WO2008044787A1 - Composition for treating cancer using dref as molecular target - Google Patents

Abstract

It is intended to provide a composition for treating cancer, characterized by containing an RNAi nucleic acid, an antisense nucleic acid or an antibody to DREF, which inhibits the in vivo function of a human cell growth regulator, DREF, or DREF gene in a cancer cell or a cancer tissue, and a screening method for an agent for treating cancer by using as an index, inhibition of expression of DREF gene or translation of DREF protein or cell growth inhibition, cell cycle arrest and/or cell death induction in a cultured human cancer cell or a DREF high-expression cell line.

Description

 Cancer therapeutic composition using DREF as molecular target

 The present invention relates to a composition for cancer treatment that targets the human cell growth regulator DREF or DREF gene as a molecular target. 'Ming

Background art

 Conventionally, various cancer treatment methods targeting genes associated with cell proliferation have been studied.

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DREF is a Drosophila transcription factor isolated by Hirose et al., Which recognizes and binds to a DNA sequence called DRE-mot if in one sequence of a gene promoter and induces the expression of that gene (non-patent literature). 1). In Drosophila, it is reported that PCNA (prol iferating cell nuclear antigen) transcription is induced in the S phase, and that several gene forces related to cell proliferation are reported to be regulated by DREF. Yes. Thereafter, a human homologue of Drosophila DREF was cloned and sequenced by Ohshima et al., And it was reported that human DREF was involved in the induction of histone HI gene (Non-patent Document 2). At this time, the expression of the human DREF gene is reduced by the two siRNAs generated against the DREF CR1 domain, thereby suppressing the histone HI gene expression (Non-patent Document 2).

 The human DREF gene is a gene that was found by Esposito et al. (Non-patent Document 3) from a cDNA library derived from a human non-inducible male teratocarcinoma cell line Tramp (Ac-like transposable element; putative Ac-like transposable element; Chromosome: 'X, Y; Location: Xp22.33; Ypll).

Sano et al. Show that DREF is one of the factors involved in cell cycle progression by knocking down the human DREF gene, and that expression of liposomal protein (RP) is reduced by knockdown. It was pointed out that it plays a role of stimulating cell proliferation through gene regulation (Non-patent Documents 4 and 5). - So far, human DREF has been suggested to be associated with cell proliferation ability S, but no detailed functional analysis has been performed in relation to cancer therapy.

 Non-patent document 1 Hirose, F. et al., J. Biol. Chera. 271: 3930-3937, 1996 Non-patent document 2 Ohshima, N. et al., J. Biol. Chem. 278: 22928-38, 2003 Non-patent document 3 Espos ito, T., et al., Human Molecular Genetics, 8 (1): 61-67,

1999

 Non-Patent Document 4 Sano, Y. et al., Abstracts of the Molecular Biology Society of Japan (issued on November 25, 2005), lecture number 1P-0873, 2005

 Non-Patent Document 5 Sano, Y. et al., 20th Annual Meeting of the International Biochemical Molecular Biology Society (issued June 18, 2006), Lecture No. 5P-A-217, 2006 Disclosure of Invention

 Refractory cancers such as lung cancer and esophageal cancer are resistant to general chemotherapy, and effective means that can be expected to cure other than surgery have not been established. For this purpose, it is necessary to develop molecular targeted therapeutics targeting genes involved in cancer onset, propagation and metastasis, and to establish treatments aimed at cure of cancer.

 An object of the present invention is to provide a therapeutic composition for cancer.

 Another object of the present invention is to provide a method of screening for a therapeutic agent for cancer. '

 The present inventors have found that human DREF is a target for cancer treatment, and have completed the present invention. .

 As described in the background art, DREF was discovered as a transcription factor of Drosophila, and it was later shown that its homolog exists in humans. The present inventors have now found that human DREF is not substantially expressed in normal cells, but is significantly expressed in cancer cells, and based on this, DREF is predicted to be a molecular target for cancer treatment. It was demonstrated that suppression of DREF expression actually leads to cancer suppression and cell death. Therefore, the present invention has the following features.

In one aspect thereof, the present invention provides an RNAi nucleus against DREF, which suppresses the in vivo function of human cell growth control factor DREF or DREF gene in cancer cells or cancer tissues. Provided is a cancer therapeutic composition comprising an acid, an antisense nucleic acid, or an antibody.

 DREF is an abbreviation for DRE (DNA replication-related element) -binding factor named by Hirose et al., Also known as ZBEDl (zinc finger, BED-type containing 1). Hereinafter, the human DREF is simply referred to as “DREF”.

In an embodiment of the present invention, DREF is a protein comprising the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence comprising a deletion, substitution or addition of one or several amino acids in the amino acid sequence. Further, DREF gene the open reading frame (0RF) nucleotide sequence encoded by an amino acid sequence of SEQ ID NO: 1, nucleotide sequence and more specifically shown in position 202 to 2 ²⁸ 6 of SEQ ID NO: 2 Including. Both amino acid and nucleotide sequences of DREF are registered with GenBank as Accession number NMJ304729. DREF in the present invention includes an amino acid sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the amino acid sequence shown in SEQ ID NO: 1. Variants having in vivo functions equivalent to DREF (for example, polymorphic variants, splice variants, degenerate variants, mutants, etc. In addition, the DREF gene in the present invention is SEQ ID NO: nucleotide sequence shown in 2 (position 202 to 228 ^6th) 95% or more, preferably 97% or more, more preferably 98% or more, including the most favorable 'Mashiku nucleotide sequence having 99% or more identity And variants similar to those described above having an in vivo function equivalent to DREF after translation.

 In this specification, the DREF gene is used with the intention of encompassing not only exons, but also introns, 3 'untranslated regions and 5' untranslated regions. '

 In another embodiment of the present invention, the suppression of in vivo function is an effect of suppressing cell proliferation, cell cycle arrest and / or cell death in cancer cells.

 As used herein, “in vivo function” refers to the action of DREF in cancer cells, such as cell proliferation, cell cycle progression, and the like.

In another embodiment of the invention, the RNAi nucleic acid or antisense nucleic acid is capable of cleaving DREF raRNA or suppressing its function. . As used herein, “RNAi” is so-called RNA interference, and is a gene having a sequence homologous to DNA or double-stranded RNA (dsRNA) added from the outside. (For example, Chisato Ushida, Protein Nucleic Acid Enzyme Vol. 46 (No. 10), pp. 1381-1386, 2001). The target RNA is mRNA or pre-mRNA encoded by the gene, and the target mRNA is degraded or suppressed in expression by RNAi.

 Specifically, suitable RNAi nucleic acids that can be used in the present invention include siRNA (small interfering RNA) or shRNA (short hairpin RNA), a DNA encoding the siRNA or sRNA, or a vector DNA containing the DNA It is. Other RNAi nucleic acids that can be used in the present invention also include miRNA (microRNA). miRNA binds to DREF mRNA 3, the untranslated region and suppresses DREF gene expression. The size of these RNAi nucleic acids is 19-30 nucleotides long, preferably 19-25 nucleotides long, more preferably 19-23 nucleotides long.

 The “antisense nucleic acid” used in the present invention includes an RNA sequence complementary to the whole or a part of DREF mRNA, DNA encoding the RNA sequence, or a vector containing the DNA sequence. .

 As used herein, “vector” refers to a regulatory sequence such as a promoter, poly T sequence, replication origin, terminator, etc., in addition to the DNA encoding the target siRNA or shRNA or DNA encoding the antisense RNA. , Selection markers, etc. can be included. When the vector of the present invention is introduced into a cancer cell, the siRNA or shRNA, or antisense RNA is produced in the cell by the expression of the DNA.

In an embodiment of the invention, the RNAi nucleic acid or antisense nucleic acid is derived from a nucleotide sequence encoding the CR2 domain or CR3 domain of DREF (SEQ ID NOs: 3 and 4, respectively). A preferable nucleotide sequence in the present invention is, for example, in the sequence of SEQ ID NO: 2 of the DREF gene, from position 1,300 to 1,500 (from position 1099 when “A” of the start codon ATG of 0RF is the first position) I ² "position), 1,900 to 2,000 position (positions 1699 to 1799 when 0RF start codon ATG" A "is first position). As used herein, “CR” refers to a highly conserved region in the sequence comparison between human DREF and Drosophila DREF. Such areas (Amino acid sequence; nucleotide sequence) is CR1 (positions 4 to 140 of SEQ ID NO: 1; positions 21 to 621 of SEQ ID NO: 2), CR2 (positions 377 to 506 of SEQ ID NO: 1; 1330 to 1719 of SEQ ID NO: 2) ) And CR3 (positions 541 to 688 of SEQ ID NO: 1; positions 1822 to 2265 of SEQ ID NO: 2), and the identities are 27.7%, 29.2%, and 21.1%, respectively (Ohshima, N. et al., J. Biol. Chem. 278: 22928-38, 2003).

 More preferred RNAi nucleic acids in the present invention include any one of the DNA sequences of SEQ ID NOs: 5 to 15, 19, 22, 25 and 28 or an RNA sequence corresponding to the DNA sequence. As used herein, the term DNA represents deoxyribonucleic acid and the term RNA represents ribonucleic acid.

 Further, a more preferable vector in the present invention includes the DNA sequence of any one of SEQ ID NOs: 20, 21, 23, 24, 26, 27, 29 and 30.

 According to the present invention, the composition of the present invention can comprise an antibody against DREF that inhibits the in vivo function of DREF. Any antibody can be used in the present invention as long as such a function can be suppressed. Preferred antibodies are DREF CR1 domain, CR2 domain or CR3 domain (SEQ ID NO: 138, 133 or 134, respectively), or a functional domain related to DNA binding of DREF in the CR1 or CR3 domain (eg, SEQ ID NO: 140 or 141), It is an antibody that binds to the epitope. As used herein, the term epitope refers to a site to which an antibody on an antigen molecule binds, and usually the number of amino acids in such a site is 8 or more, preferably 10 or more. It may consist of a target sequence or a discontinuous sequence. Therefore, the antibody of the present invention can immunologically bind to an epitope composed of 8 or more, preferably 10 or more amino acids in the amino acid sequence of DREF. 'Preferred antibodies against DREF that can be used in the present invention are human antibodies, humanized antibodies or fragments thereof.

 In another embodiment, the present invention further includes adding a candidate drug to a medium containing human cultured cancer cells, and screening a drug that suppresses the expression or translation of the DREF gene in vitro. A method for screening is provided.

Cancer cells that can be used in the present invention are cancer cells from cancer cell lines, biopsy. including. In particular, the latter cancer cells are derived from a patient and can be used to search for an effective drug for the patient. In the present invention, the type of cancer cell is not particularly limited, but cancer cells in which DREF is expressed in cancer cells but hardly or not in normal cells are desirable.

 Candidate drugs that can be used in the present invention may be any of small molecules to macromolecules, and include low molecular organic compounds, peptides, proteins, oligosaccharides, polysaccharides, lipids, and the like. In the present invention, screening can be performed using as an index the suppression of cancer cell growth, cell cycle arrest and / or cell death induction. Such an effect is not substantially observed in normal tissues or cells because DREF is hardly or not expressed in normal tissues or cells.

 This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2006-275695, which is the basis of the priority of the present application. Brief description of the drawings',

 Figure 1 shows the domain structure of the human DREF / ZBED1 gene (Xp22.33 & Ypll), and the RNAi site and sequence. '' Figure 2 shows the results of RT-PCR analysis of the RNAi effect.

 Figure 3 shows the results of Western blot analysis of the RNAi effect.

 Figure 4 shows the cell cycle analysis results.

 Figure 5A shows the cell cycle analysis results.

 Figure 5B shows the cell cycle analysis results.

 Figure 5C shows the cell cycle analysis results.

In FIG. 6, A shows the results of cell proliferation ability analysis. B shows the results of cell proliferation ability analysis. FIG. 7 shows the results of immunostaining for ₇ H2AX (Phospho-H2AX) and Phospho-ATM (Serl981). Figure 8 shows the TUNEL analysis results.

 FIG. 9 shows the results of cell death and cell cycle analysis.

 FIG. 10 shows cell death (mitotic catastroph) in M phase.

Figure 11 shows the gene expression analysis results of siDREF-, # 4 in ACC-LC-91. 1 2, _O showing expression of DREF in lung cancer patient specimens Figure 13 shows the DNA sequence of the RPL17 gene (A; SEQ ID NO: 137), which is thought to be associated with cancer growth, among the genes that are suppressed by DREF-RNAifH, and the transcriptional activation of the RPL17 gene by DREF. Mechanism (B) is shown. BEST MODE FOR CARRYING OUT THE INVENTION

1. Cancer treatment composition

 The present invention includes an RNAi nucleic acid, an antisense nucleic acid or an antibody against DREF, which suppresses the in vivo function of a human cell growth regulator DREF or DREF gene in cancer cells or cancer tissues. A composition is provided.

