JP5976922B2 - Double-stranded nucleic acid molecule, DNA, vector, cancer cell growth inhibitor, and medicine - Google Patents

Description

  The present invention provides at least one of a double-stranded nucleic acid molecule, a DNA and vector containing the double-stranded nucleic acid molecule, the double-stranded nucleic acid molecule, DNA, and a vector that can be suitably used for prevention or treatment of cancer. The present invention relates to a cancer cell growth inhibitor and a medicament containing the cancer cell growth inhibitor.

Among cancers, prostate cancer is known to be the most common cancer affecting men in the West. In Japan, the number of patients with prostate cancer has increased dramatically with the westernization of eating habits and the aging of the population.
As therapeutic techniques, surgical treatment including prostatectomy, chemotherapy with anticancer agents, and radiation therapy are widely applied clinically. In the above-mentioned treatment, surgical treatment is the first choice. However, when it is difficult to operate due to recurrence after surgery or when cancer has already been diagnosed, there is a treatment other than surgical treatment. Selected.
In general, prostate cancer growth is stimulated by androgens. Therefore, hormonal therapy that inhibits the production and function of androgen is often performed as a therapeutic method other than the surgical treatment. Although the effect of the hormone therapy is very good, there is a problem that it relapses as androgen-independent prostate cancer within several years from the start of treatment. Therefore, control of androgen-independent prostate cancer is the most important issue.

Although the detailed molecular mechanism of progression from androgen-dependent cancer to independent cancer is not yet clear, involvement of an androgen receptor (hereinafter sometimes referred to as “AR”) has been suggested. That is, it has been suggested that androgen-independent cancers are sensitive to ultra-low concentrations of androgens, antiandrogens, other steroid hormones, and the like due to AR mutation or amplification.
In relapsed prostate cancer, treatment of prostate cancer with AR antagonists is difficult or ineffective. Therefore, methods for inhibiting the binding of ligands to drugs by AR or suppressing the expression of AR using RNA interference (RNAi) technology have been studied. However, there is a limit to just targeting AR, and clinically sufficient ones have not yet been developed, and development of a method for suppressing AR more efficiently is demanded.

  To date, the transforming acid coiled-coil protein 2 (TACC2) gene, which is an androgen-responsive gene, functions as a downstream gene of AR and is a factor involved in postoperative recurrence and prognosis of prostate cancer It is shown. Specifically, the TACC2 gene is involved in microtubule stability and positively regulates the cell cycle in the mitosis phase of the cell cycle. As a result, the TACC2 gene is involved in hormone-dependent proliferation of prostate cancer, and is highly expressed in cell models that have acquired AR hypersensitivity, and is involved in the acquisition of hormone-depleting proliferation ability. Has been suggested (see, for example, Non-Patent Document 1).

  Similarly, 14-3-3ζ (hereinafter sometimes referred to as “14-3-3 zeta”), which is an androgen-responsive gene, functions as a downstream gene of AR and is associated with the onset of prostate cancer. It has been shown to be a factor with increased expression. Specifically, the 14-3-3ζ gene has an anti-apoptotic effect in the cytoplasm, promotes cancer growth through activation of the PI3K-AKT pathway, and binds to AR in the nucleus. However, it has been shown to be involved in hormone-dependent proliferation of prostate cancer by positively controlling androgen signals (see, for example, Non-Patent Document 2).

  Under such circumstances, there is a strong demand for prompt development of an excellent treatment method capable of treating cancer that has been difficult or ineffective for treatment with an AR antagonist.

Takayama K, Horie-Inoue K, Suzuki T, Urano T, Ikeda K, Fujimura T, Takahashi S, Homa Y, Ouchi Y, Inoue S. , TACC2 is an androgen-responsible cell cycle regulator promoting androgen-mediated and casting-resistant growth of promote cancer. Mol Endocrinol. 2012 May; 26 (5): 748-61. Murata T, Takayama K, Urano T, Fujimura T, Ashikari D, Obinata D, Horie-Inoue K, Takahashi S, Ochi e Ro, Y-3 H in Prostate Cancer and Promotes Prostate Cancer Cell Proliferation and Survival, Clin Cancer Res October 15, 2012 18:20 5617-5627

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the present invention can effectively suppress the growth of cancer cells by targeting at least one of the TACC2 gene and the 14-3-3ζ gene and suppressing the expression of these genes. Double-stranded nucleic acid molecule which can be suitably used for prevention or treatment, DNA and vector containing the double-stranded nucleic acid molecule, cancer cell growth inhibitor containing at least one of the double-stranded nucleic acid molecule, DNA and vector Another object is to provide a medicament containing the cancer cell growth inhibitor.

Means for solving the problems are as follows. That is,
<1> A double-stranded nucleic acid molecule for suppressing the expression of at least one of the TACC2 gene and the 14-3-3ζ gene,
(A) a sense strand having a nucleotide sequence corresponding to any target sequence of SEQ ID NO: 1 to SEQ ID NO: 18;
(B) an antisense strand having a nucleotide sequence complementary to the sense strand of (a),
A double-stranded nucleic acid molecule characterized by comprising
<2> DNA comprising the nucleotide sequence encoding the double-stranded nucleic acid molecule according to <1>.
<3> A vector comprising the DNA according to <2>.
<4> A cancer cell growth inhibitor comprising at least one of the double-stranded nucleic acid molecule according to <1>, the DNA according to <2>, and the vector according to <3>. It is.
<5> A method for inhibiting the growth of cancer cells, comprising causing the cancer cell proliferation inhibitor according to <4> to act on cancer cells.
<6> A medicament for preventing or treating cancer, comprising the cancer cell proliferation inhibitor according to <4>.
<7> A method for preventing or treating cancer, comprising administering the medicine according to <6> to an individual.

  According to the present invention, various problems in the prior art can be solved, and by suppressing the expression of these genes targeting at least one of the TACC2 gene and the 14-3-3ζ gene, the proliferation of cancer cells is effective. Of double-stranded nucleic acid molecules, DNAs and vectors containing the double-stranded nucleic acid molecules, DNAs and vectors containing the double-stranded nucleic acid molecules that can be suitably suppressed and used for prevention or treatment of cancer It is possible to provide a cancer cell growth inhibitor containing at least one, and a medicament containing the cancer cell growth inhibitor.

FIG. 1A is a graph showing the results of Test Example 1-1-1. FIG. 1B is a graph showing the results of Test Example 1-1-2. FIG. 2 is a graph showing the results of Test Example 1-2. FIG. 3 is a diagram showing the results of Test Example 2-1. FIG. 4 is a diagram showing the results of Test Example 2-2. FIG. 5A is a graph showing the results of Test Example 3-1-1. FIG. 5B is a graph showing the results of Test Example 3-1-2. FIG. 6 is a graph showing the results of Test Example 3-2. FIG. 7A is a photograph showing the results of Test Example 4-1. FIG. 7B is a graph showing the results of Test Example 4-1. FIG. 8A is a photograph showing the results of Test Example 4-2. FIG. 8B is a graph showing the results of Test Example 4-2. FIG. 9 is a graph showing the results of Test Example 5-1. FIG. 10 is a graph showing the results of Test Example 5-2. FIG. 11 is a graph showing the results of Test Example 6-1. FIG. 12 is a graph showing the results of Test Example 6-2.

(Double-stranded nucleic acid molecule)
The double-stranded nucleic acid molecule of the present invention is a double-stranded nucleic acid molecule for suppressing the expression of at least one of the TACC2 gene and the 14-3-3ζ gene. (A) SEQ ID NO: 1 to SEQ ID NO: 18 A sense strand having a nucleotide sequence corresponding to any of the target sequences; and (b) an antisense strand having a nucleotide sequence complementary to the sense strand of (a).
In the present invention, the “double-stranded nucleic acid molecule” refers to a double-stranded nucleic acid molecule obtained by hybridizing a desired sense strand and an antisense strand.

<TACC2 gene, 14-3-3ζ gene>
The TACC2 gene and the 14-3-3ζ gene are both androgen responsive genes as described above.
The base sequence of the TACC2 gene can be easily obtained through a public database such as GenBank (NCBI). For example, the human TACC2 gene is NCBI accession number NM_006997.2.
The base sequence of the 14-3-3ζ gene gene can also be easily obtained through a public database such as GenBank (NCBI). For example, the human 14-3-3ζ gene is NCBI accession number NM_001135699.1. is there.
In the present invention, since the mRNA sequence of the TACC2 gene and the 14-3-3ζ gene is a target of the double-stranded nucleic acid molecule and its expression is suppressed by the double-stranded nucleic acid molecule, the present specification Among them, the TACC2 gene and the 14-3-3ζ gene are sometimes referred to as “target genes” of the double-stranded nucleic acid molecule.
For reference, the human TACC2 gene sequence is shown in SEQ ID NO: 19, and the human 14-3-3ζ gene sequence is shown in SEQ ID NO: 20.

