CN116970606A - 用于抑制WT1基因表达的shRNA及应用 - Google Patents
用于抑制WT1基因表达的shRNA及应用 Download PDFInfo
- Publication number
- CN116970606A CN116970606A CN202310622105.XA CN202310622105A CN116970606A CN 116970606 A CN116970606 A CN 116970606A CN 202310622105 A CN202310622105 A CN 202310622105A CN 116970606 A CN116970606 A CN 116970606A
- Authority
- CN
- China
- Prior art keywords
- shrna
- breast cancer
- expression
- gene
- inhibiting
- 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.)
- Pending
Links
- 101150084041 WT1 gene Proteins 0.000 title claims abstract description 91
- 230000014509 gene expression Effects 0.000 title claims abstract description 79
- 108091027967 Small hairpin RNA Proteins 0.000 title claims abstract description 65
- 239000004055 small Interfering RNA Substances 0.000 title claims abstract description 63
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 33
- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 claims abstract description 51
- 208000022679 triple-negative breast carcinoma Diseases 0.000 claims abstract description 50
- 230000005012 migration Effects 0.000 claims abstract description 17
- 238000013508 migration Methods 0.000 claims abstract description 17
- 230000009545 invasion Effects 0.000 claims abstract description 16
- 239000003814 drug Substances 0.000 claims abstract description 14
- 239000013604 expression vector Substances 0.000 claims abstract description 13
- 206010027476 Metastases Diseases 0.000 claims description 12
- 230000009401 metastasis Effects 0.000 claims description 12
- 108091081021 Sense strand Proteins 0.000 claims description 9
- 230000000692 anti-sense effect Effects 0.000 claims description 9
- 239000002552 dosage form Substances 0.000 claims description 4
- 239000003937 drug carrier Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000002068 genetic effect Effects 0.000 claims 1
- 230000001225 therapeutic effect Effects 0.000 claims 1
- 108700020467 WT1 Proteins 0.000 abstract description 59
- 102000040856 WT1 Human genes 0.000 abstract description 57
- 108090000623 proteins and genes Proteins 0.000 abstract description 21
- 230000002829 reductive effect Effects 0.000 abstract description 10
- 241000713666 Lentivirus Species 0.000 abstract description 7
- 230000001105 regulatory effect Effects 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 description 56
- 206010006187 Breast cancer Diseases 0.000 description 29
- 208000026310 Breast neoplasm Diseases 0.000 description 29
- 108020004999 messenger RNA Proteins 0.000 description 14
- 210000001519 tissue Anatomy 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 11
- 206010028980 Neoplasm Diseases 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 230000004083 survival effect Effects 0.000 description 9
- 201000011510 cancer Diseases 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 238000004393 prognosis Methods 0.000 description 7
- 238000011529 RT qPCR Methods 0.000 description 6
- 230000007705 epithelial mesenchymal transition Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 238000012546 transfer Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 230000009368 gene silencing by RNA Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 102000000905 Cadherin Human genes 0.000 description 4
- 108050007957 Cadherin Proteins 0.000 description 4
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 4
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 4
- 108020004459 Small interfering RNA Proteins 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 230000002452 interceptive effect Effects 0.000 description 4
- 210000004072 lung Anatomy 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 238000002791 soaking Methods 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 239000008096 xylene Substances 0.000 description 4
- 101000990902 Homo sapiens Matrix metalloproteinase-9 Proteins 0.000 description 3
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000699660 Mus musculus Species 0.000 description 3
- 108050000637 N-cadherin Proteins 0.000 description 3
- 239000002033 PVDF binder Substances 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 108010065472 Vimentin Proteins 0.000 description 3
- 102000013127 Vimentin Human genes 0.000 description 3
- 210000000481 breast Anatomy 0.000 description 3
- 230000012292 cell migration Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 230000002055 immunohistochemical effect Effects 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000011580 nude mouse model Methods 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- -1 E-cadherein Proteins 0.000 description 2
- 241000237858 Gastropoda Species 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 108091030071 RNAI Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000004709 cell invasion Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 239000003531 protein hydrolysate Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 210000005048 vimentin Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OGBQILNBLMPPDP-UHFFFAOYSA-N 2,3,4,7,8-Pentachlorodibenzofuran Chemical compound O1C2=C(Cl)C(Cl)=C(Cl)C=C2C2=C1C=C(Cl)C(Cl)=C2 OGBQILNBLMPPDP-UHFFFAOYSA-N 0.000 description 1
- 208000021959 Abnormal metabolism Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000011281 clinical therapy Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000002991 immunohistochemical analysis Methods 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000006371 metabolic abnormality Effects 0.000 description 1
- 230000004066 metabolic change Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000024121 nodulation Effects 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000032361 posttranscriptional gene silencing Effects 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 210000003462 vein Anatomy 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
- C12N15/1135—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 against oncogenes or tumor suppressor genes
-
- 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
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- 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
- A61P35/04—Antineoplastic agents specific for metastasis
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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.
