variants.

The Rani gene of the present invention or a variant thereof functionally equivalent may be used by itself or in a vector for achieving the object of the present invention, and methods for introducing DNA into the vector are known in the art (Molecular Cloning: A Laboratory Manual, 3rd Ed., Sambrook and Russel, Cold Spring Harbor Laboratory Press, 2001; Current Protocols in Molecular Biology Ausubel, Brent, Kingston, More, Feidman, Smith and Stuhl eds, Greene Publ. Assoc., Wiley-Interscience, 1992 ). In one embodiment of the invention it was introduced and used as pCEV29 (Michieli et al., 1996; Yamanaka et al., 2001) vector for expression in eukaryotic cells.

Various vectors are known which can be expressed and / or amplified in cells and otherwise introduced into the chromosome or present in the cell independently of the chromosome, and the vector is suitable for linear DNA, plasmid vector, or other purposes. Contains any vector. Furthermore, such vectors include chemical conjugate vectors, viruses, including ligands or nucleic acid binding moieties (eg, polylysine binding sites) to receptors on the cell surface (eg, those described in WO 93/04701). Vector (e.g., DNA or RNA virus), fusion protein moiety (e.g., antibody moiety that recognizes the antigen of the cell, Glutathione S-transferase for ease of isolation and detection) Or a fusion protein expression vector comprising a fluorescent protein moiety, and selection of a vector appropriate for the purpose will be apparent to those skilled in the art. For example, the Rani gene of the present invention may be introduced into an expression vector used in a gene therapy system or the like, for example, an adenovirus vector, and then included in a virus particle as a carrier according to a known method.

The Rani polynucleotides according to the invention and all of the variants described above can be prepared using known organic chemical methods for polypeptide synthesis and can also be combined with each other to synthesize sequences synthesized into fragments in order to obtain the desired full length sequence. (The Peptides, Analysis, Synthesis, Biology, Vol. 1-9, Gross, Udenfriend and Meienhofer Ed. 1979-1987, Academic Press Inc.). In particular, the Rani polynucleotide, fragment thereof, or functionally equivalent variant thereof according to the present invention preferably uses genetic recombination techniques. Natural Rani polynucleotides can be expressed in a suitable host cell and then made into a cell fusion, or Rani mRNA can be translated in vitro and purified by protein isolation methods known in the art. And protein purification methods are described, for example, in Sambrook et al., Molecular Clonning: A Laboratory Mannual, Second Edition, Cold Spring Harbor Laboratory Press, 1989; Current Protocols in Molecular Biology, Ausubel et al Ed., Greene Publishing Associates and Wiley Interscience 1991). Such Rani polypeptides of the invention include, for example, purified proteins, water soluble proteins, or in the form fused with protein or amino acid residues in a form bound to a carrier for delivery or administration to a target cell.

Intracellular expression of Rani of the present invention significantly inhibited apoptosis caused by p53, TNF-a, STS and the like (about 50% or more, Figure 4). Furthermore, inhibition of endogenous Rani protein expression with siani specific siRNA induced apoptosis. These results demonstrate that the Rani gene of the present invention has a novel apoptosis inhibitory function, and the Rani protein of the present invention, which functions to inhibit apoptosis, can be used to suppress abnormally induced apoptosis. It may be useful as a therapeutic agent for a disease caused by.

Accordingly, the apoptosis modulator of the present invention is characterized in that it is for the treatment or prevention of a disease associated with excessive activity of apoptosis. Diseases associated with such hyperactivity of apoptosis are, but are not limited to, central nervous system diseases such as a number of acute and chronic degenerative diseases (eg, Alzheimer's disease, Parkinson's disease, cerebral ischemia / stroke) (Mochizuki et al., 1996; Smale et al., 1995; Thomas et al., 1995; Robertson et al., 2000), cardiovascular disease (Yue et al., 1999), and autoimmune diseases such as Graves' disease and type 2 diabetes ( O'Reilly et al., 1999; Magge et al., 1998).

The present invention is also characterized in that the Rani protein, the gene encoding it, or a variant functionally equivalent thereto is particularly associated with the HECT region of the Rani protein. In one embodiment of the present invention, it was demonstrated that only the HECT region of the C-terminal region of Rani can sufficiently inhibit apoptosis.

The present invention is based on the utility of Rani as an apoptosis inhibitor and the discovery that Rani is overexpressed in a disease associated with the underactivity of apoptosis, such as cancer cells (see FIG. 6), and in this respect the present invention relates to Rani gene or protein expression. It provides a pharmaceutical composition useful for the treatment or prevention of diseases associated with the under activity of apoptosis comprising an inhibitor as an active ingredient.

Diseases caused by the underactivation of apoptosis in the present invention include, but are not limited to, cancers in particular by inactivation of p53 (Vogelstein et al., 2000; Vousden et al., 2002). One embodiment of the present invention confirmed the overexpression of Rani in gastric cancer, liver cancer, colon cancer, and lung cancer, which means suppression of apoptosis in the cancer cells constituting the cancer tissue, apoptosis due to the loss of Rani function Suggests that induction may be effective in the treatment of cancer.

As used herein, the term "inhibitor" is not particularly limited so long as it is a substance capable of inhibiting the expression of the Rani gene, the expression of the Rani protein, and / or the activity of the expressed protein, for example, in a synthetic or genetic recombination technique. Substances obtained by the above, substances derived from nature, or derivatives thereof include, for example, antibodies, antisense nucleotides, and the like. In one embodiment of the present invention, siRNA was used as an inhibitor to effectively inhibit Rani's function as an apoptosis inhibitor.

The siRNA refers to a double-stranded RNA molecule that binds to the target mRNA and interferes with its translation, and includes a sense strand and an antisense strand. The sense strand comprises a ribonucleotide sequence corresponding to a Rani gene sequence, for example, a target sequence selected from SEQ ID NO: 1 or 2, wherein the antisense strand comprises a ribonucleotide sequence complementary to the sense strand. .

siRNA is expressed in the process of development of the living body, or the defense mechanism of the body against the virus, and when bound to the target mRNA, induces the binding of the intracellular enzyme complex that recognizes it, and degrades the corresponding mRNA (Fire, 1998; Montgomery et al., 1998; Sharp PA,, 1999).

In mammals, siRNAs are typically 20 or 21mer double-stranded RNA, consisting of 19 complementary sequences and a 3'-terminal non-complementary thymidine or uridine dimer. Such double-stranded RNA has been shown to specifically inhibit the expression of the corresponding gene upon introduction into cells (Elbashir et al., 2001).

The siRNA according to the present invention may comprise two complementary nucleotide molecules or may comprise sense and antisense sequences in one molecule (transcript), for example, in the form of a hairpin. The siRNA may be at least about 10 nucleotides in length, the longest naturally occurring Rani transcript. In particular, the siRNA is about 100 nucleotides or less in length, most particularly 15 to about 25 nucleotides. In a preferred embodiment of the present invention, the sequence represented by SEQ ID NO: 3 or 4, respectively, as the sense strand and the antisense strand constituting the double-stranded molecule effectively inhibited the function of Rani.

Complementary polynucleotide sequences can be hybridized under appropriate conditions to form several mismatched or mismatched double-stranded molecules or hairpin-structured double-stranded molecules. Given the object of the present invention, two sequences comprising up to 5 mismatches can be considered complementary.

Methods of introducing such siRNAs into cells are known, and in one embodiment of the present invention, a vector comprising its DNA template capable of transcription to siRNA is transfected into cells to express siRNA.

In one embodiment of the present invention, siRNA was designed from a site corresponding to the human Rani gene, and was used to introduce a 293 cell line in which Rani was highly expressed to specifically inhibit Rani expression. As a result, an increase in apoptosis was observed in 293 cells, and an increase in sensitivity to TNF-alpha, which is one of the apoptosis promoters, and an increase in sensitivity to known anticancer agents were confirmed (Example 7, FIG. 7 and 8 ). In particular, the phenomenon of increased sensitivity to a known anticancer agent by the inhibition of Rani is of great clinical utility, for example, a Rani inhibitor such as siRNA for Rani, together with a known anticancer agent, increases its sensitivity. When used as an adjuvant, it is possible to significantly reduce the amount of known anticancer agents with many side effects, which is very significant in terms of improving the welfare and economics of patients.

In this regard, a pharmaceutical composition useful for the treatment or prevention of a disease associated with the under activity of apoptosis comprising the Rani gene or protein expression inhibitor of the present invention as an active ingredient, when used in the treatment of cancer, may further comprise an anticancer agent. Can be.

In another aspect, the present invention provides a treatment or adjuvant for cancer comprising the Rani gene or protein expression inhibitor of the present invention as an active ingredient.

In another aspect, the present invention provides an anticancer agent comprising the combination of the Rani gene or protein expression inhibitor of the present invention and an anticancer agent. The inhibitor is characterized by increasing the sensitivity of cancer cells to anticancer agents.

The anticancer agent may be used in the art as long as it is used in combination with the Rani gene or protein expression inhibitor of the present invention to achieve the above effects, but is not limited thereto, including cisplatin, doxorubicin and etoposide. .

The present invention also provides a kit for diagnosing apoptosis-related diseases comprising a means for detecting the expression of Rani gene or Rani protein, or mutation of the Rani gene.

The inventors have found that the Rani gene or protein can not only inhibit apoptosis in cells in which apoptosis is induced, but also overexpression of apoptosis in underactivated cancer. Therefore, by measuring the expression level of the Rani gene and / or protein using a biopsy sample of the patient, it is possible to know the diagnosis and / or progression of the disease associated with cell death, the cell death that can be used the diagnostic kit of the present invention Related diseases are as mentioned above.

The expression level may be measured using a detection means known in the art, but the present invention is not limited thereto. For example, in the case of gene expression, cDNA may be separated by reverse transcription after RNA is separated from a biopsy sample. After synthesis, RT-PCR method for performing PCR (Polymerase Chanin Reaction) using appropriate primers capable of specifically amplifying Rani or real-time quantitative PCR based on the method, cDNA array method, Northern blot, etc. Can be done. In the case of protein expression, quantification by enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), high performance liquid chromatography (HPLC), western blot or mass spectrometry using an antibody against Rani can be used.

In addition, since it has been found that the Rani gene is closely related to apoptosis, certain mutations such as replacement of a specific nucleic acid sequence, deletion addition, etc. exist in the Rani coding sequence or its regulatory region such as a promoter and an enhancer. Rani's function may be reduced, or expression of genes or proteins may be reduced. Therefore, by examining the mutation of the gene including the Rani coding sequence and its regulatory region, and comparing the difference with the normal gene, it is possible to determine in advance the risk of apoptosis-related diseases. Mutations in gene sequences and differences from normal sequences can be performed using methods known in the art, including but not limited to, for example, cDNA sequencing, single strand confimration polymorphism (PCR-SSCP), Methods such as PCR-SSO (sequence-specific oligonucleotide) can be used.

The present invention also provides a method for screening a substance that can be usefully used as a therapeutic agent for apoptosis-related diseases.

Specifically, the present invention provides a method for screening one or more substances selected from the group consisting of expression regulators of Rani gene, expression regulators of Rani protein, and function regulators of Rani protein. It is preferable that the said substance is a substance obtained by synthetic | combination or genetic recombination technique, a substance derived from nature, or a derivative thereof.

The method for screening the substance comprises (1) the expression level of the Rani gene or the reporter gene by contacting the substance with (1) a transgenic control region of the Rani gene and a transformed cell or an in vitro expression system comprising the Rani gene or a reporter gene. Or (2) measuring the apoptosis inhibitory function of the Rani protein in contact with the target molecule in the presence of the Rani protein.

As the reporter gene in the step (1), for example, chloramphenicol acetyl transferase (CAT), β-galactosidase (β-Gal), luciferase (luciferase) and the like can be used. The expression regulator of the Rani gene is, for example, constructs an expression vector connecting the transcription control region (promoter, enhancer) of the Rani gene upstream or downstream of the translation region of the reporter gene and introduces it into a suitable cultured cell, and the like. Compounds obtained by synthesis or genetic recombination techniques in cells or compounds derived from nature and those

It can be screened by adding a derivative of and measuring the expression amount of reporter gene or the amount of reporter protein after a certain time. The regulatory region of the Rani gene can be obtained from a commercial genome library by performing a plaque hybridization using a fragment of the Rani cDNA as a probe by a known method. The amount of reporter protein can also be measured as enzyme activity, or an antibody or the like can be used as the expression level of the protein.

It can also be measured.

In the method of step (2), Rani or a cell extract (fraction) containing Rani purified after overexpression in a cell by recombination or the like is combined with ubiquitin in vitro, for example, the method described in Example 2 Rani expression control substances can be screened by measuring the activity of ubiquitination as described above.

Apoptosis inducing agents, and pharmaceutical compositions for treating apoptosis-related diseases according to the present invention may be administered in a suitable formulation together with a carrier, diluent or excipient known in the art. The pharmaceutical composition of the present invention may be administered via a general route as long as it can reach the desired tissue, and may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) according to the desired method. Oral administration, in particular parenteral administration, is preferred, and more particularly intravenous injection, the dosage form depending on the method of administration chosen.

Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components, as necessary. And other conventional additives such as buffers and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs. Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it may be suitably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.

In addition, other pharmaceutical delivery systems such as liposomes and emulsions well known in the art may be employed. Certain organic solvents such as dimethylsulfoxide may also be employed.

The dosage will vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the disease. For example, a therapeutically effective dose can be initially determined using in vitro assays through cell culture, and an experiment is performed in animal models to determine the IC50 (in vitro assay) in which the concentration range of blood Rani is determined in cell culture. , The concentration of the test compound in a lethal amount relative to 50% of the cultured cells in the drug treatment. Those skilled in the art will be able to determine the amount effective for treatment without undue experimentation, and this information can be used to more accurately determine the dosage useful in humans.

The pharmaceutical composition of the present invention may be provided alone or in combination with drugs such as surgery, hormonal therapy, and anticancer agents for the treatment of cancer.

Hereinafter, examples are provided to help understand the present invention. However, the following examples are merely provided to more easily understand the present invention, and the present invention is not limited to the following examples.

Example  1. Genes That Inhibit Apoptosis Cloning

1-1 cell culture and Transfection

The cells used in this example were medium containing 10% fetal bovine serum (Fetal Bovine Serum, Hyclone), high glucose, penicillin (100 unit / ml) and streptomycin (100 μg / ml), Dulbecco's Modified Eagle's Medium (DMEM (Hycolon), incubated in 37% 5% CO ₂ environment. Transfection was performed using basically Lipoectamine (Invitrogen, USA) as recommended by the manufacturer. In addition, reagents and instruments necessary for cell culture were purchased from Invitrogen.

1-2 of genes that inhibit apoptosis Cloning

A mouse testis cDNA library was introduced into Saos-2 cells (American Type Culture Collection, ATCC; Cat. No. HTB-85) derived from human osteosarcoma in which apoptosis was induced by p53. The cDNA library extracts and purifies mRNA using Trizol (Invitrogen) from mouse testis as a manufacturer's recommendation, and synthesizes cDNA using Superscipt II (Invitrogen) as a manufacturer's recommendation. Michieli et al, 1996;.. Yamanaka et al, 2001) Sfi of It was produced by cloning at the I restriction enzyme site. The pCEV29 expression vector expresses a cloned cDNA via the MuMLV-LTR (Moloney murine leukemia virus long terminal repeat) promoter and expresses a Neomycin-resistant gene that results in resistance to G418.

1 × 10 ⁶ Saos-2 cells were placed in a plate of 100 mm diameter and incubated for 2 days in 10 ml of culture solution (DMEM + 10% FBS). ml of G418 was added to the cultures and incubated. After about 2 weeks, the c418 addition was discontinued after the cDNA-non-injection was removed, with the cells forming G418 resistant colonies containing approximately 100-2,000 cells. Then, when each colony grew to about 1 mm in diameter, they were separated, and each colony was separated into two wells of two 24-well plates and cultured.

When the cells became about 50% lush, adenovirus expressing p53 (Jung et al., 2004) was infected with cells of one of the two wells and the culture was changed after about 3 hours. After 3 days, apoptosis was observed by p53 expression. One week later, cells were fixed and stained with Kimsa (Giemsa, Sigma USA) solution to compare cell growth in p53 virus-infected wells and uninfected wells.

Colonies of wells in which apoptosis was not actively induced by p53 expression were separated, transferred to 60 mm diameter plates, and grown. The cells thus isolated were again infected with p53 virus to reaffirm resistance to apoptosis induced by p53 in the same manner as described above, and cDNA introduced therefrom was isolated from the cells with confirmed apoptosis.

First, chromosomal DNA was extracted from p53-resistant cells according to a known method. In order to isolate the pCEV29 expression vector introduced into the chromosome, the chromosomal DNA (50 μg) was digested with only one restriction enzyme XhoI or NotI present in the vector, and the DNA was purified by a known phenol extraction method. After purification, about 1.2 μg of DNA was bound to T4 ligase (T4 ligase) at 16 ° C. for 24 hours, and then purified by phenol extraction. The concentration of bound DNA was confirmed by agarose gel electrophoresis, and the DNA was introduced into the E. coli DH10B cell line (Gibco-BRL, USA) by electroshock method (Gibco-BRL, USA) according to the manufacturer's recommendations. . Since the pCEV29 expression vector contains an ampicillin (ampicillin, Sigma, USA) resistance gene, resistant colonies were selected from the ampicillin containing medium. A total of 300 to 500 colonies were collected by washing the entire plate with LB medium (Luria-Bertani medium, Difco), and then ampicillin (100 μg / ml) was incubated at 37 ° C. overnight, and then the cells were collected the next day. Plasmid DNA was isolated by a known alkaline-lysing method.

After the isolated DNA was introduced back into E. coli DH10B cells, 4-5 plates were prepared to grow 50-100 colonies per plate. Single colonies were again taken from the plate and inoculated into LB medium, and then single plasmid DNA was isolated by a known alkaline-lysing method. In order to identify the cDNA inserted into the pCEV29 expression vector, primers consisting of nucleotide sequences which exist near 5 'and 3' of the insertion gene of the vector (forward: 5'-CGACTGGAGCACGAGGACACTGA-3 '; reverse: 5'-CATCAAAAATAGCCAAAAGG -3 ') and 30 cycles of 94 ° C 1min, 48 ° C 1min, 72 ° C 10min in 1 cycle using pCEV29 expression vector extracted from the isolated p53 induced apoptosis resistant Saos-2 cell lines as a template. PCR reactions were performed.

Using this method, 2.2 kb genes were isolated from 8 p53-induced apoptosis-resistant Saos-2 clones, and sequence analysis was performed using the sequencing kit (Applied Biosystmes, USA) as recommended by the manufacturer. Homology analysis by computer using (Altschul et al, 1990; http: //www,ncbi.nlm.nih.gov) shows that the sequences constitute part of GenBank AK077015 (mouse) and AB002315 (human) It was confirmed that it contains an HECT region, but the specific function as an E3-ubiquitin ligase is unknown.

 The gene was named Rani (Resistance to apoptosis induction).

Example  2. Rani Ubiquitination  Active measurement

In order to confirm the function of Rani protein, Rani was observed in the presence of the ubiquitin-active enzymes E1 and E2 to form the ubiquitin and thioester complex by performing the following experiment.

For this purpose, a sequence encoding the HECT region (Rani-H) of human-derived Rani or the HECT (Rani-Hc / a) region in which the 720th cysteine residue of the HECT region is substituted with an alanine residue is transferred to pGEX5X-3 (Pharmacia, USA). Cloning as recommended by the manufacturer, GST-Rani-H and GST-Rani-Hc / a vectors fused with Gta (Glutathione S trnasferase), respectively, were constructed and expressed and purified in E. coli (BL21). The protein was reacted with ubiquitin and ATP at 25 ° C. for 90 minutes in the presence of E1 (Calbiocem, USA) and E2 (Calbiocem, USA). After the reaction, the mixture was washed with buffer to remove excess ubiquitin, followed by 12% SDS (Sodium Dodecy Sulfate) -polyacrylamide gel electrophoresis (PAGE), and then transferred to a nylon membrane. Anti-ubiquitin (Santa Cruze Biotechnology USA) and anti-GST antibodies (Pharmingen, USA) as primary antibodies, and anti-goat-horseradish peroxidase-fusion antibodies and Chemiluminescence Detect Kit (Amersham, UK) as secondary antibodies ) Was performed using known methods and manufacturer's recommendations.

As shown in FIG. 3, the Ran1 protein coupled with ubiquitin may be separated by a difference in movement distance during electrophoresis due to its high molecular weight. The Rani protein of the present invention formed a complex with ubiquitin in the presence of E1 and E2, and the formation of such a complex resulted in a mutation of a reducing agent (DTT) and a cysteine residue to an aniline (Rani-) that prevents thioester binding to ubiquitin. Lost by Hc / a). E3-ubiquitinases involved in the ubiquitination of target proteins in cells are commonly known to form thioester bonds with ubiquitin in the presence of E1 and E2. Thus, the above results demonstrate that the Rani protein of the invention, in particular the HECT region, functions as an E3-ubiquitinase.

Example  3. Rani Cell death inhibition function

Full length Rani cDNA (9-51) of the present invention, Rani cDNA derived from humans, Rani-HECT region, and 720th cysteine residue of each HECT region cloned in Example 1 to analyze Rani's apoptosis inhibitory function The expression vector pCDNA6-v5 containing the Rani-HECT region substituted with the alanine residue was introduced into Saos-2 cells to prepare a cell line stably expressing the Rani protein or its HECT region according to the method described in Example 1. Each cell was added to ⁴ × 10 ^{4 in} a 24-well plate and 1 ml of cell culture solution was added. Adenovirus expressing p53 in each well after 1 day (Jung et al., 2004) (250 multiplicity of infection); TNF-alpha containing 50 μg / ml cyclohexamide (0.1 ng / ml And adding strosporin (25 nM) to each well, changing the culture medium after 2 hours for adenovirus, and rest the cells in each well at 24 hours after the rest. After aggregating, staining with 0.4% trypan blue (Sigma) was performed to quantify apoptosis by measuring the number of killed cells under a microscope. BclXL, known to be used (Jung et al., 2004).

As shown in FIG. 4, the Rani protein is almost identical to bclXL, previously known as apoptosis inhibitor, used as all positive controls induced by various signals known as apoptosis inducers, p53, TNF-alpha and stolosporin. It can be seen that the level inhibits apoptosis. This apoptosis inhibitory effect is lost in Rani (Rani ca, Rani-Hca), where the 720th cysteine residue of the HECT region is replaced with an alanine residue, indicating that the HECT region and the amino acid residue play an important role in the inhibition of apoptosis. Suggests that.

Example  4 Rani Protease by Caspase -9 and ka Spa -3 inhibits activity

Caspase activity was measured 24 hours after TNF-alpha (0.1 ng / ml) was added to cells expressing normal and mutant Rani genes used in Example 3 or Bcl-xL genes already known to inhibit apoptosis.

Caspase activity was determined by DEVD (corresponding to D: Aspartic acid, E: Glutamic acid, V: Valine) conjugated to 7-amino-4-trifluoromethyl coumarin (AFC), and LEHD (L: Leucine, H: Histidine). AFC was used as the substrate for caspase 3 and 9, respectively, and analyzed using the fluorescence assay kit (R & D Systems, USA) as recommended by the manufacturer. Fluorescence signals were measured using a microplate fluorescent reader at an excitation wavelength of 400 nm and an emission wavelength of 505 nm.