<Sense strand, antisense strand>
As a result of intensive studies, the inventors of the present invention have a nucleotide sequence complementary to a specific target sequence (SEQ ID NO: 1 to SEQ ID NO: 18) among the mRNA sequences of the TACC2 gene and the 14-3-3ζ gene. The present inventors have found that a double-stranded nucleic acid molecule containing an antisense strand having a remarkably excellent expression suppressing effect on the TACC2 gene and the 14-3-3ζ gene. Therefore, the double-stranded nucleic acid molecule of the present invention comprises (a) a sense strand having a nucleotide sequence corresponding to any of the target sequences of SEQ ID NO: 1 to SEQ ID NO: 18, and (b) the sense of (a) And an antisense strand having a nucleotide sequence complementary to the strand.
Here, the sense strand and the antisense strand may be RNA strands or RNA-DNA chimeric strands. The sense strand and the antisense strand can hybridize with each other to form the double-stranded nucleic acid molecule.
Of the SEQ ID NO: 1 to SEQ ID NO: 18, SEQ ID NO: 1 to SEQ ID NO: 10 are part of the TACC2 gene sequence (SEQ ID NO: 19), and SEQ ID NO: 11 to SEQ ID NO: 18 are It is a part of human 14-3-3ζ gene sequence (SEQ ID NO: 20).

Among the double-stranded nucleic acid molecules, the sense strands are SEQ ID NO: 1 to SEQ ID NO: 4, SEQ ID NO: 7 to SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 18. It preferably has a nucleotide sequence corresponding to any target sequence, and more preferably has a nucleotide sequence corresponding to any target sequence of SEQ ID NO: 2 and SEQ ID NO: 13.
When the sense strand is a sense strand other than the preferred sense strand, the expression suppression effect on the target gene of the double-stranded nucleic acid molecule may be weakened. On the other hand, if the sense strand is a more preferable sense strand, it is advantageous in that a strong expression suppression effect on the target gene can be obtained even if the amount of the double-stranded nucleic acid molecule used is small. .
The sense strand in the double-stranded nucleic acid molecule only needs to have a nucleotide sequence corresponding to the target sequence of the predetermined sequence number, and may include other nucleotide sequences. It preferably consists of a nucleotide sequence corresponding to the numbered target sequence.
In addition, the antisense strand in the double-stranded nucleic acid molecule only needs to have a nucleotide sequence complementary to the extent that it can hybridize with the sense strand, and may contain other nucleotide sequences. The nucleotide sequence complementary to the sense strand is preferably 70% or more, more preferably 80% or more, and particularly preferably 90% or more.

<Type>
The type of the double-stranded nucleic acid molecule is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include double-stranded RNA (dsRNA) and double-stranded RNA-DNA chimera. Is mentioned. Among these, double-stranded RNA is preferable.
Here, “double-stranded RNA” refers to a double-stranded nucleic acid molecule in which both sense strand and antisense strand are composed of RNA sequences, and “double-stranded RNA-DNA chimera” refers to sense Both strands and antisense strands are double-stranded nucleic acid molecules composed of a chimeric sequence of RNA and DNA.

The double-stranded RNA is particularly preferably siRNA (small interfering RNA). Here, the siRNA is a small double-stranded RNA having a length of 18 to 29 bases, which cleaves the mRNA of the target gene having a sequence complementary to the antisense strand (guide strand) of the siRNA, It has a function of suppressing gene expression.
As long as the siRNA has a sense strand and an antisense strand as described above and can suppress the expression of the target gene, the terminal structure is not particularly limited, and may be appropriately selected according to the purpose. For example, the siRNA may have a blunt end or may have a protruding end (overhang). Among these, the siRNA preferably has a structure in which the 3 ′ end of each strand protrudes from 2 to 6 bases, and more preferably has a structure in which the 3 ′ end of each strand protrudes by 2 bases.
Specific examples of the siRNA include siRNA consisting of SEQ ID NO: 21 and SEQ ID NO: 22, siRNA consisting of SEQ ID NO: 23 and SEQ ID NO: 24, siRNA consisting of SEQ ID NO: 25 and SEQ ID NO: 26, SiRNA consisting of SEQ ID NO: 27 and SEQ ID NO: 28, siRNA consisting of SEQ ID NO: 29 and SEQ ID NO: 30, siRNA consisting of SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: SiRNA consisting of 34, siRNA consisting of SEQ ID NO: 35 and SEQ ID NO: 36, siRNA consisting of SEQ ID NO: 37 and SEQ ID NO: 38, siRNA consisting of SEQ ID NO: 39 and SEQ ID NO: 40, SEQ ID NO: : SiRNA consisting of 41 and SEQ ID NO: 42, siRNA consisting of SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: SiRNA consisting of 5 and SEQ ID NO: 46, siRNA consisting of SEQ ID NO: 47 and SEQ ID NO: 48, siRNA consisting of SEQ ID NO: 49 and SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52 And siRNA consisting of SEQ ID NO: 53 and SEQ ID NO: 54, and siRNA consisting of SEQ ID NO: 55 and SEQ ID NO: 56.
Among these, siRNA consisting of SEQ ID NO: 21 and SEQ ID NO: 22, siRNA consisting of SEQ ID NO: 23 and SEQ ID NO: 24, siRNA consisting of SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 SiRNA consisting of SEQ ID NO: 28, siRNA consisting of SEQ ID NO: 33 and SEQ ID NO: 34, siRNA consisting of SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38 siRNA, siRNA consisting of SEQ ID NO: 39 and SEQ ID NO: 40, siRNA consisting of SEQ ID NO: 41 and SEQ ID NO: 42, siRNA consisting of SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: 45 and sequence SiRNA consisting of SEQ ID NO: 46, siRNA consisting of SEQ ID NO: 49 and SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: The siRNA consisting of 2 and the siRNA consisting of SEQ ID NO: 55 and SEQ ID NO: 56 are preferred, the siRNA consisting of SEQ ID NO: 23 and SEQ ID NO: 24, and the siRNA consisting of SEQ ID NO: 45 and SEQ ID NO: 46 More preferred.

The double-stranded RNA may be shRNA (short hairpin RNA). Here, shRNA is a single-stranded RNA including a dsRNA region of about 18 to 29 bases and a loop region of about 3 to 9 bases. ShRNA is expressed in base pairs by being expressed in vivo. To form a hairpin-shaped double-stranded RNA. Thereafter, shRNA is cleaved by Dicer (RNase III enzyme) to become siRNA, which can function to suppress the expression of the target gene.
The terminal structure of the shRNA is not particularly limited as in the case of the siRNA, and can be appropriately selected according to the purpose. For example, the terminal structure may have a blunt end or a protruding end (overhang). It may be a thing.

The double-stranded RNA-DNA chimera is particularly preferably a chimeric siRNA. Here, the chimera siRNA refers to a small-molecule double-stranded RNA-DNA chimera having a length of 18 to 29 bases in which a part of the RNA sequence of the siRNA is converted to DNA. Among them, a small double-stranded RNA-DNA chimera having a length of 21 to 23 bases in which 8 bases on the 3 ′ side of the sense strand of siRNA and 6 bases on the 5 ′ side of the antisense strand are converted to DNA It is preferable that The chimeric siRNA has a function of suppressing the expression of a target gene, like the siRNA. The chimera siRNA includes a mode in which a part of the sequence converted to DNA is converted back to RNA.
The terminal structure of the chimeric siRNA is not particularly limited as in the case of the siRNA, and can be appropriately selected according to the purpose. For example, it may have a blunt end or a protruding end (overhang). You may have.
The chimera siRNA (double-stranded RNA-DNA chimera) is advantageous in that it has high blood stability, low immune response induction, and low production costs.

<Modification>
In addition, the double-stranded nucleic acid molecule may have an appropriate modification depending on the purpose. For example, for the purpose of imparting resistance to a nuclease and improving stability in a culture solution or in a living body, the double-stranded nucleic acid molecule is modified with 2′O-methylation or phosphorothioated. (S) modification, LNA (Locked Nucleic Acid) modification, and the like can be applied. For example, for the purpose of increasing the efficiency of introduction into cells, the 5 ′ end or 3 ′ end of the sense strand of the double-stranded nucleic acid molecule is modified with nanoparticles, cholesterol, a cell membrane-passing peptide, or the like. You can also. In addition, there is no restriction | limiting in particular as a method of giving such a modification to the said double stranded nucleic acid molecule, A conventionally well-known method can be utilized suitably.

<How to obtain>
There is no restriction | limiting in particular as an acquisition method of the said double stranded nucleic acid molecule, Each can be produced based on a conventionally well-known method.
For example, the siRNA is chemically synthesized using a conventional DNA / RNA automatic synthesizer or the like, each of which has a base length of 18 to 29 bases corresponding to a desired sense strand and antisense strand. However, it can be produced by annealing them. In addition, a commercial product of annealed double-stranded siRNA can be obtained, or can be obtained by requesting synthesis from a siRNA synthesis contractor. Moreover, by constructing a desired siRNA expression vector, such as the vector of the present invention described later, and introducing the expression vector into the cell, siRNA can also be produced using the intracellular reaction.
The chimeric siRNA can be prepared, for example, by chemically synthesizing a sense strand and an antisense strand that are chimeric nucleic acid molecules, and annealing them.

(DNA, vector)
The DNA of the present invention is a DNA comprising a nucleotide sequence encoding the above-described double-stranded nucleic acid molecule of the present invention, and the vector of the present invention is a vector comprising the DNA.

<DNA>
The DNA is not particularly limited as long as it includes a nucleotide sequence encoding the above-described double-stranded nucleic acid molecule of the present invention, and can be appropriately selected according to the purpose. A promoter sequence for controlling transcription of the double-stranded nucleic acid molecule is preferably linked upstream (5 ′ side) of the encoded nucleotide sequence. There is no restriction | limiting in particular as said promoter sequence, According to the objective, it can select suitably, For example, pol III type | system | group promoters, such as pol II type | system | group promoters, such as a CMV promoter, H1 promoter, U6 promoter, etc. are mentioned.
Furthermore, it is more preferable that a terminator sequence for terminating the transcription of the double-stranded nucleic acid molecule is linked downstream (3 ′ side) of the nucleotide sequence encoding the double-stranded nucleic acid molecule. There is no restriction | limiting in particular also as said terminator arrangement | sequence, According to the objective, it can select suitably.
A transcription unit including the promoter sequence, the nucleotide sequence encoding the double-stranded nucleic acid molecule, and the terminator sequence is a preferred embodiment of the DNA. The transfer unit can be constructed using a conventionally known method.

