JP2000236878A - Antisense nucleic acid compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new antisense nucleic acid compound which consists of an antisense nucleic acid compound containing a specific base sequence and inhibiting the expression of HST-1 (FGF-4), functions as one of fibroblast growth factors and is useful as a therapeutic drug for orchiocus, a diagnostic drug, a reagent for investigation, or the like. SOLUTION: This antisense nucleic acid compound is a new antisense nucleic acid compound, which contains the base sequence represented by formula I or II or the base sequence substantially homologous to these sequences and inhibits the expression of HST-1 (FGF-4), functions as one of fibroblast growth factors(FGF) and is useful as a reagent for investigation for the functional analysis, or the like, of HST-1 (FGF-4), a therapeutic drug for orchiocus, a diagnostic drug, or the like. This antisense nucleic acid compound is obtained by getting the base sequence of mRNA in human HST-1 from a data base, selecting an antisense nucleic acid containing the base sequence complementary to the domain difficult to form a double strand by a widely known method and synthesizing this sequence chemically by the use of a phosphoroamidate method, or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a human fibroblast growth factor, HST-1 (or FGF-
4), which are effective as therapeutic agents, diagnostic agents and research reagents. That is, the present invention belongs to the genetic technology.

[0002]

2. Description of the Related Art HST-1 is derived from mouse-derived NIH3T.
It was discovered as a transforming gene for transforming three cells (H. Sakamoto et al., Proc. Natl. Acad.
Sci. USA 83, 3997-4001 (1986)), and subsequently, the amino acid sequence was found to be homologous to fibroblast growth factor (FGF), also called FGF-4, and It is known that the gene is highly expressed in testis tumors in relation to diseases. On the other hand, as a therapeutic agent for testicular tumor, cisplatin (CDDP:
Although cis-diammine-dichloroplatimun (II)) and its related compounds are used, there are problems of side effects such as vomiting and nephrotoxicity, and it is desired to develop a more effective treatment method with less side effects.

[0003]

The present inventors have previously reported on the effects of antisense nucleic acids on mouse-derived HST-1 (T. Ochiya et al., J. Cell Bi.
ology 130, 997-1003 (1995))
If an effective antisense nucleic acid against human HST-1 was found and HST-1 expression could be inhibited thereby, it would lead to the development of a therapeutic agent for testicular tumor.
Research was conducted for the purpose of finding an antisense nucleobase sequence that inhibits T-1 production.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies based on the above-mentioned problems, and examined various antisense nucleic acids for the gene encoding HST-1, and as a result, A nucleic acid compound having a nucleotide sequence complementary to a gene and efficiently inhibiting the production of the protein, that is, an effective antisense nucleic acid compound has been found, and its pharmacological effect has been confirmed in cultured cell systems and experimental animal systems. Was completed. That is, the present invention provides an antisense nucleic acid having the nucleotide sequence represented by SEQ ID NO: 1 or 2 or a nucleotide sequence substantially identical thereto, wherein the antisense nucleic acid is characterized by inhibiting the expression of HST-1 (FGF-4). It concerns compounds.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In the antisense nucleic acid method, it is important to find an antisense nucleobase sequence that inhibits the expression of the gene of interest.
HST-1 which has a base sequence represented by SEQ ID NO: 1 or 2 which is a base sequence complementary to the base sequence of the gene encoding No. 1 or a base sequence substantially identical thereto. Has the effect of inhibiting the expression of Here, having the base sequence represented by SEQ ID NO: 1 or 2 or a base sequence substantially the same as them means 1) the base sequence itself represented by SEQ ID NO: 1 or 2; In the base sequence represented by 2, T is converted to U, 3) those having at least 70% or more homology with 1) or 2), preferably those having at least 80% or more homology, more preferably Has a homology of 90% or more, 4) a nucleotide sequence complementary to the nucleotide sequence of the gene encoding HST-1 is added to the nucleotide sequence of 1) to 3) within a range not exceeding about 30 nucleotides. And 5) the base sequence of 1) to 3) in which some bases are deleted from the base sequence and the number of bases is 1
It is 0 or more, preferably 14 or more, more preferably 17 or more. Here, the “gene encoding HST-1” includes a structural gene defining the amino acid sequence of HST-1, an intervening sequence (intron) existing in the middle of the structural gene, and a structure involved in the expression of the factor. Base sequence upstream of gene (promoter, operator, etc.)
Or a base sequence downstream of a structural gene (such as polyA). Further, the antisense nucleic acid compound of the present invention can be used for HST.
-1 significantly inhibits the expression of -1 compared to the control, and includes those having an inhibitory effect of not more than 80%, those having an inhibitory effect of not more than 60%, and the like due to differences in base sequence. The one with the larger effect is more preferable.
The inhibitory effects of these antisense nucleic acid compounds can be confirmed in cultured cell lines and experimental animal systems. Here, “inhibiting HST-1 expression” refers to HST-1
The protein is not produced, for example, by inhibiting the translation of mRNA encoding ST-1 into HST-1;
Or it means being suppressed.