 DREF is expressed in cancer cells or tissues, but is not substantially (or little or not) expressed in normal cells or tissues. In addition to this finding, the present inventors suppressed cancer cell growth, cell cycle arrest, cell death induction by suppressing (including blocking or inhibiting) DREF gene expression or protein translation in cancer cells or tissues. It was found that such an event occurred. ^

 Cancers to be treated by the present invention are not limited to cancers capable of expressing DREF, such as lung cancer, esophageal cancer, knee cancer, stomach cancer, liver cancer, colon cancer, thyroid cancer, prostate cancer, Includes bladder cancer, kidney cancer, skin cancer, gall cancer, brain cancer, breast cancer, ovarian cancer, cervical cancer, testicular cancer, lymphoma, melanoma, sarcoma, osteosarcoma, etc. Preferred cancers are lung cancer, esophageal cancer, breast cancer, knee cancer and the like. ',

 The amino acid sequence of DREF, which is the target of the present invention, and the nucleotide sequence of the DREF gene are known, for example, as SEQ ID NO: 1 and SEQ ID NO: 2 (open reading frame (0RF): positions 202 to 228δ), respectively. Both DREF amino acid and nucleotide sequences are registered in GenBank as Accession number 丽 _004729. The present invention also includes suppressing the in vivo function of a DREF or a mutant of the DREF gene, for example, a mutant based on mutation, polymorphism, alternative splicing, degeneracy and the like. ·

Specifically, DREF in the present invention is a protein comprising the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence containing one or several amino acid deletions, substitutions or additions in the amino acid sequence. . Or DREF in the present invention is In vivo function equivalent to DREF, comprising an amino acid sequence having 95% or more, preferably 97% or more, more preferably 98% or more, most preferably 99% or more identity with the amino acid sequence shown in SEQ ID NO: 1. The variant which has is also included.

 The DREF gene in the present invention corresponds to the nucleotide sequence encoded by the amino acid sequence of SEQ ID NO: 1, more specifically the nucleotide sequence shown in SEQ ID NO: 2, particularly DREF 0RF at positions 202 to 2286. Contains nucleotide sequence. The DREF gene in the present invention is a DNA further comprising a nucleotide sequence containing a deletion, substitution or addition of one or several nucleotides in the nucleotide sequence shown at positions 202 to 2286 of SEQ ID NO: 2. Alternatively, the DREF gene in the present invention has 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the nucleotide sequence shown in positions 202 to 2286 of SEQ ID NO: 2. And a variant having an in vivo function equivalent to DREF after translation. '

 As used herein, the term “several” means an integer of about 10 or less, such as 9, 8, 7, 6, 5, 4, 3, 2.

 DREF has in vivo functions such as cell proliferation and cell cycle progression, but the present invention makes it possible to suppress the in vivo functions of DREF in cancer and lead to cancer degeneration and cell death.

 In the present invention, examples of the agent that suppresses the expression or protein translation of the target DREF gene include RNAi nucleic acid, antisense nucleic acid and antibody against DREF. Each of these drugs will be described more specifically below.

1.1 RNAi nucleic acids

 The RNAi nucleic acid usable in the present invention is a continuous 19 to 30 nucleotide sequence derived from the nucleotide sequence of the DREF gene (consisting of exons, introns, 3 ′ untranslated region and 5 ′ untranslated region) or the nucleotide sequence of DREF mRNA. It is a DNA or RNA containing nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides.

RNAi methods that can be used in the present invention include 1) direct introduction of short double-stranded RNA (siRNA) into cells, or 2) incorporation of short-hairpin RNA (shRNA) into various expression vectors, Introduce the vector into the cell, or 3) Insert a short double-stranded DNA corresponding to siRNA between the promoters into a vector with two promoters aligned in the opposite direction to express s iRNA. This includes methods such as preparation and introduction into cells.

 The RNAi nucleic acid includes s iRNA, shRNA or miRNA that enables cleavage of DREF mRNA or suppression of its function. The RNAi nucleic acid may also be a DNA encoding these RNAs or a vector containing the DNA, which, when used in vivo, makes it possible to produce siRNA, shRNA or miRNA.

 In the present invention, a preferred RNAi nucleic acid is a continuous 19 to 30 nucleotides, preferably 19 to 25 nucleotides derived from nucleotide sequences (SEQ ID NOs: 3 and 4, respectively) encoding the CR2 domain or CR3 domain of DREF. Is DNA containing 19-23 nucleotides, or RNA corresponding to the DNA. Further preferred CR2 domain or CR3 domain target sites are, for example, positions 1,300 to 1,500 in the sequence of SEQ ID NO: 2 of the DREF gene (0RF open-start codon ATG “A” is the first Position 1099 to 1299), 1,900 to 2,000 (0RF start codon ATG

If “A” is the first place, it will be 169.9 to 1799).

 The RNAi nucleic acid that can be used in the present invention is, for example, the DNA sequence of any of the following SEQ ID NOs: 5 to 15, 19, 22, 25, and 28, or the RNA sequence corresponding to the DM sequence (that is, the DNA sequence) Array with T in U replaced by U). The number after the sequence is the position of the start site on 0RF of DREF (however, the start codon of 0RF

Number ATG base “A” as the first. ).

 CTTGGTGGAGGACAGCAACAA 1122 (SEQ ID NO: 5)

 AGCAACAACCACCACCTCATG 1135 (SEQ ID NO: 6)

 CAACCACCACCTCATGCTGGAG 1139 (SEQ ID NO: 7)

 TGGCCGAGATCTTCTGCCAGAC 1642 (SEQ ID NO: 8)

 GGTGGTGGAGGAGCTGAGCAAC 1700 (SEQ ID NO: 9)

 GTCCCAGAAGGTGCTTGGCCTC 1727 (SEQ ID NO: 10)

 CCCAGAAGGTGCTTGGCCT 1730 (SEQ ID NO: 11)

GAAGGTGCTTGGCCTCAACGA.;,. 1734 (SEQ ID NO: 12)- GGTGCTTGGCCTCAACGAAGAC 1736 (SEQ ID NO: 13)

 GGTGCTTGGCCTCAACGAAGA 1737 (SEQ ID NO: 14)

 TGCTTGGCCTCAACGAAGA 1739 (SEQ ID NO: 15)

 GCAACAACCACCACCTCATGC 1136 (SEQ ID NO: 19)

 GCAACTTCAAGTCCCAGAAGG 1718 (SEQ ID NO: 22)

 GTACCCCACCATCAGCATGGT 1245 (SEQ ID NO: 25)

 GGTGCTTGGCCTCAACGAAGA 1737 (SEQ ID NO: 28)

Particularly preferred sequences are SEQ ID NO: 19 (RNAi # 3 (positions 1136 to 1156)), SEQ ID NO: 22 (RNAi # 4 (positions 1718 to 1738) :), SEQ ID NO: 25 (RNAi # 5 (positions 1245 to 1265)) ), And SEQ ID NO: 28 (RNAi # 6 (positions 1737 to 1757)), or an RNA sequence corresponding to the DNA sequence (that is, a sequence in which T in the DNA sequence is replaced with U). The number range shown in parentheses together with the SEQ ID No. is when the base number “A” (position ² ) of the start codon ATG of DREF 0RF is numbered as the first in the nucleotide sequence of SEQ ID No. 2. Represents the position of. ·

 Another example of an RNAi nucleic acid for DREF is the DNA sequence of SEQ ID NO: 33-132 below, or the RNA sequence corresponding to that DNA sequence (ie, a sequence in which T in the DNA sequence is replaced with U).

 CAAGAGCAAGGTGTGGAAGTATT 63 (SEQ ID NO: 33)

 GTGGAAGTATTTCGGCTTCGA 75 '(SEQ ID NO: 34)

 GGGATGCATCCTGCAGTGGAAG 107 (SEQ ID NO: 35)

 GATGCATCCTGCAGTGGAAGA .110 (SEQ ID NO: 36)

 GATGCATCCTGCAGTGGAA 110 (SEQ ID NO: 37)

 CCCAGATCGCCTACTCCGGAAA 154 (SEQ ID NO: 38)

 CCAGATCGCCTACTCCGGAAA 156 (SEQ ID NO: 39)

 CAGATCGCCTACTCCGGAA 157 (SEQ ID NO: 40)

 AGATCGCCTACTCCGGAAACA 158 (SEQ ID NO: 41)

 GTCCTACCACCTGGAGAAGAAC 188 (SEQ ID NO: 2)

 CCTACCACCTGGAGAAGAA 191 (SEQ ID NO: 43)

CTGCGAGTTCGTCAAGAGCAAC 224 (SEQ ID NO: 44) GCGAGTTCGTCAAGAGCAA 227 (SEQ ID NO: 45) CGTGCTGGGCCTCATCTGCGAG 386 (SEQ ID NO: 46) CCCAGCCTCCATCGTGGACGAG 416 (SEQ ID NO: 47) CAGCCTCCATCGTGGACGA 419 (SEQ ID NO: 48) CACCTTCAAGGTGCTGCTGAAG 440 (SEQ ID NO: 49) CCTTCAAGGTGCTGGA 491 (SEQ ID NO: 52) CTCTACCAAGGCCATCCCTGAG 503 (SEQ ID NO: 53) CGTCCGGGAGGTGATCCTGAAG 536 (SEQ ID NO: 54) TCCGGGAGGTGATCCTGAA 538 (SEQ ID NO: 55) CCGGGAGGTGATCCTGAAGGAG 539 (SEQ ID NO: 56) GGGAGGTGATCCTGAAGGA GT GG (GT ACC GG (SEQ ID NO: 59) CACCGACATGTGGAGGAGTGA 591 (SEQ ID NO: 60) CGACATGTGGAGGAGTGAGAA 594 (SEQ ID NO: 61) GTGGAGGAGTGAGAATCAGAAC 599 (SEQ ID NO: 62) ATCAGAACCGCGCCTACGTCAC 613 (SEQ ID NO: 63) TGGGCTCCCGCTGCCTGAAGAC 682 (TTC GCGACT CTCG (SEQ ID NO: 66) CATCACGCGAGTGCTCTATGAGG 738 (Allocation No. 67) GCTCTATGAGGTCTTCATCGAG 749 (SEQ ID NO: 68) TTCGGGGCCACCACCAACTATGG 793 (SEQ ID NO: 69) CACCAACTATGGCAAGGACATC 803 (SEQ ID NO: 70) CCAACTATGGCAAGGACAT 806 (SEQ ID NO: 71) AGGACATCGTGAAGGCGTGCT 818 (SEQ ID NO: 72) CTGCCGCAAACTGGTGGAGTAC 941 (SEQ ID NO: 73) GCCGCAAACTGGTGGAGTACT 944 (SEQ ID NO: 74)

GCCGCAAACTGGTGGAGTA 944 (SEQ ID NO: 75)

CCGCAAACTGGTGGAGTACTTCC 945 (SEQ ID NO: 76)

GTGGAGTACTTCCAGCAGTCT 955 (SEQ ID NO: 77)

CGTGGCCATGTACATGCTCTA 978 (SEQ ID NO: 78)

GTGGCCATGTACATGCTCTAT 979 (SEQ ID NO: 79)

GGCCATGTACATGCTCTATGAG 980 (SEQ ID NO: 80)

CCATGTACATGCTCTATGA 982 (SEQ ID NO: 81)

CATGTACATGCTCTATGAGAAG 983 (SEQ ID NO: 82)

CATGCTCTATGAGAAGCAGAAG 989 (SEQ ID NO: 83)

TGCTCTATGAGAAGCAGAA 992 (SEQ ID NO: 84)

GCTGCAGCGCCTCAAGGAGCAG 1079 (SEQ ID NO: 85)

CCGAGATGCTGTCGGCCTCCAG 1222 (SEQ ID NO: 86)

GAGATGCTGTCGGCCTCCA 1225 (SEQ ID NO: 87)

GCTGCTGCACATGCTCCTGAAC 1271 (SEQ ID NO: 88)

TGCTGCACATGCTCCTGAA 1274 (SEQ ID NO: 89)

TGCACATGCTCCTGAACACCAC 1276 (SEQ ID NO: 90)

GGCCAAGGAGGTCATCGCCAAG 1337 (SEQ ID NO: 91)

CCAAGGAGGTCATCGCCAA 1340 (SEQ ID NO: 92)

CATCGCCAAGGAGCTTTCCAAG 1349 (SEQ ID NO: 93)

CAAGGAGCTTTCCAAGACCTA 1356 (SEQ ID NO: 94)

GACGCCCGAGATCGACATGTTTC 1383 (SEQ ID NO: 95)

ACGCCCGAGATCGACATGTTTCT 1384 (SEQ ID NO: 96)

GCCCGAGATCGACATGTTTCTCA 1386 (SEQ ID NO: 97)

CCGAGATCGACATGTTTCTCAAC 1388 (SEQ ID NO: 98)