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Virology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种用于抑制WT1基因表达的shRNA在制备治疗三阴性乳腺癌的药物中的应用。所述用于抑制WT1基因表达的shRNA为shRNA‑1、shRNA‑2或shRNA‑3。本发明基于WT1基因的shRNA序列,设计筛选出3条shRNA可以显著降低WT1基因在三阴性乳腺癌细胞中的表达。通过构建相应的shRNA慢病毒表达载体,之后用含目标shRNA序列的慢病毒感染三阴性乳腺癌细胞MDA‑MB‑231以及BT549细胞后,能有效调控和干扰WT1基因的表达,从而抑制三阴性乳腺癌细胞侵袭和/或迁移的能力;因此该WT1基因靶向的shRNA,具有降低WT1的表达,有效抑制三阴性乳腺癌细胞的侵袭和/或迁移的能力。本发明为三阴性乳腺癌的治疗提供了一个新的基因靶点和药物,具备很高的临床实际应用价值。
Description
技术领域
本发明属于医药分子生物学技术领域,具体涉及一种用于抑制WT1基因表达的shRNA及应用。
背景技术
乳腺癌是最常见的女性恶性肿瘤,其发病率和死亡率呈逐年上升趋势。尽管近年来在乳腺癌治疗方面取得了进展,但侵袭转移仍然是导致乳腺癌患者死亡的主要原因。在乳腺癌亚型中,三阴性乳腺癌侵袭性强,预后最差。其中,肿瘤转移是TNBC(三阴性乳腺癌)患者预后差生存率低的重要原因,也是其临床治疗的瓶颈所在。因此,研究侵袭转移相关基因在三阴性乳腺癌中的作用,对于寻找三阴性乳腺癌有效治疗靶点,改善三阴性乳腺癌患者预后具有重要的理论意义和临床应用价值。
RNA干扰技术(RNAinterference,RNAi)是物种在进化上的一种高度保守的基因防御机制,即一种依赖于双链RNA特异性短序列实现转录后的基因沉默的技术。目前实验室常用的RNAi技术主要有siRNA寡核苷酸载体与shRNA慢病毒质粒表达载体。siRNA是短的双链RNA,大小在19-29nt左右,3’端有两个游离的碱基,可激活RNA的干扰,通过与目标mRNA互补序列结合,特异性地实现mRNA降解。但是siRNA在细胞中的表达是瞬时的,发挥作用的时间比较短。相对于siRNA,shRNA可以通过病毒介导的转染保持稳定,能够减少脱靶效应。shRNA包括两个短的反向重复序列,中间由茎环结构进行分割,组成一个类似发夹的结构。shRNA首先被插入到慢病毒载体上形成重组慢病毒质粒,慢病毒质粒与其他辅助质粒借助于293FT细胞形成慢病毒,最终用慢病毒转染细胞发挥shRNA的沉默作用。
WT1作为重要的转录调控因子,在癌症发生发展过程中也起到了重要的作用。对WT1与肿瘤相关的研究发现,不同类型的癌症均伴随着WT1在不同程度上的异常表达,WT1已经被证实在某些癌症中存在的高表达现象,并且和患者的低存活率密切相关。癌细胞转移是肿瘤恶化的标志,WT1可通过促进肿瘤细胞的迁移和浸润从而增强癌症的转移能力。但WT1在乳腺癌尤其是三阴性乳腺癌中的表达及功能尚未见任何报道。
肿瘤细胞的异常代谢是维持肿瘤细胞生长的必要条件,WT1在肿瘤细胞中具有促进代谢活动的作用,干扰WT1蛋白的活性使肿瘤代谢改变很有可能运用于癌症的治疗。但是目前没有关于shRNA干扰WT1基因在三阴性乳腺癌组织中表达情况及作用研究的相关报道。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此,本发明的主要目的在于提供一种用于抑制WT1基因表达的shRNA,其能有效抑制WT1基因在三阴性乳腺癌细胞中的表达,以及其能显著抑制三阴性乳腺癌细胞的侵袭和/或迁移能力;本发明所要解决的另一个技术问题在于提供抑制WT1基因的shRNA在制备抗三阴性乳腺癌的药物中的应用。
本发明的目的是通过以下技术方案实现的:
一种用于抑制WT1基因表达的shRNA,所述shRNA为shRNA-1、shRNA-2或shRNA-3;其中所述shRNA-1包含:
正义链:
5′-CCGGGCAGCTAACAATGTCTGGTTACTCGAGTAACCAGACATTGTTAGCTGCTTTTT-3′:
反义链:
5′-GATCCAAAAAGCAGCTAACAATGTCTGGTTACTCGAGTAACCAGACATTGTTAGCTGC-3′;
其中所述shRNA-2包含:
正义链:
5′-CCGGGCAGTGACAATTTATACCAAACTCGAGTTTGGTATAAATTGTCACTGCTTTTT-3′;
反义链:
5′-GATCCAAAAAGCAGTGACAATTTATACCAAACTCGAGTTTGGTATAAATTGTCACTGC-3′;
其中所述shRNA-3包含:
正义链:
5′-CCGGCCAGGCTGCAATAAGAGATATCTCGAGATATCTCTTATTGCAGCCTGGTTTTT-3′:
反义链:
5′-GATCCAAAAACCAGGCTGCAATAAGAGATATCTCGAGATATCTCTTATTGCAGCCTGG-3′。
本发明还提供了一种shRNA慢病毒表达载体,所述shRNA慢病毒表达载体包含前述shRNA;
本发明还提供了所述用于抑制WT1基因表达的shRNA或所述shRNA慢病毒表达载体的用途,所述shRNA或shRNA慢病毒表达载体能够抑制三阴性乳腺癌细胞中WT1基因的表达,从而显著抑制三阴性乳腺癌细胞的侵袭和/或迁移能力。
所述用于抑制WT1基因表达的shRNA或shRNA慢病毒表达载体的用途,用于获取抑制三阴性乳腺癌细胞转移的信息。
所述用于抑制WT1基因表达的shRNA或shRNA慢病毒表达载体的用途,可以基于该shRNA序列开发制备抑制三阴性乳腺癌细胞迁移和/或浸润的基因药物。
其中,所述药物包含前述shRNA或前述shRNA慢病毒表达载体以及药学上可接受的载体。
其中所述药物的剂型为任何药物治疗学上可接受的剂型。
其中所述药物的剂量为任何药物治疗学上可接受的剂量。
本发明基于WT1基因的shRNA序列,设计筛选出3条shRNA可以显著降低WT1基因在三阴性乳腺癌细胞中的表达。通过构建相应的shRNA慢病毒表达载体,之后用含目标shRNA序列的慢病毒感染三阴性乳腺癌细胞MDA-MB-231以及BT549细胞后,能有效调控和干扰WT1基因的表达,从而抑制三阴性乳腺癌细胞侵袭和/或迁移的能力;因此该WT1基因靶向的shRNA,具有降低WT1的表达,有效抑制三阴性乳腺癌细胞的侵袭和/或迁移的能力。本发明为三阴性乳腺癌的治疗提供了一个新的基因靶点和药物,具备很高的临床实际应用价值。
与现有技术相比,本发明至少具有以下优点:
1)本发明首次提出利用RNA干扰技术开发用于抑制WT1基因表达的shRNA,可以显著抑制三阴性乳腺癌细胞的侵蚀和/或迁移,是治疗三阴性乳腺癌的一种新型靶向药物制剂;
2)本发明所提供的用于抑制WT1基因表达的shRNA通过细胞实验证实,在抑制三阴性乳腺癌细胞的侵袭和/或迁移具有显著效果,能有效发挥抑制WT1基因表达的作用,对三阴性乳腺癌起到基因治疗的目的,为三阴性乳腺癌的临床治疗提供新的靶向治疗药物。
附图说明
为了更清楚地说明本发明具体实施方式,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍。
图1为WT1在乳腺癌组织中的表达情况及其与预后的关系;其中A为WT1 mRNA在乳腺癌和乳腺正常组织中的表达情况(METABRIC);其中图B为WT1 mRNA在乳腺癌和乳腺正常组织中的表达情况(TCGA数据库);C为免疫组化分析WT1在乳腺癌组织和癌旁组织的蛋白表达情况(WT1抗体:12609-1-AP);D为根据Kaplan-Meier plot分析WT1 mRNA在高低表达乳腺癌患者的生存差异(无复发生存率RFS);E为根据Kaplan-Meier plot分析WT1 mRNA在高低表达乳腺癌患者的生存差异(无远处转移生存率DMFS);F为WT1蛋白表达水平与乳腺癌患者预后关系。
图2为WT1在三阴性乳腺癌中的表达水平。其中A为WT1在乳腺癌亚型TNBC中较Luminal亚型显著高表达且较高于HER2+亚型;B为WT1在乳腺癌亚型TNBC中的表达情况;C、D为在TNBC亚型细胞系(MDA-MB231、BT549、Hs578t、HCC1806)中,WT1的mRNA和蛋白表达水平情况。
图3为shRNA干扰WT1效率鉴定的qRT-PCR(用于检测细胞中基因表达水平)和蛋白免疫印迹结果。
图4为shRNA干扰WT1表达的细胞侵袭迁移实验结果。其中A&B为在MDA-MB-231细胞中对照sh-Control、干扰sh-WT1-1(shRNA-1)以及sh-WT1-3(shRNA-3)组迁移及侵袭能力分析;C&D)为在BT549细胞中对照sh-Control、干扰sh-WT1-1(shRNA-1)以及sh-WT1-3(shRNA-3)组迁移及侵袭能力分析。
图5为shRNA-3干扰WT1表达后上皮间质转化EMT相关标志基因的蛋白表达情况。
图6为shRNA-3干扰WT1表达后TNBC细胞在动物体内的肺转移情况。
具体实施方式
下面结合附图和实施例对本发明作进一步详述,以下实施例只是描述性的,不是限定性的,不能以此限定本发明的保护范围。
除非另有说明,本文中所用的专业与科学术语与本领域熟练人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法或材料也可应用于本发明中。除了特别指明,以下实施例中所使用的技术手段和操作方法为本领域技术人员所熟知的常规手段和方法,所使用原料均为市售商品。