<Vector>
The vector is not particularly limited as long as it contains the DNA, and can be appropriately selected according to the purpose. Examples thereof include a plasmid vector and a virus vector. The vector is preferably an expression vector capable of expressing the double-stranded nucleic acid molecule.
There is no restriction | limiting in particular as an expression mode of the said double stranded nucleic acid molecule, According to the objective, it can select suitably, For example, as a method of expressing siRNA as a double stranded nucleic acid molecule, two short single stranded RNAs are used. Examples thereof include a method of expression (tandem type) and a method of expressing single-stranded RNA as shRNA (hairpin type).
The tandem siRNA expression vector includes a DNA sequence encoding a sense strand constituting the siRNA and a DNA sequence encoding an antisense strand, and upstream (5 ′ side) of the DNA sequence encoding each strand. And a promoter sequence, and a terminator sequence linked to the downstream (3 ′ side) of the DNA sequence encoding each strand.
In the hairpin siRNA expression vector, the DNA sequence encoding the sense strand constituting the siRNA and the DNA sequence encoding the antisense strand are arranged in opposite directions, and the sense strand DNA sequence and the antisense strand DNA The sequence includes DNAs that are connected to each other via a loop sequence, and that has a promoter sequence linked upstream (5 ′ side) and a terminator sequence linked downstream (3 ′ side).
Each of the vectors can be constructed using a conventionally known technique. For example, the vector can be constructed by ligating the DNA to a cleavage site of a vector that has been cleaved with a restriction enzyme in advance.

  By introducing (transfecting) the DNA or the vector into a cell, the promoter is activated and the double-stranded nucleic acid molecule can be generated. For example, in the tandem vector, the sense strand and the antisense strand are generated when the DNA is transcribed in a cell, and siRNA is generated by hybridizing them. In the hairpin vector, the DNA is transcribed in a cell to first generate hairpin RNA (shRNA), and then siRNA is generated by processing with a dicer.

(Cancer cell growth inhibitor)
The cancer cell growth inhibitor of the present invention is a cancer cell growth inhibitor (sometimes referred to as “tumor growth inhibitor”) for suppressing the growth of cancer cells, and the double-stranded nucleic acid molecule of the present invention described above. , DNA, and vector, and other components as necessary.

<Double-stranded nucleic acid molecule, DNA, vector>
Details of the double-stranded nucleic acid molecule are as described in the item of the double-stranded nucleic acid molecule of the present invention described above. Since the double-stranded nucleic acid molecule can effectively suppress the expression of at least one of the target TACC2 gene and 14-3-3ζ gene, the cancer cell growth for suppressing the growth of cancer cells It is suitable as an active ingredient of an inhibitor. The details of the DNA and vector are as described in the item of the DNA and vector of the present invention.
The content of at least one of the double-stranded nucleic acid molecule, DNA, and vector in the cancer cell growth inhibitor is not particularly limited and can be appropriately selected depending on the purpose. The cancer cell growth inhibitor may be at least one of the double-stranded nucleic acid molecule, DNA, and vector.
Among the double-stranded nucleic acid molecule, DNA, and vector, two comprising a sense strand having a nucleotide sequence corresponding to the target sequence of SEQ ID NO: 2 and an antisense strand having a nucleotide sequence complementary to the sense strand At least one of a single-stranded nucleic acid molecule, a DNA containing a nucleotide sequence encoding the double-stranded nucleic acid molecule, and a vector containing the DNA; a sense strand having a nucleotide sequence corresponding to the target sequence of SEQ ID NO: 13; An embodiment comprising at least one of a double-stranded nucleic acid molecule comprising an antisense strand having a nucleotide sequence complementary to the sense strand, a DNA comprising a nucleotide sequence encoding the double-stranded nucleic acid molecule, and a vector comprising the DNA Is preferred.

<Other ingredients>
The other component is not particularly limited and may be appropriately selected depending on the purpose. For example, a physiological condition for diluting at least one of the double-stranded nucleic acid molecule, DNA, and vector to a desired concentration. Examples include diluents such as saline and culture solutions, and transfection reagents for introducing (transfecting) at least one of the double-stranded nucleic acid molecules, DNA, and vectors into the cells of interest.
There is no restriction | limiting in particular also as content of the said other component in the said cancer cell growth inhibitor, According to the objective, it can select suitably.

<Cancer cells>
There is no restriction | limiting in particular as a cell used as the application object of the said cancer cell growth inhibitor, Although it can select suitably according to the objective, A prostate cancer cell is mentioned suitably. The prostate cancer cells may be hormone therapy resistant prostate cancer cells.
The cancer cells may be cells cultured outside the body, or may be cells present in the body of a patient suffering from cancer.
The prostate cancer cells cultured outside the body are not particularly limited and can be appropriately selected according to the purpose. For example, LNCaP cells (derived from Adenocarcinoma), PC-3 cells (derived from Adenocarcinoma), DU145 cells ( carcinoma origin).
These cells can be obtained from, for example, ATCC (American Type Culture Collection).

<Action>
The cancer cell growth inhibitor can be allowed to act on the cells, for example, by introducing (transfecting) them into the cancer cells. The introduction method is not particularly limited and can be appropriately selected from conventionally known methods according to the purpose. For example, a method using a transfection reagent, a method using electroporation, a method using magnetic particles And methods using virus infection.
There is no restriction | limiting in particular as the quantity of the said cancer cell growth inhibitor made to act with respect to a cancer cell, Although it can select suitably according to the kind of cell, the grade of a desired effect, etc., for example, 1 * 10 <^6>. The amount of the active ingredient (the double-stranded nucleic acid molecule) is preferably about 0.1 μg, more preferably about 5 μg, and particularly preferably about 15 μg with respect to the number of cells.

<Cancer cell growth suppression method>
Since the cancer cell growth inhibitor contains at least one of the double-stranded nucleic acid molecule, DNA, and vector, expression of at least one of the TACC2 gene and the 14-3-3ζ gene is caused by acting on a cancer cell. Cancer cell growth can be effectively inhibited through inhibition. Therefore, the present invention is referred to as a cancer cell growth inhibition method (“tumor growth inhibition method”), which comprises allowing at least one of the double-stranded nucleic acid molecule, DNA, and vector to act on cancer cells. Also). There is no restriction | limiting in particular as said cancer cell, Although it can select suitably according to the objective, A prostate cancer cell is mentioned suitably. The prostate cancer cells may be hormone therapy resistant prostate cancer cells.

(Medicine)
The medicament of the present invention is a medicament for preventing or treating cancer, and includes the above-described cancer cell proliferation inhibitor of the present invention, and further includes other components as necessary.

<Cancer growth inhibitor>
Details of the cancer cell growth inhibitor are as described in the item of the cancer cell growth inhibitor of the present invention. Since the cancer cell growth inhibitor contains at least one of the above-described double-stranded nucleic acid molecule, DNA, and vector of the present invention, expression suppression of at least one of the target TACC2 gene and 14-3-3ζ gene is suppressed. Through this, it is possible to effectively suppress the growth of cancer cells. That is, the cancer cell proliferation inhibitor can be suitably used as a medicament for preventing or treating cancer. There is no restriction | limiting in particular as said cancer, Although it can select suitably according to the objective, Prostate cancer is mentioned suitably. The prostate cancer may be hormone therapy resistant prostate cancer.
There is no restriction | limiting in particular as content of the said cancer cell growth inhibitor in the said pharmaceutical, According to the objective, it can select suitably. The medicament may be the cancer cell growth inhibitor itself.

Here, as the double-stranded nucleic acid molecule serving as the active ingredient of the medicament, an unmodified double-stranded nucleic acid molecule itself may be used, but in order to appropriately obtain preventive or therapeutic effects, It is preferable to use a double-stranded nucleic acid molecule in a form suitable for administration of
For example, the double-stranded nucleic acid molecule is preferably modified in that the stability of the double-stranded nucleic acid molecule in vivo can be improved. The type of modification that can be applied to the double-stranded nucleic acid molecule is not particularly limited, and examples thereof include 2′O-methylation modification, phosphorothioate (S) modification, and LNA (Locked Nucleic Acid) modification. Further, for the purpose of increasing the efficiency of introduction into target cells, for example, the 5 ′ end or 3 ′ end of the sense strand of the double-stranded nucleic acid molecule is modified with nanoparticles, cholesterol, a cell membrane-passing peptide, etc. It is also preferable. The method for modifying the double-stranded nucleic acid molecule is not particularly limited, and a conventionally known method can be appropriately used.
Moreover, it is also preferable that the double-stranded nucleic acid molecule forms a complex with a liposome, a polymer matrix, or the like in that the introduction efficiency into the target cell can be increased. There is no restriction | limiting in particular also as the method of forming the said composite body, A conventionally well-known method can be utilized suitably.

<Other ingredients>
The other components are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include pharmaceutically acceptable carriers. There is no restriction | limiting in particular as said support | carrier, For example, it can select suitably according to a dosage form etc. Moreover, there is no restriction | limiting in particular also as content of the said other component in the said pharmaceutical, According to the objective, it can select suitably.

<Dosage form>
The pharmaceutical dosage form is not particularly limited, and can be appropriately selected according to a desired administration method as described later, for example, oral solid preparations (tablets, coated tablets, granules, powders, capsules). Agents), oral liquids (internal solutions, syrups, elixirs, etc.), injections (solutions, suspensions, solid preparations for erection, etc.), ointments, patches, gels, creams, external powders Examples include sprays and inhaled powders.

Examples of the oral solid preparation include, for example, excipients and further additives such as binders, disintegrants, lubricants, colorants, flavoring and flavoring agents as necessary, in addition to the active ingredients. Can be manufactured.
Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone and the like. It is done. Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose. Examples of the lubricant include purified talc, stearate, borax, and polyethylene glycol. Examples of the colorant include titanium oxide and iron oxide. Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like.

The oral solution can be produced by a conventional method, for example, by adding additives such as a flavoring / flavoring agent, a buffering agent, and a stabilizer to the active ingredient.
Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like. Examples of the buffer include sodium citrate. Examples of the stabilizer include tragacanth, gum arabic, and gelatin.

As the injection, for example, a pH adjuster, a buffer, a stabilizer, a tonicity agent, a local anesthetic, etc. are added to the active ingredient, and subcutaneous, intramuscular, intravenous use are performed by a conventional method. Etc. can be manufactured.
Examples of the pH adjusting agent and the buffering agent include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid, and the like. Examples of the isotonic agent include sodium chloride and glucose. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.

As the ointment, for example, a known base, stabilizer, wetting agent, preservative and the like may be blended with the active ingredient and mixed by a conventional method.
Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, paraffin and the like. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and the like.

  As the patch, for example, a cream, gel or paste as the ointment can be applied to a known support by a conventional method. Examples of the support include woven fabric, nonwoven fabric, soft vinyl chloride, polyethylene, polyurethane and other films made of cotton, suf, and chemical fibers, and foam sheets.

<Administration>
The medicament is suitable for prevention or treatment of cancer. Therefore, the said medicine can be conveniently used by administering to the patient suffering from cancer.

  The animal to which the pharmaceutical is administered is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans, mice, rats, cows, pigs, monkeys, dogs, and cats. Among them, human is particularly preferable.

  There is no restriction | limiting in particular as the administration method of the said pharmaceutical, For example, according to the dosage form of the said pharmaceutical, the kind of disease, a patient's condition, etc., either local administration or systemic administration can be selected. For example, in local administration, the active ingredient of the medicine (the double-stranded nucleic acid molecule) can be administered by directly injecting it to a desired site (for example, a tumor site). For the injection, a conventionally known method such as injection can be appropriately used. Further, in systemic administration (for example, oral administration, intraperitoneal administration, administration into blood, etc.), the active ingredient of the medicament (the double-stranded nucleic acid molecule) is stable to a desired site (for example, a tumor site). In addition, it is preferable to appropriately apply a conventionally known drug delivery technique so that it can be efficiently delivered.

There is no restriction | limiting in particular as dosage of the said medicine, Although it can select suitably according to the age of a patient who is an administration subject, a body weight, the grade of the desired effect, etc., for example per day administration to an adult The amount of the active ingredient (the double-stranded nucleic acid molecule) is preferably 1 mg to 100 mg.
Further, the number of administrations of the medicine is not particularly limited, and can be appropriately selected according to the age, weight, desired degree of effect, etc. of the patient to be administered.

  There is no restriction | limiting in particular as the administration time of the said medicine, According to the objective, it can select suitably, For example, with respect to the said disease, you may administer prophylactically and may administer therapeutically. . Among these, since the medicament is excellent in the effect of inhibiting cancer cell growth and preventing tumor growth due to proliferation of the cancer cells, it is desirable that the medicament is administered at the earliest possible stage of the disease. Conceivable.

<Prevention or treatment method>
Since the medicament contains the cancer cell growth inhibitor, administration to an individual suffering from cancer has an effect on the growth of cancer cells through suppression of the expression of at least one of the TACC2 gene and the 14-3-3ζ gene. Can be suppressed and cancer can be prevented or treated. Therefore, the present invention also relates to a method for preventing or treating cancer, which comprises administering the medicine to an individual. There is no restriction | limiting in particular as said cancer, Although it can select suitably according to the objective, Prostate cancer is mentioned suitably. The prostate cancer may be hormone therapy resistant prostate cancer.

  Test examples of the present invention will be described below, but the present invention is not limited to these test examples.

(Production Example 1: Preparation of double-stranded nucleic acid molecule (siRNA))
A double-stranded nucleic acid molecule (siRNA) of the present invention for suppressing the expression of at least one of the TACC2 gene and the 14-3-3ζ gene was prepared as follows.
The siRNA of the present invention against the TACC2 gene (siTACC2 # 1 to # 10) and the siRNA of the present invention against the 14-3-3ζ gene (si14-3-3 zeta # 1 to # 8) are targeted for the respective siRNAs. A gene sequence (described below) and a complementary sequence thereof were synthesized as a double-stranded RNA (manufactured by Sigma-Aldrich) having an overhang of 2 bases at the 3 ′ end. These can prevent the off-target effect derived from the sense strand.
Moreover, siRNA (siControl # 1 (manufactured by RNAi), siControl # 2 (manufactured by Ambion, 4390843)) having no off-target effect on all known genes was prepared as a negative control.
Moreover, siTACC2 # 0 (manufactured by Invitrogen, HSS116289) was prepared as a positive control of siRNA for the TACC2 gene.

The target gene sequence of each siRNA and the sequence of the prepared siRNA are shown below.
<SiTACC2 # 1>
-Target gene sequence-
5′-GTACCCTTAAGCGAACTAAA-3 ′ (SEQ ID NO: 1)
-Sequence of siRNA-
--Sense strand--
5′-GUACCCUUAAGCGAACUAAA-3 ′ (SEQ ID NO: 21)
--Antisense strand--
5′-UUAGUUCCGCUUAAGGGGUACUA-3 ′ (SEQ ID NO: 22)
The siRNA sequence corresponds to the 932th to 954th positions in NM_006997.2.
<SiTACC2 # 2>
-Target gene sequence-
5′-CTTAACTGTTGCGTGCAATAT-3 ′ (SEQ ID NO: 2)
-Sequence of siRNA-
--Sense strand--
5′-CUUAACUGUUGCGUGCAAUAU-3 ′ (SEQ ID NO: 23)
--Antisense strand--
5′-AUUGCACGCAACAGGUUAAGUC-3 ′ (SEQ ID NO: 24)
The siRNA sequence corresponds to positions 3436 to 3458 in NM_006997.2.
<SiTACC2 # 3>
-Target gene sequence-
5′-CGTGCCCTCAGAGCTCATAAGAAT-3 ′ (SEQ ID NO: 3)
-Sequence of siRNA-
--Sense strand--
5′-CGUGCCUCAGACCGCUAAGAAU-3 ′ (SEQ ID NO: 25)
--Antisense strand--
5′-UCUUAGCCGUCUGAGGCACAGAG-3 ′ (SEQ ID NO: 26)
The siRNA sequence corresponds to positions 730 to 752 in NM_006997.2.
<SiTACC2 # 4>
-Target gene sequence-
5′-CCATTGCTAAAGGTACTTACA-3 ′ (SEQ ID NO: 4)
-Sequence of siRNA-
--Sense strand--
5'-CCAUUGCUAAAGGUACUUACA-3 '(SEQ ID NO: 27)
--Antisense strand--
5′-UAAGGUACCUUAGCAAUGGGG-3 ′ (SEQ ID NO: 28)
The siRNA sequence corresponds to positions 1553 to 1575 in NM_006997.2.
<SiTACC2 # 5>
-Target gene sequence-
5′-CGGAGGAAGTCCCACGGATTCC-3 ′ (SEQ ID NO: 5)
-Sequence of siRNA-
--Sense strand--
5′-CGGAGGAAGUCCACGGAUUCC-3 ′ (SEQ ID NO: 29)
--Antisense strand--
5′-AAUCCCGUGGACUCUCCUCGUG-3 ′ (SEQ ID NO: 30)
The siRNA sequence corresponds to positions 772 to 794 in NM_006997.2.
<SiTACC2 # 6>
-Target gene sequence-
5′-GTGGTGCACTTTACTACTCTGG-3 ′ (SEQ ID NO: 6)
-Sequence of siRNA-
--Sense strand--
5′-GUGGUGCACUUGACUAAUCUGG-3 ′ (SEQ ID NO: 31)
--Antisense strand--
5′-AGAUAGUCAAGUGCACCACAG-3 ′ (SEQ ID NO: 32)
The siRNA sequence corresponds to positions 2534 to 2556 in NM_006997.2.
<SiTACC2 # 7>
-Target gene sequence-
5′-CGAGAAACTTTGACAACACTCC-3 ′ (SEQ ID NO: 7)
-Sequence of siRNA-
--Sense strand--
5′-CGAGAAACUUGACAACACUCC-3 ′ (SEQ ID NO: 33)
--Antisense strand--
5′-AGUGUUGUCAAGUUUCUCGGGG-3 ′ (SEQ ID NO: 34)
The siRNA sequence corresponds to positions 1491 to 1513 in NM_006997.2.
<SiTACC2 # 8>
-Target gene sequence-
5′-GGACCTGTCCCACCTTTGTAAA-3 ′ (SEQ ID NO: 8)
-Sequence of siRNA-
--Sense strand--
5′-GGACCUGGUCCACCUUGUUAAA-3 ′ (SEQ ID NO: 35)
--Antisense strand--
5′-UACAAAGGGUGGACAGGUCCGA-3 ′ (SEQ ID NO: 36)
The siRNA sequence corresponds to positions 2064 to 2086 in NM_006997.2.
<SiTACC2 # 9>
-Target gene sequence-
5′-GCCTTAAGGAGGTGTAAACTTG-3 ′ (SEQ ID NO: 9)
-Sequence of siRNA-
--Sense strand--
5′-GCCUUAAGGAGUGUUAAACUUG-3 ′ (SEQ ID NO: 37)
--Antisense strand--
5′-AGUUUACACUCCUCUUAAGGCAA-3 ′ (SEQ ID NO: 38)
The siRNA sequence corresponds to positions 3783 to 3805 in NM_006997.2.
<SiTACC2 # 10>
-Target gene sequence-
5′-CTGCCGTCTTCGATGAAGACA-3 ′ (SEQ ID NO: 10)
-Sequence of siRNA-
--Sense strand--
5′-CUGCCGUCUUCUGAUGAACA-3 ′ (SEQ ID NO: 39)
--Antisense strand--
5′-UCUUCAUCGAAGACGGCAGAG-3 ′ (SEQ ID NO: 40)
The siRNA sequence corresponds to positions 629 to 651 in NM_006997.2.

<Si14-3-3 zeta # 1>
-Target gene sequence-
5′-GTGAGACATCGGATACCACAGG-3 ′ (SEQ ID NO: 11)
-Sequence of siRNA-
--Sense strand--
5′-GUGGACAUCGGAAUCCCAAGG-3 ′ (SEQ ID NO: 41)
--Antisense strand--
5'-UUGGGGUUCCCGAUGUCCACAA-3 '(SEQ ID NO: 42)
In addition, the sequence of the siRNA corresponds to the 834th to 856th positions in NM_001135699.1.
<Si14-3-3 zeta # 2>
-Target gene sequence-
5'-CAGCACGCTCATAATAGCCAATT-3 '(SEQ ID NO: 12)
-Sequence of siRNA-
--Sense strand--
5′-CAGCACCGCUAAUAUUGCAAUU-3 ′ (SEQ ID NO: 43)
--Antisense strand--
5′-UUGCAUAUAUAGCGUGUGUC-3 ′ (SEQ ID NO: 44)
The siRNA sequence corresponds to the 792nd to 814th positions in NM_001135699.1.
<Si14-3-3 zeta # 3>
-Target gene sequence-
5′-CCGTTACTGTGCTGAGGTTGC-3 ′ (SEQ ID NO: 13)
-Sequence of siRNA-
--Sense strand--
5′-CCGUUACUGUGGCUGAGUGUGC-3 ′ (SEQ ID NO: 45)
--Antisense strand--
5′-AACCUCAGCCCAAGUAACGGUA-3 ′ (SEQ ID NO: 46)
The siRNA sequence corresponds to positions 531 to 553 in NM_001135699.1.
<Si14-3-3 zeta # 4>
-Target gene sequence-
5′-GTTAATAAGGTTTGGCATAGGT-3 ′ (SEQ ID NO: 14)
-Sequence of siRNA-
--Sense strand--
5′-GUUAUAAGUGGUUUGGCAUAGU-3 ′ (SEQ ID NO: 47)
--Antisense strand--
5′-UAUGCCAAACACUUAUAACUU-3 ′ (SEQ ID NO: 48)
The sequence of the siRNA corresponds to the 1139th to 1161st positions in NM_00113569.1.
<Si14-3-3 zeta # 5>
-Target gene sequence-
5′-GTAGCATTAACTGTAAGTTTT-3 ′ (SEQ ID NO: 15)
-Sequence of siRNA-
--Sense strand--
5′-GUAGCAAUUAACUGUUAAGUUUU-3 ′ (SEQ ID NO: 49)
--Antisense strand--
5′-AACUUACAGUUAUAUGCUACCC-3 ′ (SEQ ID NO: 50)
The siRNA sequence corresponds to positions 2242 to 2264 in NM_00113569.1.
<Si14-3-3 zeta # 6>
-Target gene sequence-
5'-GCACGCCTATAATAGCCAATTAC-3 '(SEQ ID NO: 16)
-Sequence of siRNA-
--Sense strand--
5′-GCACCGCUUAAUAUGCAUAUAC-3 ′ (SEQ ID NO: 51)
--Antisense strand--
5′-AAUUGCAAUAUAUAGCGUGCUG-3 ′ (SEQ ID NO: 52)
In addition, the sequence of the siRNA corresponds to the 794th to 816th positions in NM_001135699.1.
<Si14-3-3 zeta # 7>
-Target gene sequence-
5'-CCTACCTATCCCTGAATGGTCT-3 '(SEQ ID NO: 17)
-Sequence of siRNA-
--Sense strand--
5′-CCUACCUAUCCUGAAUGGUCU-3 ′ (SEQ ID NO: 53)
--Antisense strand--
5′-ACCAUUCAGGAUAGGUAGGGGU-3 ′ (SEQ ID NO: 54)
The sequence of the siRNA corresponds to the 2578th to 2600th positions in NM_001135699.1.
<Si14-3-3 zeta # 8>
-Target gene sequence-
5′-GTAGTAATTGTGGGGTACTTTA-3 ′ (SEQ ID NO: 18)
-Sequence of siRNA-
--Sense strand--
5′-GUAGUAAAUUGUGGGGUACUUUA-3 ′ (SEQ ID NO: 55)
--Antisense strand--
5′-AAGUACCCACAAUAUACUACAC-3 ′ (SEQ ID NO: 56)
The siRNA sequence corresponds to positions 1288 to 1310 in NM_00113569.1.

The sequence of siControl # 1 used for the negative control is shown below.
<SiControl # 1>
-Sense strand-
5′-GUACCCGCACGUCAUUCGUAUC-3 ′ (SEQ ID NO: 57)
-Antisense strand-
5′-UACGAAUGACGUGCGGUACGU-3 ′ (SEQ ID NO: 58)

(Test Example 1-1-1: Examination of target gene expression suppression effect at the mRNA level by siRNA)
The siTACC2 # 1 to # 10 obtained in Production Example 1 were transfected into prostate cancer cells, and after 48 hours, total RNA was collected and quantitative real-time PCR was performed. The inhibitory effect (knockdown effect) on TACC2 gene expression was examined.
In addition, siControl # 2 and siTACC2 # 0 were used as controls.
Details of the experimental method are shown below.

[cell]
As the prostate cancer cells, androgen receptor (AR) positive human prostate cancer-derived LNCaP cells (source: ATCC (American Type Culture Collection), No. CRL-1740) were used.

[Cell culture]
LNCaP cells were prepared by using RPMI-1640 (Sigma) containing 10% fetal bovine serum (FBS, Sigma), 100 μg / mL streptomycin, 100 U / mL penicillin (Invitrogen) as a cell culture medium. The cells were cultured at 37 ° C. in an incubator containing 5% carbon dioxide gas.

[Transfection]
Puncture 1 × 10 ⁵ LNCaP cells / well in a 6-well plate, and then add RNAi MAX (Invitrogen) and OPTI-MEM (Invitrogen) to the next day (number of cells: 3 × 10 ⁵ / hole). Used to transfect siRNA. The amount of siRNA introduced was adjusted to 10 nM.

[Measurement of gene expression level]
After transfection and culturing for 48 hours, total RNA was recovered from the cells using ISOGEN (Nippon Gene Co., Ltd.). Using 500 ng of the total RNA, cDNA was synthesized by Prime script RT reagent kit (manufactured by Takara Bio Inc.).
The cDNA was diluted 10-fold, and 2 μL of the cDNA was used for quantitative real-time PCR. The quantitative real-time PCR is performed using Step one real-time PCR (Applied biosystems) and KAPA SYBR Fast PCR kit (Nippon Genetics Co., Ltd.), and the GAPDH gene which is an internal control. The expression level was measured.
The expression level of the TACC2 gene was calculated from the number of cycles of the expression level for the GAPDH gene using the ΔΔCt method.

The results are shown in FIG. 1A. In FIG. 1A, “Reagent” indicates the result of the siRNA not transfected (reagent only), and “siControl” indicates the result of “siControl # 2” transfected.
From the result of FIG. 1A, siTACC2 # 1- # 10 of this invention suppressed the expression of the TACC2 gene compared with Reagent and siControl.

(Test Example 1-1-2: Examination of target gene expression suppression effect at the mRNA level by siRNA)
In Test Example 1-1-1, the test was performed in the same manner as in Test Example 1-1-1, except that the amount of siRNA introduced was changed from 10 nM to 50 nM.
The results are shown in FIG. 1B. In FIG. 1B, “Reagent” indicates the result of the siRNA not transfected (reagent only), and “siControl” indicates the result of “siControl # 2” transfected.
From the result of FIG. 1B, it was confirmed that siTACC2 # 1 to # 3, # 5, # 7, and # 8 of the present invention have a knockdown effect equivalent to or higher than siTACC2 # 0.

(Test Example 1-2: Examination of target gene expression suppression effect at the mRNA level by siRNA)
In Test Example 1-1-1, except that siRNA was si14-3-3 zeta # 1 to # 8, control was siControl # 2, and the expression level of 14-3-3ζ gene was measured, Test Example 1- The test was performed in the same manner as in 1-1.
The results are shown in FIG. In FIG. 2, “Reagent” indicates the result of the siRNA not transfected (reagent only), and “siControl” indicates the result of “siControl # 2” transfected.
From the result of FIG. 2, si14-3-3 zeta # 1 to # 8 of the present invention suppressed the expression of the 14-3-3ζ gene at the mRNA level as compared with Reagent and siControl. Among these, si14-3-3 zeta # 1, # 3, # 4, and # 6 were particularly effective.

(Test Example 2-1: Examination of target gene expression suppression effect at the protein level by siRNA)
The siTACC2 # 1 to # 10 obtained in Production Example 1 were transfected into prostate cancer cells, and protein samples were collected 48 hours later and subjected to Western blot analysis, whereby the TACC2 gene in the prostate cancer cells of each siRNA was obtained. The inhibitory effect on expression (knockdown effect) was examined.
As a control, siControl # 2 was used.
Details of the experimental method are shown below.

[cell]
As the prostate cancer cells, androgen receptor (AR) positive human prostate cancer-derived LNCaP cells (source: ATCC (American Type Culture Collection), No. CRL-1740) were used.

[Cell culture]
LNCaP cells were prepared by using RPMI-1640 (Sigma) containing 10% fetal bovine serum (FBS, Sigma), 100 μg / mL streptomycin, 100 U / mL penicillin (Invitrogen) as a cell culture medium. The cells were cultured at 37 ° C. in an incubator containing 5% carbon dioxide gas.

[Transfection]
Puncture 1 × 10 ⁵ LNCaP cells / well in a 6-well plate, and then add RNAi MAX (Invitrogen) and OPTI-MEM (Invitrogen) to the next day (number of cells: 3 × 10 ⁵ / hole). Used to transfect siRNA. The amount of siRNA introduced was adjusted to 50 nM.

[Western blot analysis]
After transfection and culturing for 48 hours, whole cells from cells using NP40 lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, Protease Inhibitor Cocktail (manufactured by Nacalai Tesque)] The extract was collected.
The protein concentration of the whole cell extract was prepared by BCA assay (Pierce).
10 μg of the whole cell extract adjusted for the protein concentration was electrophoresed on an 8% SDS-PAGE gel, and blotted onto Immobilon-P Transfer Membrane (Millipore).
Thereafter, the anti-TACC2 antibody (Upstate, 07-228) diluted 1,000 times as the primary antibody was reacted overnight, and then the peroxidase diluted 5,000 times as the secondary antibody. The anti-rabbit IgG antibody (manufactured by Sigma) was reacted for 1 hour. Further, as an internal control, anti-β-actin diluted with 500-fold (Sigma, A5316) was allowed to react overnight, and then as a secondary antibody, peroxidase-conjugated anti-mouse IgG diluted 5,000-fold. An antibody (manufactured by GE Healthcare Japan, NA931-100UL) was reacted for 1 hour.
Next, the antigen-antibody complex was reacted with an ImmunoCruz Western blotting detector system (manufactured by Santa Cruz Biotechnology) and photographed on an X-ray film.

The results are shown in FIG. In FIG. 3, “Reagent” indicates the result of the siRNA not transfected (reagent only), and “siControl” indicates the result of “siControl # 2” transfected. The upper row shows the expression level of the TACC2 gene, and the lower row shows the expression level of β-actin as an internal control.
From the results of FIG. 3, siTACC2 # 1 to # 3, # 6, and # 9 suppressed the expression of the TACC2 gene at the protein level than Reagent and siControl. Among these, # 1 was the most effective.

(Test Example 2-2: Examination of target gene expression suppression effect at the protein level by siRNA)
In Test Example 2-1, the siRNA was si14-3-3 zeta # 1 to # 8, the amount of the introduced siRNA was changed from 50 nM to 10 nM, and the primary antibody was diluted 1,000-fold anti-14-3- 3 except that the zeta antibody (manufactured by Santa Cruz Biotechnology, C-16) was used and anti-β-actin (manufactured by Sigma, A5316) was diluted 1,000 times. And tested.
The results are shown in FIG. In FIG. 4, “Reagent” indicates the result of the siRNA not transfected (reagent only), and “siControl” indicates the result of “siControl # 2” transfected. The upper row shows the expression level of 14-3-3ζ gene, and the lower row shows the expression level of β-actin, which is an internal control.
From the results of FIG. 4, si14-3-3 zeta # 1 to # 4 and # 6 to # 8 suppressed the expression of the 14-3-3ζ gene at the protein level compared to Reagent and siControl. Among these, # 3 was the most effective.

(Test Example 3-1-1: In vitro cell proliferation inhibitory effect of siRNA)
By transfecting prostate cancer cells with siTACC2 # 1 to # 10 obtained in Production Example 1 and measuring the subsequent cell growth rate, each siRNA has a prostate cancer cell proliferation inhibitory effect (knockdown effect). investigated.
In addition, siControl # 2 and siTACC2 # 0 were used as controls.
Details of the experimental method are shown below.

[cell]
As the prostate cancer cells, androgen receptor (AR) positive human prostate cancer-derived LNCaP cells (source: ATCC (American Type Culture Collection), No. CRL-1740) were used.

[Cell culture]
LNCaP cells were prepared by using RPMI-1640 (Sigma) containing 10% fetal bovine serum (FBS, Sigma), 100 μg / mL streptomycin, 100 U / mL penicillin (Invitrogen) as a cell culture medium. The cells were cultured at 37 ° C. in an incubator containing 5% carbon dioxide gas.

[Transfection]
The day before transfection, the LNCaP cells were subcultured to 3 × 10 ³ cells / well in a 96-well plate. On the day of transfection, RNAi MAX (manufactured by Invitrogen) and OPTI-MEM (manufactured by Invitrogen) were used. SiRNA was transfected. The amount of siRNA introduced was adjusted to 10 nM.

[Cell proliferation test]
The cell proliferation test was performed by reacting for 2 hours using Cell titer 96 (manufactured by Promega) 1 day, 3 days, and 5 days after transfection, and measuring the cell proliferation ability at an absorbance of 490 nm using a microplate reader. . The test was conducted via PES (phenazine ethersulfate) through the tetrazolium salt [MTS; 3- (4,5-dimethylthiazole-2-yl) -5- (3-carbophenylphenyl) -2- (4-sulfophenyl) -2H- The number of viable cells is measured based on a reduction reaction that converts tetrazolium, inner salt] into a formazan product that is a coloring substance.

The results are shown in FIG. 5A. In FIG. 5A, “Reagent” indicates the result of the siRNA not transfected (reagent only), and “siControl” indicates the result of “siControl # 2” transfected. Moreover, the bar graph in the column of each siRNA etc. shows the result 1 day, 3 days, and 5 days after transfection in order from the left.
From the results of FIG. 5A, siTACC2 # 1 to # 4 and # 7 to # 9 were suppressed in cell growth more than Reagent and siControl.

(Test Example 3-1-2: In vitro cell growth inhibitory effect of siRNA)
In Test Example 3-1-1, the test was performed in the same manner as Test Example 3-1-1 except that the amount of siRNA introduced was changed from 10 nM to 50 nM.
The results are shown in FIG. 5B. In FIG. 5B, “Reagent” indicates the result of the siRNA not transfected (reagent only), and “siControl” indicates the result of “siControl # 2” transfected. Moreover, the bar graph in the column of each siRNA etc. shows the result 1 day, 3 days, and 5 days after transfection in order from the left.
From the results of FIG. 5B, siTACC2 # 1 to # 4 and # 7 to # 10 were confirmed to have a cell growth inhibitory effect equivalent to or higher than siTACC2 # 0. Among these, it was observed that the cell growth inhibitory effect was particularly strong in siTACC2 # 2 and # 9.

(Test Example 3-2: In vitro cell growth inhibitory effect of siRNA)
In Test Example 3-1-1, the siRNA was si14-3-3 zeta # 1 to # 8, the control was siControl # 2, and the cell proliferation test was performed 1, 4, 5, and 6 days after transfection. The test was performed in the same manner as in Test Example 3-1-1 except that the test was performed.
The results are shown in FIG. In FIG. 6, “Reagent” indicates the result of the siRNA not transfected (reagent only), and “siControl” indicates the result of “siControl # 2” transfected. Moreover, the bar graph in the column of each siRNA etc. shows the result after 1 day, 4 days, 5 days, and 6 days after transfection in order from the left.
From the result of FIG. 6, si14-3-3 zeta # 1 to # 8 significantly suppressed the cell growth more than Reagent and siControl # 2. Among these, in particular, si14-3-3 zeta # 1 to # 3, # 5, # 6, and # 8 were found to strongly suppress cell proliferation after 4 days from transfection.

(Test Example 4-1: In vivo tumor growth inhibitory effect of siRNA)
Of siTACC2 obtained in Production Example 1, siTACC2 # 2, which was highly effective in suppressing the expression at the mRNA level and cell growth in the test, was transplanted with tumor cells subcutaneously in nude mice. The growth inhibitory effect on the tumor was examined.
As a control, siControl # 1 was used.
Details of the experimental method are shown below.

[cell]
As the tumor cells, androgen receptor (AR) -positive human prostate cancer-derived LNCaP cells (source: ATCC (American Type Culture Collection), No. CRL-1740) were used.

[mouse]
As the mouse, a male 8-week-old nude mouse (BALB / cAJcl-nu / nu) purchased from CLEA Japan was used.

[Subcutaneous transplantation of tumor cells]
The LNCaP cells were adjusted to 1 × 10 ⁷ per nude mouse and mixed with 100 μL of PBS. The mixture was mixed with 100 μL of Matrigel (BD bioscience). The mixture was injected subcutaneously into mice using a 25G needle.

[Administration of siRNA]
After transplanting tumor cells into mice, administration of siRNA was started when the tumor volume reached 100 mm ³ .
The siRNA was administered by locally injecting a solution (100 μL) mixed with 15 μL of RNAi MAX (manufactured by Invitrogen) and phenol red-free OPTI-MEM (85 μL) so that the siRNA was 5 μg / animal. It was done by doing.
The administration was repeated twice a week for 4 weeks.

[Measurement and evaluation of tumor growth]
Mouse tumor size was measured once a week.
The tumor volume was measured at two locations, the major axis (r1) and the minor axis (r2, r3). “{Major axis (r1) (mm) × minor axis (r2) (mm) × minor axis (r3) (Mm)} / 2].

The results are shown in FIGS. 7A and 7B.
FIG. 7A is a photograph taken 4 weeks after the start of siRNA administration, anesthetized with abatine and taken so that the size of the tumor can be seen (left side: siControl # 1 administered, right side: siTACC2 # 2 administered).
FIG. 7B is a graph showing changes in tumor volume. In FIG. 7B, “◇” indicates the result in the mouse (n = 7) administered with “siControl # 1”, and “□” indicates the result in the mouse (n = 8) administered with “siTACC2 # 2”. Show. In the figure, “***” represents p <0.001.
From the results of FIGS. 7A and 7B, it was revealed that administration of siTACC2 # 2 significantly suppressed the proliferation of LNCaP cells in mice.

(Test Example 4-2: In vivo tumor growth inhibitory effect of siRNA)
The test was conducted in the same manner as in Test Example 2-1, except that siRNA was changed to si14-3-3 zeta # 3, which had the highest inhibitory effect in the above test examples.
The results are shown in FIGS. 8A and 8B.
FIG. 8A is a photograph taken 4 weeks after the start of siRNA administration, anesthetized with abatine and taken so that the size of the tumor can be seen (left side: administered siControl # 1, right side: si14-3-3 zeta # 3). Administration).
FIG. 8B is a graph showing changes in tumor volume. In FIG. 8B, “◇” indicates the result in mice (n = 7) administered with “siControl # 1”, and “Δ” indicates mice administered with “si14-3-3 zeta # 3” (n = The result in 7) is shown. In the figure, “**” represents p <0.01, and “***” represents p <0.001.
From the results of FIGS. 8A and 8B, it was revealed that administration of si14-3-3 zeta # 3 significantly suppressed the growth of LNCaP cells in mice.

(Test Example 5-1: Comparison of siTACC2 # 2 with previously reported siRNA)
SiTACC2 # 2 obtained in Production Example 1 and siRNA against TACC2 gene described in “Takayama et al., Mol Endocrinol 26: 748-761, 2012” (hereinafter sometimes referred to as “previously reported literature 1”). (See below) and examined the inhibitory effect (knockdown effect) on TACC2 gene expression in prostate cancer cells.
In addition, siControl # 2 and siTACC2 # 0 were used as controls.

<SiRNA described in literature 1>
(1) siTACC2s20765 (siTACC2 (ME) # 1 in literature 1 already published, manufactured by lifetechnologies)
(2) siTACC2s20767 (siTACC2 (ME) # 2 in literature 1 already published, manufactured by lifetechnologies)

The details of the experimental method are as follows.
[cell]
As the prostate cancer cells, androgen receptor (AR) positive human prostate cancer-derived LNCaP cells (source: ATCC (American Type Culture Collection), No. CRL-1740) were used.

[Cell culture]
LNCaP cells were prepared by using RPMI-1640 (Sigma) containing 10% fetal bovine serum (FBS, Sigma), 100 μg / mL streptomycin, 100 U / mL penicillin (Invitrogen) as a cell culture medium. The cells were cultured at 37 ° C. in an incubator containing 5% carbon dioxide gas.

[Transfection]
On the day before transfection, 3 × 10 ⁵ LNCaP cells / well were seeded in a 6-well plate, and siRNA was transfected using RNAi MAX (Invitrogen) and OPTI-MEM (Invitrogen) on the day of transfection. did. The amount of siRNA introduced was adjusted to be 0.05 nM.

[Measurement of gene expression level]
72 hours after transfection, total RNA was recovered from the cells using ISOGEN (Nippon Gene Co., Ltd.). Using 500 ng of the total RNA, cDNA was synthesized by Prime script RT reagent kit (manufactured by Takara Bio Inc.).
The cDNA was diluted 10-fold, and 2 μL of the cDNA was used for quantitative real-time PCR. The quantitative real-time PCR is performed using Step one real-time PCR (Applied biosystems) and KAPA SYBR Fast PCR kit (Nippon Genetics Co., Ltd.), and the GAPDH gene which is an internal control. The expression level was measured.
The expression level of the TACC2 gene was calculated from the number of cycles of the expression level for the GAPDH gene using the ΔΔCt method.

FIG. 9 shows the results of calculating the expression level of the TACC2 gene in the group treated with siTACC2s20765, siTACC2s20767, siTACC2 # 0, or siTACC2 # 2 with respect to the expression level of the TACC2 gene in the group treated with siControl # 2. In FIG. 9, the vertical axis represents the ratio (%) with respect to the group treated with siControl # 2.
In FIG. 9, “1” indicates the result of siTACC2s20765, “2” indicates the result of siTACC2s20767, “3” indicates the result of siTACC2 # 0, and “4” indicates the result of siTACC2 # 2. . In FIG. 9, “**” indicates “P <0.01”, and “*” indicates “P <0.05”.

  From the results of FIG. 9, siTACC2 # 2 showed a higher gene expression suppression effect than other siRNAs at a low concentration of 0.05 nM. Therefore, the superiority of the knockdown effect of siTACC2 # 2 was confirmed.

(Test Example 5-2: Comparison between si14-3-3 zeta # 3 and reported siRNA)
In si14-3-3 zeta # 3 obtained in Production Example 1 and “Murata et al., Clin Caner Res 18: 5617-27, 2012” (hereinafter, referred to as “previously reported document 2”). For the described 14-3-3ζ gene and siRNA (see below), the suppression effect (knockdown effect) on 14-3-3ζ gene expression in prostate cancer cells was examined.
As a control, siControl # 2 was used.

<SiRNA described in literature 2>
(1) si14-3-3 zeta (previously reported document 2, manufactured by lifetechnologies)

The details of the experimental method are as follows.
[cell]
As the prostate cancer cells, androgen receptor (AR) positive human prostate cancer-derived LNCaP cells (source: ATCC (American Type Culture Collection), No. CRL-1740) were used.

[Cell culture]
LNCaP cells were prepared by using RPMI-1640 (Sigma) containing 10% fetal bovine serum (FBS, Sigma), 100 μg / mL streptomycin, 100 U / mL penicillin (Invitrogen) as a cell culture medium. The cells were cultured at 37 ° C. in an incubator containing 5% carbon dioxide gas.

[Transfection]
On the day before transfection, 3 × 10 ⁵ LNCaP cells / well were seeded in a 6-well plate, and siRNA was transfected using RNAi MAX (Invitrogen) and OPTI-MEM (Invitrogen) on the day of transfection. did. The amount of siRNA introduced was adjusted to be 0.05 nM.

[Measurement of gene expression level]
72 hours after transfection, total RNA was recovered from the cells using ISOGEN (Nippon Gene Co., Ltd.). Using 500 ng of the total RNA, cDNA was synthesized by Prime script RT reagent kit (manufactured by Takara Bio Inc.).
The cDNA was diluted 10-fold, and 2 μL of the cDNA was used for quantitative real-time PCR. The quantitative real-time PCR is performed using Step one real-time PCR (Applied biosystems) and KAPA SYBR Fast PCR kit (Nihon Genetics Co., Ltd.). The expression level of a certain GAPDH gene was measured.
The expression level of the 14-3-3ζ gene was calculated from the number of cycles of the expression level for the GAPDH gene using the ΔΔCt method.

FIG. 10 shows the expression level of 14-3-3ζ gene in the group treated with siControl # 2 and 14 in the group treated with si14-3-3 zeta (previously reported literature 2) or si14-3-3 zeta # 3. The result of having calculated the expression level of the 3-3ζ gene is shown. In FIG. 10, the vertical axis represents the ratio (%) with respect to the group treated with siControl # 2.
In the horizontal axis in FIG. 10, “1” indicates the result of si14-3-3 zeta (Report 2), and “2” indicates the result of si14-3-3 zeta # 3. In FIG. 10, “**” indicates “P <0.01”.

  From the result of FIG. 10, si14-3-3 zeta # 3 showed a higher gene expression suppression effect than other siRNAs at a low concentration of 0.05 nM. Therefore, the superiority of the knockdown effect of si14-3-3 zeta # 3 was confirmed.

(Test Example 6-1: Examination of cell growth inhibitory effect on hormone therapy-resistant prostate cancer cells)
By transfecting siTACC2 # 2 obtained in Production Example 1 into hormone therapy-resistant prostate cancer cells, and measuring subsequent cell proliferation, cells against siTCCC2 # 2 against hormone therapy-resistant prostate cancer cells were obtained. The growth inhibitory effect was examined.
As a control, siControl # 1 was used.
Details of the experimental method are shown below.

