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  • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

• the present invention relates to a means for binding to StAR protein, which is a cholesterol transport promoting factor, to suppress the production of a StAR-binding protein that regulates the function of StAR protein.
• StAR protein which is a cholesterol transport promoting factor
• StAR protein acute regulatory protein plays an important role in transporting cholesterol from the outer mitochondrial membrane (Endocr. Rev. 17, 221-224 (1996)). StAR protein is thought to promote steroid hormone production in the cytoplasm (Recent Prg Horm Res 54, 369-94 (1999)) 0
• Apoptosis is a biological action that removes degenerated or malignant cells from living organisms. Cancer cells have been produced by radiation or chemotherapy and have been used to treat cancer. However, their specificity for cancer cells was low and they were not effective. Therefore, it has been suggested that apoptosis can be introduced into cancer cells by regulating the function of stAR protein, which is a transporter of cholesterol that constitutes the cell membrane (Proc Natl Acad Sci USA 99, 6943-6948 (2002)).
• RNA interference thigh A interference technique
• Cancer cells divide and proliferate faster than normal cells, and metabolize intracellular substances faster than normal cells. Therefore, the present invention focuses on the metabolism of cholesterol constituting the cell membrane, and binds to StAR protein, which is a cholesterol transport promoting factor, The aim was to induce apoptosis specifically in cancer cells by suppressing the production of proteins that regulate the function of StAR proteins and impairing cell functions.
• StAR protein which is a cholesterol transport promoting factor
• the present inventors screened a protein that interacts with the StAR protein in the process of studying the mechanism of promoting the production of steroid hormones of the StAR protein in the cytoplasm of steroid hormone-producing cells.
• One of the clones contained a 2.3 kb transgene, and it was confirmed that this clone interacted with the StAR protein.
• the translation product of this transgene was found to be a StAR protein binding protein (SBP, DDBJ Accession nuirfoer AB112474 (80..2428), AB112475 (431..2779), SEQ ID NO: 1).
• RNA interference technology (Nature, 391, 806-811 (1998); Table 2002-516062). It was introduced into cancer cells. As a result, it was confirmed that the expression of the StAR-binding protein was suppressed, and further, the appearance of apoptotic cells was confirmed.
• the present invention relates to a base having a length of not more than 23 bases, including 187 to 205 or 474 to 494, in the base sequence of SEQ ID NO: 1 (human StAR binding protein gene).
• This base sequence of 23 bases or less is preferably a base sequence of 187 to 205 or 474 to 494 of the base sequence of SEQ ID NO: 1.
• the present invention is a method for suppressing the expression of the SBP gene in a cancer cell, comprising introducing the oligoribonucleotide, its complementary oligoribonucleotide, or a double-stranded RNA comprising the same into a cancer cell. .
• the present invention includes the above-mentioned oligoribonucleotide, its complementary oligoribonucleotide, or a double-stranded RNA comprising the same, wherein the oligoribonucleotide, its complementary oligoribonucleotide, or a double-stranded RNA comprising these Is a kit for treating cancer, which comprises means for introducing s into a cancer cell.
• FIG. 1 shows the results of Burdan Atsushi of the StAR protein and clone 4.
• FIG. 2 shows SBP gene expression in human tissues.
• FIG. 3 shows SBP gene expression in various cell lines.
• HepG2 indicates human hepatoma cells
• KGN indicates human granulosa cancer cells
• H295R indicates human adrenal carcinoma cells
• MCF-7 indicates human breast cancer cells.
• FIG. 4 shows the effect of SBP on steroidogenesis. The value was expressed as an increase in the amount of predanenolone produced by co-introducing g of SBP with the cytochrome P450 sec system.
• FIG. 5 shows the amount of predanenolone produced by H295R cells treated with siRNA-SBP-I and siRNA-SBP-II.
• FIG. 6 shows the amount of predanenolone produced by KGN Itoda spores treated with siRNA-SBP-I and siRNA-SBP-II.
• FIG. 7 shows the appearance of apoptotic cells due to the introduction of siRNA (SBP II).
• A shows siRNA-Scramble
• B shows siRNA-SBPII gene-introduced gene
• C shows B-DNAase-treated one.