以下结合附图和实施例对本发明的技术方案做进一步的说明。下列实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等人,分子克隆实验室手册(NewYork:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。
实验材料:shRNA慢病毒购自中国吉凯公司,Anti-WT1(12609-1-AP),MMP9(10375-2-AP),E-cadherin(20874-1-AP),N-cadherin(22018-1-AP),Snail1(13099-1-AP)和Vimentin(10366-1-AP)购自中国武汉三鹰公司和Anti-beta Actin(ab8227)购自英国Abcam公司,Tanswell小室购自美国康宁公司,4%多聚甲醛固定液购自广州Biosharp生物科技有限公司,结晶紫染色液购自中国碧云天生物有限公司,Trizol购自中国TaKaRa公司,Trizol试剂购自美国Invitrogen公司,Takara逆转录试剂盒和荧光定量试剂盒购自日本Takara公司。
实施例1
WT1基因在三阴性乳腺癌患者中表达情况分析
WT1在乳腺癌中的表达情况生信分析:在METABRIC和TCGA数据库的mRNA表达数据以及临床信息,将其分为癌和癌旁两组分析WT1的表达情况,如图1A和1B,结果表明WT1mRNA在乳腺癌组织中的表达水平显著高于正常乳腺组织。
免疫组织化学染色步骤:取多聚甲醛固定的乳腺癌组织,进行石蜡包埋,连续切片,压在载玻片上;将免疫组化片,放在80℃烘箱内,烘片30min;在二甲苯I和二甲苯II中各浸泡15min;然后依次放入无水乙醇、95%乙醇、80%乙醇、70%乙醇各2min;最后将片子放入超纯水中浸泡清洗;将片子放在EDTA抗原修复液中,高压锅煮至冒气后,继续加热3min,离开热源,打开高压锅盖并冷却;PBST冲洗3次后,使用免疫组化笔圈出组织,再用H2O2室温孵育20min;用抗体稀释液稀释一抗,4℃孵育过夜;第二天,室温复温30min;PBST冲洗3次后,孵育二抗20min;PBST小心冲洗后,用DAB显色,观察颜色变化,当显示棕色时,用自来水终止反应;苏木精染30s,自来水冲洗,显微镜下观察,若染色不深,可重复苏木精染色;然后依次放入梯度乙醇浸泡4min,再放入二甲苯I和二甲苯II各浸泡10min。中性树脂封片,通风橱晾干后,显微镜下观察拍照;如图1C所示,可以发现WT1蛋白在乳腺癌组织中的表达明显高于癌旁组织。
预后分析:使用Kaplan–Meier数据库在乳腺癌预后中的影响,根据基因表达中值确定WT1对BC患者无复发生存率(RFS)和无远处转移生存率(DMFS)的影响。P<0.05表示有统计学意义的差异。WT1高表达患者的无复发生存率(图1D)和无远处转移生存率(图1E)显著低于低表达患者。根据WT1的免疫组化结果将组织芯片中乳腺癌患者分成高低表达2组,结果分析表明WT1高表达不利于乳腺癌患者预后(图1F)。
TCGA数据库分析WT1 mRNA在不同乳腺癌组织亚型中的表达情况:使用UALCAN数据库分析WT1在乳腺癌不同亚型中的表达情况,P<0.05表示有统计学意义的差异。
CCLE数据库中WT1在Basal以及Non-basal亚型乳腺癌细胞中的表达情况:使用CCLE数据库分析WT1在Basal以及Non-basal亚型乳腺癌细胞中的表达情况。结果如图2所示,WT1在乳腺癌亚型TNBC中较Luminal亚型显著高表达且较高于HER2+亚型(图2A),在CCLE数据库分析发现WT1在Basal较Non-basal中显著高表达,表明WT1在乳腺癌亚型TNBC中特异性高表达(图2B);在TNBC亚型细胞系(MDA-MB231、BT549、Hs578t、HCC1806)中,WT1的mRNA和蛋白表达水平显著高表达(图2C&D)。结果表明WT1在三阴性乳腺癌中的表达更高,可能参与调控三阴性乳腺癌的侵袭迁移能力。
实施例2:
针对WT1基因shRNA的设计、筛选和验证
根据WT1(Gene ID:7490,Human)transcript variant 4,mRNA(NM_024426.6)序列设计三种shRNA序列,由上海吉凯基因医学科技股份有限公司股份合成,其中所述shRNA-1包含:
正义链:
5′-CCGGGCAGCTAACAATGTCTGGTTACTCGAGTAACCAGACATTGTTAGCTGCTTTTT-3′(SEQIDNo.1):
反义链:
5′-GATCCAAAAAGCAGCTAACAATGTCTGGTTACTCGAGTAACCAGACATTGTTAGCTGC-3′(SEQIDNo.2);
其中所述shRNA-2包含:
正义链:
5′-CCGGGCAGTGACAATTTATACCAAACTCGAGTTTGGTATAAATTGTCACTGCTTTTT-3′(SEQID No.3);
反义链:
5′-GATCCAAAAAGCAGTGACAATTTATACCAAACTCGAGTTTGGTATAAATTGTCACTGC-3′(SEQID No.4);
其中所述shRNA-3包含:
正义链:5′-CCGGCCAGGCTGCAATAAGAGATATCTCGAGATATCTCTTATTGCAGCCTGGTTTTT-3′(SEQ ID No.5):
反义链:5′-GATCCAAAAACCAGGCTGCAATAAGAGATATCTCGAGATATCTCTTATTGCAGCCTGG-3′(SEQ ID No.6)。
基于上述抑制WT1基因表达的序列,由中国吉凯公司进一步设计并合成用于抑制WT1基因表达的shRNA-1/shRNA-2/shRNA-3质粒,并包装为shRNA-1/shRNA-2/shRNA-3慢病毒的形式用于细胞、动物和治疗等实验。
WT1 shRNA的筛选:
细胞培养条件及培养基:MDA-MB-231和BT549细胞株分别用含10%胎牛血清的DMEM和RPMI 1640(Gibco)培养在5%CO2,37℃的培养箱中。
细胞转染:按说明书分别将shRNA-1、shRNA-2和shRNA-3慢病毒分装待用;将细胞消化后,铺在6孔板中,当细胞密度汇合到30-50%时进行转染;准备四个无菌1.5mL的EP管,分别在四个EP管中加入转染试剂P 40μl和对应的shRNA-1、shRNA-2、shRNA-3及阴性对照shRNA溶液5μl吹打混匀,使shRNA和转染试剂形成转染复合物,再加入6孔板对应的孔中,轻晃摇匀;48h后,收集细胞提取mRNA和总蛋白,qRT-PCR免疫印迹法验证敲低效率,结果如图3所示。可以看出,与对照组比较,转染shRNA-1(KD1)、shRNA-2(KD2)和shRNA-3(KD3)的实验组的WT1蛋白的表达量都降低,从蛋白的相对表达量能清楚得看出,转染shRNA1和shRNA3的实验组对WT1蛋白表达的干扰最大,效果最好。因此可以将干扰WT1基因表达的shRNA1和shRNA3应用在干扰WT1基因表达中,用于后续研究。
实施例3:
qRT-PCR实验证明WT1干扰片段在乳腺癌细胞的表达以及shRNA-1和shRNA-3干扰效果:
取待测细胞去除培养基后,用PBS洗去残留培养基,将1mL Trizol加入培养皿中裂解细胞。冰上孵育10min后4℃,12000rpm离心10min,小心吸取上清液到新的无酶EP管中。加入200μl氯仿,剧烈振荡15秒,室温孵育5min。4℃,12000rpm离心15min。混合液分三层,包括最下面的苯酚-氯仿有机相,中间相和上层水相。小心吸取上层水相到新的无酶EP管(大约60%体积的Trizol),不要吸到中间相。加入等体积的异丙醇,轻轻地颠倒混匀约10次,室温放置30min。4℃,12000rpm离心10min。弃上清,用1mL 75%乙醇洗涤RNA沉淀两次。4℃,12000rpm离心5min,弃上清,室温下风干5min。将RNA溶于30μl DEPC水中。取2μl稀释5倍,取2μl RNA稀释液测量RNA浓度以及在260nm和280nm吸光度,260nm/280nm比率为2.0左右表明RNA的纯度较好。取5μl RNA稀释液通过琼脂糖凝胶电泳检测RNA的完整性,28S/18S≈2意味着RNA具有较高的完整度。根据PrimeScriptTMRT reagent Kit with gDNAEraser(PerfectReal Time)试剂盒操作说明进行反转录。根据TBPremix Ex TaqTMII(Tli RNaseHPlus)试剂盒进行qPCR实验。
结果如图3所示,图3为shRNA干扰WT1效率鉴定的qRT-PCR(用于检测细胞中WT1基因表达水平)和蛋白免疫印迹结果;通过qRT-PCR及Western blotting筛选在MDA-MB-231以及BT549细胞中WT1靶点的干扰效率。*:P<0.05,**:P<0.01。结果表明,与对照组比较,转染shRNAKD1(shRNA-1)、KD2(shRNA-2)和KD3(shRNA-3)的实验组的WT1 mRNA和蛋白的表达量都降低,但综合WT1蛋白的相对表达量表明,转染shRNA-1和shRNA-3的实验组对WT1蛋白表达的干扰效果最好。
实施例4:
Transwell分析实验证明WT1干扰片段shRNA-1和shRNA-3抑制乳腺癌细胞水平的侵袭迁移能力:
准备带小室的24孔板,将基质胶提前一天从-20℃放入4℃冰箱,24孔板、枪头、小室都提前预冷,所有操作都需在冰上进行。将转染后的细胞消化离心后,弃去上清,收集细胞沉淀,加入1mL无血清培养基重悬,计数后取100μl体积内含5×104个细胞,置于孔径为8.0μm的Transwell小室中,进行或不进行Matrigel处理,并在下室中装入含10%FBS的完全培养基600μl。温育18小时后,吸去上室细胞悬液,用4%多聚甲醛固定细胞20-30min,然后用结晶紫染色5min。用显微镜随机选择5个视野来计数迁移或侵袭细胞。结果如图4所示,A图和C图为迁移实验的细胞状态,迁移实验的统计学结果显示shRNA-1和shRNA-3干扰WT1转染的三阴性乳腺癌细胞MDA-MB-231和BT549后迁移能力显著减弱,细胞数量极显著降低(P<0.01),B图和D图为侵袭实验的细胞状态,侵袭实验的统计学结果显示shRNA-1和shRNA-3干扰WT1转染的三阴性乳腺癌细胞MDA-MB-231和BT549后侵袭能力明显减弱,细胞数量显著降低(P<0.01)。
实施例5
Western blotting实验检测WT1干扰片段shRNA-3对上皮间质转化EMT过程的影响::
收集sh-control组,shRNA-3组细胞,吸去培养液,置于冰上。加入1mL4℃预冷的PBS(0.01M pH 7.2~7.3)洗去残留培养基。按1mL裂解液加10μlPMSF(100mM)摇匀置于冰上。在6孔板细胞培养板每个孔中加入150μl含PMSF的裂解液,然后于冰上裂解30min后收集蛋白裂解液。将蛋白裂解液置于冰浴中,采用超声波破碎(功率200W,破碎1min,间歇30s)。破碎液于4℃12000rpm/min下离心30min,收集上清夜。用BCA定量法测定蛋白浓度,根据所测样品的吸光值用裂解液稀释至同一浓度。加入5×loading buffer 100℃水浴变性10min,保存于-80℃。根据蛋白分子量大小不同,选用所使用的10%和12%浓度分离胶和5%浓度浓缩胶。将配制好得SDS-PAGE胶放入电泳槽中,加足够的电泳液。将样品涡旋混匀,每个孔道加入总体积20μl的蛋白,先用80V跑30min,待蛋白Marker开始分离后将电压调至120V跑1h。待溴酚兰刚跑出时停止电泳进行转膜。按照一定比例配置好转膜液,将滤纸和海绵垫浸入转膜液中。与胶等大的PVDF膜在无水甲醇中激活30s后放入转膜液。在转膜夹两面放入各放一张海绵垫,然后将两片滤纸再分别放在海绵垫上面。在两层滤膜之间依次放入胶和PVDF膜(胶对应转膜板黑色面/仪器黑色面-连接电源负极,PCDF膜对应转膜板透明面/白色面/仪器红色面-连接电源正极),转膜时要注意赶走膜与胶之间的气泡,转膜仪器正负极与电源相对应。使用80V转150min。将PVDF膜放入3% BSA封闭液中,置于摇床上室温下封闭2h。TBST洗2次,每次5min。将膜按照所需要的目标蛋白所处位置剪切下来并做好标记,加入目标蛋白抗体,4℃过夜孵育。吸去一抗,用TBST洗涤3次,每次5min。用对应的二抗室温孵育1h。吸去二抗后用TBST洗涤3次,每次5min。1:1配制显影液,取适量显影液于显影仪台面上,将膜用镊子夹起来正反面充分和显影液接触,注意正面朝上。
结果如图5所示,图5为shRNA-3干扰WT1表达后上皮间质转化EMT相关标志基因的蛋白表达情况。实验结果如图5所示,MMP9、E-cadherin、N-cadherin、snail和Vimentin蛋白表达量明显下调;即通过shRNA-3干扰抑制WT1基因后,MDA-MB-231细胞中转移相关蛋白MMP9、E-cadherin、N-cadherin、Vimentin和snail表达量随WT1表达的下调而同步下调。因此,可以将干扰RNAshRNA-3应用到制备抑制细胞迁移和或侵袭相关基因表达的靶向药物中。
实施例6
动物模型实验检测WT1干扰片段shRNA-3抑制TNBC转移的效果:
收集sh-control组,shRNA组细胞,用PBS配置成1×106个/mL浓度的细胞悬液。取100μl细胞悬液注射入6-8周龄的裸鼠尾部静脉血管。待裸鼠体重明显变轻后处死取裸鼠肺部脏器拍照并制作石蜡切片进行HE染色。显微镜下检测并统计5个视野中肺部结节形成情况。
结果如图6所示,图6为shRNA-3干扰WT1表达后TNBC细胞在动物体内的肺转移情况。实验结果如图6所示,通过shRNA-3干扰抑制WT1基因后,MDA-MB-231细胞在裸鼠体内的肺转移结节随WT1表达的下调显著降低。
综上所述,本发明所提供的用于抑制WT1基因表达的shRNA可作为靶向药物,通过shRNA抑制WT1基因表达,能抑制三阴性乳腺癌细胞迁移和侵袭能力,对于临床开发三阴性乳腺癌靶向药物具有较好的应用前景。
以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围,其均应涵盖在本发明的权利要求和说明书的范围当中。
Claims (9)
1.一种用于抑制WT1基因表达的shRNA,其特征在于,所述shRNA为shRNA-1、shRNA-2或shRNA-3;其中所述shRNA-1包含:
正义链:
5′-CCGGGCAGCTAACAATGTCTGGTTACTCGAGTAACCAGACATTGTTAGCTGCTTTTT-3′:
反义链:
5′-GATCCAAAAAGCAGCTAACAATGTCTGGTTACTCGAGTAACCAGACATTGTTAGCTGC-3′;
其中所述shRNA-2包含:
正义链:
5′-CCGGGCAGTGACAATTTATACCAAACTCGAGTTTGGTATAAATTGTCACTGCTTTTT-3′;
反义链:
5′-GATCCAAAAAGCAGTGACAATTTATACCAAACTCGAGTTTGGTATAAATTGTCACTGC-3′;
其中所述shRNA-3包含:
正义链:
5′-CCGGCCAGGCTGCAATAAGAGATATCTCGAGATATCTCTTATTGCAGCCTGGTTTTT-3′:
反义链:
5′-GATCCAAAAACCAGGCTGCAATAAGAGATATCTCGAGATATCTCTTATTGCAGCCTGG-3′。
2.根据权利要求1所述的用于抑制WT1基因表达的shRNA,其特征在于,所述shRNA为shRNA-1或shRNA-2,其中shRNA-1和shRNA-2如权利要求1所述。
3.一种shRNA慢病毒表达载体,其特征在于,所述shRNA慢病毒表达载体包含权利要求1所述的shRNA。
4.权利要求1或2所述的用于抑制WT1基因的shRNA或权利要求3所述的shRNA慢病毒表达载体在三阴性乳腺癌细胞中WT1基因表达中非诊断治疗目的的应用。
5.根据权利要求4所述的应用,其特征在于,所述应用为抑制三阴性乳腺癌细胞的侵袭或/和迁移能力。
6.权利要求1或2所述的用于抑制WT1基因的shRNA或权利要求3所述的shRNA慢病毒表达载体在制备抑制三阴性乳腺癌细胞转移的基因药物中的应用。
7.根据权利要求6所述的应用,其特征在于,所述药物包含权利要求1或2所述的shRNA或权利要求3所述的shRNA慢病毒表达载体以及药学上可接受的载体。
8.根据权利要求6所述的应用,其特征在于,所述药物的剂型为任何药物治疗学上可接受的剂型。
9.根据权利要求6所述的应用,其特征在于,所述药物的剂量为任何药物治疗学上可接受的剂量。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310622105.XA CN116970606A (zh) | 2023-05-30 | 2023-05-30 | 用于抑制WT1基因表达的shRNA及应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310622105.XA CN116970606A (zh) | 2023-05-30 | 2023-05-30 | 用于抑制WT1基因表达的shRNA及应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116970606A true CN116970606A (zh) | 2023-10-31 |
Family
ID=88478547
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310622105.XA Pending CN116970606A (zh) | 2023-05-30 | 2023-05-30 | 用于抑制WT1基因表达的shRNA及应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116970606A (zh) |
-
2023
- 2023-05-30 CN CN202310622105.XA patent/CN116970606A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Wang et al. | Long noncoding RNA B3GALT5-AS1 suppresses colon cancer liver metastasis via repressing microRNA-203 | |
Guo et al. | Enhanced expression of TGFBI promotes the proliferation and migration of glioma cells | |
Yuan et al. | miR-542-3p inhibits colorectal cancer cell proliferation, migration and invasion by targeting OTUB1 | |
Lv et al. | Long noncoding RNA MNX1-AS1 knockdown inhibits cell proliferation and migration in ovarian cancer | |