GGTGGAGAATCGCGTGGTGGAA 1475 (SEQ ID NO: 99)

TGGAGAATCGCGTGGTGGA 1478 (SEQ ID NO: 100)

GGAGAATCGCGTGGTGGAAGA 1479 (SEQ ID NO: 101)

GGCCAAGGGCCTGCTGGACAAG 1499 (SEQ ID NO: 102) CCAAGGGCCTGCTGGACAA 1502 (SEQ ID NO: 103)

GCCCGCCAGCGTCATCAACAAC 1616 (SEQ ID NO: 104)

CGGCCAGCGTCATCAACAACAT 1618 (SEQ ID NO: 105)

CCGCCAGCGTCATCAACAA 1619 (SEQ ID NO: 106:

CGCCAGCGTCATCAACAACAT 1620 (SEQ ID NO: 107:

CGCCAGCGTCATCAACAACATGC 1620 (SEQ ID NO: 108)

CATCAACAACATGCTGGCCGAG 1628 (SEQ ID NO: 109:

ACAACATGCTGGCCGAGATCT 1634 (SEQ ID NO: 110:

GCTGCCCAAGGTGCTGCAGAAG 1799 (SEQ ID NO: 111:

TGCCCAAGGTGCTGCAGAA 1801 (SEQ ID NO: 112:

GCCCAAGGTGCTGCAGAAGTAC 1802 (SEQ ID NO: 113:

CCAAGGTGCTGCAGAAGTA 1805 (SEQ ID NO: 1 14

TGCAGAAGTACTGGTGCGTGAC 1813 (SEQ ID NO: 115

TGCAGAAGTACTGGTGCGTGA 1814 (SEQ ID NO: 1 16

CAGAAGTACTGGTGCGTGA 1816 (SEQ ID NO: 117

CGTGGTCAGCGCCAAGAGGAAC. 1880 (SEQ ID NO: 118

GGACGAGCAGGTGTTTCTGTAT 1922 (SEQ ID NO: 119

GGACGAGCAGGTGTTTCTGTAT 1922 (SEQ ID NO: 120

ACGAGCAGGTGTTTCTGTA 1924 (SEQ ID NO: 121

ACGAGCAGGTGTTTCTGTATGAG 1924 (SEQ ID NO: 122

GCAGGTGTTTCTGTATGAGAAC 1928 (SEQ ID NO: 123

AGGTGTTTCTGTATGAGAA 1931 (SEQ ID NO: 124

TGGCGTCAGCGGCGGTTTCTTTG 2037 (SEQ ID NO: 125

GGCGTCAGCGGCGGTTTCTTTGG 2038 (SEQ ID NO: 126

CAGCGGCGGTTTCTTTGGCATT 2042 (SEQ ID NO: 127

GCGGCGGTTTCTTTGGCATTAG 2044 (SEQ ID NO: 128

GCGGCGGTTTCTTTGGCAT 2045 (SEQ ID NO: 129

CGGTTTCTTTGGCATTAGGGAC 2048 (SEQ ID NO: 130

GTTTCTTTGGCATTAGGGA .2051 (SEQ ID NO: 131 TAGGGACAGCAGCTTCCTGTAG 2063 (SEQ ID NO: 132)

 In a preferred embodiment of the invention, the RNAi nucleic acid is a continuous 19-30 nucleotides, preferably 19-25, from the sequence of the mRNA or variant thereof encoded by the DRSF gene comprising the nucleotide sequence of SEQ ID NO: 2. An siRNA comprising a nucleotide, more preferably a 19-23 nucleotide sense strand sequence and its complementary sequence, the antisense strand sequence. Here, the siRNA can suppress the expression of the DREF gene or mRNA in vivo or in vitro. A preferred example of the corresponding DNA sequence comprising the siRNA is a contiguous 19-30 nucleotide, preferably 19-25 nucleotides derived from the nucleotide sequence encoding the CR2 domain or CR3 domain of DREF (SEQ ID NOs: 3 and 4, respectively). More preferably, the DNA sequence comprising 19 to 23 nucleotides, more preferably, the nucleotide sequence of SEQ ID NO: 2, positions 1,300 to 1,500 (0RF starting codon ATG "A" is the first position 1999 to 1299), 1,900 to 2,000 (from 1699 to 1799 if the 0RF start codon ATG “A” is the first), 19-30 DNA sequences comprising nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides, more preferred examples are shown by SEQ ID NOs: 5-15, 19, .22, 25, 28, 33-132 DNA sequence.

 In the present invention, the sequence of the DREF RNAi nucleic acid is not limited to the above sequence, and for example, in the nucleotide sequence of SEQ ID NO: 2 as long as it suppresses the in vivo function of the DREF gene, the corresponding mRNA, or a variant thereof. This includes sequences prepared by shifting 1 to 5 bases in the 5 'direction or 3' direction of the sequences of SEQ ID NOs: 5 to 15, 19, 22, 25, 28, 33 to 132, or the above-mentioned specific The sequence may be different from the sequence of, for example, 1 to 3 bases. '

 The siRNA of the present invention can be synthesized using a well-known chemical synthesis technique based on the DREF sequence that is the target of siRNA as exemplified above. For example, it can be synthesized chemically using an automated DNA (/ RNA) synthesizer using DNA synthesis technology such as solid phase phosphoamidite method, or a siRNA-related contract synthesis company (for example, Funakoshi Corporation) It is also possible to synthesize by outsourcing to companies such as Dharmacon and Ambion.

According to an embodiment of the present invention, the siRNA of the present invention is a duplex that is a precursor thereof. It can also be derived from RNA (shRNA) via processing by the intracellular RNase Dicer.

 shRNA is a double-stranded RNA having a loop between the sense strand sequence and the antisense strand sequence of siRNA, preferably from 1 to 6, preferably 2 to 4 poly U at its 3 ′ end. Including overhangs. shRNA is processed into siRNA by a dicer belonging to the RNase III family, and then siRNA is made into a single strand, and its antisense strand RNA is complexed with a molecule having RNA cleavage activity, etc. RISC (RNA- Induced Silencing Complex), which cleaves the target mRNA having a sequence complementary to the siRNA sequence, thereby suppressing the expression of the DREF gene.

 Therefore, both the above siRNA of the present invention and its precursor shRNA can be used as an active ingredient of the composition of the present invention. ·

 It is desirable that the selected RNAi nucleic acid does not exhibit a so-called off-target action in clinical use. Off-target action refers to the action of suppressing the expression of another gene that is partially homologous to the RNAi nucleic acid used in addition to the target m gene. To avoid the off-target effect, the candidate RNAi nucleic acid has the ability to confirm that there is no cross-reaction using a gene chip in advance, or there is no sequence identity using a database such as GenBank (NCBI) Or it is necessary to confirm that there is little. Fortunately, however, human DREF is not substantially expressed in normal cells in humans, but is significantly expressed in cancer cells, so it is expected that the problem of aftergetting can be avoided.

When introducing the RNAi nucleic acid of the present invention into a patient's body, it is preferable to use a vector capable of directly injecting the nucleic acid into the affected area or capable of expressing the nucleic acid. Alternatively, siRNA or vectors can be complexed with ribosomes such as lipofectamine, lipofectin, selfectin, other positively charged ribosomes (eg, positively charged cholesterol), or microcapsules, and this complex can be used. (For example, Nakanishi Mamoru et al., Protein Nucleic Acid Enzyme, 44-11, 48-54, 1999; Clinical Cancer research 59: 4325-4333, 1999; Wu et al., J. Biol. Chem. 262: 4429, 1987) . Since the cell membrane of mammals is negatively charged, positively charged ribosomes are preferably used. The positively charged ribosome-nucleic acid complex is taken up into the cell by engineered cytosis. After being inserted, the nucleic acid can be further transferred to the cytoplasm or nucleus. Alternatively, the therapeutic RNAi nucleic acid of the present invention can be encapsulated in nanoparticles having a particle size of about 500 nm or less. Examples of nanoparticles include hollow nanoparticles formed from, for example, hepatitis B virus envelope L particles, and nucleic acids are encapsulated in the particles by electroporation. This nucleic acid-encapsulated particle can be delivered to the liver when administered into the blood (T. Yamada et al., Nature Biotech 21 (8): 885-890, 2003) and may be useful in the treatment of liver cancer.

 Alternatively, when encapsulating the therapeutic RNAi nucleic acid of the present invention in a liposome, the nucleic acid is treated with protamine sulfate to cause condensation to form a nucleic acid-protein complex, which is then encapsulated in a positively charged lipid or polymer micelle. You can also

 • Liposome-nucleic acid complexes can be obtained, for example, by the reverse phase evaporation method (F: Szoka et al., Biochim. Biophys. Acta, 601: 559, 1980), Bonoretex shaking method, Calcium fusion-EDTA chelate method (Yasufumi Kaneda, Experimental Medicine 22卷 No. 14 (extra number), pages 14-152, 2004).

 Other methods for introducing a nucleic acid such as the siRNA or vector of the present invention into a target cancer cell include a microinjection method and a viral vector method.

 The microinjection method is a method in which a nucleic acid is directly microinjected into a cell with a fine injection needle.

 The viral vector method is a method of introducing a nucleic acid into a cell using a viral vector, in which a recombinant virus incorporating an expression unit that expresses siRNA, shRNA or miRNA is prepared, and the cell is infected. It is a method of expressing in cells. Examples of viruses include adenovirus, adeno-associated virus, retrovirus and the like. According to an embodiment of the present invention, a nucleic acid that can be used in the present invention includes an expression vector comprising a DNA sequence encoding an RNAi nucleic acid such as siRNA or shRNA under the control of a promoter.

One example of an expression vector is a hairpin vector. This vector includes DNA encoding a hairpin RNA in which the sense strand RNA sequence and the antisense strand RNA sequence are covalently bonded via a single-stranded loop sequence, wherein the DNA is intracellularly contained. The hairpin RNA is formed by transcription and processed by a dicer. A vector that forms the siRNA.

 At the end of the hairpin DNA encoding siRNA, a poly-T sequence consisting of 1 to 6, preferably 1 to 5, T for transcription termination signal sequence, or for overhanging, for example, Four or five poly T sequences are linked. Short hairpin RNA (shRNA) as a siRNA precursor transcribed from vector DNA should have an overhang consisting of 3 to 4 U at the end of its antisense strand. By this, sense strand RNA and antisense strand RNA can be more stable against nuclease degradation. There is one endogenous dicer in humans, which has the role of converting long dsRNA and precursor miRNA into siRNA and mature miRNA, respectively.

 Examples of the loop sequence according to the present invention include 5'-UUCAAGAGA-3 '(SEQ ID NO: 135), 5'-CUUCCUGUCA-3' (SEQ ID NO: 136), 5'-UUCCAG-3 '(SEQ ID NO: 137), etc. However, it is not limited to these.

 The combinations of DNA sequences incorporated into vectors for expressing RNAi # 3, RNAi # 4, RNAi # 5 and RNAi # 6, which are particularly preferred among the RNAi nucleic acids of the present invention, are shown in SEQ ID NOs: 20 and 21, respectively. SEQ ID NO: 23 and 24, SEQ ID NO: 26 and 27, SEQ ID NO: 29 and 30. The underline indicates the sequence corresponding to the sense strand and antisense strand of siRNA.

 RNAi # 3 oligo (SEQ ID NOS: 20 and 21, respectively): '

RNAi # 4 oligo (SEQ ID NOS: 23 and 24, respectively):

 RNAi # 5 oligo (SEQ ID NOS: 26 and 27, respectively):

si5F: 5, -TACCCCACCATCAGCATGGTCTTCCTGTCAACCATGCTGATGGTGGGGTACTTTTTG-3 '

RNAi # 6 oligo (SEQ ID NO: 29 and 30 respectively):

si6F: 5-( si6R: 3,-The expression vector of the present invention can contain a promoter on the 5th side of the hairpin DNA. Examples of promoters are pol III promoters, eg, U6 promoter or HI promoter from human or mouse, pol II promoter, or cytomegaloinoles promoter.

 Another example of the expression vector of the present invention is a tandem vector. This vector comprises a DNA sequence encoding the sense strand RNA sequence constituting the siRNA and a DNA sequence encoding the antisense strand RNA sequence, and a promoter at the 5 ′ end of each strand, DNA comprising a poly T sequence linked to the 3 'end of each strand, wherein the DNA hybridizes with the sense strand RNA and the antisense strand RNA after transcription in the cell to form the siRNA It is a vector.

 An example of a promoter in a tandem vector is a pol HI promoter, such as a human or mouse-derived U6 promoter or HI promoter, or a site megaloinoles promoter.

 Also, Poly T 酉 己 歹 IJ :! A poly T sequence consisting of ˜6, preferably 1-5 T, such as 4 or 5 poly T sequences.

 The tandem vector of the present invention can be introduced into a cell, transcribed into RNA corresponding to the sense strand and the antisense strand, and hybridized with each other to produce the desired siRNA. '

 According to an embodiment of the present invention, the hairpin type and tandem type vector is a plasmid vector or a winores vector.

 The plasmid vector may be prepared using the methods described in the Examples below or the methods described in the literature, or commercially available vector systems such as the piGENE ™ U6 vector and the piGENE ™ Hl vector (Takarabai Bio Inc.). Company) (Τ · R.

Brummelkamp et al., Science (2002), 296: 550-553; N. S. Lee et al., Nature Biotech.

(2002), 20: 500-505; Tsuji Miyagishi et al., Nat. Biotechnol. (2002), 20: 497-500; P. J.

Paddison et al., Genes & Dev. (2002), 16: 948-958; T. Tusch, Nature Biotech (2002),

20: 446-448; CP Paul et al., Ature Biotech. (2002), 20: 505-508; edited by Yoshikazu Tahira, RNAi Experimental Protocol, Yodosha, 2003). In general, a plasmid vector is a drug resistance gene (eg, neomycin resistance gene, ampicillin resistance gene, puromycin resistance gene, hygromycin resistance gene) in addition to the DNA sequence and promoter encoding the siRNA of the present invention. It may contain transcription termination sequences, unique restriction sites or multiple cloning sites, and replication origins.

As the virus vector, for example, an adenovirus vector, an adeno-associated virus vector, a lentiwinores betater, a retrowinoles vector (such as a leukemia virus vector), a herpes virus vector, or the like can be used. The virus vector is preferably of a type lacking self-replicating ability, for example, so as not to cause disease when used in humans. For example, in the case of an adenovirus vector, a self-replication ability-deficient adenovirus vector lacking the E1 gene and E3 gene (eg, pAdeno-Χ from Invitrogen) can be used. Construction of viral base click terpolymer can utilize literature methods (U.S. Patent No. 525 ² 4 § 9 No., and International Publication W094 / 13788). ',

 As described above, the plasmid vector of the present invention forms a complex with a positively charged liposome such as lipophectamine, lipofectin, self-actin, or positively charged cholesterol, and is introduced into the patient's body in a force-pellated state. (Mr. Nakanishi et al., Supra; Wu et al., Supra). In addition, viral vectors can be introduced into cells by introducing them into affected areas and infecting cells (L. Zender et al., Proc. Natl. Acad. Sci. USA (2003), 100: 77797-7802; H. Xia et al., Nature Biotech. (2002), 20: 1006-101.0; XF 'Qin et al., Proc. Natl. Acad. Sci. USA (2003), 100: 183-188; GM Barton et al., Proc. Natl. Acad., Sci. USA (2002), 99: 14943-14945; JD Homme 1 Nature Med. (2003), 9: 1539-1544). In particular, it has been confirmed that adenovirus vectors or adeno-associated virus vectors can introduce genes into various cell types with very high efficiency. Since this vector is also not incorporated into the genome, its effect is transient and is considered to be safer than other viral vectors.

In the DNA encoding siRNA incorporated into the vector of the present invention, the sense strand sequence and the antisense strand sequence constituting the siRNA are covalently linked via a single strand loop sequence. The hairpin RNA is encoded, and the hairpin RNA is formed by transcription in a cell and processed by a dicer to form the siRNA. Another example of DNA encoding siRNA incorporated into the vector of the present invention comprises a DNA sequence encoding a sense strand sequence constituting siRNA and a DNA sequence encoding an antisense strand sequence, and A promoter is linked to the 5 'end of each strand, and a poly T sequence is linked to the 3' end of each strand, and the sense strand RNA and the antisense strand RNA hybridize after transcription in the cell. Thus, the siRNA is formed.

1.2 Antisense nucleic acid

Another nucleic acid as an active ingredient of the composition of the present invention is an antisense nucleic acid. This antisense nucleic acid is an RNA sequence containing a sequence complementary to the mRNA sequence corresponding to the DREF gene containing the nucleotide sequence of SEQ ID NO: 2 (positions 202 to 2286), or a partial sequence thereof, or a sequence. Either the nucleotide sequence of number 2 (positions ² to ²²⁸⁶ ), or a partial sequence thereof, and DNA containing a complementary sequence. ,

 The partial sequence is a sequence consisting of about 30 or more, 50 or more, 70 or more, 100 or more, 150 or more, 200 or more, or 250 or more and a full length of, for example, 50 to 150 nucleotides in the DREF gene or mRNA sequence. Can be included.

 In addition to natural nucleotides, antisense nucleic acid nucleotides

It can contain modified nucleotides having groups such as (fluorine, chlorine, bromine or iodine), methyl, carboxymethyl or thio groups.

 Antisense nucleic acids can be synthesized using well-known DNA / RNA synthesis techniques or DNA recombination techniques. When synthesizing by DNA recombination technology, the vector DNA containing the sequence of SEQ ID NO: 2 is mirrored, and the target sequence is amplified by polymerase chain reaction (PCR) using primers that squeeze the sequence to be amplified. If necessary, it can be cloned into a vector to produce antisense DNA. Alternatively, the DNA having the amplified target sequence thus obtained is inserted into a vector, the vector is introduced into a eukaryotic or prokaryotic cell, and the transcription system is used for antisense.

RNA can be obtained. As the vector, the above-described virus vector or plasmid vector can be used. - Whether the antisense nucleic acid of the present invention is DNA or RNA, it can suppress translation into protein by binding to DREF mRNA.

 In order to deliver the antisense nucleic acid of the present invention to a patient, the antisense nucleic acid may be encapsulated in a liposome such as a positively charged ribosome as described above, or the antisense nucleic acid may be, for example, a strong pol II or It may be incorporated into a vector (the above-mentioned plasmid or viral vector) so as to be under the control of the pol III promoter. 13

 The present invention further provides a composition for treating cancer comprising an antibody or a fragment thereof that suppresses the in vivo function of a DREF protein comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof.

 The target protein of the present invention is a protein encoded by the DREF gene, and is a protein involved in human cancer cell proliferation and cell cycle progression. Therefore, inhibition or suppression of the function of the protein expressed in cancer cells can result in suppression of cancer growth and cell death. A drug for this purpose is an antibody against the DREF protein or part thereof.

 Preferably, the antibody against DREF is an antibody against the CR1 domain, CR2 domain or CR3 domain of DREF (SEQ ID NO: 135, 133 or 134, respectively), or a portion thereof. An example of a particularly preferred antibody is an antibody against a DRE (DNA replication-related element) binding region on DREF. The DNA-binding domain of DREF is called “BED zinc finger” and has a protein structure with one zinc ion. This domain is part of CR1 and contains the 20th to 74th sequence (SEQ ID NO: 140) of the DREF amino acid sequence of SEQ ID NO: 1. Antibodies to the domain containing this domain of DREF can inhibit DREF DNA binding and consequently inhibit DREF's in vivo function.

 BED. Zinc finger amino acid sequence (SEQ ID NO: 140):

 RAKSKVWKYFGFDTNAEGCILQWKKIYCRICMAQIAYSGNTSNLSYHLEK 丽 PEE

Another domain associated with DNA binding is a part of CR3 of DREF, and includes the 571st to 651th sequence (SEQ ID NO: 141) of the DREF amino acid sequence of SEQ ID NO: 1. This domain is an activator's family (hAT element superf amily) Called the dimerization domain. Antibodies to the region of DREF containing this domain inhibit DREF dimer formation, resulting in inhibition of DREF DNA binding and inhibition of DREF in vivo function.

Amino acid sequence of the dimerized domain (sequence number 141):

EQVFLYENA

 For the production of an antibody useful in the present invention, the amino acid sequence and nucleotide sequence of the following CR1, CR2, and CR3 domains are used to make (poly) peptides using known peptide synthesis techniques or DNA recombination techniques. Can be synthesized to produce the desired antibody.

CR1 amino acid sequence (SEQ ID NO: 138):

 The nucleotide sequence of CR1 (SEQ ID NO: 139)

 CAGGAGCTGACGGCCGCCGTGCTGGGCCTCATCTGCGAGGGGCTGTACCCA CR2 amino acid sequence (SEQ ID NO: 133):

EDSNNHHLMLEASEWATIEGLVELLQPFKQVAEIV EVIAKELSKTYQETPEIDMFLNVATFLDPRYKRLPFLSAFERQQVENRVVEEAKGLLD CR2 nucleotide sequence (SEQ ID NO: 3): GTGGTGGAAGAGGCCAAGGGCCTGCTGGAC

CR3 amino acid sequence (SEQ ID NO: 134):

 FFGI

CR3 nucleotide sequence (SEQ ID NO: 4):

 TTCTTTGGCATT

Such antibodies include polyclonal antibodies, monoclonal antibodies, recombinantly produced antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, Fab, F (ab ') ₂ , scFv, Fv, two These include bispecific antibodies and synthetic antibodies. The characteristics and structure of the antibody are described in Toru Otsuki et al., Experimental Medicine Vol. 22 No. 14 (Extra) 125-: 130, 2004, Yodosha (Tokyo, Japan). Can be used for.

Preferred antibodies suitable for use in the present invention are human or humanized antibodies, particularly human or humanized monoclonal antibodies, that cause little or no side effects due to anaphylaxis. The antibody class and subclass may be of any type. For example, the type of antibody, IgGヽIgMヽIgEヽIgD, IgA, include IgG had _{I g G 2, IgG 3,} IgG 4, IgA have IgA _2. An antibody may also be derivatized by pegylation, acetylation, glycosylation, amidation, and the like.

 Human antibodies can be generated by, for example, phage display library (pharge display library).

¾ (TC Thomas et al., Mol. Immunol. 33: 1389-1401, 1996) or methods using human antibody-producing animals (eg mice, ungulates, etc.) (I. Ishida et al., Cloning Stem Cell 4: 91- 102

2002).

Human antibody-producing mice contain, for example, human antibody-producing genes in human artificial chromosomes After introducing the human chromosome fragment, using the microcell method, for example, an artificial chromosome is integrated into the genome of a mouse embryonic stem cell, transplanted into the uterus of a foster parent mouse, a chimeric mouse is born, a male or female chimeric mouse is bred, or a chimeric mouse And a wild-type mouse of the same species, for example, by producing a homozygous offspring mouse that contains a human antibody gene and is therefore capable of producing a human antibody (for example, Table 02/092812, International W0 98/24893, International W0 96/34096, etc.). This human antibody-producing transgenic mouse is immunized with the target DREF protein of the present invention as an antigen, and then the spleen is removed and fused with this spleen cell and mouse myeloma cell by conventional techniques to form a hybridoma. The desired monoclonal antibody can be prepared (G. Kohler and C. Milstein, Nature 256: 495-497, 1975).

 In the phage display library method, DNA encoding the antibody of interest is screened from a library of immunoglobulin genes obtained directly from untreated human lymphocytes, and phage particles are placed between this DNA and the antibody chain. To establish a physical association, which involves enriching affinity-screened phage that present antibodies with affinity for the target. Using this method, an antibody having a binding affinity for a target can be synthesized in a large amount by an ordinary method (for example, JP 20013-527832).

A humanized antibody can be obtained, for example, by binding a human IgG complementarity determining region (CDR) produced from a mouse immunized with human DREF protein to human IgG. Such humanized antibodies can be produced using genetic recombination techniques. Techniques for humanizing antibodies are described, for example, in US Pat. Nos. 6639055 and 5530101. '' In the present invention, DREF protein variants include all naturally occurring variants in human individuals, such as variants based on polymorphisms or mutations, or selective Includes variants by splicing. The variant has a sequence in which one or several amino acids are substituted, deleted or added in the amino acid sequence of SEQ ID NO: 1, and has an in vivo function involved in cancer cell growth. In this specification, “several” means 10 or less, 8 · or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 Means individual. Alternatively, the variant comprises a sequence having% identity of 95% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence of SEQ ID NO: 1. As used herein '' _0/0 identity "may be determined based on the possible known BLAST program introduction of gaps (BL ASTX and BLASTN) In a sequence alignment (e.g. SF Altschul et al, J. Mol. Bio 215: 403-410, 1990, etc.).

 The antibody or fragment thereof of the present invention can be used as a therapeutic agent for cancer alone or in a form to which a drug such as an anticancer drug (chemotherapeutic agent, radioactive substance, etc.) is bound. Anti-cancer agents include, but are not limited to, chemotherapeutic agents such as taxol, cytarabine, cisplatin, etoposide, doxonorevicin, daunonorevicin, vinblastine, paclitaxe ^^, and radioactive metals such as radioactive indium, technetium, and ytterbium. . Binding of the anticancer agent to the antibody can be performed by a method including binding of the anticancer agent to any site of the antibody constant region, for example, by covalent bonding through a linker or by coordinate bonding with a metal ion.

 Delivery of the antibody or fragment thereof to the patient is usually appropriate, either alone or in the form of the antibody or fragment thereof encapsulated in liposomes (preferably positively charged liposomes), microcapsules or nanoparticles. It can be administered by a parenteral route (for example, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, topical administration, etc.) in combination with an appropriate carrier, excipient or diluent. '1.4 Composition

 The compositions of the invention can be used to treat patients with cancer, in particular cancers that express DREF. Examples of such cancers include, but are not limited to, lung cancer, esophageal cancer, sputum cancer, stomach cancer, liver cancer, colon cancer, thyroid cancer, prostate cancer, bladder cancer, kidney cancer, skin cancer, gall cancer, brain tumor Breast cancer, ovarian cancer, cervical cancer, testicular cancer, lymphoma, melanoma, sarcoma, osteosarcoma, etc. More preferred cancers include lung cancer, esophageal cancer, breast cancer, vaginal cancer and the like.

The dose of the nucleic acid in the composition of the present invention is not limited to the following when converted to siRNA molecules or antisense nucleic acid molecules, but is 1η100 to 100 _μ投 与, preferably 10ηΜ to 50Μ per dosage unit. More preferably, it is 100ηΜ to 20 °. The dose of the antibody or fragment thereof in the composition of the present invention is not limited to the following, but is about 1 to about 100 mg / ml, about 5 to about 70 mg / ml, about 10 to 50 mg per dosage unit. / ml.

 However, the above dose or dose may vary depending on the patient's condition, age, gender, severity, etc., and the dose or dose should be determined at the discretion of a specialist.

 The composition of the present invention usually contains a pharmaceutically acceptable carrier, excipient or diluent such as sterile water, physiological saline, buffer solution, non-aqueous liquid (eg almond oil, vegetable oil, ethanol, etc.), etc. Can be included. The composition further comprises a pharmaceutically acceptable stabilizer (for example, amino acids such as methionine), a preservative (methyl P-hydroxybenzoate, sorbic acid), an isotonic agent (for example, sodium chloride), an emulsifier ( For example, lecithin, gum arabic), a suspending agent (for example, a cellulose derivative) and the like can be contained. .

'Preferred pharmaceutical preparations are solutions, suspensions, emulsions, liposome encapsulants, etc. The composition of the present invention can be administered parenterally, for example intravenously, intraperitoneally, intramuscularly. Administration, subcutaneous administration, local administration, etc. Local administration includes direct injection into the affected area under surgery or endoscopy, etc. In addition, based on a treatment plan determined by a specialist One to several doses of the composition of the present invention to the patient at time intervals such as 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 6 months, 8 months, 1 year or 2 years Can be administered separately.

 When the RNAi nucleic acid, antisense nucleic acid, or antibody of the present invention is applied to cancer, particularly lung cancer, it can induce cell death together with cell cycle arrest strongly in lung cancer cells. In particular, in some lung cancer cells, induction of cell death in M phase called mitotic catastroph was observed. On the other hand, in normal lung-derived cell lines, cell cycle arrest was mild compared to lung cancer.

 The cancer treatment method targeting DREF of the present invention can be used as a tailor-made medicine optimal for cancer patient cases by combining with the screening method for therapeutic agents described below.

2. Screening of cancer drugs The present invention further suppresses the expression or translation of the DREF gene by adding a candidate drug to a culture medium containing human cultured cancer cells or a human cell line in which the DREF gene has been transfected so that it can be strongly expressed (normal or non-normal). There is provided a method for screening a drug for treating cancer, comprising screening a drug in vitro.

 Specifically, drug screening is performed by inhibiting cancer cell growth, cell cycle arrest, and / or cell death in cancer cells or mammalian cells that strongly express DREF (eg, humans, mice, etc., preferably humans). Can be used as an index.

 Specifically, the method of the present invention comprises preparing a human cultured cancer cell or a cell line that strongly expresses DREF, culturing the cell in the presence of a candidate drug, and suppressing the expression of the DREF gene or mRNA. Or screening candidate drugs for cell proliferation inhibition, cell cycle arrest and Z or cell death induction of the cell or cell line.

 The human cultured cancer cells include known cancer cell lines, cancer cells derived from biopsies from cancer patients, etc., and can be used in the present invention. Examples of cancer cell lines are all lung cancer cell lines such as ACC-LC-91 (H. Osada et al., Mol. Carcinog., 2005, 44: 233-241), A549 (ATCC, Rockvill, MD), PC10 (Immuno-Biological Research Institute, Gunma, Japan), Calu6 (ATCC, Rockville, MD).

 A cell line that strongly expresses DREF can be prepared, for example, as follows. A lentivirus was prepared by inserting the DREF cDNA into a lentivirus vector (eg CSII-CMV-MCS-IRES2-Bsd vector (obtained from Dr. Hiroyuki Miyoshi of RIKEN (Kodama, Japan)). Infect the respiratory epithelium-derived cell line BEAS2B and selectively culture with Blasticidin (Invitrogen) to select a strain that constantly expresses DREF strongly. Medium (Sigma) can be used The growth rate can be measured by MTT Atsey etc. using TetraColorOne ™ (Seikagaku, Tokyo, Japan) etc. DREF strong expression cell lines (eg BEAS2B- The DREF strain) was observed to increase growth rate about 3 times compared to the control strain after 6 days of culture.

In the method of the present invention, the degree of suppression of DREF gene or raRNA expression can be determined by a comparison experiment with a control to which no candidate drug is added. The expression level is determined by a well-known method (for example, phenol / chloroform / isoform) from cancer cells or DREF strongly expressing cell lines. Total RNA or mRNA or poly A (+) RNA obtained by the amyl alcohol method, guanidinium / CsCl method, oligo dT cellulose column chromatography, etc.) or from the RNA by reverse transcriptase-PCR (RT-PCR) method For synthesized cDNA, quantitative RT-PCR method, hybridization method using fluorescently or radioactively labeled probe (eg Northern hybridization, Southern hybridization, DNA microarray, tissue microarray, etc.) (Translated by Atsushi Saigo et al., Molecular Biology Experiment Protocol I, II, III, 1997, Maruzen). Alternatively, the expression level is determined by measuring the intracellular level of the protein encoded by the DREF gene (SEQ ID NO: 1) by immunoassay using an antibody against the protein or a fragment thereof, Western hyperprecipitation method, tissue staining method, etc. It can be determined indirectly by measurement.

 The probe is a nucleotide sequence of SEQ ID NO: 2 (positions 202 to 2286) or a sequence complementary thereto, or a sequence thereof, for example, about 20 or more, about 30 or more, 50 or more, 70 or more, 100 or more, 150 or more, 200 A DNA having a sequence consisting of 250 or more nucleotides. The probe is preferably a labeled probe bound with a fluorescent or radioactive label. Fluorescent labels include, for example, fluoresamine, rhodamine, their derivatives, Cy3, Cy5, etc. Radiolabels include, for example, radioactive phosphorus or thio atoms.

 Hybridization can be performed under low hybridization, medium hybridization or high hybridization conditions, preferably under high hybridization conditions. For example, 2-6 X SSC at about 45-50 ° C

(1 X SSC is 150 mM sodium chloride / 15 mM sodium taenate) followed by a high pre-degradation, followed by 0.2-2 X SSC / 0.1-1% SDS at about 50-65 ° C. (For example, Ausubel et al., Curent Protocols in Molecular Biology,

1995, John Wiley and Sons, US; translated by Kei Saigo et al., Molecular Biology Experiment Protocol

I, II, III, 1997, Maruzen). Or 6 X SSC ヽ Denhardt's solution at 60-65 ° C, 0.2%

After hybridization in SDS, it consists of cleaning with 0.2X SSC, 0.1% SDS at 60-65 ° C. The hybridization temperature can generally be 5 to 10 ° C lower than the melting temperature (Tm). Tra is, for example, the formula: Tm (° C) = 81.5 ⁺ 16.6 (log ₁₀ [Na ⁺ ]) +0.41 (% G + C)-(600 / N) (where N is the number of hybrid bases) , [Na ⁺ ] is the concentration of Na + in the hybridization buffer.) Q. Sarabrook et al., Molecular Cloning: A Laboratory Mannual 2 Cold Spring Haroor Laboratory, Cold Spring Harbor Laboratory Press, 1989).

 Candidate agents include, but are not limited to, small molecules, peptides, polypeptides, proteins, nucleosides, oligonucleotides, polynucleotides, nucleic acids (DNA or RNA), and the like.

 An immunoassay is an analysis method that uses an antigen-antibody reaction. For example, an enzyme-linked antibody method (for example, ELISA), a fluorescent antibody method, a solid phase method, a homogenous method, a sandwich method, a piotine / avidin system, etc. It can be carried out. As the solid phase, for example, a commercially available plastic plate (for example, a 96-well plate made of polystyrene) can be used. These methods are well known in the art and their conventional techniques can be used in the present invention.

 If the expression of DREF gene or mRNA is significantly inhibited or suppressed in human cultured cancer cells, or DREF strongly expressing cell lines, compared to the control without addition of the candidate drug due to the presence of the candidate drug, the candidate The drug can be identified as a cancer therapeutic.

 Quantitative RT-PCR is performed, for example, by PCR using mRNA or poly A (+) RNA in the vertical form in the presence of thermostable polymerases such as Taq polymerase and using a PREF derived from the DREF gene sequence. Can do. The size of the primer is about 15-30 nucleotides, preferably 17-25 nucleotides. At this time, the expression level of the DREF gene is determined relative to the expression level of the housekeeping gene with no expression fluctuation.

 Candidate drug screening can also be performed by examining cell growth inhibition, cell cycle arrest, or cell death in human cultured cancer cells, or DREF strongly expressing cell lines.

Cell proliferation can be measured by the following method. On the next day after gene transfer, repopulate the cells and select with puromycin / zg / ml) for 2 days, then continue with puromycin (0.5 μg / ml) for 10 days. Then, replace with TetraColorOne ™ (Seikagaku, Tokyo, Japan) 5% medium and incubate at 37 ° C for 1 hour. The medium is then collected and removed. 0D450nm is measured using a reader (in this case, OD630nm is used as a control), and the number of living cells is determined (colorimetric assay). ₀ After that, the cells are fixed with methanol, and then 5% Gierasa (Sigma-Aldrich) Stain with aqueous solution.

(mi tot ic catastroph άΧ)、 apoptosisノ Ιιこ rけ て計測する。 Cell cycle and cell death can be measured by the following methods. The transgenic cells are collected after puromycin selection and suspended in a hypotonic solution (75 mM KC1) (37 ° C, 20 minutes). Then, fix the cells by adding about 3 volumes of fixative (methanol: glacial acetic acid = 3: 1 mixture) with stirring. After removing the supernatant, resuspend in the fixative. Drop 1 or 2 drops of cell suspension on a slide glass and air dry. Then, stain the nuclear chromosome with 5% Giemsa aqueous solution. The cells were observed with a stereomicroscope BHT-323 (Olympus), and the cells were interphase (G9 / G1, S, G2), prophase, metaphase, metaphase, late phase / terminal phase (Anaphase / telophase) ; ヽ

(mi tot ic catastroph άΧ), measure by apoptosis.

 3. Gene groups affected by suppression of DREF gene expression

 A group of genes whose expression changes when DREF gene expression is suppressed by RNAi nucleic acid was analyzed using a microarray. ·

 Due to the suppression of DREF gene expression by DREF-RNAi # 4, changes in the expression of genes shown in Table 1 (see below) were observed.

 Due to the suppression of DREF gene expression by DREF-RNAi # 3, changes in the expression of genes shown in Table 2 (see below) were observed. '

 Many genes have been found to be highly related to cancer development, cancer progression, and cancer metastasis. These analyzes also indicate that DREF is an important factor for various cancers. Therefore, it can be said that the method of the present invention using DREF as a molecular target provides an effective method for the treatment of cancer.

 The following examples further illustrate the present invention, but the scope of the present invention is not limited by these examples. Example

 (experimental method)

1. Human cancer cell line Human lung cancer cell line (ACC-LC- 91 or A549), were cultured in 5% C0 ₂ presence with 5% fetal calf serum (Invitrogen) added RPMI 1640 medium (Sigma), it was used in the following experiments. Human normal airway epithelial cell lines (BEAS2B or HPL1) are 1% fetal bovine serum (Invitrogen;), 5 μg / ml bovine insulin (Sigma), 5 μg / ral human transferrin (Sigma), with 0. 1 mu M hydrocortisone (S i gma)及Pi 0. 2 nM preparative RHO de thyronine (S i gma) was added a Ham F12 medium (Sigma) and cultured in 5% C0 ₂ the presence of the following Used in the experiment.

 2. Transformation and DREF expression vectors

For DREF expression vector and RNAi-vector gene transfer into cells (tra.nsf ect ion), seed 3 x 10 ⁵ cells in a 3.5 cm culture dish and mix DNA and Lipofectamine 2000 (Invitrogen) the next day. And added to the medium. '' When a vector with a puromycin-resistant gene such as RNAi vector is transfected, puromyc in dihydrochlori de (Sigma-Aldrich, St (Louis, M0) 2 g / ml medium was added, and after 2 days of selection, the transfected cells were collected, and RNA and protein samples were prepared or FACS analysis was performed. The DREF expression vector was obtained by inserting the 0RF portion of the DREF cDNA obtained by RT-PCR into the expression vector pcDNA3 (Invitrogen). Dr. Fumiko Hirose (Hyogo Prefectural University, Hyogo, Japan) More distributed.

 3. RNAi vector RNAi vector '

 As shown in Figure 1, one of the two known RNAi sites (# 1, # 2) as a control (# 1) and four new RNAi sites (# 3, # 4, # 5 and # 6) ) DNA oligonucleotide was prepared by requesting Greiner Japan (Tokyo, Japan). Its base sequence is as follows (note that the number at the RNAi site represents the position number of the nucleic acid residue when the base “A” of the start codon in DREF 0RF is the first position).

RNAi # l (SEQ ID NO: 16)

 5,-AATTCTGCGAGTTCGTCAAGA-3 '(221st to 241st)

Ol igo for RNAi # l (for HI promoter) (SEQ ID NOs: 17 and 18)

H1F1: H1R1:

 3, shouting

 RNAi # 3 (ffi column number 19)

 5, -GCAACAACCACCACCTCATGC-3 '(1136th ~ 1156th)

Ol igo for RNAi # 3 (SEQ ID NO: 20 and 21)

si3F: 5 '-CAACAACCACCACCTCATGCCTTCCTGTCAGCATGAGGTGGTGGTTGTTGCTTTTTG-3'

RNAi # 4 (SEQ ID NO: 22)

 5,-GCAACTTCAAGTCCCAGAAGG-3 '(from 1718 to 1738).

Ol igo for RNAi # 4 (SEQ ID NO: 23 and 24)

s i4F

s i4R

 RNAi # 5 (SEQ ID NO: 25)

 5 '-GTACCCCACCATCAGCATGGT-3' (1245th ~ 1265th)

Ol igo for RNAi # 5 (SEQ ID NO: 26 and 27)

si5F

 RNAi # 6 (SEQ ID NO: 28)

 5, -GGTGCTTGGCCTCAACGAAGA-3 '(1737-1757)

Ol igo for RNAi # 6 (SEQ ID NOs: 29 and 30)

s i6F:

blank ol igo (for control) (Apal-EcoRI digested pU6-puro) (SEQ ID NO: 31 and 32) 'blankF: 5'-CGATATCTTTTTTG-3 '

blankR: 3 '-CCGGGCTATAGAAAAAACTTAA-5'

 (Note 1) Since the first G of RNAi # 3 ~ ^ reads the G on the vector, the ol igo sequence does not include the first G of the RNAi site and starts from the second residue.

 (Note 2) The underlined portion indicates the DREF RNAi site.

Plasmid vector with puromycin resistance gene (pCMV-puro) [Note] This vector The filter was prepared by cutting off the unnecessary part 1. Okb length from Clontech's plasmid pIRESpuro2 (5.2 kb length). In addition, an RNAi vector containing the mouse U6 gene promoter, a promoter transcribed by RNA polymerase III, was prepared (pU6-puro) (Osada., .. et al., Cancer Res. 65: 10680-5, 2005). Similarly, an RNAi betater inserted with the human HI gene promoter was prepared (pHl-RNApuro).

 To prepare RNAi vector, ol igo for RNAi # l was inserted after digestion of pHl-RNApuro with Bglll-Hindlll. RNAi # 3 to # 6 were inserted after pU6-puro was digested with Apal, treated with T4 DNA polymerase, and digested with EcoRI.

 Blank ol igo inserted pU6-puro after Apal-EcoRI was erased.

4. RT-PCR analysis ''

 Total RNA was recovered from the transfected cells using the RNeasy Mini Kit (Qiagen). RT-PCR was performed using the One-step RT-PCR kit (Qiagen) according to the attached instruction manual, and the RT-PCR product was subjected to TBE-agarose gel electrophoresis to examine the expression of the DREF gene (Figure 2). ).

 PCR primers:

 DREF F1: 5, -TGGCAAGGACATCGTGAAGG (positions 813 to 832 of hDREF 0RF; SEQ ID NO: 135) DREF R1: 5 '-GACAGCAACAACCACCACCT (1132 to 1151 of hDREF 0RF; SEQ ID NO: 136)'

 5. Western blot analysis

 DREF protein was detected using an antibody against HA-tag / myc-tag to which DREF-cDNA was added. The cells after gene transfer are lysed with 4% SDS solution and heat-treated, and then the protein is electrophoresed on SDS-PAGE and Immobilon using the semi-dry electrotransfer system Ti ^ ns-Blot SD cell (Bio-Rad). -Transferred to Millipore. The membrane is overlaid with anti-HA-tag antibody (Santa cruz) and anti-myc-tag antibody (9E10, Santa cruz), washed and then overlaid with secondary antibody (Cell Signaling) bound with horseradish peroxidase, and ECL Western DREF protein was detected by Blotting Detection Reagents (GE Healthcare).

6. FACS analysis ·- The cells after gene transfer are suspended in IGEPAL (registered trademark) CA-630 (0.5% Sigma-Aldrich), Propidiura iodide (20 μg / ml, Sigma-Aldrich), PBS solution, and DNA in the naked nucleus is transferred. After staining, the amount of DNA was quantified using FACSCalibur (Becton-Dickinson), and the cell cycle (G0 / G1, S, G2 / M) was analyzed using the cell cycle analysis software ModFit (BD Biosciences).

 7. Cell proliferation analysis

 On the day after gene transfer, repopulate the cells and select with puromycin (2 μg / ml) for 2 'days, and then continue with puromycin (0.5 g / ml) for 10 days. Then, replace with TetraColor One (Seikagaku Kogyo, Tokyo, Japan) 5% medium and incubate at 37 ° C for 1 hour. Thereafter, the medium was collected, and 0D450nm was measured with a plate reader (in this case, 0D630nm was used as a control), and the number of viable cells was determined (coloimetric assay). Thereafter, the cells were fixed with methanol and then stained with 5% Giemsa (Sigma-Aldrich) aqueous solution.

8. Immunostaining.

 'The day after gene transfer, seed cells on a cover glass placed in a culture dish and select puromycin (2 µg / ml) for 2 days. Then, cells were fixed with 3.7% Formaldehyde (10% Formalin) (Wako, Osaka, Japan) PBS solution (room temperature, 8 minutes), and washed with TBS solution (150 mM NaCl, 50 mM Tris (pH 7.6)) Apply cell permeabilization with 0.5% TritonX (Wako) TBS solution (room temperature, 8 minutes). After that, various primary antibodies (anti-T / H2AX (Upstate, Lake Placid, NY), phospho-ATM-Serl981 (Cell Signaling, Danvers, MA), ji phospho-hdstoneH3 (Upstate, Lake Placid, NY)) were added, A fluorescent dye-conjugated secondary antibody (Molecular Probe—Invitrogen) such as Alexa Fluor 488 was added together with DAPI (Sigma—Aldrich). After washing, the mounting medium PermaFluor (Thermo Shandon, Pittsburgh, PA) was added, placed on a slide glass, and observed with a confocal laser fluorescence microscope Radiance OO (BIO-RAD).

 9. TUNEL analysis

As in the immunostaining experiment, seed the transgenic cells on a cover glass and select puromycin for 2 days. Then, the cells are fixed with 3.7% Formaldehyde (10% Formalin) (Wako) PBS solution (room temperature, 10 minutes). After treatment with 1% hydrogen peroxide-containing methanol solution (room temperature, 2 minutes) and 0.1% TritonX-100-containing PBS solution (room temperature, 5 minutes), Using the In Situ Cell Death Detection Kit, Fluorescein (Roche), label the intracellular DNA cleavage site with Fluorescein-binding nucleic acid. Then, if necessary, immunostain with anti-phospho-histone H3, etc., and after washing, add fluorescent mounting agent Shandon PermaFluor (Thermo Electron Corp., MA, USA) and place it on a slide glass. Observed at '

1 0. Cell death and cell cycle analysis

 The transgenic cells are collected after puromycin selection and suspended in hypotonic solution (75 mM KCl) (37 ° C, 20 minutes). Then, fix the cells by adding about 3 volumes of fixative (methanol: glacial acetic acid = 3: 1 mixture) while stirring. After removing the supernatant, resuspend it in the fixative. Drop 1 or 2 drops of cell suspension on a slide glass and air dry. Thereafter, nuclear chromosomes were stained with 5% Giemsa aqueous solution. Observed with a stereomicroscope BHT-323 (Olympus), the cells were interphase (Go / Gl, S, G2), early cell division (Prophase), metaphase (Metaphase), late phase / terminal (Anaphase / telophase), and dead cells (mitot i (? catastroph and apoptos is)).

 1 1. Microarray

 Hiroyuki Miyoshi of RIKEN BioResource Center (Wako City, 'Saitama, Japan) from Lentiviral Systems (pENTR4-Hl, CS-RfA-EG, pCAG-HIVgp, pCMV-VSV-G -Receive distribution of RSV- Rev). The U6 prmoter-RNAi-oligo part of the pU6-puro-RNAi vector is inserted into the pENTR4-HI vector and then incorporated into the CS-RfA-EG vector using LR clonase (Invitrogen). Transfect 293T cells with transfectamin2000 together with pCAG-HIVgp and pCMV-VSV-G-RSV-Rev. The next day, replace with medium supplemented with Forskol in (Sigma-Aldrich) lCM and incubate for 24 hours. Collect the culture supernatant. The culture supernatant is ultracentrifuged at 19, 400 rpm for 2 hours in a Swing rotor SW28, the lentiviral particle sediment is collected, dissolved in the medium so as to be concentrated about 50 times, and used as a virus stock (-70 ° C save). Lentivirus on ACC-LC-91 cells

Add 50 X enriched stock (1.5 μ 1 per 1 X 10 ⁴ cells). Collect cells at 36, 54, and 72 hours after addition, and perform FACS analysis and RNA sample preparation. CDNA is synthesized from 250 ng of the obtained 36 hour RNA sample using MMLV-RT (Agilent Technologies, Palo Alto, Calif.) And an oligo dT primer to which a T7 promoter sequence is added. Then T7 Synthesis of Cy3 / Cy5-labeled cRNA using Low RNA Fluorescent Linear Amplification Kit (Agi lent Technologies) including RNA polymerase and Cyanine 3 (Cy3)-CTP and Cyanine 5 (Cy5)-CTP (PerkinElmer, Quebec, Canada) ₀ Cy3 / Cy5-labeled cRNA was amplified on an Agilent oligonucleotide microarray (Whole human genome 44K), and the amount of fluorescent dye was quantified and analyzed with an array scanner (Agi lent Technologies). Two combinations of RNAi # 3 and RNAi # 4 RNA samples and control lentiviral RNA samples were exchanged (Color swapping) and performed twice, and the mean change in the genetic change after RNAi was examined.

 (Result)

 Figure 1 shows the domain structure, RNAi site and sequence of the human DREF / ZBED1 gene. puromycin resistance '! "Plasmic vector with a ginger is child ^ pCMV-puro) Insert the mouse U6 promoter, which is a promoter transcribed by RNA polymerase III, and then insert a short-hairpin type ol igo to DREF- The RNAi system was constructed (pCMV-puro-siDREF # l to # 6) The DREF-RNAi system was transfected into a human lung cancer cell line, and then the transfected cells were selected by puromyc in treatment for 24-48 hours. In addition, the lentivirus vector was used for RNAi, and a short hairpin type.ol igo was inserted into the lentivirus vector in the same manner, and the gene was introduced into 293T cells according to a standard method. lentivirus particles expressing short hairpin RNA were collected and added to the cell culture for analysis.

 Figure 2 shows the results of RT-PCR analysis of the RNAi effect. Introduce RNAi vector pCMV-puro-s iDREF # l, # 3, # 4, '# 5, # 6 into lung cancer cell A549, and after puromycin treatment for 48 hours, collect cells, extract RNA, RT- DREF expression was examined by PCR. Compared to the control vector that does not express DREF-siRNA (pCMV-puro_) U6blank, all pCMV-puro-siDREF vectors showed decreased expression due to RNAi.

 Figure 3 shows the results of Western blot analysis of the RNAi effect. To further investigate the RNAi effect, we used a DREF full-length cDNA expression vector and a DREF full-length cDNA expression vector that had undergone a silent mutation in the RNAi sites of # 3 and # 4, respectively, and became RNAi resistant.

Changes in the expression of DREF protein were examined by Western blot. For detection, an anti-HA polyclonal antibody, which is an antibody against HA-tag inserted at the N-terminus of DREF cDNA, was used. siDREF # 3 In # 4, both wild-type DREF proteins showed a marked decrease. DREF (# 3mut, # 4mut) with # 3 and # 4 silent mutations was resistant to RNAi.