[cell]
According to the method described in “Takayama K. et al., Molecular Endocrinology 26: 748-761, 2012”, androgen receptor (AR) positive human prostate cancer-derived LNCaP cells (source: ATCC (American Type Culture)) No. CRL-1740) was cultured in a hormone-depleted medium for 9 months or longer to produce hormone therapy-resistant prostate cancer cells (hereinafter sometimes referred to as “LTAD cells”).

[Cell culture]
LTAD cells were charcoal-adsorbed 10% fetal bovine serum (FBS, manufactured by Sigma), 100 μg / mL streptomycin, 100 U / mL penicillin (manufactured by Invitrogen), and phenol red-free RPMI-1640 (manufactured by Sigma). It was used as a cell culture medium and cultured at 37 ° C. in an incubator containing 5% carbon dioxide gas in the air.

[Transfection]
In 96-well plates, LTAD cells were seeded at 5,000 cells / well, and 24 hours later, siRNA was transfected using RNAi MAX (Invitrogen) and OPTI-MEM (Invitrogen). The amount of siRNA introduced was adjusted to 1 nM.

[Cell proliferation test]
Five days after transfection, a cell proliferation test was performed using a living cell count reagent SF (manufactured by Nacalai Tesque).

The results are shown in FIG. In FIG. 11, the vertical axis indicates the absorbance at 490 nm, and “siControl” indicates the result of transfection of “siControl # 1”. “**” indicates P <0.01.
From the results of FIG. 11, it was shown that siTACC2 # 2 significantly suppresses cell proliferation even in LTAD cells.

(Test Example 6-2: Examination of cell growth inhibitory effect on hormone therapy-resistant prostate cancer cells)
Si14-3-3 zeta # 3 obtained in Production Example 1 was transfected into prostate cancer cells resistant to hormone therapy, and the subsequent cell proliferation was measured, whereby si14-3-3 zeta # 3 was measured. The inhibitory effect on cell proliferation of hormone-resistant prostate cancer cells was investigated.
As a control, siControl # 1 was used.
Details of the experimental method are shown below.

[cell]
According to the method described in “Takayama K. et al., Molecular Endocrinology 26: 748-761, 2012”, androgen receptor (AR) positive human prostate cancer-derived LNCaP cells (source: ATCC (American Type Culture)) No. CRL-1740) was cultured in a hormone-depleted medium for 9 months or longer to produce hormone therapy-resistant prostate cancer cells (hereinafter sometimes referred to as “LTAD cells”).

[Cell culture]
LTAD cells were charcoal-adsorbed 10% fetal bovine serum (FBS, manufactured by Sigma), 100 μg / mL streptomycin, 100 U / mL penicillin (manufactured by Invitrogen), and phenol red-free RPMI-1640 (manufactured by Sigma). It was used as a cell culture medium and cultured at 37 ° C. in an incubator containing 5% carbon dioxide gas in the air.

[Transfection]
In 96-well plates, LTAD cells were seeded at 5,000 cells / well, and 24 hours later, siRNA was transfected using RNAi MAX (Invitrogen) and OPTI-MEM (Invitrogen). The amount of siRNA introduced was adjusted to 1 nM.

[Cell proliferation test]
Five days after transfection, a cell proliferation test was performed using a living cell count reagent SF (manufactured by Nacalai Tesque).

The results are shown in FIG. In FIG. 12, the vertical axis indicates the absorbance at 490 nm, and “siControl” indicates the result of transfection of “siControl # 1”. “*” Indicates P <0.05.
From the results of FIG. 12, it was shown that si14-3-3 zeta # 3 significantly suppresses cell proliferation even in LTAD cells.

From the above Test Example 1 to Test Example 6, it was shown that the double-stranded nucleic acid molecule of the present invention has a high inhibitory effect on the expression of the TACC2 gene and 14-3-3ζ gene in cancer cells. Furthermore, since these double-stranded nucleic acid molecules have been shown to have a high tumor growth inhibitory effect in vivo, they can be suitably used as an active ingredient of a medicament for preventing or treating cancer. Conceivable.
The double-stranded nucleic acid molecule of the present invention targets at least one of the TACC2 gene and the 14-3-3ζ gene that exist as downstream signals of the AR, unlike the therapeutic method performed by suppressing the conventional AR itself. Therefore, it can be suitably used for cancer that has become refractory to hormone therapy.
Moreover, since there is clinical data that the expression of the TACC2 gene and the 14-3-3ζ gene is markedly increased in prostate cancer, the double-stranded nucleic acid molecule of the present invention can also suppress the progression of cancer. Excellent effect is expected.

Examples of the aspect of the present invention include the following.
<1> A double-stranded nucleic acid molecule for suppressing the expression of at least one of the TACC2 gene and the 14-3-3ζ gene,
(A) a sense strand having a nucleotide sequence corresponding to any target sequence of SEQ ID NO: 1 to SEQ ID NO: 18;
(B) an antisense strand having a nucleotide sequence complementary to the sense strand of (a),
A double-stranded nucleic acid molecule characterized by comprising
<2> The sense strand corresponds to any of the target sequences of SEQ ID NO: 1 to SEQ ID NO: 4, SEQ ID NO: 7 to SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 18. The double-stranded nucleic acid molecule according to <1>, wherein the double-stranded nucleic acid molecule is a sense strand having a nucleotide sequence.
<3> The two strands according to any one of <1> to <2>, wherein the sense strand is a sense strand having a nucleotide sequence corresponding to any of the target sequences of SEQ ID NO: 2 and SEQ ID NO: 13. A strand nucleic acid molecule.
<4> The double-stranded nucleic acid molecule according to any one of <1> to <3>, which is at least one of a double-stranded RNA and a double-stranded RNA-DNA chimera.
<5> The double-stranded nucleic acid molecule according to any one of <1> to <4>, which is at least one of siRNA and chimeric siRNA.
<6> The double-stranded nucleic acid molecule according to any one of <1> to <5>, which is an siRNA.
<7> DNA comprising a nucleotide sequence encoding the double-stranded nucleic acid molecule according to any one of <1> to <6>.
<8> A vector comprising the DNA according to <7>.
<9> At least one of the double-stranded nucleic acid molecule according to any one of <1> to <6>, the DNA according to <7>, and the vector according to <8>. It is a cancer cell proliferation inhibitor.
<10> Double-stranded nucleic acid molecule comprising a sense strand having a nucleotide sequence corresponding to the target sequence of SEQ ID NO: 2 and an antisense strand having a nucleotide sequence complementary to the sense strand, the double-stranded nucleic acid molecule At least one of DNA containing a nucleotide sequence encoding the DNA, and a vector containing the DNA;
A double-stranded nucleic acid molecule comprising a sense strand having a nucleotide sequence corresponding to the target sequence of SEQ ID NO: 13 and an antisense strand having a nucleotide sequence complementary to the sense strand, and encodes the double-stranded nucleic acid molecule At least one of DNA containing a nucleotide sequence and a vector containing the DNA;
The cancer cell proliferation inhibitor according to <9>, comprising
<11> The cancer cell proliferation inhibitor according to any one of <9> to <10>, wherein the cancer cells are prostate cancer cells.
<12> The cancer cell growth inhibitor according to <11>, wherein the prostate cancer cells are hormone therapy resistant prostate cancer cells.
<13> A method for inhibiting the growth of cancer cells, comprising causing the cancer cell proliferation inhibitor according to any one of <9> to <12> to act on the cancer cells.
<14> The method for inhibiting cancer cell proliferation according to <13>, wherein the cancer cells are prostate cancer cells.
<15> The cancer cell growth suppression method according to <14>, wherein the prostate cancer cells are hormone therapy-resistant prostate cancer cells.
<16> A medicament for preventing or treating cancer, comprising the cancer cell proliferation inhibitor according to any one of <9> to <12>.
<17> The medicament according to <16>, wherein the cancer is prostate cancer.
<18> The medicament according to <17>, wherein the prostate cancer is hormone therapy-resistant prostate cancer.
<19> A method for preventing or treating cancer, comprising administering the pharmaceutical according to any one of <16> to <18> to an individual.
<20> The cancer prevention or treatment method according to <19>, wherein the cancer is prostate cancer.
<21> The method for preventing or treating cancer according to <20>, wherein the prostate cancer is hormone therapy-resistant prostate cancer.

  The double-stranded nucleic acid molecule of the present invention can effectively suppress the growth of cancer cells through the suppression of the expression of at least one of the TACC2 gene and the 14-3-3ζ gene. Useful as an active ingredient.

Claims (7)

A double-stranded nucleic acid molecule for suppressing the expression of the TACC2 gene,
(A) a sense strand having a nucleotide sequence corresponding to the target sequence of SEQ ID NO: 2;
(B) an antisense strand having a nucleotide sequence complementary to the sense strand of (a),
A double-stranded nucleic acid molecule comprising: The double-stranded nucleic acid molecule according to claim 1, wherein the sense strand is a sense strand consisting of a nucleotide sequence corresponding to the target sequence of SEQ ID NO: 2. The double-stranded nucleic acid molecule according to claim 1, which is an siRNA. DNA comprising a nucleotide sequence encoding the double-stranded nucleic acid molecule according to any one of claims 1 to 3. A vector comprising the DNA according to claim 4. A cancer cell growth inhibitor comprising at least one of the double-stranded nucleic acid molecule according to any one of claims 1 to 3, the DNA according to claim 4, and the vector according to claim 5. A medicament for preventing or treating cancer, comprising the cancer cell proliferation inhibitor according to claim 6.