• the RNA fragment used in the present invention may be a sense or antisense target RNA, but these are easily degraded by RNase and are considered to be inferior in effect. It is preferably used.
• This double-stranded RNA is usually used to synthesize two strands of sense and antisense separately and then hybridize them to form a double strand.
• this RNA fragment is 21 to 23 bases, but in general, 21 bases are preferably used, and in the examples described later, 21 bases are also effective. It is functioning.
• the target cell is a cancer cell such as a human.
• Oligoribonucleotides “corresponding to” the specific nucleotide sequence of these SBP genes are defined as the specific nucleotide sequence of the SBP gene of mRNA that is transcribed and generated. It means RNA complementary to the portion corresponding to the sequence, specifically, it means that T in the specific DNA sequence of this SBP gene is replaced with U.
• Means for introducing the RNA fragment into cells is not particularly limited, and examples thereof include a calcium phosphate method, a microinjection method, a protoplast fusion method, electroporation, and a method using a viral vector. It is convenient to use commercially available transfusion reagents based on this.
• the siRNA of the present invention and a method using the same include solid cancer: epithelial cancer (stomach cancer, lung cancer, liver cancer, pancreatic cancer, etc.), non-solid cancer: leukemia, malignant lymphoma, malignant sarcoma (osteosarcoma, fibrosarcoma, etc.)
• Anticancer drugs anti-neoplastic agents, steroid hormone-dependent malignant tumor hormone therapy (breast cancer, endometrial cancer, prostate cancer, ovarian cancer, etc.), estrogen-dependent benign drugs (endometriosis) , Fibroids, etc.), promotion and suppression of sperm maturation, ovulation inducers, contraceptives, treatment of precocious puberty, treatment of gender identity disorder, etc.
• the plasmid and cells used in this example were prepared and cultured as described below. Plasmid construction
• GAL4-StAR mutants (GAL4-R193X, GAL4-Q253X, GAL4-frameshi ft) were prepared.
• PG51uc (Promega Corp., Madison, Wis.) Contains the CAT gene or the norreciferase gene as reporters.
• RACE the EcoRI fragment of the complete coding region obtained by PCR from testis CDNA was inserted into a pTarget vector (Promega Corp.) to obtain an expression plasmid (pSBP).
• COS-1 cells and human G2 cells were obtained from RIKEN cell bank.
• Human adrenocortical carcinoma H295R cells were obtained from Dr. Okamoto of Osaka University.
• Human MCF-7 breast cancer cells were obtained from ATCC (Man assas, VA).
• Human granular layer-like tumor KG cells were obtained from Dr. Nishi of Kyushu University.
• COS-1 cells were grown in 35 plastic dishes and cultured in DMEM medium supplemented with 10 fetal bovine serum and SOwg / ml gentamicin.
• KGN cells were cultured in DMEM / F12 medium containing 10 fetal bovine serum and 50 ⁇ g / ml gentamicin.
• H295R Itoda spores were cultured in DMEM / F12 medium containing 2% ULTR ⁇ SERG (BioSepra, Cergy-Pontoise, France) and 1% ITS Premix (Becton Dickinson and Co., Franklin Lakes, NJ).
• DMEM / F12 medium containing 2% ULTR ⁇ SERG (BioSepra, Cergy-Pontoise, France) and 1% ITS Premix (Becton Dickinson and Co., Franklin Lakes, NJ).
• ITS Premix Becton Dickinson and Co., Franklin Lakes, NJ.
• Example 1 proteins that interact with StAR proteins were identified using the GAL4-based yeast two-hybrid system.
• Yeast Two-hybrid interaction screening was performed as follows.
• the cells were cultured at 30 ° C for 5 days in a selective synthetic medium (SD) lacking tryptophan.
• HEC and NUCB2 express the LacZ phenotype. Based on the prediction of the intercellular localization of the proteins encoded by these clones, clone 4 was selected and analyzed.
• clone 4 contained a 2.3 kb insertion gene encoding a putative cytoplasmic protein, 197 a 2 nt open-read frame encoding a protein consisting of 657 amino acids, and a 62 nt untranslated sequence at the 3 terminus. It was found to have. Clone Insert size Okb) Identity Colony coloi
• plasmids expressing GAD-clone 4 fusion protein and expression of GAL4-N-62-StAR and GAL4-StAR mutant fusion protein were expressed.
• Galactosidase activity was determined using an X-gal filter assay Clone 4 fused to GAL4 reverse combination and N- Transfection was performed using a reverse combination of 62-StAR fused to GAD.As shown in Table 2, yeast transfected with StAR and clone 4 hybrid vector showed LacZ phenotype, Mutants and clones 4 Hybrid Beck Trans Hue transfected yeast in over showed LacZ phenotype This is, N in yeast -. 62 - StAR is indicates that interacting with clone 4. Table 2
• pull-down evaluation was performed to examine the direct interaction between StAR protein and clone 4.
• the pull-down evaluation was performed according to the following procedure.
• the EcoRI fragment obtained by PCR was inserted into a pCI vector (Promega Corp.) to prepare a plasmid expression clone.
• the translated protein was synthesized in vitro using a TNT-linked reticulocyte lysis system based on T7 RNA polymerase (Promega Corp.).
• the EcoRI fragment obtained by PCR using StAR cDNA as type I was inserted into a pET38b plasmid having a His tag (Novagen, San Diego, CA) at the C-terminus, and His-tagged CBD-N-62.
• -A plasmid was constructed to express the STAR fusion protein (lacking the 62 amino acid end).
• CBD is a Cellulose Binding Domain system IJ that has the property of binding specifically to cellulose, and immobilizes the fusion protein on an inert carrier such as cellulose or chitin without chemical modification. be able to.
• N-62 was subjected to His tag bound to -STAR fusion tank
• Nono 0 click protein 250 mu 1 of buffer (50mM potassium phosphate, pH 7.4, 150m M KC1, Im MgC12, Incubated for 3 hours with 35S methionine-labeled translated clone 501 in 10% glycerol, 0.1% Triton-X>
• the resin was collected by microcentrifugation and washed three times.
• the suspension was suspended in 2XSDS sample buffer of ⁇ , heated for 5 minutes, pelleted, and the supernatant was subjected to SDS- ⁇ AGE and autoradiography.
• Fig. 1 shows the results.
• the translated protein of clone 4 was found to interact with the CBD-N-62-StAR fusion protein and not with CBD.
• This clone 4 is called StAR binding protein (SBP) (DDBJ Accession number AB112474 (80..2428), AB112475 (431..2779), arrangement lj number 1).
• SBP StAR binding protein
• Northern blots were performed using SBP and actin cDNA as probes for 2 // g of poly A and RNA isolated from each tissue, respectively. SBP gene expression was detected in all tissues tested. As shown in FIG. 2, the expression level of the transcript was significantly higher in tissues having a size of 2.4 kb or 3.8 kb.
• HepG2 cells human hepatocellular carcinoma
• KGN cells human granulosa cell carcinoma
• H295R cells human adrenal carcinoma cells
• MCF-7 cells RT-PCR was performed using mRNA extracted from (human breast cancer cells).
• RT-PCR was performed according to the following procedure.
• the mRNA used was isolated from Hep G2 cells, KGN Itoda spores, H295R cells, and MCF-7 cells.
• For the synthesis of complementary DNA use 150 pmol of oligo dT as a primer, l / g total RNA and 200 units of SUPERSCRIPT Rnase H (Life Technologies, Inc./BRL, Washington, DC). , 37. C for 60 minutes.
• the reaction solution 20 il containing reverse transcriptase comprising 50mM Tris-HCI (pH 8.3) , 75mM KC1, 3m MgCl 2, 20m dithiothreitol and each 0.5mM of dATP, dCTP, dGTP, and ⁇ Pi dTTP.
• SBP was amplified using the synthetic oligonucleotides of SEQ ID NO: 2 and SEQ ID NO: 3 as primers.
• This PCR reaction solution (50 ⁇ l) contains 10 mM Tris-HCI (pH 8.3), 50 L KC1, 1.5 mM MgCl 2 , 0.2 mM dNTPs, and 10 pmol of each primer.
• the PCT reaction was performed for 35 cycles of a cycle consisting of denaturation at 94 ° C for 45 seconds, secondary annealing at 55 ° C for 45 seconds, and extension at 72 ° C for 1 minute.
• COS-1 meniscus was cotransfected with F2, cytochrome P450 cholesterol side chain cleavage system (Dr Walter L Miller of the University of California) ⁇ pStAR (pSPOR T StAR CDNA) and pSBP using FuGENE 6 .
• Cells F2 / StAR / SDP
• F2 / StAR / SDP were incubated for 48 hours after transfection.
• Several culture dishes were treated with 22R-hydroxy-cholesterol during the last 24 hours of culture. Forty-eight hours after transfection, the medium was collected and predanenolone was performed in Imnoassay. The results of this assay were normalized to the serum predanenolone concentration produced by cultures with 22R-hydroxy-cholesterol to correct for variations in transfection efficiency.
• the amount of pregnenolone produced by cotransfected COS-1 cells depends on the amount of predenolone produced by cells transfected with F2, StAR or empty vector (F2 / StAR).
• the amount of genolone was 138.
• SBP expression was suppressed by RNA interference, and steroid hormones were detected.
• Two target sequences in the SBP gene were selected, and the effect of the siRNA was measured by RT-PCR.
• RNA interference was performed in the following procedure.
• siRNA was constructed using the ribooligonucleotide pairs SBP-I and SBP-II.
• SBP-1 is 5'-CGGGAUGUUUCCAGUGACAdTdT-3 '(SEQ ID NO: 4) and 5'- UGU CACUGGAAACAUCCCGdTdT-3' (Distribution No. 5)
• SBP-II is 5'-GAACUUGGAAGAGG GGAGGCAdTdT-3 '(Distribution) J number 6) and 5 '-UGCCUCCCCUCUUCCAAGUUCdTdT-3'.
• RNA-I scrambled ribooligonucleotide pair
• These oligonucleotides were annealed according to the Dharmacon protocol.300 pmol of each strand was transferred to cells using 15 ⁇ l CD metafectene (Biontex Laboratories GmbH, Kunststoff, Germany) according to the manufacturer's instructions.
• the transfection of H395R cells was performed using 15 ⁇ of triostane (Mochida Pharmaceutical Co., Ltd.) to suppress the enzymatic activity of 3/3 hydroxysteroid dehydrogenase (3] 3-HSD) after transfection.
• 3-S-HSD transforms pregnenolone into progesterone 48 hours after transfection cells are harvested and treated with steroid form. Having conducted the radio I Takeno mediation Si of total RNA is extracted, the primer one for GAPDH (sense: SEQ ID NO: 10; anti sense: SEQ ID NO: 11). was performed using RT- PCR.
• SBP gene The expression level of SBP gene was reduced, as was the level of siRNA-SBP-I and siRNA-SBP-I. This indicates that treatment of the target sequence of the SBP gene with siRNA reduced expression of the SBP gene, resulting in a decrease in steroid hormone production.
• H295R cells were cultured in a culture medium DMEM / F12 containing 2% ULTROSER G (BioSepra) and 1% Premix (Betaton Dickinson). The day before gene transfer, a 24 mm x 24 mm cover glass was spread over a 35 mm plastic culture dish, and the cells were subcultured so that they were confluent (40-50%). To suppress the expression of SBP mRNA, siRNA-1 or siRNA-II was transfected using metafectene. After culturing for 24 hours after gene transfer, the cultured cells were fixed with 4% formaldehyde human phosphate buffered saline for 30 minutes.
• Apoptotic cells were detected using the DeadEnd Fluorometric TUNEL system (Promega, Inc.) according to the protocol of the system. After washing twice with phosphate buffered saline, the back side of the cover glass was placed on a slide glass and observed with a microscope. Observation was performed with a fluorescence microscope (Axiophot, Carl Zeiss) under the conditions of an excitation wavelength of 450-49 O nm and an absorption filter of 51-565 nm. Cells that fluoresce are apoptotic cells with fragmented DNA. A digital camera (DXM 1200, Nikon Corporation) was attached to the microscope and images were taken. The captured images were processed using Adobe's Photoshop 5.0 (Adobe Systems). The magnification of the image is 400 times.
• siRNA-SBP II The result of transfection of siRNA-SBP II is shown in FIG. 7 (B).
• siR A-SBP II introduction More apoptotic cells appear.

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