As shown in FIG. 5, TNF-alpha increases the activity of caspases 9 and 3 as evidence of apoptosis. However, in cells expressing Rani, the activity of the caspase was reduced despite TNF-alpha treatment. It can be seen that it is suppressed. Inhibition of this activity does not appear in the case of Ranic / a in which a specific amino acid in the HECT region is substituted, indicating that the caspase inhibitory activity is due to Rani.

Example  5. In cancer tissue Rani Overexpression of

Since overexpression of Rani is associated with inhibition of apoptosis, the expression patterns of Rani were investigated in cancers, a representative disease in which inhibition of apoptosis was observed. To this end, Tyramide Signal Amplification Kit (NEN Life Science) as an immunization and signal amplifier on tissue microarrays (US Biomax, Inc, USA) containing samples from normal and cancerous tissues of the stomach, colon and liver is recommended by the manufacturer. Assays were performed as is. In this experiment, 61, 124, 57, and 184 samples were used for gastric cancer, colorectal cancer, liver cancer and lung cancer and their respective normal tissues.

In summary, first, the Rani contained in the tissue was exposed by heating with a microwave in citric acid buffer at pH 6.0. Antibodies to Rani include those specific to the Rani protein sequence. Antibodies (Zymed, USA) prepared using epitopes of peptides covering the site were used. After binding the antibody to the tissue sample, it was developed with diaminobenzidine (Sigma, USA) and counterstained with hematoxylin A. An unimmunized rabbit serum was used as a negative control. The results were read independently by three pathologists and categorized the response to Rani antibodies in four steps as follows: (1) negative, 0-5%; (2) low, 5-30%; (3) medium, 30-50%; (4) high, 50% or more. Since Rani expression was read as negative or low in normal tissues, it was considered as overexpressing the medium and high reactivity in the present results.

As shown in FIG. 6, Rani overexpression was observed in cancer tissues compared to normal tissues, and Rani was detected in 48%, 46%, and 33% of the tissues of the stomach, colon, and liver tissues, respectively. Overexpression was observed.

The results suggest that inactivation of Rani in cancer tissue may be effective in the treatment of cancer.

Example  6. Rani siRNA Cancer cell killing effect

Example  6-1 siRNA  making

siRNA nucleic acid sequences were designed using the Ambion website (www. ambion.com/techlib/misc/siRNA_finder.html). In summary, starting with the AUG sequence of the Rani cDNA and scanning in the 3 'direction to yield the AA sequence, this sequence and then 19 nucleic acid sequences were selected as potential siRNA target sites, 5' and 3 'untranslated and translated The sequence at the starting site (within 75 base) was excluded from the target. The selected sequence was compared with the sequence of a known database, and the homology with the coding sequence of other genes was excluded. The target sequence of Rani used in this example is 5'-AATTGGTCCCTGAGAACCTTT-3 ', and the siRNA forming sequence is as follows: sense, 5'-UUGGUCCCUGAGAACCUUUTT-3' (SEQ ID NO: 3); antisense, 5'-AAAGGUUCUCAGGGACCAATT-3 '(SEQ ID NO: 4). Scrambled siRNA was purchased from PROLIGO as a control siRNA that did not affect Rani gene expression. Knockout RNAi System (BD-Clontech, USA) BD was used as recommended by the manufacturer for the intracellular expression of siRNAs having a hairpin structure consisting of the sense and antisense sequences. In summary, oligonucleotides comprising the sense and antisense sequences were synthesized according to the manufacturer's recommendations and cloned into pSIREN-Retro Q (BD-Clontech, USA) to express siRNAs targeting Rani (RNAi-Ready). ) Was prepared and used for transfection of cells.

Example  6-2 RNA interference  analysis

After 293 cells were seeded in each well of a 24-well plate at a concentration of 3 x 10 ⁴ cells and incubated overnight, the cells were incubated at 40 pmole per well, using Lipofectamine 2000 (Invitrogene) as recommended by the manufacturer. The concentration was treated for 24 hours. After treatment, cells were stained with 0.4% trypan blue (Sigma) to quantify cell death by measuring the number of cells killed under a microscope.

As shown in Figure 7, it can be seen that the apoptosis is significantly increased compared to untreated cells by the introduction of siRNA. This, together with the results of Figure 8, suggests that by inhibiting Rani expression can be effectively used for killing in cells, especially cancer cells.

Example  7 Rani siRNA Induction of apoptosis and increased sensitivity to anticancer drugs

SiRNA-treated cells (colon cancer cell line H1299), known as anticancer agents Bristol-Myers Squibb Company, USA, doxorubicin (ALZA Pharmaceuticals, USA), and etoposide (the same method as described in Example 6-2) Bristol-Myers Squibb Company, USA) was treated at concentrations of 73 μM, 2 μM, and 5 μM, respectively, to investigate the apoptosis effect.

As shown in Figure 8, compared with the treatment with siRNA alone or anticancer agent alone, when combined with the son, it can be seen that the effect is improved up to about four times.

These results suggest that not only Rani siRNA but also can be effectively used for anticancer treatment when used in combination with known anticancer agents.

Since the Rani protein of the present invention effectively inhibits apoptosis by inhibiting the activity of protease caspase that executes apoptosis, and furthermore, since the Rani inhibitor can effectively induce apoptosis in cancer cells in which apoptosis is suppressed, Rani can be usefully used as a therapeutic agent for diseases associated with abnormal cell death, such as degenerative neurological diseases, autoimmune diseases and cancer.

<110> GENCROSS <120> A NOVEL USE OF Rani AS AN APOPTOSIS REGULATOR <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 5402 <212> DNA <213> Homo sapiens <220> <221> misc_feature 222 (463) .. (2943) <223> Coding sequence 463 to 2943 <400> 1 gccggttccc aggccgggtg gagaccaact ctggggtctc ttggtgggag agtggggagc 60 ccgtcttctg ctgagtgctg ccgccccttc ccaggacagc ggaggtggaa cttcgtcgcg 120 ctgcaacccc cggctcggga tcctggggcg tcctttggca gttgattgca ccctgcacta 180 gagagaagtc caggaatgat atattcttgg gctgtatttc tctaccctgg agttcatcca 240 gttcatccca gaaacactga cctgattact gtgacaaagt ctgatggata gaccctttct 300 gttgaccttg tggcgttttt ctttcatgtg gaagttggaa gacaaggtga aaggggccaa 360 aagttacctg ctttggtgaa taggagtgct ctacttggtt ctcagaacat gggccctcgt 420 ttaaagctgc agctgtgatc ctcggctgtc tgttggcatt gacgggacct gatgttttac 480 gttattggtg gaatcacagt gtctgtggtt gcattcttct tcacaattaa gttcctcttt 540 gagcttgccg cacgtgtagt cagcttcctc cagaatgagg accgcgagcg ccgaggggac 600 cggactattt atgactacgt gcggggaaat tacctggatc cccggtcttg caaagtctcc 660 tgggattgga aggaccccta tgaggtgggc cacagcatgg ccttccgagt gcatttattc 720 tataagaacg ggcagccttt ccctgcacat cggcctgtgg gactaagagt tcacatctct 780 catgtcgagc tagcagtgga aattccagtg acccaggaag tccttcagga gcccaattcc 840 aacgtagtaa aagtggcctt cactgtgcgc aaggctgggc gttatgaaat cacagtgaag 900 cttggtggat taaatgtggc atatagtccc tactacaaaa tttttcaacc tggaatggtg 960 gttccttcta agaccaaaat tgtgtgccac ttttctactc ttgtattgac ctgtgggcag 1020 ccgcacaccc ttcaaatagt accccgagat gagtatgata atcccaccaa caattccatg 1080 tccttgagag atgagcacaa ttacaccttg tccattcatg agctcggccc tcaagaagaa 1140 gagagtactg gtgtctcatt tgagaaatca gtaacatcca acaggcagac tttccaggtg 1200 ttcttgcgac tcaccctgca ttctcgaggc tgcttccatg cttgcatttc ataccaaaat 1260 cagccaatca ataatggtga atttgacatt attgtcctaa gtgaggatga gaagaatatc 1320 gtcgaacgca atgtgtccac ttcaggcgtg agcatttact ttgaggctta tctttataat 1380 gctaccaact gtagcagcac tccatggcac ctgccaccca tgcacatgac ctcttcccag 1440 cgccggccat ccactgctgt tgacgaggaa gatgaagact cgccctctga gtgccacacc 1500 cctgagaagg tgaagaaacc gaagaaggtg tactgctatg tgtcaccaaa gcaattctca 1560 gtgaaggagt tctacctgaa gatcatcccc tggcgccttt acaccttccg agtgtgtcca 1620 ggaacaaaat tttcatacct tggtcctgac cctgtccata agctgctcac actggtggtg 1680 gatgatggca ttcaacctcc tgtggagctc agctgtaagg agaggaacat tctagcagcc 1740 acttttatcc gctccctgca taagaacata ggaggctctg agacctttca ggacaaggtg 1800 aactttttcc agcgagagct tcggcaggta catatgaaaa gaccacattc caaagtcacc 1860 ctgaaggtca gcagacatgc cttgttggaa tcgtctctga aagccactcg gaatttctcc 1920 atctcagatt ggagcaagaa ctttgaggtt gttttccagg atgaagaagc tctggactgg 1980 ggagggcctc gccgggaatg gtttgagcta atctgcaaag cactatttga taccaccaat 2040 cagctcttca cccggttcag tgacaacaac caagcattag tgcatcccaa ccctaatcgc 2100 cccgctcatc tgcgcctgaa aatgtatgag tttgcgggac ggctcgtggg caagtgtctc 2160 tatgagtcct ctctaggagg agcctacaag cagttggtcc gagctcgctt cacccgctct 2220 ttcctggccc aaatcatagg actgcgtatg cattacaagt actttgaaac agatgaccca 2280 gaattctaca aatctaaagt ttgttttatc ctcaacaatg acatgagtga gatggagctg 2340 gtctttgcag aagagaaata taataaatca ggtcaattgg ataaggttgt agaactcatg 2400 acaggtggag ctcaaactcc agtcaccaat gcgaataaaa tcttctattt aaatttgctg 2460 gcccaatatc ggctggccag tcaagtgaaa gaggaggtgg aacatttcct aaaaggcctg 2520 aatgaattgg tccctgagaa ccttttggct atttttgatg agaatgagct tgagctgctg 2580 atgtgtggga ctggagacat cagtgtgtct gacttcaaag cccatgcagt agttgttggt 2640 ggctcatggc atttcagaga aaaggtcatg aggtggtttt ggactgtggt ttccagtctg 2700 acccaggagg agttggctcg gctacttcag ttcacaacag gctcctctca gctaccacct 2760 ggaggctttg ccgccctctg tccctcattt cagattattg ccgctccgac ccatagcacg 2820 ctgcctactg cacacacatg ttttaaccag ctgtgcctcc ctacatatga ctcctatgaa 2880 gaggtgcaca ggatgctgca gctggccatc agcgagggtt gcgagggctt tggcatgctc 2940 tgaccactct cctgtcatcc agttggctcc catgctctct ggagcttctg ggcgcaagtt 3000 acagacatca taaccactga tcctaacaca cataaccatc agccagaaga tgccgcatgc 3060 tcccctgtgt ctggaggatt ttgtcaccta caagccttgt ctttacctca cctgctccct 3120 gcccatatct accacaggcc actttggcat ggtatgtaag ctgagctctt cattctgtca 3180 tgagaagagg accatgctgc tatcatttat ttggtccttt gaagatctca gtagctgagg 3240 gagatggcac acggggctca gccctgttgg gaaactggtg tggagaccct taaatccaca 3300 ctgtgctcca aaactccctc tgctgatact ccttggagac accctctttg gccctcacta 3360 cttgaccaga ctggtacttg agtccttctc atgggtgggg tgattgcctc ttctcatcag 3420 gagccaggag agagggggac agataggagg tggcccatag gagcagtccc gctgcacaat 3480 ggtaggcata ggccatggca ctggactgcc tctaaggact gctaaaaaga atattttttt 3540 gtggtgtcag aactggaaaa agcactttcc cttcgggcat ttctggaaat gattattaat 3600 cacaaagaag aactctgtaa gctttttctt gaattgtagc cagtgagaaa agcagataga 3660 ctgaagaata tgaaggatag ctgagctgta gcctccagag tggggcatgc ctaggcatat 3720 ggctggcttg gagactactg atgcttttcc ctgagtttgt tattggcact gaagtatggc 3780 cggcttgggc cactgacttc cccattatgt agtctgctaa aagctgggga tcctttagca 3840 ttctactgaa gaaaattttg tagcaaaaga ttagaacagt aagaatagta tgccagcaat 3900 ccctgattct ctctctgctt ggtgttctct ggcagcacta agacaaagga acagggacta 3960 ggagtttacg tgcttatcac aggtccttgg cgtcaggaca ctagggatga acatggaggt 4020 gatatgttac acagtaaaca cctgccaacc cctgtactcc cctttgctcc atcagttatc 4080 aggaaggaga actaaggagg gaggaagctt attaggttca ctgttgaatg atgattgaag 4140 agtacctgct gctgtatctt ggaagatgac aacatccttt ttcattactg tttgattgaa 4200 aataatttca agattcaaaa tctcattgac ttcccaaatt tgtgttttta agaaggcttt 4260 gctgcaagct accattccca atggggcagt caactctgag aatattacaa gccatcttta 4320 ttgccaaaac cagcaagtac acagagtcct gaaacaaggc aggactttgg caatgccttg 4380 gcctcctgga agcatgtctg agccctcctg ttcgcaggga tcatgaggaa caagccctcc 4440 tgagctgcta cagttcctgc ctgacctcag tctttcgcag tcatcatttg tcctttcttg 4500 gcacatgggt gctgacctca ctcatccttg gggtccggga tccagcatca gggtctctat 4560 accccaggat cctccatgcc acatggtcac tgctctcctc agggaccagg acaaggtgct 4620 gctgcttgcc caaatgtttt cccatgggat atgtaccgga aggtttatca ccaccctggg 4680 aaacataatg ctgccccttg ggcccagaaa ggggtttcca gctgggggca cgtggactgg 4740 ttctgctgtt tttggcagtc gcttttctga attctcccct ccggcagcct tccagagact 4800 gatcctggag attgagttga ttgtcttggc tagacctgtc attttaagct ctagtcatag 4860 cactttttca gagcatctta ggcaccattg caacccaacc agggaagctg catccctgtg 4920 gtggtcctta ggcaccagtc tttgttaaac aaaacccttt ggcactattg tggttttcta 4980 ttctctgtct gaactctatt caaaagtatc tttgctctct tgggcctttt cttttactgt 5040 tttgtttttt ttttctaatc ctgctttcat actagccagt gtggggaaaa ggtacaatat 5100 gtcaaagaga tgagagagtg ttatttcttg ggcaattttc tattagtgtt tcttattttg 5160 gccagttctt ttatttatgt ccttgtgacc caggtacttg gggggccagc tacccttctg 5220 gccttttagc gtctttgaag gagaccagac atgagtgaat acctaggaga gtgtcagcat 5280 gtttctggaa aattggcaga gaccaagccc tgctgcagat tcgtcaggcc aggtgaaagg 5340 gccaggcagt tgcagctgat gatgtaaata ttttgtacag tagataaata aatgtttaaa 5400 ag 5402 <210> 2 <211> 3816 <212> DNA <213> Mus musculus <220> <221> misc_feature 165 (165) .. (2636) <223> Coding seqnence 165 to 2636 <400> 2 agtctgctcc tggcttcctt tggatgatgt agaactctca gctccttctt gagcaccatg 60 ccttcctggt tgctgccatg cttcccgcca tgatgataat ggactgaact tcaaaacctt 120 tgcagctgtg atcctcggct gtccggtggc gttgaagaga cctgatgttt tacgttattg 180 gtggaatcat agtgtctgtg gttgcattct tcttcacaat taagttcctc tttgagcttg 240 ccgcacgtgt agtcagcttt cttcagaatg aagaccgtga gcgccgaggg gaccggacta 300 tttatgacta cgtgcgggga aattacctgg atccccggtc ctgcaaagtg tcctgggatt 360 ggaaggaccc ctatgaggtg ggccacagca tggccttccg agtgcatttg ttctataaga 420 atggacagcc tttccctgca catcggcccg tgggactgcg ggttcacatc tctcacgttg 480 aactagcagt ggacattcca gtgacccagg aagtcctcca ggagccgaat tctaacgtgg 540 tgaaggtggc cttcactgtg cgcaaggctg ggcgctatga gatcactgtg aagttgggtg 600 gcttaaatgt ggcctatagt ccctactaca aaatttttca gcctggaatg gtggttcctt 660 ccaaaacaaa aattgtgtgc catttttcta ctctggtgtt gacgtgtggg cagccacaca 720 ctctgcagat agtgccccga gatgagtatg acaacccaac caacaattcc atgtccctga 780 gagatgagca cagctacagc ttagccattc acgagcttgg tcctcaagaa gaagagaata 840 atgaagtctc atttgagaag tcagtgacgt ccaacaggca gacttgccag gtgttcttgc 900 ggctcaccct gcattcccga ggttgcttcc atgcctgcat ttcatatcag aaccaaccca 960 tcaataatgg cgaatttgac attattgtcc ttagtgagaa tgagaagaat attgttgaac 1020 gaaatgtgtc cacctcagga gtgagcattt actttgaggc ttacctttac aatgctaaca 1080 actgtaccag cacaccatgg catcttcctc ccatgcacat gagctcttct caacgccggc 1140 cctccactgc tattgaggag gacgatgaag actcaccctc agagtgtcac acccctgaga 1200 aggtgaagaa accgaagaag gtgtactgct atgtgtcacc aaagcaattc tctgtgaagg 1260 aattctacct gaaaatcatc ccctggcgcc tctacacctt ccgagtgtgc ccaggaacaa 1320 agttttcata ccttggccct gaccctgttc acaagctcct cacgctggtg gtggatgacg 1380 gcattcagcc tccagtggag ctcagctgta aagaaaggaa tatcctagca gccactttca 1440 tccgttcctt gcacaagaac attggaggct ctgagacctt tcaagacaag gtgaactttt 1500 tccagagaga gcttcggcag gtacatatga agagacctca ttccaaagtc actctgaagg 1560 tcagcagaca cgccttgttg gagtcgtctt tgaaggccac tcggaacttt tccatctcag 1620 actggagcaa gaactttgaa gtagttttcc aggatgagga agctttggat tggggagggc 1680 ctcgccggga atggtttgag ctaatctgca aagcactgtt tgacaccacc agtcagctct 1740 tcgcccggtt cactgacagc aaccaagcac tagtgcatcc taaccctaac cgcccggctc 1800 acctgcgctt gaagatgtat gagtttgcag ggcggctggt gggcaagtgt ttgtatgaat 1860 cctctctagg gggagcctac aagcagttgg tccgtgcccg gttcactcgg tctttcctgg 1920 cccaaatcat aggactccgt atgcattaca agtactttga aacggatgac ccagaattct 1980 ataagtccaa ggtgtgcttt atcctcaaca atgacatgag tgagatggag ctggtctttg 2040 cagaagagaa atacaacaaa tcaggtcaac tggataagat tgtagaactc atgacaggcg 2100 gagctcaaac cccagtcacc aatgcaaata aaatcttcta tttaaacctg ctggctcagt 2160 atcggctggc cagtcaagtg aaagaggagg tggagcattt cctgaaaggc ttgaatgagc 2220 tggttcctga gaacctcctg gctatttttg atgagaatga gcttgagctg ctgatgtgtg 2280 ggactggaga catcaatgta tctgacttca aagcccatgc tgtagtggtt ggtggatcct 2340 ggcatttcag agaaaaggtc atgaggtggt tttgggctgt ggtttccagt ctgacccagg 2400 aggaactggc taggctgctt cagtttacaa caggctcttc tcagctccca cctggaggat 2460 ttgctgccct ctgtccctca tttcagatta ttgctgctcc aacccatagc acgcttccta 2520 ctgcacacac atgttttaac caactgtgcc tccctacata tgactcatat gaagaggtgc 2580 acaggatgct acagctggcc atcagtgagg gttgcgaggg ctttggcatg ctctgaccgc 2640 tcctctgtca cctgcttggc tcccaagctc tctagagcat ctggacacat gttaacaagc 2700 atagccactg accttaacca caaccattag ccagaagata ctgcatgctc ccttgtgtct 2760 ggagtattct gtcatctgca agccctgttc ttacctcacc tgtccacgtt caccacaggc 2820 cacttgagca cgtggcacct atctgagctc tactcagtca tgagaggagg accatgctgc 2880 tgtcacttcg ttggtccctt gaagatctct gtgaccggat ctgcctccgt ggctgggaga 2940 cattcataac acagaggact cagttctgtt ggaaagccag catgtgagac ccttccacca 3000 tgcctcattc cagccctcct tgagctcact acttgaccag actgggtaat gcatcacctc 3060 ttctcatcag tggtcaggag aagtgcagct agaaagtggc ccacacaagc cgtctgattt 3120 gtcctcccac ataatggcag gcacaggcca tggcactgga ttagttctca tggggactgg 3180 tagtacaaaa ggatgtcttt aaggtgacag aactggaaaa gatactccct gtgggcattt 3240 ttaaagatga tcattaatct ataaggaatt tgctaagctt tctcttgaat ttgagccagt 3300 gagaaaagca gtcagaagaa tgtgaaggat gtctgcgctg cagcctccag tgctgggtac 3360 gcttctctcg tggggaaggg ccgtgtccag gcatgtggat ggcttgaaga ctagctgatg 3420 ctttcctctg aattgttctt tgcactgaag gaggacttgg actgacctcc gacttacata 3480 gtttgttaag agctgtcttt agcattctgc tggaagaatt ttgggagcaa cagactggga 3540 cagagcctgc cagctgtggt ggtgatcttt ggcagtgcta aggcaaagga gcggtgaagc 3600 cactggggac agacaaggtg gagacccagc agcagtaaat acctagcaat tccaaggact 3660 tggtttcatc agttaccagg aagtggggga accatggagg gagaacgcta tattgggttc 3720 acagttacat ggtgatcaaa gagaacttct cagtctccag gaagatagca tcttttcatg 3780 agtgtttgaa tgaaaacaac acgaaggctc aatacc 3816 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Sense strand for Rnai siRNA, all the "T" s except last two "T" s          are U in siRNA <400> 3 ttggtccctg agaacctttt t 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Antisense strand for RNAi siRNA, all the "T" s except last two          "T" s are U in siRNA <400> 4 aaaggttctc agggaccaat t 21

Claims (15)

A RANI gene consisting of SEQ ID NO: 1 or SEQ ID NO: 2; Or a cell death regulator comprising the RANI protein encoded by the gene of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient, used for the treatment, prevention, or diagnosis of gastric cancer, colorectal cancer, liver cancer, or lung cancer. delete delete delete A pharmaceutical composition for preventing or treating gastric cancer, colorectal cancer, liver cancer or lung cancer, comprising an RANI gene or protein expression inhibitor composed of the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 as an active ingredient. The pharmaceutical composition for preventing or treating cancer according to claim 5, wherein the inhibitor increases the sensitivity of cancer cells to anticancer agents. delete delete delete delete delete delete delete delete delete

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