Figure 4 shows the results of cell cycle analysis (1). Lung cancer cell line ACC- LC- 91 to _{P CMV-puro-s iDREF #} l, # 3, # 4, # 5, introduced # 6 (U6bl ank of及Pi control vector), after puromycin selection, the PI staining Cell cycle analysis (DNA content analysis) was performed with FACS. s iDREF- # 3 and # 5 showed a decrease in the S phase, and a trend toward cell cycle arrest (G1 arrest) at the G1-S transition stage. In # 4, G1 decreased significantly, G2 / M increased, and cell cycle arrest at a very strong G2 / M step was observed. A slight increase in G2 / M was also seen in # 3, and # 3 was thought to cause a strong G1 stop and a relatively mild G2 / M stop.

 Henceforth, we analyzed mainly # 3 and # 4, which showed strong cell cycle arrest.

 Figures 5A, 5B, and 5C show the results of cell cycle analysis (2). Similarly, pCMV-puro-siDREF, # 3, # 4 (and U6blank) were introduced into several lung cancer cell lines, and the cell cycle was analyzed. In ACC-LC-172, as in ACC-LC-91, # 3 causes a strong G1 arrest and a relatively mild G2 / M arrest, while # 4 shows a strong G2 / M cell cycle arrest. It was. Calu6 'ACC-LC-319 also showed a tendency to stop G2 / M in # 4. A549- # 4 'PC10- # 3 and-# 4 showed a decrease in S and a tendency to stop G1. In addition, cell cycle arrest was hardly observed in the normal lung-derived cell lines BEAS2B and HPL1.

 6A and 6B show the results of cell proliferation ability analysis. Puromycin-selection cell number after 10 days was analyzed with MTT assay (using cell proliferation kit). s iDREF- # 3 and # 4 showed a much stronger cell growth inhibitory effect than '4, and # 4 was considered to be stronger. # 1 had only a weak antiproliferative effect.

 FIG. 7 shows the results of staining with yH2AX (Phospho-Η2ΑΧ) and Phospho-ATM (Serl981). Considering the possibility of DNA damage induction as a mechanism of growth inhibition in the MTT analysis, ATM of the kinase activated in response to DNA damage (Serl981 phosphorylation is an activation index), and phosphorylation by this ATM Oxidized histone Η2ΑΧ (γ Η2ΑΧ = phospho- Η2ΑΧ) was examined by immunofluorescence. In both ACC-LC-91 and ACC-LC-172, γ 2ΑΧ signal increased with # 3 and # 4, especially in # 4. Of ACC-LC-91

Phospho- ATM (Serl981) staining showed a marked increase in signal, especially s iDREF- # 3, # 4

In # 4, DNA damage was induced, ATM activation, ATM such as Ή2ΑΧ, substrate phosphorylation, cell periphery It seems that the signal of the phase stop is working. Compared with ACC-LC-91, the γ Η2 に よ る signal from # 3 of A549 · PC10 was almost the same level (data not shown). Compared to ACC-LC-91, the signal of γ Η2ΑΧ by # 4 of A549.PC10 was Α549 (Sat) and PC10 (+) and ACC-LC-91 (++) (data shown) )

 Figure 8 shows the TUNEL analysis results. Induction of apoptosis (cell death) was examined by TUNEL analysis to detect DNA fragmentation. Compared to U6blank, apoptosis induction was seen at # 3 and # 4. Apoptosis frequency was different between # 3 and # 4, and # 3 was almost the same in 4 strains of A549, PC10, ACC-LC-91 and ACC-LC-172, but in # 4, A549 It is almost the same as PC10, and ACC-LC-91 is almost the same as ACC-LC-172, and ACC-LC-91 showed significantly greater apoptosis induction than A549 (some data not shown) ).

 Figure 9 shows changes in the cell cycle. Since it is not easy to distinguish between G2 and M in FACS, cells are examined by Giemsa staining to examine the cell cycle (interphase (interphase = G1, S, G2). M phase (prophase, metaphase, anaphase-telophase)) did. In ACC-LC ^ 91, siDREF # 3 and # 4 showed an increase in M period. In # 4, the number of M-phase cells that caused strong chromatin aggregation increased, indicating that cell death induction (mitotic catastroph) occurred in M-phase.

 Figure 10 shows cell death (mitotic catastroph) in M phase. Phospho-histon H3 (H3-P) and γH2AX, which are phosphorylated in M phase, were immunostained to investigate the relationship between DNA damage and cell death induction and the cell cycle. ACC-LC-91 showed that most of Η3-Ρ positive Μphase cells were γΗ2ΑΧ-positive, indicating that cell death was induced by DNA damage in the Μ phase. FIG. 11 shows the results of gene expression analysis of siDREF-# 3 and # 4. ACC-LC-91 was infected with siDREF # 3 and siDREF # 4 lentivirus to induce RNAi. Changes in gene expression caused by siDREF # 3 and # 4, with RNA collected 36 hours after infection, before cell cycle arrest (54 hours and 72 hours), and using control lentivirus-infected RNA as control RNA It was investigated. The genes that change at this stage are likely to be genes that are directly regulated by DREF and cause cell cycle arrest and cell death induction. Figure 12 shows the expression of DREF in lung cancer patient specimens.

Total RNA was collected from lung cancer patient samples using the RNeasy Mini Kit (Qiagen). From RNA 5 g, RT was performed using Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT, Invitrogen) to synthesize cDNA. Using the cDNA (equivalent to 20 ng of RNA), the amount of DREF gene expression and the amount of 18S ribosomal RNA expression as an internal control were quantified using ABI-PRISM 7900 (ABI) using the DREF primer and 18S primer shown in Figure 2. did. The significance test was performed by the StatView (SAS) t test.

 In all lung cancers, DREF expression was observed, whereas in normal lung, DREF expression was absent. In addition, when comparing DREF expression among histological types in lung cancer, DREF expression differs for each histological type, especially adenocarcinoma and lung cancer of other histological types (other than adenocarcinoma and squamous cell carcinoma) There was a significant difference between and (P = 0.012). These facts suggested an association between DREF expression and the development of lung cancer.

 Fig. 13 shows the DNA sequence (A) of RPL17 gene, which is considered to be related to cancer growth, among the genes that are suppressed by DREF-RNAi # 4, and the mechanism of transcriptional activation by DREF of RPL17 gene. (B) is shown. A sequence almost identical (90%) to the DNA binding motif TGTCGYGAYA (Y = C or T) of the DREF protein was found at + 7n1: ~ + 16nt from the transcription start point. DREF binds to this sequence to induce RPL17 DREF expression, and DREF-RNAi # 4 reduces DREF expression, so it is thought that RPL17 gene expression also decreases. The results of gene expression analysis using the microarray in Fig. 11 are shown in Table 1 (# 4) and Table 2 (# 3). These genes whose expression changes at an early stage of RNAi induction (36 hours) are directly regulated by DREF, and are likely to be cell groups that cause cell cycle arrest and cell death induction. It is considered to be a target.

table 1

(A group of genes whose expression is suppressed by DREF-RNAi # 4) ― ―

 Ofncia 丄 Symbol: SRRM1 and Name: serine / arginine: repetitive matrix 1 Gene aliases: 160-KD; P0P101; SRM160; GC39488.

Summary: Abnormal splicing of the cell surface molecule CM4 is associated with cancer malignancy and invasive potential. SRRMl / SRtnl60 is a co-activator of splicing and induces CD splicing abnormalities in an oncogene RAS-dependent manner (Mol Cell Biol. 2006 Jan; 26 (1): 362-70). It also binds to the oncogene TLS / FUS (Exp Cell Res. 2003 Feb 15; 283 (2): 184-95). The association with cancer development is strongly suggested.

Official Symbol: C0R01C and Name: coronin, actin binding protein, 1C Gene aliases: HCRNN; coronin-3

 Summary: A gene family with TO40 domains involved in various processes such as signal transduction, apoptosis induction, pre-mRNA maturation, and cytoskeleton formation.

Official Symbol: SON and Name: SON DNA binding protein

 Gene aliases: S0N3; BASS1; DBP-5; 賺 BP; C21orf50; FLJ21099; FLJ3391 KIAA1019

 Summary: Identified as a transcriptional regulator of human hepatitis B virus (HBV). Oncogene MYC family · Binds to M0S. It has a function to protect cells from Apoptosi s. A strong association with cancer development is strongly suggested. .

Oif icial symbol: S ^ RS16 and Name: splicing factor, ar'ginine / serine-rich 16 (suppressor-of-white- apricot homolog, Drosophila)

Gene aliases: CLASP; SWAP2; FLJ90109

 Su thigh ary: Controls splicing before becoming mature mRNA after gene transcription.

Official Symbol: RPL17 and Name: ribosoraal protein L17

Gene aliases: rpL23; GC117162

 Summary: It is a ribosome component of the protein synthesis mechanism, and protein synthesis is enhanced in cancer; increased expression of ribosomal protein is a poor prognostic factor for cancer. This suggests an association with cancer development.

Oxf icial Symbol: ZBED1 and Name: zinc finger, BED-type containing 1 Gene aliases: ALTE; TRAMP; KIAA0785

Summary: DREF gene itself

Official Symbol: CTDSP1 and Name: CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase 1

 Gene aliases: SCP1; NLIIF (nuclear LIM interac tor-interacting factor) A phosphatase that regulates the activity of RNA polymerase II that transcribes the gene. It controls the expression of a wide range of gene groups.

Official Symbol: XRCCl and Name: X-ray repair complementing defective repair in Chinese hamster cells 1

Gene aliases: RCC

Summary: Genes essential for DNA single strand break repair, nucleotide excision repair, etc. It is suggested to be associated with cancer development.

Official Symbol: WDR81; and Name: WD repeat domain 81-Gene aliases: FLJ23776; FLJ33817

 Summary: One of the gene families with the WD40 domain involved in various processes such as signal transduction, apoptosis induction, pre-mRNA maturation, and cytoskeleton formation.

Official Symbol: DDX21 ； and Name: DEAD (Asp-Glu Ala- Asp) box polypeptide 21

Gene aliases: GUA; GURDB; RH-II / GU; RH-II / GuA; DKFZp686F21172

 Summary: An RNA helicase with a DEAD box domain that regulates RNA conformation. Involved in the control of a wide range of functions related to RNA, such as the initiation of translation into proteins and splicing.

Official Symbol: WDS0F1; and Name: WD repeats and S0F1 domain containing Gene aliases: Gm83; HSPC064; GC126859; MGC138247; DKFZP56400463

Summary: This gene family is a gene family with a WD40 domain involved in various processes such as signal transduction, apoptosis induction, pre-mRNA maturation, and cytoskeleton formation.

Official Symbol: CDV3; and Name: CDV3 homo log (mouse)

Gene aliases: H41.

 Summary: It was identified as a gene that promotes oncogene erb-B2 in breast cancer that highly expresses it, and was also identified as a causative gene that causes cardiac hypertrophy in CVD3 mice. It is thought to promote cell growth and is strongly suggested to be related to cancer development.

Official Symbol "· TIRAP and Name: toll-interleukin 1 receptor (TIR) domain containing adaptor protein

 Gene aliases: Mai; wyatt; FLJ42305

 Summary: Binds to Toll-like receptor (TLR) —2 and TLR—4 and transduces NF—κΒ activation signal with TRAF6. It is suggested to be associated with epilepsy.

Official Symbol: TRADD and Name: TNFRSF1A-associated via death domain Gene aliases: Hs.89862; MGC11078

 Summary: It binds to TNF receptor (TNF-R / TNFRSF1A) that induces cell death, regulates apoptosis induction and NF-κΒ activation, binds to adapter protein TRAF2, and suppresses apoptosis induction by TRAF2 To do. It is suggested to be associated with cancer development.

Official Symbol: TSG101; and Name: tumor susceptibility gene 101

Gene aliases: TSG10; VPS 23

 Summary: Controls microtubule polymerization and binds to stathmin molecules that are implicated in the onset of cancer, and controls cell proliferation and differentiation. It is a tumor suppressor gene whose gene mutation is found in breast cancer.

Off icia 丄. Symbol: EGRl; and Name: early growth response 1

Gene aliases: TIS8; AT225; G0S30; NGFI-A; ZNF225; KROX-24; ZIF-268 Summary: EGRl is a transcriptional regulatory molecule of C2H2_zinc-finger type that regulates the expression of genes involved in differentiation and cell division It is thought to be involved in cancer development.

Official Symbol: FANCti; Oihi Name: Fanconi anemia, complementation group E Gene aliases: FAE; FACE

 Summary: One of the genes responsible for Fanconi anemia, a component of DNA repair mechanism.

Official Symbol: RAD23A and Name: RAD23 homo log A (S. cerevisiae)

Gene aliases: HHR23A; MGC111083

 Summary: Recognize DNA damage and participate in the mechanism of nucleotide excision repair.

Official Symbol: ITPKA; and Name: inositol 1, 4, 5-trisphosphate 3 kinase A Summary: Inositol 1, 4, 5-trisphosphate (IP3) is an intracellular signal concentration of calcium that is an important signal pathway ITPKA phosphorylates this IP3 to inositol 1, 3, 4, 5-tetrakisphosphate (IP (4)) and suppresses the increase in the strength concentration. It is deeply involved in the regulation of intracellular signals.

Official Symbol: BIRC3 · 'Pai Name: baculoviral IAP repeat-containing 3 Gene aliases: AIP1; API2; MIHC; CIAP2; HAIP1; HIAP1; MALT2; RNF49

Summary: It is a molecule that suppresses apoptosis, and gene amplification has also been reported in cancer. The association with cancer development is strongly suggested.

Official Symbol ： P0LD1 ； and Name: polymerase (DNA directe.d), delta 1, catalytic subunit 125kDa

Gene aliases: POLD

 Summary: An enzyme that replicates DNA during cell growth and is essential for cell growth. The difference with the onset of cancer is strongly suggested.

Official Symbol: TRAF7; and Name: TNF receptor-associated factor 7 Gene aliases: RFWD1; RNF119; MGC7807; DKFZp586I021

 Summary: It binds to the TNF family receptor involved in cell growth control and cell death induction, and is involved in signal transduction. This suggests a difference from the onset of cancer.

Official Symbol: WDR34; and Name: WD repeat domain 34

Gene aliases: MGC20486; bA216B9.3; RP11-216B9.5 '

 Summary: This is a gene family with WD40 domain involved in various processes such as signals, apoptosis induction, pre-mRNA maturation, and cytoskeleton formation.

Oii'icial Symbol: DVLl; Name Name: dishevelled, dsh nomolog 1 (Drosophila) Summary: Onset of cancer 'Wnt- -catenin signaling pathway that has been reported to be involved in growth, suppresses -catenin degradation and Wnt --Increase catenin signal. 'Relevant to cancer development is strongly suggested.

Table 2 (A group of genes whose expression is suppressed by DREF-RNAi # 3) _

Official Symbol: EGR1; Oihi Name: early growth response 1

Gene aliases: TIS8; AT225; G0S30; NGFI— A; ZNF225; KROX-24; ZIF-268

Summary: EGR1 is a C2H2-zinc-finger transcriptional regulatory molecule that regulates the expression of genes involved in differentiation and cell division and is thought to be involved in cancer development.

Official Symbol: CTBP1 ； and Name: C-terminal binding protein 1

Gene aliases: BARS; MGC104684

 Su 脑 ary: C-terminal binding molecule of adenovirus oncogene E1A. This CtBP gene family (CtBPl'CtBP2) is a target gene when the tumor suppressor gene ARF induces apoptosis induction independent of p53. Overexpression of CtBP induces p53-independent apoptosis induction of this ARF. Inhibits (Mol Cell Biol. 2006 Mar; 26 (6): 2360-72 ·). Therefore, this CtBPl is considered an oncogene.

Official Symbol: SMARCE1 ； and. Name: SWI / SNF related, matrix associated, actin dependent regulator of chromatin, subfamily e, member 1

Gene aliases: BAF57

 Summary: SWI / SNF complex that regulates chromatin structure and regulates gene expression. It also functions as a co-activator for nuclear hormone receptors such as androgen receptors that promote prostate cancer growth. It is deeply involved in the regulation of gene expression and essential for prostate cancer growth (Mol Cell Bi. L. 2005 Mar; 25 (6): 2200-15). The association with cancer development is strongly suggested.

Official Symbol: BAT2D1 ； and Name: BAT2 domain containing 1

Gene aliases: XTP2; BAT2-iso, KIAA1096

 Summary: Genes with gene amplification and overexpression in urological tumors, suggesting a relationship with cancer development (DNA Cell Biol. 2002 Oct; 21 (10): 707-15).

Official Symbol: HAX1; and Name: HCLS1 associated protein X-1

Gene aliases: HS1BP1; HCLSBP1.

Summary: This is a substrate of the representative kinase-type oncogene Src family and functions by binding to PKD2, the gene responsible for polycystic kidney disease. It is suggested to be associated with cancer development.

Ofrcial Symbol: PPAT ； and Name: phosphoribosyl pyrophosphate ami aotransf erase

 Gene aliases: GPAT; ATASE

 Summary: One of the biosynthetic enzymes of purine nucleic acid, which is a material for DNA, and is related to cell growth.

Official Symbol: STK40; Oihi Name: serine / threonine kinase 40

Gene aliases: SHIK; SgK495; MGC4796; RP11-268J15.4

Summary: Repression of transcriptional regulation by oncogene p65RELA and typical tumor suppressor gene p53 (Biochem Biophys Res Cora 匪. 2003 Oct 3; 309 (4): 774-8) ₀ The association with cancer development is suggested.

Official Symbol: SUV39H2 ； and Name: suppressor of variegation 3-9 homolog 2 (Drosophila)

 Gene aliases: F1J23414

 Summary: The SUV39H family (SUV39H1, SUV39H2) gene induces the methylation of histone H3-lysine9, which induces heterochromatin formation. It binds to RB2 / pl30 of the typical tumor suppressor gene Retinoblastoma (RB) family and promotes transcriptional regulation by RB2. It is suggested to be associated with cancer development. ,

Ofricial Symbol: PTGS2; Oi ame: prostaglandin-endoperoxide synthase 2

^ prostaglandin G / H synthase and cyclooxygenase

 Gene aliases: C0X2; COX-2; PHS-2; PGG / HS; PGHS-2; hCox-2

 Summary: Although it is a molecule involved in prostaglandin biosynthesis, its expression has been promoted in cancer, and its relation to cancer onset / progress and metastasis has been strongly suggested.

Ofricial Symbol: PARPl; and Name: poly (ADP-ribose) polymerase family, memoer 1

 Gene aliases: PARP; PPOL; ADPRT; ADPRT1; PARP-1; pADPRT-1

 Summary: It is an enzyme that converts nuclear molecules into poly (ADP-ribosyl) by processes such as cell proliferation, differentiation, canceration, and DNA damage reaction, and its relationship with cancer development is strongly suggested.

Official Symbol: CEBPD ； and Name: CCAAT / enhancer binding protein (C / EBP), delta

 Gene aliases: CELF; CRP3; C / EBP-delta; NF-] 6- beta

 Summary: A representative transcription factor that is involved in the expression of genes related to immune and inflammatory responses.

Official Symbol: HK2 ； and Name: hexokinase 2

Gene aliases: HKII; HXK2; DKFZp686 1669

 Summary: It is an enzyme involved in glucose degradation and is involved in the enhancement of pudosaccharide degradation in cancer cells with a high growth rate. The association with cancer development is strongly suggested.

Official Symbol: C1QDC1 ； and Name: Clq domain containing 1

 Gene aliases: EEG1; EEG-1; FLJ11391; FLJ22569; MGC102894

It is a gene whose expression is induced during cell differentiation and suppresses cell proliferation (JBiol Chem.

2004 Jan 16; 279 (3): 1916-21).

Ofticial Symbol: GCET2 jOihi Name: germinal center expressed transcript 2 Gene aliases: HGAL; GCAT2; MGC40441

 Summary: This gene is upregulated in B-cell malignant lymphoma and is induced by interleukin-4. It is suggested to be related to the onset of tumors such as lymphoma.

Official Symbol ： ACTG1 ； and Name: actin, gamma 1

Gene aliases: ACT; ACTG; DFNA20; DFNA26; beta-actin

 Summary: One of the representative cytoskeletal actin molecules involved in cell motility. It is also suggested that it is associated with cancer invasive metastasis.

Official Symbol: PLAGL2; and Name: pleiomorphic adenoma gene-like 2 Gene aliases: FLJ23283

Summary: Molecules belonging to the PLAG family activated by chromosomal translocations in tumors In also involved in the development of leukemia (Blood 2005 Apr 1; 105 ( 7):. 2900-7) 0 associated with cancer development is suggested strongly.

Official Symbol: NC0A6 ； and Name: nuclear receptor coact ivator 6

 Gene aliases: NRC; AIB3; ASC2; PRIP; TRBP; RAP250; KIAA0181

 Summary: It binds to a nuclear hormone receptor and functions as a co-activator. It also binds to histone acetylase and methylase that control chromatin structure and regulates transcription of a wide range of genes.

Official Symbol: UHRF1 ； and Name: ubiquitin-l ike, containing PHD and RING finger domains, 1

 Gene aliases: Np95; ICBP90; RNF106; huNp95; FLJ21925; MGC138707

 Summary: A gene that binds to the promoter's CCAAT box, regulates transcription, and correlates with cell growth. Promotion of cancer cell growth (Mol Biol Cell. 2005 Dec; 16 (12): 5621-9) Anti-apoptotic action (Ann NY Acad Sci. 2004 Dec; 1030: 355-60.) Has been reported. The association with the onset of cancer is strongly suggested. .

Official Symbol: DUSP6; and Name: dual specificity phosphatase 6

 Gene aliases: MKP3; PYST1

 Summary: A phosphatase that suppresses the action of kinase molecules and is a MAP kinase family.

 It suppresses the function of (MAP / ERK, SAPK / JNK, p38) and is considered a tumor suppressor gene.

Official Symbol: SFRS8; and Name: spl icing factor, arginine / serine-rich 8 (suppressor-of- white-apricot homolog, Drosophila)

 Gene al iases: SWAP

 Summary: It has been reported that splicing of fibronectin, CD45, etc., which is abnormal in cancer, is controlled by controlling splicing before gene transcription after gene transcription. It is suggested to be associated with cancer development.

Industrial applicability.

 The cancer treatment targeting DREF according to the present invention has a very strong suppression of cell proliferation, induction of cell death in M phase, and G2 / M phase without substantially affecting normal tissues. It has a very strong cell cycle arresting effect in cancer and is very effective for cancer regression and cell death. ,

 The present invention enables cancer treatment using DREF as a molecular target. That is, since the therapeutic agent of the present invention is very effective for cancer regression and cell death, it can be effectively used for the treatment of cancer expressing DREF.

All publications, patents and patent applications cited in this specification are used as is for reference. Incorporated herein.

Claims

The scope of the claims 1. A cancer therapeutic composition comprising an RNAi nucleic acid, an antisense nucleic acid or an antibody against DREF, which suppresses the in vivo function of a human cell growth regulator DREF or DREF gene in cancer cells or cancer tissues.  2. The composition according to claim 1, wherein the DREF comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence containing a deletion, substitution or addition of one or several amino acids in the amino acid sequence.  3. The composition according to claim 1, wherein the DREF gene comprises a nucleotide sequence represented by positions 202 to 2286 of SEQ ID NO: 2, or a nucleotide sequence having 95% or more identity with the nucleotide sequence.  4. The composition according to claim 1, wherein the suppression of in vivo function is cell growth suppression, cell cycle arrest and Z or cell death induction in cancer cells.  5. The composition according to claim 1, wherein the RNAi nucleic acid or antisense nucleic acid is capable of cleaving DREF mRNA or suppressing its function.  6. The composition according to claim 1, wherein the RNAi nucleic acid is siRNA or shRNA, DNA encoding the siRNA or shRNA, or a vector containing the DNA.  7. The composition according to claim 1, wherein the antisense nucleic acid is an antisense RNA, a DNA encoding the antisense RNA, and a vector containing the DNA. '  8. The composition according to claim 1, wherein the RNAi nucleic acid or antisense nucleic acid is derived from the nucleotide sequence of the DREF gene.  9. The composition according to claim 1, wherein the RNAi nucleic acid or the antisense nucleic acid is derived from a nucleotide sequence (SEQ ID NO: 3 and 4) encoding the CR2 domain or CR3 domain of DREF, respectively.  10. The composition according to claim 1, wherein the RNAi nucleic acid comprises a DNA sequence of any one of SEQ ID NOs: 5 to 15, 15, 22, 25 and 28, or an RNA sequence corresponding to the DNA sequence.  11. The composition according to claim 6, wherein the vector comprises the DNA sequence of any one of SEQ ID NOs: 20, 21, 23, 24, 26, 27, 29 and 30. 1 2. The antibody against DREF is the CR1 domain, CR2 domain or CR3 domain of DREF. The composition according to claim 1, which is an antibody that binds to the main (SEQ ID NO: 138, 133, or 134, respectively) or an epitope of a functional domain related to DNA binding of DREF in the CR1 or CR3 domain.  1 3. The composition according to claim 1 or 12, wherein the antibody against DREF is a human antibody, a humanized antibody or a fragment thereof.  , 1 4. Cancer treatment, including screening in vitro a drug that suppresses the expression or translation of the DREF gene by adding a candidate drug to a medium containing human cultured cancer cells or a mammalian cell line that strongly expresses DREF. A method of screening drugs.  15. The method according to claim 14, wherein cell growth inhibition, cell cycle arrest and / or cell death induction of the cell or cell line is used as an index.