Wu et al. | Oncogene FOXK1 enhances invasion of colorectal carcinoma by inducing epithelial-mesenchymal transition | |
Wang et al. | miR-206 inhibits cell proliferation, migration, and invasion by targeting BAG3 in human cervical cancer | |
Gong et al. | Long noncoding RNA linc00462 promotes hepatocellular carcinoma progression | |
Zhou et al. | MicroRNA-134 inhibits tumor stem cell migration and invasion in oral squamous cell carcinomas via downregulation of PI3K-Akt signaling pathway by inhibiting LAMC2 expression | |
CN111718995B (zh) | 一种鼻咽癌转移诊断和/或预后评估的生物标记 | |
Hong et al. | Long noncoding RNA LINC00460 conduces to tumor growth and metastasis of hepatocellular carcinoma through miR-342-3p-dependent AGR2 up-regulation | |
Yang et al. | Knockdown of lncRNA GHET1 inhibits osteosarcoma cells proliferation, invasion, migration and EMT in vitro and in vivo | |
Chen et al. | LncRNA LINC00944 promotes tumorigenesis but suppresses Akt phosphorylation in renal cell carcinoma | |
Gan et al. | Retracted Paper-Long Non-Coding RNA ZEB1-Antisense 1 Affects Cell Migration and Invasion of Cervical Cancer by Regulating Epithelial-Mesenchymal Transition via the p38MAPK Signaling Pathway | |
CN109288855B (zh) | 试剂在制备药物中的用途、干涉片段、抑制肝癌肿瘤干细胞自我更新方法和治疗肝癌药物 | |
Sun et al. | Targeting TGF-β1 suppresses survival of and invasion by anaplastic thyroid carcinoma cells | |
CN109055374B (zh) | 特异性抑制OCT1基因表达的shRNA及应用 | |
Zhu et al. | Differential effects of GLI2 and GLI3 in regulating cervical cancer malignancy in vitro and in vivo | |
Shi et al. | RPS15a silencing suppresses cell proliferation and migration of gastric cancer | |
Cui et al. | DGCR8 promotes the metastasis in triple-negative breast cancer by epigenetically regulating TGF-β. | |
CN104878098A (zh) | 人stim1基因的用途及其相关药物 | |
CN116970606A (zh) | 用于抑制WT1基因表达的shRNA及应用 | |
Si et al. | LncRNA LINC00649 aggravates the progression of cervical cancer through sponging miR‐216a‐3p | |
CN112220926B (zh) | Golt1b抑制剂在制备治疗结直肠癌药物中的应用 | |
CN113491772B (zh) | P4hb抑制剂在治疗或预防肿瘤恶病质中的用途 | |
CN108949974B (zh) | E3泛素连接酶asb3在制备肝癌治疗药物中的应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |