JPWO2005092393A1 - Wt1遺伝子の発現を抑制するマイクロrnaおよびその利用 - Google Patents
Wt1遺伝子の発現を抑制するマイクロrnaおよびその利用 Download PDFInfo
- Publication number
- JPWO2005092393A1 JPWO2005092393A1 JP2006511568A JP2006511568A JPWO2005092393A1 JP WO2005092393 A1 JPWO2005092393 A1 JP WO2005092393A1 JP 2006511568 A JP2006511568 A JP 2006511568A JP 2006511568 A JP2006511568 A JP 2006511568A JP WO2005092393 A1 JPWO2005092393 A1 JP WO2005092393A1
- Authority
- JP
- Japan
- Prior art keywords
- gene
- cells
- rna
- expression
- microrna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 101150084041 WT1 gene Proteins 0.000 title claims abstract description 40
- 230000014509 gene expression Effects 0.000 title abstract description 34
- 108700011259 MicroRNAs Proteins 0.000 title description 48
- 230000010261 cell growth Effects 0.000 claims abstract description 14
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 35
- 239000013598 vector Substances 0.000 claims description 18
- 230000000295 complement effect Effects 0.000 claims description 14
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- 239000003966 growth inhibitor Substances 0.000 claims description 9
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 6
- 230000024245 cell differentiation Effects 0.000 claims description 6
- 239000000411 inducer Substances 0.000 claims description 6
- 102000040650 (ribonucleotides)n+m Human genes 0.000 claims description 5
- 239000004480 active ingredient Substances 0.000 claims description 5
- 230000030833 cell death Effects 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 239000002679 microRNA Substances 0.000 abstract description 27
- 206010028980 Neoplasm Diseases 0.000 abstract description 15
- 201000011510 cancer Diseases 0.000 abstract description 11
- 108020004705 Codon Proteins 0.000 abstract description 6
- 108091070501 miRNA Proteins 0.000 abstract description 6
- 230000008685 targeting Effects 0.000 abstract description 6
- 230000002401 inhibitory effect Effects 0.000 abstract description 5
- 210000004027 cell Anatomy 0.000 description 53
- 108700025700 Wilms Tumor Genes Proteins 0.000 description 25
- 108090000623 proteins and genes Proteins 0.000 description 20
- 102000004169 proteins and genes Human genes 0.000 description 16
- 108020004999 messenger RNA Proteins 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 102000040856 WT1 Human genes 0.000 description 6
- 108700020467 WT1 Proteins 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 102000008935 SMN Complex Proteins Human genes 0.000 description 5
- 108010049037 SMN Complex Proteins Proteins 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 230000001629 suppression Effects 0.000 description 5
- 108091024449 let-7e stem-loop Proteins 0.000 description 4
- 108091044227 let-7e-1 stem-loop Proteins 0.000 description 4
- 108091071181 let-7e-2 stem-loop Proteins 0.000 description 4
- 208000032839 leukemia Diseases 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 108020005345 3' Untranslated Regions Proteins 0.000 description 3
- 108010029485 Protein Isoforms Proteins 0.000 description 3
- 102000001708 Protein Isoforms Human genes 0.000 description 3
- 208000005718 Stomach Neoplasms Diseases 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000013329 compounding Methods 0.000 description 3
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 3
- 206010017758 gastric cancer Diseases 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000009422 growth inhibiting effect Effects 0.000 description 3
- 201000011549 stomach cancer Diseases 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 102000008167 DEAD Box Protein 20 Human genes 0.000 description 2
- 108010060424 DEAD Box Protein 20 Proteins 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- 241000244206 Nematoda Species 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 108091030071 RNAI Proteins 0.000 description 2
- 108020004459 Small interfering RNA Proteins 0.000 description 2
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 2
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 2
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- -1 fluidity promoters Substances 0.000 description 2
- 235000013355 food flavoring agent Nutrition 0.000 description 2
- 230000009368 gene silencing by RNA Effects 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 108091023663 let-7 stem-loop Proteins 0.000 description 2
- 108091063478 let-7-1 stem-loop Proteins 0.000 description 2
- 108091049777 let-7-2 stem-loop Proteins 0.000 description 2
- 238000001638 lipofection Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 210000003643 myeloid progenitor cell Anatomy 0.000 description 2
- 108091027963 non-coding RNA Proteins 0.000 description 2
- 102000042567 non-coding RNA Human genes 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 239000008177 pharmaceutical agent Substances 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- 239000011701 zinc Substances 0.000 description 2
- 229910052725 zinc Inorganic materials 0.000 description 2
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 101100364969 Dictyostelium discoideum scai gene Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 206010053759 Growth retardation Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000711408 Murine respirovirus Species 0.000 description 1
- 101100364971 Mus musculus Scai gene Proteins 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 239000012722 SDS sample buffer Substances 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 208000021712 Soft tissue sarcoma Diseases 0.000 description 1
- 208000000102 Squamous Cell Carcinoma of Head and Neck Diseases 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 208000008383 Wilms tumor Diseases 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 206010006007 bone sarcoma Diseases 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 208000013549 childhood kidney neoplasm Diseases 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 239000007888 film coating Substances 0.000 description 1
- 238000009501 film coating Methods 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 239000007902 hard capsule Substances 0.000 description 1
- 201000000459 head and neck squamous cell carcinoma Diseases 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 230000002700 inhibitory effect on cancer Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 101150072601 lin-14 gene Proteins 0.000 description 1
- 101150111214 lin-28 gene Proteins 0.000 description 1
- 108091053735 lin-4 stem-loop Proteins 0.000 description 1
- 108091032363 lin-4-1 stem-loop Proteins 0.000 description 1
- 108091028008 lin-4-2 stem-loop Proteins 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000011242 molecular targeted therapy Methods 0.000 description 1
- 210000002161 motor neuron Anatomy 0.000 description 1
- 230000017095 negative regulation of cell growth Effects 0.000 description 1
- 230000017066 negative regulation of growth Effects 0.000 description 1
- 201000008026 nephroblastoma Diseases 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 239000007901 soft capsule Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 208000002320 spinal muscular atrophy Diseases 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Oncology (AREA)
- Hematology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
[1] 以下の(a)から(c)のいずれかを有効成分として含有する、細胞増殖抑制剤
(a)WT1遺伝子の転写産物に相補的な一本鎖RNA
(b)(a)のRNAをコードするDNA
(c)(b)のDNAが挿入されたベクター
[2] 一本鎖RNAが、WT1遺伝子の転写産物における配列番号:1に記載の塩基配列に相補的である、[1]に記載の細胞増殖抑制剤
[3] 一本鎖RNAが、配列番号:2に記載の塩基配列からなるRNAである、[1]または[2]に記載の細胞増殖抑制剤
[4] 以下の(a)から(c)のいずれかを有効成分として含有する細胞分化誘導剤または細胞死誘導剤。
(a)WT1遺伝子の転写産物に相補的なRNAと該RNAに相補的なRNAとを含む二重鎖RNA
(b)(a)の二重鎖RNAをコードするDNA
(c)(b)のDNAが挿入されたベクター
[5] 一本鎖RNAが、WT1遺伝子の転写産物における配列番号:1に記載の塩基配列に相補的である、[4]に記載の薬剤。
[6] 一本鎖RNAが、配列番号:2に記載の塩基配列からなるRNAである、[4]または[5]に記載の薬剤。
本発明のmiRNAは、WT1遺伝子の転写産物の塩基配列を基に標的となる配列を選択し、化学的合成方法等によって、適宜調製することができる。
なお本明細書において引用された全ての先行技術文献は、参照として本明細書に組み入れられる。
WT1 mRNAのstop codon直前から3'UTRの配列に対しmicroRNAとして働きうる可能性のあるmicroRNAをデータベース(NCBI GenBank)から検索し、候補としてmicroRNA (miR) 115を選択した。
RNAを日本バイオサービスに依頼し合成した。これを100μMの濃度でRNAse free waterに溶解して分注し、使用するまで-80℃で凍結保存した。合成したRNAの配列を以下に記す。mir−115: 5' −uga agc gga gcu gga a-3'(配列番号:2)
Luciferase AS 5' −ucg aag uau ucc gcg uac guu -3'(配列番号:4)。
WT1遺伝子を高発現するgastric cancer cell line AZ−521細胞をFBS10%含Dulbecco's Modified Medium (DMEM)中で培養した。microRNAによる細胞の処理は、トリプシナイズしたAZ-521細胞を1.2x105 cells/2mlに調製後、6well plateにまき24時間後にRNA(final conc.2μM)をRNAi Fect(QIAGEN)を用いて細胞に導入した。細胞数はトリプシナイズした後、算定板を用いて算定した。
上記実施例3と同様に培養、トリプシナイズしたAZ-521を1.2x105cells/2mlに調製後6well plateにまき、24時間後にRNAi Fect (QIAGEN)存在下でmiR-115またはLuciferase AS(濃度2μM)で12時間処理した。
結果を図4に示す。miR-115処理により細胞内でのタンパクの発現が効率よく抑制されていた。
miR-115がWT1特異的にAZ-521細胞の増殖を抑制しているかどうかを確認するため、WT1を強制発現させたAZ-521細胞をmiR-115で処理し、WT1発現抑制効果を検討した。
強制発現用として、CMVプロモーターを持つ発現ベクターpcDNA3.1(Invitrogen)にWT1 17AA(+)KTS(+)を挿入したベクターpcDNAWT1A(+/+)を作製した。AZ-521細胞への導入はvector 2μgをFugene6 (Roche)を用いてLipofection した。
miR115がWT1mRNAの仮想標的配列を介してWT1タンパクの発現を抑制していることを証明するために、GFPタンパクの3'側にmiR115の仮想標的配列を挿入したベクターを構築し、GFPタンパクの細胞内での発現に対するmiR115の効果を解析した。
次に、この2種類のベクター(pEGFP-115TS vector、 pEGFP-115mTS)を制限酵素Alw44Iにて切断しリニアにした後、 エレクトロポレーション法にてAZ-521胃癌細胞に導入した。G418を含む選択培地で2週間以上培養した後、FACS解析によりEGFPを過剰発現する細胞株をそれぞれ樹立した(AG115TS、AG115mTS)。
ウェスタンブロット解析は、以下の方法で行った。miR-115あるいはコントロールとして使用した Let7e で処理した細胞をSDS sample bufferに溶解し、タンパクをSDS-PAGEにて分離後、PVDFメンブレンに転写した。1次抗体に抗GFPモノクローナル抗体(Santa Cruz Biotechnology)、抗GAPDH抗体(Chemicon)を、2次抗体にALP conjugated antimouse 抗体(Santa Cruz Biotechnology)を用いてBCIP-NBT キットにて発色した。
Claims (6)
- 以下の(a)から(c)のいずれかを有効成分として含有する、細胞増殖抑制剤。
(a)WT1遺伝子の転写産物に相補的な一本鎖RNA
(b)(a)のRNAをコードするDNA
(c)(b)のDNAが挿入されたベクター - 一本鎖RNAが、WT1遺伝子の転写産物における配列番号:1に記載の塩基配列に相補的である、請求項1に記載の細胞増殖抑制剤。
- 一本鎖RNAが、配列番号:2に記載の塩基配列からなるRNAである、請求項1または2に記載の細胞増殖抑制剤
- 以下の(a)から(c)のいずれかを有効成分として含有する細胞分化誘導剤または細胞死誘導剤。
(a)WT1遺伝子の転写産物に相補的なRNAと該RNAに相補的なRNAとを含む二重鎖RNA
(b)(a)の二重鎖RNAをコードするDNA
(c)(b)のDNAが挿入されたベクター - 一本鎖RNAが、WT1遺伝子の転写産物における配列番号:1に記載の塩基配列に相補的である、請求項4に記載の薬剤。
- 一本鎖RNAが、配列番号:2に記載の塩基配列からなるRNAである、請求項4または5に記載の薬剤。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2006511568A JP4762133B2 (ja) | 2004-03-29 | 2005-03-28 | Wt1遺伝子の発現を抑制するマイクロrnaおよびその利用 |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2004096877 | 2004-03-29 | ||
JP2004096877 | 2004-03-29 | ||
JP2006511568A JP4762133B2 (ja) | 2004-03-29 | 2005-03-28 | Wt1遺伝子の発現を抑制するマイクロrnaおよびその利用 |
PCT/JP2005/005790 WO2005092393A1 (ja) | 2004-03-29 | 2005-03-28 | Wt1遺伝子の発現を抑制するマイクロrnaおよびその利用 |
Publications (2)
Publication Number | Publication Date |
---|---|
JPWO2005092393A1 true JPWO2005092393A1 (ja) | 2008-02-07 |
JP4762133B2 JP4762133B2 (ja) | 2011-08-31 |
Family
ID=35055999
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2006511568A Expired - Fee Related JP4762133B2 (ja) | 2004-03-29 | 2005-03-28 | Wt1遺伝子の発現を抑制するマイクロrnaおよびその利用 |
Country Status (4)
Country | Link |
---|---|
US (1) | US20080038819A1 (ja) |
EP (1) | EP1738772A4 (ja) |
JP (1) | JP4762133B2 (ja) |
WO (1) | WO2005092393A1 (ja) |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8299234B2 (en) | 2004-03-29 | 2012-10-30 | Haruo Sugiyama | siRNA hat inhibits WT1 gene expression and uses thereof |
US8192938B2 (en) | 2005-02-24 | 2012-06-05 | The Ohio State University | Methods for quantifying microRNA precursors |
ES2387999T3 (es) * | 2006-03-29 | 2012-10-05 | International Institute Of Cancer Immunology, Inc. | ARNsi específico de la isoforma WT1 17AA(-) y uso del mismo |
US7955848B2 (en) | 2006-04-03 | 2011-06-07 | Trustees Of Dartmouth College | MicroRNA biomarkers for human breast and lung cancer |
US8207325B2 (en) | 2006-04-03 | 2012-06-26 | Univ. of Copenhagen | MicroRNA biomarkers for human breast and lung cancer |
WO2008029790A1 (fr) * | 2006-09-04 | 2008-03-13 | Kyowa Hakko Kirin Co., Ltd. | Nouvel acide nucléique |
US20100099746A1 (en) * | 2006-12-18 | 2010-04-22 | Kyowa Hakko Kirin Co., Ltd. | Novel nucleic acid |
WO2009148137A1 (ja) * | 2008-06-04 | 2009-12-10 | 協和発酵キリン株式会社 | 肥満細胞の脱顆粒を制御する核酸 |
MA40253A (fr) * | 2014-07-15 | 2017-05-24 | Juno Therapeutics Inc | Cellules modifiées pour thérapie cellulaire adoptive |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH09104629A (ja) * | 1995-06-01 | 1997-04-22 | Kishimoto Chuzo | ウイルムス腫瘍遺伝子(wt1)に対するアンチセンスオリゴヌクレオチド誘導体を含んで成る白血病細胞増殖阻害剤 |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0841068B1 (en) * | 1995-06-01 | 2006-07-12 | Kishimoto, Tadamitsu | Leukemic cell growth inhibitor containing antisense oligonucleotide derivative against wilms' tumor gene (wt1) |
EP0846949B1 (en) * | 1996-04-16 | 2005-06-29 | Kishimoto, Tadamitsu | Method of detecting solid cancer cells and histological heterotypia and method of examining tissue for bone marrow transplantation and peripheral blood stem cell transplantation |
JP4789095B2 (ja) * | 1997-07-16 | 2011-10-05 | 治夫 杉山 | ウイルムス腫瘍遺伝子(wt1)に対する発現阻害物質を含んで成る固形腫瘍治療剤 |
US6232073B1 (en) * | 1999-02-05 | 2001-05-15 | Virginia Commonwealth University | Nucleic acid marker for cancer |
WO2003061386A1 (en) * | 2002-01-03 | 2003-07-31 | Board Of Regents, The University Of Texas System | Wt1 antisense oligos for the inhibition of breast cancer |
US20050118625A1 (en) * | 2003-10-02 | 2005-06-02 | Mounts William M. | Nucleic acid arrays for detecting gene expression associated with human osteoarthritis and human proteases |
US8299234B2 (en) * | 2004-03-29 | 2012-10-30 | Haruo Sugiyama | siRNA hat inhibits WT1 gene expression and uses thereof |
-
2005
- 2005-03-28 EP EP05727699A patent/EP1738772A4/en not_active Withdrawn
- 2005-03-28 WO PCT/JP2005/005790 patent/WO2005092393A1/ja active Application Filing
- 2005-03-28 US US10/594,706 patent/US20080038819A1/en not_active Abandoned
- 2005-03-28 JP JP2006511568A patent/JP4762133B2/ja not_active Expired - Fee Related
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH09104629A (ja) * | 1995-06-01 | 1997-04-22 | Kishimoto Chuzo | ウイルムス腫瘍遺伝子(wt1)に対するアンチセンスオリゴヌクレオチド誘導体を含んで成る白血病細胞増殖阻害剤 |
Also Published As
Publication number | Publication date |
---|---|
EP1738772A4 (en) | 2007-12-26 |
EP1738772A1 (en) | 2007-01-03 |
US20080038819A1 (en) | 2008-02-14 |
JP4762133B2 (ja) | 2011-08-31 |
WO2005092393A1 (ja) | 2005-10-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP4762133B2 (ja) | Wt1遺伝子の発現を抑制するマイクロrnaおよびその利用 | |
Kikuchi et al. | Effects of PAX3-FKHR on malignant phenotypes in alveolar rhabdomyosarcoma | |
JP6895175B2 (ja) | エクソソーム分泌阻害剤 | |
WO2007084954A2 (en) | Selective inhibition of ig20 splice variants to treat cancers | |
JP2007530431A (ja) | 膵臓癌を治療するための組成物および方法 | |
WO2019093502A1 (ja) | がん促進因子発現抑制剤、その有効成分のスクリーニング方法、該方法に有用な発現カセット、診断薬、及び診断方法 | |
WO2012121178A1 (ja) | 腫瘍血管形成阻害剤 | |
US9597346B2 (en) | Methods for reducing protein levels in a cell | |
WO2010136846A1 (en) | Differentiation therapy for sarcomas | |
US8603997B2 (en) | siRNA that inhibits WT1 gene expression and uses thereof | |
EP2464730B1 (en) | miRNA COMPOUNDS FOR TREATMENT OF PROSTATE CARCINOMA | |
JP4467559B2 (ja) | 細胞増殖を阻害する組成物および方法 | |
US20170016001A1 (en) | Asymmetric interfering rna compositions that silence k-ras and methods of uses thereof | |
Xiang et al. | Silencing of Notch3 using shRNA driven by survivin promoter inhibits growth and promotes apoptosis of human T-cell acute lymphoblastic leukemia cells | |
US20200370050A1 (en) | Cell proliferation inhibitor and cancer treatment or prevention pharmaceutical composition including cell proliferation inhibitor | |
CN115768891A (zh) | 用于抑制tdp-43和fus聚集的组合物和方法 | |
JP7450268B2 (ja) | がん幹細胞マーカー及びがん幹細胞標的薬 | |
EP2212421B1 (en) | Mirna, sirna and use thereof in therapy | |
KR102397455B1 (ko) | PD-1의 발현을 억제하는 비대칭 siRNA | |
JP2011188849A (ja) | 抗腫瘍効果を有するmiR−7発現プラスミド | |
JP2024019894A (ja) | ガレクチン-4陽性胃がん治療用医薬組成物 | |
JP2012171894A (ja) | 腫瘍縮小剤 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20080207 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20101208 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20110204 |
|
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20110509 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20110607 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20140617 Year of fee payment: 3 |
|
R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
LAPS | Cancellation because of no payment of annual fees |