[0006] The antisense nucleic acid compound of the present invention is designed based on the concept of the antisense nucleic acid method as described in detail below, prepared based on the concept, and its effects are determined based on the function of HST-1. It was obtained by evaluation. That is,
First, the nucleotide sequence information of the gene encoding HST-1 is obtained. Next, among nucleic acid compounds complementary to the partial base sequence of the gene, a nucleic acid compound expected to have an effect as an antisense nucleic acid is selected, and the selected nucleic acid compound is prepared by a synthetic chemical technique or the like. . In the cultured cell line expressing HST-1, it is tested whether the nucleic acid compound thus prepared efficiently inhibits the production of HST-1 and a nucleic acid compound containing a nucleotide sequence that inhibits cell growth Was selected as an antisense nucleic acid compound.
Further, the thus selected antisense nucleic acid compounds were subjected to experiments in experimental animal systems, and confirmed to suppress the growth of tumor cells. In the present invention, the effect was determined to be effective when the effect of the nucleic acid compound synthesized as described above was greater than the effect of the control nucleic acid compound. Here, the control nucleic acid compound refers to a nucleic acid of a sense strand (sense nucleic acid) corresponding to the antisense nucleic acid, or an antisense nucleic acid having another base sequence.

The antisense nucleic acid method (1) Selection of antisense nucleic acid The base sequence of HST-1 is obtained. This can be obtained as information described in a paper, as information stored in a gene database such as GenBank, or as a result of direct nucleotide sequence analysis. next,
Among the nucleic acid compounds complementary to the partial base sequence of the gene, a nucleic acid compound expected to have an effect as an antisense nucleic acid is selected. This method includes a method based on the prediction of the secondary structure of the mRNA encoding the protein of interest or its precursor (Nataka Kiyoshi, Naoki Sugimoto, Antisense 1
Vol., Pp. 6-11 (1997)), a method for searching for the possibility of forming a double chain and selecting from regions where a double chain is difficult to form (JP-A-10-52285; Uchida Kiyoshi, Sugimoto Naoki, Antis
ense 1, pp. 6-11 (1997); Kiyoshi Uchida, Chemistry and Industry 50 1758-1761 (1997); Kiyoshi Uchida, Toagosei Research Annual Report TREND First issue 52-56
Page (1998); Kiyoshi Uchida, Antisense Volume 2, pages 3-7 (1998)), and a method of selecting from near the start of translation (Kiyoshi Uchida, Naoki Sugimoto, Antisense 1, Vol. 6-11 (199)
7)) and the like. (2) Preparation of nucleic acid compound The nucleic acid compound includes natural oligodeoxyribonucleotides, phosphorothioate-type oligodeoxyribonucleotides, phosphorodithioate-type oligodeoxyribonucleotides, methylphosphonate-type oligodeoxyribonucleotides, and phosphoramidate-type oligodeoxyribonucleotides. Oligodeoxyribonucleotides, H-phosphonate-type oligodeoxyribonucleotides, triester-type oligodeoxyribonucleotides, α-anomeric oligodeoxyribonucleotides, oligoribonucleotides corresponding to each of the above-mentioned oligodeoxyribonucleotides, peptide nucleic acids, and other artificial Nucleic acids and nucleic acid-modifying compounds can be mentioned. Natural-type and phosphorothioate-type oligodeoxyribonucleotides are represented by mR NA
Is a preferred nucleic acid compound in that it becomes a substrate for RNase H when it forms a double chain by binding to, and has little nonspecific expression inhibition and easy synthesis. The synthesis of a natural nucleic acid compound is performed, for example, by using ABI (Applied Biosystems In
c.) 381A DNA synthesizer or 394 DNA / RNA
Using a synthesizer, the phosphoramidite method (ABI's procedure, edited by F. Eckstein, Oligonucleotides and Ana
logues: A Practical Approach, see IRL Press, 1991). The phosphoramidite method is a method in which a modified deoxyribonucleoside or a modified ribonucleoside is bound to a phosphoramidite protected with a cyanoethyl group or the like at the 3 ′ end of the modified deoxyribonucleoside, another modified deoxyribonucleoside, a modified ribonucleoside, an oligo-modified deoxyribonucleotide. This is a method for synthesizing nucleic acid-related compounds such as oligodeoxyribonucleotides and oligoribonucleotides, which is based on condensation with the 5 ′ end of oligo-modified ribonucleotides. Then, in the final cycle of the synthesis, the synthesis is terminated in a state where the protecting group (such as a dimethoxytrityl group) for the sugar hydroxyl group at the 5 ′ end is bonded. After the oligomer synthesized at room temperature is cleaved from the support, the base portion and the phosphate portion are deprotected. Thus, a crude product of a natural oligonucleic acid is obtained. Phosphorothioate type nucleic acid compounds are also A
It can be synthesized by a phosphoramidite method using a synthesizer manufactured by BI. The processing after the final cycle of the synthesis is the same as in the case of the natural type described above. The resulting crude product of the nucleic acid compound can be purified by a conventional purification method, for example, ethanol precipitation, or reversed-phase chromatography, ion-exchange chromatography, and high-performance liquid chromatography based on gel filtration chromatography. (HP
LC), various chromatography such as supercritical chromatography, and further, electrophoresis can be used for purification. In addition, cartridges manufactured based on the principle of reversed-phase chromatography [for example, Seppak Plus (filled with tC18) (Longbody / EN
V); Waters]. Phosphorothioate-type nucleic acid compound (20-mer, about 3 mg in crude product)
Is the same as in the case of the above-mentioned natural type. The purity of the natural type and phosphorothioate type nucleic acid compounds is as follows:
It can be examined by HPLC analysis or capillary electrophoresis. The synthesized nucleic acid compound is used in an evaluation experiment in a cultured cell system or an experimental animal system described below. (3) Evaluation Experiment in Cultured Cell System An evaluation experiment of a nucleic acid compound can be performed using a culture cell system. That is, using an antisense nucleic acid compound such as a phosphorothioate type oligodeoxyribonucleotide or a natural type oligodeoxyribonucleotide having a base sequence expected to have an antisense nucleic acid effect,
The effect on proliferation of cells expressing ST-1 can be examined in cultured cell lines. That is, antisense nucleic acid compounds such as phosphorothioate-type oligodeoxyribonucleotides or natural-type oligodeoxyribonucleotides 0.01 to 10 which express HST-1 in a human-derived cell under sterile conditions.
0 μM, preferably 0.1 to 10 μM, as required, DMRIE-C reagent, lipofectin reagent, lipofectamine reagent, DOTAP reagent, Tfx reagent, artificially synthesized lipid vesicle, liposome, membrane fusion reagent, polymer micellization reagent ,
It is added together with a polymer carrier or other intracellular transfection reagent, and its effect is determined from the degree of cell proliferation. Alternatively, the expression of the target protein may be inhibited by ELISA or Western blotting using an antibody against HST-1, or by encoding HST-1.
By examining mRNA by RT-PCR (reverse transcriptase-polymerase chain reaction) or the like, the protein expression inhibitory effect of the used antisense nucleic acid compound can be confirmed. (4) Evaluation experiment in experimental animal system The evaluation experiment of the nucleic acid compound can also be performed using an experimental animal system. That is, an antisense nucleic acid compound such as a phosphorothioate type oligodeoxyribonucleotide or a natural type oligodeoxyribonucleotide having a nucleotide sequence expected to have an antisense nucleic acid effect is used together with an appropriate introduction reagent as necessary, and described in (3). The cells are transplanted into the testis of a nude mouse or the like, and whether or not the growth of cancer cells is suppressed is observed. As another method, HST-
An appropriate cancer cell expressing 1 is transplanted, and then an antisense nucleic acid and a control nucleic acid are administered at an appropriate time together with an appropriate transfection reagent as needed, and the effect on cancer cell proliferation is observed.

[0008]

[Example 1]

(1) Selection of Antisense Nucleic Acid First, the nucleotide sequence of human HST-1 mRNA was obtained from the public gene database, GenBank. next,
The possibility of forming a double strand was searched, and an antisense nucleic acid having a complementary base sequence was selected for a region where a double strand was difficult to form using the method proposed previously (JP-A-10-5).
2285; Uchida Kiyoshi, Sugimoto Naoki, Antisense Volume 1, 6-
11 (1997); Uchida, Chemistry and Industry 50 17
58-1761 (1997); Toagosei Research Annual Report T
REND first issue 52-56 (1998);
Antisense 2: 3-7 (1998)). That is, as a result of searching for the nucleotide sequence represented by SEQ ID NO: 5 with respect to human HST-1, the following four types of nucleic acid compounds have a complementary nucleotide sequence to a region where a double strand is difficult to form. Then, it was selected as a candidate for an antisense nucleic acid compound. <Nucleic acid compound name><Basesequence><SEQID> A4196T CTCGTAGGCGTTGTAGTTGT (SEQ ID NO: 1) A5773V CTGGTAGCAAGCTAAGGTCATT (SEQ ID NO: 2) A4206V GTACTTGTAGGACTCGTAGGCG (SEQ ID NO: 3) A5990V CAAGTGTCTTCTTTCTTCCAGG (SEQ ID NO: 4)

(2) Preparation of Nucleic Acid Compound A phosphorothioate nucleic acid compound corresponding to the above sequence was prepared by the phosphoramidite method.

(3) Evaluation experiment using a cultured cell line NEC-8 cells derived from human testicular tumor were cultured at 4 × / well.
Spread on a 96-well plate so that it has 103 cells
The cells were cultured at 7 ° C. under 5% CO ₂ for 4 hours (Dulbecco's modified Eagle medium (DMEM) containing 10% FCS). After removing the medium, 0.2-1.0 μM antisense nucleic acid (or nucleic acid of the sense sequence as a control) and 0.2 μl of D
Medium containing MRIE-C (Life Technologies) (10
100 μl of (DMEM containing% FCS) was added to each well. After this was cultured for 4 days at 37 ° C. in 5% CO ₂ , 10 μl of TetraColor ONE (Seikagaku Corporation) was added to each well, and the cells were cultured at 37 ° C. in 5% CO ₂ for 2 hours. Then, the absorbance at 450 nm and 630 nm (control) of each well of the 96-well plate was measured using a microplate reader. Assuming that the absorbance at 450 nm based on the absorbance at 630 nm was proportional to the number of living cells, the effects of the antisense nucleic acid and its control nucleic acid on cell proliferation were evaluated. As a result, A4196
The most significant inhibition of cell growth was observed when T was used (44% when the antisense nucleic acid concentration was 0.2 μM).
% Inhibition rate), and when A5773V was used, a cell growth inhibitory effect was observed (antisense nucleic acid concentration was 0.1%).
30% inhibition rate when 2 μM). These effects are concentration-dependent (85% for A4196T and 6% for A5773V when 1 μM antisense nucleic acid is used).
8%), and the maximum inhibitory effect was 11% for the nucleic acid of the sense sequence as a control even when 1 μM was added. A4196T and its control sense sequence, A419
FIG. 1 shows the result of examining the concentration dependency of 6T-S (sense). In addition, as the cells used, in addition to the above, NEC-14 cells producing HST-1 (derived from human testis tumor) and NCC-IT cells (human Embryona
l Carcinoma (testis tumor)), the same results as described above were obtained (FIG. 2). However, cells that do not produce HST-1 (HeLa: derived from human cervical carcinoma; Hep
G2: from primary hepatoblastoma hepatoblastoma; N
When IH3T3: NIH / Swiss mouse fetus was used, 0.2 μM of A4196T was added (DMRIE-C
(In the presence of co-presence), 1
0% or less. From this fact, the growth inhibitory effect of A4196T can be understood as the effect of the antisense nucleic acid on HST-1 (FIG. 3).

(4) Evaluation experiment using an experimental animal system NEC-8 cells (1 × 105 cells) were transferred to nude mice (B
alb / c nu / nu) and transplanted to the testis three to four weeks later.
A% tumor-forming system was established. Using this system, the effect of the antisense nucleic acid was examined as follows. As described in (3), the antisense nucleic acid (A41) was added to NEC-8 cells grown to sub-confluence.
96T) or a nucleic acid (A4
196T-S (sense)) (each 0.2 μM) was added to DM
It was allowed to act for 4 hours in the presence of RIE-C (37 ° C., 5% C
O ₂ ). Thereafter, the cells were collected, and 1 × 10 5 cells were administered to one side of testes of 8-week-old nude mice. as a result,
As shown in FIG. 4, NEC treated with antisense nucleic acid
The growth rate of cancer when -8 cells were transplanted was clearly suppressed as compared to the growth rate of NEC-8 cells treated with the control nucleic acid and the cells not treated. Metastases to the lymph nodes of the renal hilum were 6/10 in the control group (no nucleic acid administration group) and 7
/ 10 (sense nucleic acid administration group), whereas no metastasis was observed in the antisense nucleic acid administration group (0/10, Table 1).

[0012]

[Table 1]

Next, the antisense nucleic acid was administered after the tumor was formed, and the effect was examined. That is, untreated NEC-8 cells were transplanted into nude mouse testes in the same manner as described above (1 × 10 5 cells / unilateral testis), and 10 days after tumor formation was observed, the antisense nucleic acid or its control was Was injected once into the testis tumor site (0.2 μl).
M (in the presence of DMRIE-C). as a result,
When the antisense nucleic acid is administered, the following 10 days
Growth of tumors in the testes and distant metastasis to the hilar lymph nodes were suppressed (FIG. 5). Furthermore, suppression of distant metastasis to the renal hilar lymph nodes was also observed 30 days after tumor formation was observed (20 days after antisense nucleic acid administration) (Table 2). That is, metastases to the lymph nodes of the renal hilum were 5/6 (non-nucleic acid-administered group) and 4/6 (sense-nucleic acid-administered group) in the control group, whereas 1/6 in the antisense nucleic acid-administered group. / 6.

[0014]

[Table 2]

Further, the antisense nucleic acid was continuously administered three times after the tumor was formed, and the effect was examined. That is, untreated NEC-8 cells were transplanted into nude mouse testes in the same manner as described above (1 × 10 5 cells / unilateral testis), and 10, 15, and 20 days after tumor formation began to be observed, The sense nucleic acid or its control nucleic acid was injected into the testicular tumor three times in total (each time, 50 μl of 0.2 μM (in the presence of DMRIE-C)). As a result, 30 days after tumor formation was observed (20 days after antisense nucleic acid administration), not only distant metastasis to lungs and hilar lymph nodes (Table 3), but also tumor growth was suppressed (FIG. 6). ).

[0016]

[Table 3]

[0017]

According to the present invention, HST-1 (FGF-
An antisense nucleic acid compound that inhibits the expression of a gene encoding 4) is provided. Since the antisense nucleic acid compound of the present invention inhibits the production of HST-1 (FGF-4), it is useful for functional analysis of HST-1 (FGF-4). Therefore, it can be used for research as an inhibitory reagent for gene function analysis. In addition, HST-1 (FGF-
Since 4) is overexpressed in tumors such as testicular tumors, the antisense nucleic acid compound of the present invention can be used as a therapeutic agent for testicular tumors, for example. Furthermore, by using the antisense nucleic acid compound of the present invention, it can be developed as a diagnostic agent targeting tumors and morphogenesis.

[0018]

[Sequence list] SEQ ID NO: 1 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Antisense nucleic acid Sequence: CTCGTAGGCG TTGTAGTTGT (SEQ ID NO: 1) SEQ ID NO: 2 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Antisense nucleic acid Sequence: CTGGTAGCAA GCTAAGGTCA TT SEQ ID NO: 3 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Antisense nucleic acid Sequence: GTACTTGTAG GACTCGTAGG CG SEQ ID NO: 4 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Straight Linear Sequence type: Antisense nucleic acid Sequence: CAAGTGTCTT CTTTCTTCCA GG SEQ ID NO: 5 Sequence length: 6616 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic DNA Sequence: GGATCCAGAA GGCATCCCCG A GTGGCTACT CCAATGGAGT GGCTTCTCCA TTCAGGCAAA CCTGAATGGG ATAAGTCATT GGCAGGAAGA TCTGGGGCCG GGGGTCATCC AGTGGGAAGG GGAGAGATGA CGCGGTCAGC ATGGCGGGAA CACAGGAGCA GAAAGGAAGC AGGTGGGAAG CCAGGTCAAG GGCCAGGGGC ACGGAAAGGG GTCAGATGCA GATAAGTGAG TGCTTCCTGG TGCATCCTTC ATCCGCAATT CATCCTTACC TGTGCTTTTG TTGCCTCCAT TGCACAGCTG AGGAGGCCAG GGCCTGCGGA GGTTGAGAGT GTGCTCAGGG AGCCCCCGGA GCAAAGTGGA AGCCAGATTC CAGATCAGTT CTGCTGGGAA TTCCCAGCTC CCAAAAGCCC TGCTGGCTGT CAGTCCCCAG TCACCACAAG CACCTATCCT GTGTGGGTGG GCCTGCAGTT CTGGGAGATA TATCAGCTGC CTGCAGCGTC CTTTGCTGAA CTCACAGCAA ATAGGAGAGA CAGGGAGGGG TCCTTGGGAA GCCCTAAATT GAGCTTGCTG TGGGAGTCCT GGGAAGAAAG GAGCCTCATC CTATCAAAAG CCGGGGGGAA GACATCAGAG TCCCTCTGCT CAGGTCAGCT GGCACAGGTG GGTCTCCAGG CCTGGGTCTC ACTTCCCCAG AGGGTGTGTT CGGGTGGCCC CAGGCTGAGG GAGGAAAGCC CACCTCCCAT GTCATTTTGC AAATGGGGAG TCAGGGACCT AGAGATGGAA AGACAACACA GCAAGTGAGG GATGGGTTCT AGGTCCCCTG CACCCTGCAC CCTGCACCCT GGCCAACGAT GTCTATTTGG CACCAGATCT GCAGGCTCAT CTGGGGGACC CCAGGACCCA GAGGCAGCCG GGTTGCATCT CGAAGCTGTG AGCTGCAGCC CAGGAAGGTC CAGGTCTGGG TGGCGCTGCC CAAGCAGGCT GCAGGCCCAA GGAGGAACAA AGATCCTCTC AAGGGGTGCG GAGCTGAGGT TCCGGTCCTG CCAAAGCCAC TTGATGACCC CCAAGTGCCC CCCTTTCTGC ACCTCAGAGA AGAGCCCTCA AGCCTCCCAG GTCCCCTCCA GGGGCACGAA TAAGCCCCAG CAGGGTTCTG AAGGGGTCCC AGGAATCTCC CTGTGGGGAT GCGGTGGAGG TGGAGGAGGC TGCGGTGGCC TGGGGACATC TCTGGTCACA GGTGCTGGTG GTATGAGAGA TGGGGTAGGC ACCAAGCCCC CTGCAGCTGT GGCTAGGCGG GCCTGCAGGA AGGGCCAGGC AGGCTCCTCA GGGACCACAA AGAACAGGGG TTTTCACACC TAGGTGGGCC TGCATCTAGC TAGGCCAGTC CCCATCAGGC CATAATGGGC ACAGTGGGAG GTAGAACCAT GAGTGAGAGA GGGGAGGCTT CCAGAGGCCT GGCCTGGGTC CCTGCTAGAT TGAGGGCTCT GGCTATGGTA CATGGATATT TCTGCTGTGG AATCAAAGGA GCAGGGGATG CTGAATATCC CCTCTGGCCC TATGCCCTGC TACCTGTCCT TTCACGGAAG GGTGTGTGTG TAGGGGGTGC AGGACCAGGC CTCCCTGGGT GCATCTCTGC CACCTTGCCC TTTGGCTCAG GTGGACCTCC ACCAGGTATT CAGAACTCCA GCCCAGAAAC GCGCCAAGCC TGTGGGGCCA AGACCTAGGG GGTGGGGGTG GCCTCCCTCC CGCCTGTAGC CAAAGGGTCC TCCCTTGCCC AGCCAGGCCC CGGTGTCGCT TACTGCTCTT ATCCACCCCT CCTTCCCAGG CCGGTCCTCA AGGCCCCAGC AAAGGAACCA AGTTCCCGTG AGCCTCCGAA AGGCGAAGGG CAGGCAGCAG CCGCTGGCTT CTGCGCCCAC TAGGAGCTTC GGATGCCCGA GTTAGGGCTG CGCCAAGGCG GCCGGAGCAG AGAGGGAGAC GGGGACGGGG ACAGGCAGGG ACAAAGTGCA AGAGGCAAAA CTGGCTGAAA AGCAGAAGTG TAGGAGCCGC CAAGGGGCGG GACGAACAGG TCCGTGGGCC GGGCGGAGCC AAGGGTGGGG GCCGGGGTCC CTCCAGGTGG CACTCGCGGC GCTAGTCCCC AGCCTCCTCC CTTCCCCCGG CCCTGATTGG CAGGCGGCCT GCGACCAGCC GCGAACGCCA CAGCGCCCCG GGCGCCCAGG AGAACGCGAA CGGCCCCCCG CGGGAGCGGG CGAGTAGGAG GGGGCGCCGG GCTATATATA TAGCGGCTCG GCCTCGGGCG GGCCTGGCGC TCAGGGAGGC GCGCACTGCT CCTCAGAGTC CCAGCTCCAG CCGCGCGCTT TCCGCCCGGC TCGCCGCTCC ATGCAGCCGG GGTAGAGCCC GGCGCCCGGG GGCCCCGTCG CTTGCCTCCC GCACCTCCTC GGTTGCGCAC TCCCGCCCGA GGTCGGCCGT GCGCTCCCGC GGGCCGCCAC AGGCGCAGCT CTGCCCCCCA GCTTCCCGGG CGCACTGACC GCCTGACCGA CGCACGGCCC TCGGGCCGGG ATGTCGGGGC CCGGGACGGC CGCGGTAGCG CTGCTCCCGG CGGTCCTGCT GGCCTTGCTG GCGCCCTGGG CGGGCCGAGG GGGCGCCGCC GCACCCACTG CACCCAACGG CACGCTGGAG GCCGAGCTGG AGCGCCGCTG GGAGAGCCTG GTGGCGCTCT CGTTGGCGCG CCTGCCGGTG GCAGCGCAGC CCAAGGAGG C GGCCGTCCAG AGCGGCGCCG GCGACTACCT GCTGGGCATC AAGCGGCTGC GGCGGCTCTA CTGCAACGTG GGCATCGGCT TCCACCTCCA GGCGCTCCCC GACGGCCGCA TCGGCGGCGC GCACGCGGAC ACCCGCGACA GTGAGTGGCG CGGCCAGGCG CGAAGGGGCG GGGGCGGGGG GCAACGGCCG CCGGGCCAAC CCGCTCAGTC ACACTCTGAG ACCCTCGGCG GGCACCTGCT CGGGGGCCCC GGGAACCGGG GCGGACTCGG GCTCCGGTCC CTTCTGACGC GGGGCTGGGG ACGCAGACAC TCTTGGCTCC GGCAGCCCAG CGCAACCCCT GAGGTCGGGC GCCGCCTCCC GCCTTCAGAA ACTCGGGCTC CGAGCGCCGA ATTCCAGCGC CTTCGCCCGT GGGCACAGGG CGCGCGGTGC AGCCACAGGG GGCCCGAGAC ACGCGCCCCG GCCTGGCCCA GGCTGGGGAA CCGCTGGGGT CGGGCTCGCG TCTGAAGGTC CGGGACTGGG TGCGGCCGCC GGGGGTCCCC TACACAGGCA AGCTAATCTG AGCTAGCGCA GGCTTGGGCT CCGGAGGCCC TAGAGGGCAG CTTGGGCTCT GGAGGCCCTT GGGGGCGGCT GCGCCGGGAA CCCTGGCCCT TTATCCCCAA CCCCACCCCA GAAATAGGGT CCCCGGAGGC GAACAAGCCG AGGGGCGGAG TGGGCCAGGG ATCACCTGCC CCGCAATGAC CTGCGCCCCG CCCCCAGGCC TGCTGGAGCT CTCGCCCGTG GAGCGGGGCG TGGTGAGCAT CTTCGGCGTG GCCAGCCGGT TCTTCGTGGC CATGAGCAGC AAGGGCAAGC TCTATGGCTC GGTGAGTACC GCAGGGGTCT GGCTAGGCAC CTAGTTGGGA ACAGCGGA CA TGGCTAGCAG GCTCGTGGCT TCTCCAGCCC CACCTGTGCC TGGGTCTTGG AGGGGTGGCA GGGTCACCAG GTCACGGGAC CGGCAGGCCT CCCCAGACAA AGGAAGCAGC CCCAAGGCAG GAACAATGAG GTTCCTGCCA TCCCTGAGTG GGCCCCTCCC AGACCGAGGA AAGGGCGCTA TTGAGAGCCC TTCCCTTCTC TAGTCCAGAG GGGTAGGTCT CAGTGTTGGA ACTGCGGGCT TGAGGCTGGA CACGCAGGGA ATGAATTCTC TGGCTGCTAG GTGCAGGGCA GGTGGTGAGA GCACCAGCTG TTGTGGGCTG GCCATGTCCC CTTCTCACCC TGTGTGGGTC TTGACACCTT AACTGCTCAG CAGAGACATC TCAGCCCAGG GTGGGGGGTG GGACAGAAGG GGGTTCTGAC CCCTGGCTTC AGGCTGGGTA CCTTGCCCAA GAGGTGCCCC AGCCCTGACA CTGCCCTGCT TTGCTGCAGC CCTTCTTCAC CGATGAGTGC ACGTTCAAGG AGATTCTCCT TCCCAACAAC TACAACGCCT ACGAGTCCTA CAAGTACCCC GGCATGTTCA TCGCCCTGAG CAAGAATGGG AAGACCAAGA AGGGGAACCG AGTGTCGCCC ACCATGAAGG TCACCCACTT CCTCCCCAGG CTGTGACCCT CCAGAGGACC CTTGCCTCAG CCTCGGGAAG CCCCTGGGAG GGCAGTGCCG AGGGTCACCT TGGTGCACTT TCTTCGGATG AAGAGTTTAA TGCAAGAGTA GGTGTAAGAT ATTTAAATTA ATTATTTAAA TGTGTATATA TTGCCACCAA ATTATTTATA GTTCTGCGGG TGTGTTTTTT AATTTTCTGG GGGGAAAAAA AGACAAAACA AAAAACCAAC TCTGACTTTT CTGGTGC AAC AGTGGAGAAT CTTACCATTG GATTTCTTTA ACTTGTCAAA AGTTGTCACG AGTGTGCTGC TATTCTGTGT TTTAAAAAAA GGTGACATTG GATTCCGATG TCATCCCCTG TAGTATGGCG TGGAGCATCT CTGTCTGGAA AGGCCCGCCT GAGGCTTGGG CAGCCAGTTC AGGGAGCTCC CAGGCTTGGC TCTCGGCTAG CATCCTCAGA GGCCCACTCC CTTTGTGCCC TGTTGCTATT AATCGGGACA TATCGGTTTA CTTCGGGTAC AGAAAGTGCG GTGTTGAAGT CCTCGCTGCC ACTCTGTTTT TAGATCTGCC AAGACTGACC TTTGAACTTT CCTGTAGTCA ATCTTCCTCG ATCTACCAGA TGGGAGAGAC CCTTGGACAA CTTTATAAAC TCCTGTTTGC CTTTTTTGGA TCAGCGACAG CCCCCATCGC TGTGACTATT GGGGAAAAGA CGAAGCTCTT TCATAAATTC CATGGAGAGG AATCAATATC CCACTGGAAG GCTAGAAATG GACAAGATAG TGTATTTGCA ATCACAAACA AAACCCTAGT GATGAAAAAT AATTTGTGAT GGCAGATGCT TCTGATGGTG TGATAGAATA TGTTTTTGAA AACAAACCAT CGAACCCCCC GCCCCACCCC CAAAACGGGC TTCCCTGTGT TTAGGGAGCT TTGGGCTAGA ACTAGCTACG ATTTTTAGGT GAAATGTCCT TGTAATTGTA CAAAGCACTT GGTGCAGTGT TTGCGTGGAG CAGCCTGCTG CTTTCTGATG CATTCCCTGT TTAAGTGCGT TTAACATCTA CCTCACAAGC CCTGAAACCC CAGGCAAAAC CCACAGAAAG CTCATACCCG GTGCAGGAGT TTGCCATCCC AAGTGGCTTT TTTTCCATAT GTAGCC AAAA AGGATTGCAG ATAGCGTCGG TGCGTCCCAT TCGAACCTTG TCACGTTTGA GCTATCTTTA CCCTGTGATT TACTTTTAGT AAGGGTGATC ATGGTGAAAA TATTTGCAGA CAGCTGTTAC AGTACACTAT ATGGTCACCA AGTAACCTTA TATTTTTCTT TATATATTTT ACAAATGTAA CCCCTGTCAT TGAAGCAACC GTGGAAGAGG CAGGGTCGGT GATGTTTAAA AAAAGTTCCG AGGTGATGGC AAACATTTAA TTTTAATGAA TGACTTTTTA GAGTTTATAC AAAATGACCT TAGCTTGCTA CCAGAAATGC TCCGAATGTT TCGTCAAGAC TTTAATACTC TCCTAGGATG TTTCTGAACT GTCTCCCGAA TTAACTTTAT GGGAGTCTAC AGACAGCAAG ACTGGAAAAT CTGATTGGAG TTTTTGTCTT TCACATTCCT TTTGAAAACT CTTTGTTCGA ATGCAAATCA TCGACTTAAA ATACTATTCT TAACCAAGGC CTGGAAGAAA GAAGACACTT GCAAAGCCGC TAAGACAGGA CCACACATCT TAAACTGCTG TTCCTACCAT GCACTAAACT GTTTTTAAGT TTTAAACCAC ACCCTAGGCT CCAGGAGTGT TCAGGAAAGA TGGTGTTTGT AGGTCTCCAT GCTGTTTGGC GTTGGGGGGT GTGGAGGGAT CATCCGTCGA CTTTCTGAAT TTTAATGTAT TCACTTAGTA ACAAACCATG ATTGTCTTAA ATGCCTTAAA TTATTATGAG ATTTCTTGTC TCAGAGCCCA ATCAGATTGT CAGGAATTAA CATGTGTTAG GTTTGATCAC CCTTGACCAC TTCTTATAGA TATTTCTTCA ACAAATCATG TGTGATGCCT GTAGGAACAC AACTGTACCT TTAAA ATATT GTTTTCATAT TGCTGTGATG GGGATTCGAG GTTCCTGTAT GTGCCACTGT TTTCAGAATC TGTAGTTTTA TACAGGTGCC GACCCTCGTT GTGATGTATG TGCTGTGCAC ATTGACATGC TGACCGACAA TGATAAGCGT TTATCGTGTATAAAAAGACAGACGATCAGGAGATACGAAGATCAATCGAA

[Brief description of the drawings]

FIG. 1 is a graph showing the concentration dependence of antisense nucleic acid (A4196T) on NEC-8 cell proliferation.

FIG. 2. Antisense nucleic acid (A4196T)
HST-1 (FGF-4) expressing cells (NEC-8, NE
It is a figure which shows the growth suppression effect of C-14 and NCC-IT).

FIG. 3: H by antisense nucleic acid (A4196T)
ST-1 (FGF-4) expressing cells (NEC-8) and HST-1 (FGF-4) non-expressing cells (HeLa, He
It is a figure which shows the growth suppression effect of pG2 and NIH3T3).

FIG. 4 is a view showing the effect of transplanting NEC-8 cells treated with an antisense nucleic acid (A4196T) into testes of nude mice.

FIG. 5 shows transplantation of NEC-8 cells into testes of nude mice,
It is a figure which looked at the effect which administered antisense nucleic acid (A4196T) 10 days after that.

FIG. 6 shows transplantation of NEC-8 cells into testes of nude mice,
10 is a view showing the effect of continuously administering the antisense nucleic acid (A4196T) three times every five days from 10 days later.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hiromi Sakamoto 2-2-24 Minamihanaen, Hanamigawa-ku, Chiba-shi, Chiba (72) Inventor Takashi Sugimura 3-13-7, Kichijoji-Higashicho, Musashino-shi, Tokyo F-term (reference) 4B024 AA01 AA12 BA80 CA20 HA17 4C084 AA13 NA06 ZB262 4C086 AA03 EA16 NA06 ZB26

Claims (1)

[Claims] 1. An HST having a nucleotide sequence represented by SEQ ID NO: 1 or 2 or a nucleotide sequence substantially identical thereto.
An antisense nucleic acid compound which inhibits expression of -1 (FGF-4).