CN110923319B - Ptpre作为靶标在制备或筛选抗肝癌药物中的用途及其相关药物 - Google Patents
Ptpre作为靶标在制备或筛选抗肝癌药物中的用途及其相关药物 Download PDFInfo
- Publication number
- CN110923319B CN110923319B CN201911327688.3A CN201911327688A CN110923319B CN 110923319 B CN110923319 B CN 110923319B CN 201911327688 A CN201911327688 A CN 201911327688A CN 110923319 B CN110923319 B CN 110923319B
- Authority
- CN
- China
- Prior art keywords
- ptpre
- liver cancer
- smad3
- gene
- expression
- 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.)
- Active
Links
- 208000014018 liver neoplasm Diseases 0.000 title claims abstract description 85
- 201000007270 liver cancer Diseases 0.000 title claims abstract description 84
- 239000003814 drug Substances 0.000 title claims abstract description 27
- 239000003560 cancer drug Substances 0.000 title claims abstract description 20
- 229940079593 drug Drugs 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 101000606535 Homo sapiens Receptor-type tyrosine-protein phosphatase epsilon Proteins 0.000 title claims abstract 11
- 102100039665 Receptor-type tyrosine-protein phosphatase epsilon Human genes 0.000 title claims abstract 10
- 238000012216 screening Methods 0.000 title abstract description 8
- 101710143111 Mothers against decapentaplegic homolog 3 Proteins 0.000 claims abstract description 65
- 206010027476 Metastases Diseases 0.000 claims abstract description 47
- 230000009401 metastasis Effects 0.000 claims abstract description 41
- 230000014509 gene expression Effects 0.000 claims abstract description 27
- 101150065794 Ptpre gene Proteins 0.000 claims abstract description 26
- 230000009545 invasion Effects 0.000 claims abstract description 22
- 241000700605 Viruses Species 0.000 claims abstract description 19
- 239000004055 small Interfering RNA Substances 0.000 claims abstract description 18
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 17
- 239000013598 vector Substances 0.000 claims abstract description 17
- 108091027967 Small hairpin RNA Proteins 0.000 claims abstract description 11
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 claims abstract description 11
- 102000004887 Transforming Growth Factor beta Human genes 0.000 claims abstract description 10
- 108090001012 Transforming Growth Factor beta Proteins 0.000 claims abstract description 10
- 230000035755 proliferation Effects 0.000 claims abstract description 9
- 102100025748 Mothers against decapentaplegic homolog 3 Human genes 0.000 claims abstract description 7
- 230000033228 biological regulation Effects 0.000 claims abstract description 6
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 6
- 206010073071 hepatocellular carcinoma Diseases 0.000 claims description 14
- 241000713666 Lentivirus Species 0.000 claims description 12
- 239000013612 plasmid Substances 0.000 claims description 12
- 238000004806 packaging method and process Methods 0.000 claims description 11
- 238000001514 detection method Methods 0.000 claims description 9
- 238000001727 in vivo Methods 0.000 claims description 8
- 230000002452 interceptive effect Effects 0.000 claims description 4
- 239000004480 active ingredient Substances 0.000 claims description 3
- 239000003147 molecular marker Substances 0.000 claims description 3
- 239000000411 inducer Substances 0.000 claims description 2
- 230000010534 mechanism of action Effects 0.000 claims 1
- CDKIEBFIMCSCBB-UHFFFAOYSA-N 1-(6,7-dimethoxy-3,4-dihydro-1h-isoquinolin-2-yl)-3-(1-methyl-2-phenylpyrrolo[2,3-b]pyridin-3-yl)prop-2-en-1-one;hydrochloride Chemical compound Cl.C1C=2C=C(OC)C(OC)=CC=2CCN1C(=O)C=CC(C1=CC=CN=C1N1C)=C1C1=CC=CC=C1 CDKIEBFIMCSCBB-UHFFFAOYSA-N 0.000 abstract description 58
- 102000049939 Smad3 Human genes 0.000 abstract description 58
- 230000026731 phosphorylation Effects 0.000 abstract description 16
- 238000006366 phosphorylation reaction Methods 0.000 abstract description 16
- 230000000694 effects Effects 0.000 abstract description 15
- 102000014172 Transforming Growth Factor-beta Type I Receptor Human genes 0.000 abstract description 13
- 108010011702 Transforming Growth Factor-beta Type I Receptor Proteins 0.000 abstract description 13
- 230000007115 recruitment Effects 0.000 abstract description 6
- 230000004913 activation Effects 0.000 abstract description 4
- 230000007246 mechanism Effects 0.000 abstract description 4
- 102000004190 Enzymes Human genes 0.000 abstract description 3
- 108090000790 Enzymes Proteins 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 108091005735 TGF-beta receptors Proteins 0.000 abstract 1
- 102000016715 Transforming Growth Factor beta Receptors Human genes 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 116
- 210000001519 tissue Anatomy 0.000 description 36
- 238000012546 transfer Methods 0.000 description 20
- 230000002018 overexpression Effects 0.000 description 18
- 238000002474 experimental method Methods 0.000 description 16
- 230000035772 mutation Effects 0.000 description 15
- 210000004072 lung Anatomy 0.000 description 14
- 206010028980 Neoplasm Diseases 0.000 description 12
- 210000004185 liver Anatomy 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 229920000936 Agarose Polymers 0.000 description 10
- 108060001084 Luciferase Proteins 0.000 description 10
- 239000011324 bead Substances 0.000 description 10
- 230000029918 bioluminescence Effects 0.000 description 10
- 238000005415 bioluminescence Methods 0.000 description 10
- 239000005089 Luciferase Substances 0.000 description 9
- 239000013642 negative control Substances 0.000 description 9
- 230000000638 stimulation Effects 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 8
- 101150063416 add gene Proteins 0.000 description 8
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 102000005962 receptors Human genes 0.000 description 8
- 108020003175 receptors Proteins 0.000 description 8
- 238000004393 prognosis Methods 0.000 description 7
- 238000010276 construction Methods 0.000 description 6
- 238000011534 incubation Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 239000006166 lysate Substances 0.000 description 5
- 230000009261 transgenic effect Effects 0.000 description 5
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 4
- 238000012404 In vitro experiment Methods 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 101710120037 Toxin CcdB Proteins 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 239000013592 cell lysate Substances 0.000 description 4
- 239000008004 cell lysis buffer Substances 0.000 description 4
- 238000010195 expression analysis Methods 0.000 description 4
- 239000013613 expression plasmid Substances 0.000 description 4
- 230000002055 immunohistochemical effect Effects 0.000 description 4
- 238000011065 in-situ storage Methods 0.000 description 4
- 239000012139 lysis buffer Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 238000011580 nude mouse model Methods 0.000 description 4
- 230000002685 pulmonary effect Effects 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 230000019491 signal transduction Effects 0.000 description 4
- 238000002054 transplantation Methods 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 3
- 101150077909 Smad3 gene Proteins 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000000875 corresponding effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002991 immunohistochemical analysis Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- 238000011729 BALB/c nude mouse Methods 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 2
- 102000002727 Protein Tyrosine Phosphatase Human genes 0.000 description 2
- 230000001476 alcoholic effect Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 230000009400 cancer invasion Effects 0.000 description 2
- 230000004709 cell invasion Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 244000144993 groups of animals Species 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 238000013115 immunohistochemical detection Methods 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108020000494 protein-tyrosine phosphatase Proteins 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 208000000419 Chronic Hepatitis B Diseases 0.000 description 1
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 1
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 102000007374 Smad Proteins Human genes 0.000 description 1
- 108010007945 Smad Proteins Proteins 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 102000004060 Transforming Growth Factor-beta Type II Receptor Human genes 0.000 description 1
- 108010082684 Transforming Growth Factor-beta Type II Receptor Proteins 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000002115 aflatoxin B1 Substances 0.000 description 1
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 description 1
- 229930020125 aflatoxin-B1 Natural products 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 208000016350 chronic hepatitis B virus infection Diseases 0.000 description 1
- 238000000749 co-immunoprecipitation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000010864 dual luciferase reporter gene assay Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 208000010710 hepatitis C virus infection Diseases 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108091006082 receptor inhibitors Proteins 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Food Science & Technology (AREA)
- Hospice & Palliative Care (AREA)
- General Physics & Mathematics (AREA)
- Oncology (AREA)
- Epidemiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供了PTPRE作为靶标在制备或筛选抗肝癌药物中的用途。本发明通过大量的实验数据证明,PTPRE能够促进肝癌细胞的侵袭与转移,通过进一步的机制研究发现PTPRE参与TGF‑β/SMAD3信号通路的调节,PTPRE能够介导TGF‑β受体TGFBR1对SMAD3的招募,促进其磷酸化激活。且发现PTPRE的两个酶活位点对于SMAD3磷酸化激活至关重要。因此针对PTPRE为靶标,可以筛选药物,该药物通过抑制PTPRE基因的表达或者抑制PTPRE蛋白的酶活从而抑制其对TGF‑β/SMAD3信号通路的调节,进而能够抑制肝癌细胞的增殖、侵袭与转移。且本发明的PTPRE基因的shRNA、慢病毒载体及慢病毒能够抑制PTPRE的表达,因此可作为抗肝癌药物。
Description
技术领域
本发明涉及分子生物学技术领域,尤其涉及PTPRE作为靶标在制备或筛选抗肝癌药物中的用途及其相关药物。
背景技术
肝细胞癌(HCC)是世界上最常见的恶性肝癌之一。其发病机制是极其复杂的,可以有多种原因,包括慢性乙型肝炎病毒或丙型肝炎病毒感染、接触黄曲霉毒素B1、酒精性和非酒精性脂肪性疾病等。尽管最近的研究发现了许多影响肝癌发展的基因和途径,但治疗和患者预后仍不能令人满意。
TGF-β是一种多向性、多效性的细胞因子,以自分泌或旁分泌的方式通过细胞表面的受体信号转导途径调节细胞的增殖、分化、凋亡,对细胞外基质的合成、创伤的修复、免疫功能等有重要的调节作用。成熟的TGF-β是通过二硫键连接而成的分子量为25×103的同质二聚体。TGF-β是参与慢性肝脏疾病和癌症的发病机理的重要的多功能细胞因子。TGF-β/smad信号通路为:TGF-β与TGF-βII型受体结合后,再激活募集TGF-βI型受体组合后形成二聚体形式的受体复合物。TGF-βII型受体磷酸化TGF-βI型受体的甘氨酸-丝氨酸富集区域(GS序列)并活化TGF-βI型受体的丝氨酸/苏氨酸活性。活化的TGF-βI型受体又磷酸化受体相关smad蛋白。其中smad2和smad3直接被TGF-βI型受体磷酸化,使得构象发生改变从而从受体复合物中释放出来,进入细胞核,参与细胞核内相关基因的调控。然而,smad2-smad3如何介导TGF-β在肝癌入侵与转移中的机制仍不完全清楚,需要进一步研究。
PTPRE(Protein Tyrosine Phosphatase Receptor Epsilon)受体型蛋白酪氨酸磷酸酶epsilon,属于蛋白酪氨酸磷氨酸家族,催化蛋白的酪氨酸去磷酸化反应,从而调节信号通路的转导。但是目前PTPRE在肝癌入侵与转移中尚无相关报道。
发明内容
本发明的目的在于克服现有技术之缺陷,提供PTPRE作为靶标在制备或筛选抗肝癌药物中的用途及其相关药物。
本发明是这样实现的:
本发明的目的之一在于提供PTPRE作为靶标在制备或筛选抗肝癌药物中的用途。
优选地,所述抗肝癌药物包括抑制肝癌细胞的增殖、侵袭与转移的药物。
所述抗肝癌药物的作用机理包括:抑制PTPRE基因的表达或者抑制PTPRE蛋白的酶活从而抑制其对TGF-β/SMAD3信号通路的调节。
本发明的目的之二在于提供PTPRE作为分子标志物在制备肝癌细胞的增殖、侵袭与转移的检测试剂盒中的用途。实验表明PTPRE的表达与肝癌细胞的增殖、侵袭与转移成正相关,因此可以作为分子标志物。所述检测试剂盒可包括PTPRE的多克隆抗体或单克隆抗体,或者实时定量PCR检测试剂盒等等,只要能够检测PTPRE的常规方法均在本发明的保护范围之内。
本发明的目的之三在于提供一种抗肝癌药物该抗肝癌药物包括抑制PTPRE的表达的药物或抑制PTPRE酶活的药物。
具体地,所述抗肝癌药物包括能够抑制PTPRE的表达或抑制PTPRE酶活的药物(不限于有机合成药物、或者中药组合物等),以及抑制PTPRE表达的核酸分子药物。
所述抑制PTPRE表达的核酸分子药物包括PTPRE基因的shRNA,所述PTPRE基因shRNA中含有能够在严紧条件下与PTPRE基因杂交的核苷酸序列。其包括序列如SEQ IDNO.1-3任一所示的shRNA。
所述抗肝癌药物还包括可诱导shRNA在体内表达的诱导剂。
本发明的目的之四在于提供抗肝癌的慢病毒载体药物,其有效成分含有PTPRE基因干扰慢病毒载体。所述慢病毒载体的表达区序列如SEQ NO.1-3任一所示。
本发明的目的之五在于提供一种抗肝癌的慢病毒药物,其有效成分含有PTPRE基因干扰慢病毒,所述PTPRE基因干扰慢病毒由所述PTPRE基因干扰慢病毒载体在慢病毒包装质粒、细胞系的辅助下,经过病毒包装而成。
含有治疗有效量的所述shRNA或者所述PTPRE基因干扰慢病毒,以及药学上可接受的载体、稀释剂或赋形剂组成的药物组合物也在本发明的保护范围之内。
本发明具有的有益效果是:
本发明通过大量的实验数据证明,PTPRE能够促进肝癌细胞的侵袭与转移,通过进一步的机制研究发现PTPRE能够介导受体TGFBR1对SMAD3的招募,使其磷酸化激活。且发现PTPRE的两个酶活位点对于SMAD3磷酸化至关重要。因此针对PTPRE为靶标,可以筛选药物,该药物通过抑制PTPRE基因的表达或者抑制PTPRE蛋白的酶活从而抑制其对TGF-β/SMAD3信号通路的调节,进而能够抑制肝癌细胞的增殖、侵袭与转移,因此本发明为药物筛选提供一个新的靶标。且本发明的PTPRE基因的shRNA、慢病毒载体及慢病毒能够抑制PTPRE的表达,因此可以作为抗肝癌药物。
附图说明
图1为本发明实施例1的SMAD3在体外的检测结果;其中图1A为SMAD3过表达细胞系与SMAD3敲减细胞系的鉴定结果;图1B和图1E为过表达细胞系与敲减细胞系的划痕实验结果;图1C和图1F为过表达细胞系与敲减细胞系的转移实验结果;图1D和图1G为过表达细胞系与敲减细胞系的侵袭实验结果;
图2为本发明实施例1的SMAD3在体内的检测结果;其中图2A为肝癌组织和癌旁组织中的SMAD3的免疫组化结果和表达分析检测结果,图中横坐标中N代表癌旁组织,C代表肝癌组织;图2B为SMAD3高表达和低表达的病人的预后结果;图2C为SMAD3过表达后肝癌肺转移的结果;图2D为SMAD3敲减后肝癌肺转移的结果;图2E为SMAD3过表达后肝癌肝内转移的结果;图2F为SMAD3敲减后肝癌肝内转移的结果;
图3为本发明实施例1的PTPRE在体外的检测结果;其中图3A为PTPRE过表达细胞系与PTPRE敲减细胞系的鉴定结果;图3B为过表达细胞系与敲减细胞系的划痕实验结果;图3C为过表达细胞系与敲减细胞系的转移实验结果;图3D为过表达细胞系与敲减细胞系的侵袭实验结果;
图4为本发明实施例1的PTPRE在体内的检测结果;图4A为PTPRE过表达后肝癌肺转移的结果;图4B为PTPRE敲减后肝癌肺转移的结果;图4C为PTPRE过表达后肝癌肝内转移的结果;图4D为PTPRE敲减后肝癌肝内转移的结果;图4E为肝癌组织和癌旁组织中的PTPRE的免疫组化结果和表达分析检测结果,图中横坐标中N代表癌旁组织,C代表肝癌组织;图4F为PTPRE高表达和低表达的病人的预后结果;
图5是实施例3中PTPRE与TGFBR1、SMAD3之间存在相互作用示意图;其中图5A为PTPRE分别与TGFBR1,以及SMAD3共转后的Co-IP结果;图5B为内源性PTPRE与TGFBR1相互作用的结果;如图5C为内源性PTPRE与SMAD3相互作用的结果;
图6是实施例3中PTPRE介导受体TGFBR1对SMAD3的招募的示意图;其中图6A、6B为PTPRE分别与TGFBR1,以及SMAD3共转后的正拉、反拉的的Co-IP结果;图6C为PTPRE促进TGF-β诱导的SMAD3的磷酸化的结果;
图7为实施例4中PTPRE的突变体系的建立结果;其中图7A表明PTPRE的两个突变位点Y164A、D250A在TGF-β刺激下对SBE4荧光素酶的活性调控结果;其中图7B为PTPRE的DM突变(Y164A、D250A同时突变)与野生型WT、空载VEC在TGF-β刺激下对SBE4荧光素酶的活性调控结果;图7C为DM突变与野生型WT、空载VEC构建的稳定细胞系的鉴定结果;图7D为PTPRE-WT、PTPRE-DM以及VEC稳定细胞系在不同时间的TGF-β刺激下对SMAD3磷酸化水平的调控结果;图7E和7F为DM突变与野生型WT、空载VEC的稳定细胞系的体外迁移和侵袭实验结果。
具体实施方式
实施例1 SMAD3促进肝癌细胞的增殖、转移与侵袭
一、体外实验
1、SMAD3过表达细胞系与敲减细胞系的构建
(1)设计SMAD3-shRNA靶序列(5’-GAGCCTGGTCAAGAAACTCAA-3’,如SEQ NO.4所示);交由上海生工生物公司合成含有SMAD3-shRNA序列的引物构建pLKO.1-shRNA质粒。重组慢病毒表达载体进行病毒包装:培养293T细胞,取上述pLKO.1-shRNA质粒与慢病毒包装质粒pMD2.G(Addgene#12259),psPAX2(Addgene#12260)一起共转染293T细胞(三者比例为4:1:3),按照病毒包装辅助试剂盒操作说明书进行操作,48h后收集病毒上清,1500g离心10min后取上清经0.45μM滤膜过滤,于-80℃保存。将包装成功的慢病毒分别感染细胞LM3,得到PTPRE敲减细胞系LM3。
(2)SMAD3过表达细胞系的构建:构建含有PTPRE编码序列的慢病毒载体pLenti-SMAD3。所述pLenti-SMAD3载体采用常规克隆方法构建,然后将pLenti-SMAD3与慢病毒包装质粒pMD2.G(Addgene#12259),psPAX2(Addgene#12260)一起共转染293T细胞(三者比例为4:1:3),按照病毒包装辅助试剂盒操作说明书进行操作,48h后收集病毒上清,1500g离心10min后取上清经0.45μM滤膜过滤,于-80℃保存。将包装成功的过表达慢病毒分别感染细胞7721,得到SMAD3过表达细胞系7721。
图1A为SMAD3过表达细胞系与敲减细胞系的鉴定结果;由图1A可知SMAD3过表达细胞系及敲减细胞系构建成功。
2、Transwell与细胞划痕实验检测SMAD3对肝癌细胞系转移能力的影响
图1B和图1E为过表达细胞系与敲减细胞系的划痕结果;图1C和图1F为过表达细胞系与敲减细胞系的转移结果;结果表明过表达细胞系的肝癌细胞转移能力增加;敲减细胞系的肝癌细胞转移能力降低。表明SMAD3促进肝癌细胞的转移。
3、Matrigel实验检测SMAD3对肝癌细胞系侵袭能力的影响
图1D和图1G为过表达细胞系与敲减细胞系的侵袭结果;结果表明过表达细胞系的肝癌细胞侵袭能力增加;敲减细胞系的肝癌细胞侵袭能力降低。表明SMAD3促进肝癌细胞的侵袭。
二、临床样本及体内实验
1、病人肝癌组织和癌旁组织SMAD3的表达情况
选取病人肝癌组织和癌旁组织,分别做免疫组化检测SMAD3的表达。肝癌组织和癌旁组织由华中科技大学同济医学院附属同济医院提供。整个采集及后续实验过程符合医学伦理道德要求并严格遵循病例资料的保密原则。组织样品经手术取出后,经福尔马林固定、石蜡包埋后切片备用。
图2A为肝癌组织和癌旁组织中的SMAD3的免疫组化结果和表达分析检测结果,图中横坐标中N代表癌旁组织,C代表肝癌组织;由图可知,SMAD3在肝癌组织的表达量明显高于癌旁组织。
图2B为SMAD3高表达和低表达的病人的预后结果;表明肝癌组织中SMAD3的表达量越高病人的预后效果越差。
2、肺转移模型以及肝内转移模型验证SMAD3基因的促转移功能
(1)肺转移模型结果
肺转移模型中,将含荧光素酶的HCC细胞(1×106)注入5周龄雄性BALB/C裸小鼠尾静脉。每组6只老鼠。每只小鼠注射后第1、4、5、6周分别捕获荧光素酶生物发光。所有小鼠于注射后6周处死。每只小鼠的肺分离固定,进行H&E染色。在显微镜下计数各组转移灶的平均数量。所述肺转移模型中实验动物分为4组分别为:分别转入实施例1制备得到敲低SMAD3的稳转细胞系及其阴性对照组;转入SMAD3基因的稳转细胞系及其阴性对照组(空载)。
图2C为SMAD3过表达后肝癌肺转移的结果;由图2C的左图可知,SMAD3过表达后生物发光强度和荧光数量明显大于对照组;图2C的右图可知,SMAD3过表达后转移灶的平均数量明显大于对照组。
图2D为SMAD3敲减后肝癌肺转移的结果;由图2D的左图可知,SMAD3敲减后生物发光强度和荧光数量明显小于对照组;图2D的右图可知,SMAD3敲减后转移灶的平均数量明显小于对照组。
(2)肝内转移模型结果
在原位移植和肝内转移模型中,将含有荧光素酶的HCC细胞(1×106)注射到5周大的BALB/C雄性裸小鼠皮下,形成肿瘤异种移植物。将皮下移植瘤模型的肿瘤结节分离并切成1mm3块,植入裸鼠(雄性,5周龄)肝左外叶,模拟原发性HCC。每组6只老鼠。植入8周后处死小鼠,计数肝内可见肿瘤结节。所述原位移植和肝内转移模型中实验动物也分为4组分别为:分别转入敲低SMAD3稳转细胞系及其阴性对照组;转入SMAD3基因的稳转细胞系及其阴性对照组(空载)。
图2E为SMAD3过表达后肝癌肝内转移的结果;由图2E可知,SMAD3过表达后生物发光强度和荧光数量明显大于对照组。
图2F为SMAD3敲减后肝癌肝内转移的结果;由图2F可知,SMAD3敲减后生物发光强度和荧光数量明显小于对照组。
综上可知,体内实验与体外实验均证明SMAD3能促进肝癌细胞的侵袭与转移。
实施例2 PTPRE促进肝癌细胞的侵袭与转移
一、体外实验
1、PTPRE过表达细胞系与敲减细胞系的构建
(1)PTPRE shRNA的靶序列如表1所示,交由上海生工生物公司合成含有SMAD3-shRNA序列的引物构建得到pLKO.1-shRNA质粒。
表1
将质粒pMD2.G(Addgene#12259),psPAX2(Addgene#12260)和pLKO.1-shRNA质粒(三者比例为1:3:4)共转染293T细胞。按照慢病毒包装辅助试剂盒操作说明书进行操作,48h后收集病毒上清,1500g离心10min后取上清经0.45μM滤膜过滤,用PEG-8000沉淀,于-80℃保存。将包装成功的慢病毒分别感染细胞ALEX、LM3,构建PTPRE敲减的ALEX、LM3细胞系。
(2)PTPRE过表达细胞系的构建:构建含有PTPRE编码序列的慢病毒载体pLenti-PTPRE,所述载体的编码区的序列如SEQ NO.5所示。所述pLenti-PTPRE载体采用常规克隆方法构建,然后将pLenti-PTPRE与慢病毒包装质粒pMD2.G(Addgene#12259),psPAX2(Addgene#12260)一起共转染293T细胞(三者比例为4:1:3),按照病毒包装辅助试剂盒操作说明书进行操作,48h后收集病毒上清,1500g离心10min后取上清经0.45μM滤膜过滤,于-80℃保存。将包装成功的过表达慢病毒分别感染细胞7721、HLF,得到PTPRE过表达的7721细胞系和HLF细胞系。
图3A为PTPRE过表达细胞系与敲减细胞系的鉴定结果;由图3A可知PTPRE过表达及敲减细胞系构建成功。
2、Transwell与细胞划痕实验检测PTPRE对肝癌细胞系转移能力的影响
图3B为过表达细胞系与敲减细胞系的划痕结果;图3C为过表达细胞系与敲减细胞系的转移结果;结果表明过表达细胞系的肝癌细胞系转移能力增加;敲减细胞系的肝癌细胞系转移能力降低。表明PTPRE促进肝癌细胞的转移。
3、Matrigel实验检测PTPRE对肝癌细胞系侵袭能力的影响
图1D为过表达细胞系与敲减细胞系的侵袭结果;结果表明过表达细胞系的肝癌细胞系侵袭能力增加;敲减细胞系的肝癌细胞系侵袭能力降低。表明PTPRE促进肝癌细胞的侵袭。
二、临床样本及体内实验
1、病人肝癌组织和癌旁组织的PTPRE表达情况
选取病人肝癌组织和癌旁组织,分别做免疫组化检测PTPRE的表达。肝癌组织和癌旁组织由华中科技大学同济医学院附属同济医院提供。整个采集及后续实验过程符合医学伦理道德要求并严格遵循病例资料的保密原则。组织样品经手术取出后,经福尔马林固定、石蜡包埋后切片备用。
图4E为肝癌组织和癌旁组织中的PTPRE的免疫组化结果和表达分析检测结果,图中横坐标中N代表癌旁组织,C代表肝癌组织;由图可知,PTPRE在肝癌组织的表达量明显高于癌旁组织。图4F为PTPRE高表达和低表达的病人的预后结果;表明肝癌组织中PTPRE的表达量越高,病人的预后效果越差。
2、肺转移模型以及肝内转移模型验证PTPRE基因的促转移功能
(1)肺转移模型结果
肺转移模型中,将含荧光素酶的HCC细胞(1×106)注入5周龄雄性BALB/C裸小鼠尾静脉。每组6只老鼠。每只小鼠注射后第1、4、5、6周分别捕获荧光素酶生物发光。所有小鼠于注射后6周处死。每只小鼠的肺分离固定,进行H&E染色。在显微镜下计数各组转移灶的平均数量。所述肺转移模型中实验动物分为4组分别为:分别转入实施例1制备得到敲低PTPRE的稳转细胞系及其阴性对照组;转入PTPRE基因的稳转细胞系及其阴性对照组(空载)。
图4A为PTPRE过表达后肝癌肺转移的结果;由图4A的左图可知,PTPRE过表达后生物发光强度和荧光数量明显大于对照组;图4A的右图可知,PTPRE过表达后转移灶的平均数量明显大于对照组。
图4B为PTPRE敲减后肝癌肺转移的结果;由图4B的左图可知,PTPRE敲减后生物发光强度和荧光数量明显小于对照组;图4B的右图可知,PTPRE敲减后转移灶的平均数量明显小于对照组。
(2)肝内转移模型结果
在原位移植和肝内转移模型中,将含有荧光素酶的HCC细胞(1×106)皮下注射到5周大的BALB/C雄性裸小鼠的侧边,形成肿瘤异种移植物。将皮下移植瘤模型的肿瘤结节分离并切成1mm3块,植入裸鼠(雄性,5周龄)肝左外叶,模拟原发性HCC。每组6只老鼠。植入8周后处死小鼠,计数肝内可见肿瘤结节。所述原位移植和肝内转移模型中实验动物也分为4组分别为:分别转入敲低PTPRE稳转细胞系及其阴性对照组;转入PTPRE基因的稳转细胞系及其阴性对照组(空载)。
图4C为PTPRE过表达后肝癌肝内转移的结果;由图4C可知,PTPRE过表达后生物发光强度和荧光数量明显大于对照组。
图4D为PTPRE敲减后肝癌肝内转移的结果;由图4D可知,PTPRE敲减后生物发光强度和荧光数量明显小于对照组。
综上可知,体内实验于体外实验均证明PTPRE能促进肝癌细胞的侵袭与转移。
实施例3
1、通过免疫共沉淀确定相互作用
构建Flag-PTPRE、HA-TGFBR1、HA-SMAD3表达质粒(本发明的Flag、HA为常见的Flag、HA标签),将Flag-PTPRE表达质粒与HA-TGFBR1共转、Flag-PTPRE表达质粒与HA-SMAD3表达质粒共转后30小时,收取细胞,加入适量细胞裂解缓冲液(含蛋白酶抑制剂),冰上裂解30min,细胞裂解液于4℃,最大转速离心15min后取上清得到细胞总蛋白。取少量裂解液以备Western blot分析(input分析),剩余裂解液加1μg相应的抗体(剩余裂解液分为2份,一份加入1ug抗HA抗体共同孵育,另外一份加入1ug IgG共同孵育)加入到细胞裂解液,4℃缓慢摇晃孵育过夜;取10μl protein G琼脂糖珠,用适量裂解缓冲液洗3次,每次3,000rpm离心3min;将预处理过的protein G琼脂糖珠加入到和抗体孵育过夜的细胞裂解液中4℃缓慢摇晃孵育2-4h,使抗体与protein G琼脂糖珠偶连;免疫沉淀反应后,在4℃以3,000rpm速度离心3min,将琼脂糖珠离心至管底;将上清小心吸去,琼脂糖珠用1ml裂解缓冲液洗3-4次;最后加入30μl的2×SDS上样缓冲液,沸水煮5分钟;Western blotting分析。
图5A为Flag-PTPRE分别与HA-TGFBR1,以及HA-SMAD3共转后的Co-IP结果;表明与阴性对照组相比,PTPRE特异的与TGFBR1、SMAD3发生相互作用。
2、内源的相互作用
向肝癌细胞加TGF-β刺激,刺激1小时时间后收取细胞,加入适量细胞裂解缓冲液(含蛋白酶抑制剂),冰上裂解30min,细胞裂解液于4℃,最大转速离心30min后取上清得到细胞总蛋白。取少量裂解液以备Western blot分析(input分析),剩余裂解液加5μg相应的抗体加入到细胞裂解液4℃缓慢摇晃孵育过夜;取10μl protein G琼脂糖珠,用适量裂解缓冲液洗3次,每次3,000rpm离心3min;将预处理过的琼脂糖珠加入到和抗体孵育过夜的细胞裂解液中4℃缓慢摇晃孵育2-4h,使抗体与琼脂糖珠偶连;免疫沉淀反应后,在4℃以3,000rpm速度离心3min,将琼脂糖珠离心至管底;将上清小心吸去,琼脂糖珠用1ml裂解缓冲液洗3-4次;最后加入30μl的2×SDS上样缓冲液,沸水煮5分钟;Western blotting分析。
由图5B可知,加TGF-β刺激后PTPRE与TGFBR1的结合增强;由图5C可知,加TGF-β刺激后PTPRE才能与SMAD3结合。
2、PTPRE介导受体TGFBR1对SMAD3的招募
将实验组1:Flag-PTPRE与HA-TGFBR1、HA-SMAD3共转293T细胞;同上做外源性Co-IP实验。
图6A、6B为共转后的Flag正拉、HA反拉的的Co-IP结果;结果表明PTPRE介导受体TGFBR1对SMAD3的招募。
3、shRNA-PTPRE对SMAD磷酸化水平影响实验
为了进一步研究shRNA-PTPRE对SMAD3的磷酸化水平影响。我们将过表达和敲减PTPRE的细胞进行了TGF-β和受体抑制剂刺激实验。
结果如图6C所示,实验结果可以看出,PTPRE能够促进SMAD3的磷酸化。
实施例4 PTPRE蛋白的酶活与SMAD3的磷酸化的关系
1、构建PTPRE的两个突变位点Y164A(PTPRE的第164位氨基酸由Y突变成A)、D250A(PTPRE的第250位氨基酸由D突变成A)的过表达质粒,荧光素酶活性检测采用双荧光素酶报告基因检测系统(Promega,Madison,WI,USA),GloMax 20/20光度仪(Promega)测定荧光素酶的相对活性。图7A表明,与野生型WT相比,PTPRE的两个突变位点Y164A、D250A能够降低SBE4荧光素酶报告基因的活性。所述PTPRE-WT的序列如SEQ NO.5所示,PTPRE-Y164A的序列如SEQ NO.6所示,PTPRE-D250A的序列如SEQ NO.7所示,PTPRE-DM的序列如SEQ NO.8所示。
2、基于上述结果构建PTPRE的Y164A、D250A同时突变即DM突变的质粒。图7B表明,与野生型WT相比,PTPRE的DM突变完全丧失对SBE4荧光素酶活性的调控作用。
3、构建稳定细胞系,图7C为PTPRE-WT稳定细胞系与PTPRE-DM稳定细胞系中PTPRE的表达量。表明PTPRE-WT稳定细胞系与PTPRE-DM稳定细胞系构建成功。
4、检测PTPRE-WT稳定细胞系与PTPRE-DM稳定细胞系中SMAD3的磷酸化水平、以及SRC的磷酸化水平;结果如图7D所示,TGF-β刺激后,PTPRE-WT稳定细胞系的SMAD3的磷酸化水平和SRC的磷酸化水平明显大于PTPRE-DM稳定细胞系,表明PTPRE蛋白两个突变位点Y164A(PTPRE的第164位氨基酸由Y突变成A)、D250A(PTPRE的第250位氨基酸由D突变成A)对SMAD3的磷酸化激活、SRC的磷酸化激活至关重要。
5、检测PTPRE-WT稳定细胞系与PTPRE-DM稳定细胞系的迁移和侵袭,结果如7E和7F所示。结果表明PTPRE-WT稳定细胞系的细胞迁移和侵袭能力明显大于PTPRE-DM稳定细胞系。
所述仅为本发明的较佳实施例而已,并不限制本发明,凡在本发明之内,所作的任何修改、等同替换、改进等,均应包括在本发明的保护范围之内。
序列表
<110> 华中科技大学同济医学院附属同济医院
<120> PTPRE作为靶标在制备或筛选抗肝癌药物中的用途及其相关药物
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ctgcgaccat cgtcatgtta a 21
<210> 2
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gctaccgaca gaaggactat t 21
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ccgagtgatc ctttccatga a 21
<210> 4
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gagcctggtc aagaaactca a 21
<210> 5
<211> 2103
<212> DNA
<213> 野生型PTPRE(PTPRE-WT)
<400> 5
atggagccct tgtgtccact cctgctggtg ggttttagct tgccgctcgc cagggctctc 60
aggggcaacg agaccactgc cgacagcaac gagacaacca cgacctcagg ccctccggac 120
ccgggcgcct cccagccgct gctggcctgg ctgctactgc cgctgctgct cctcctcctc 180
gtgctccttc tcgccgccta cttcttcagg ttcaggaagc agaggaaagc tgtggtcagc 240
accagcgaca agaagatgcc caacggaatc ttggaggagc aagagcagca aagggtgatg 300
ctgctcagca ggtcaccctc agggcccaag aagtattttc ccatccccgt ggagcacctg 360
gaggaggaga tccgtatcag atccgccgac gactgcaagc agtttcggga ggagttcaac 420
tcattgccat ctggacacat acaaggaact tttgaactgg caaataaaga agaaaacaga 480
gaaaaaaaca gatatcccaa catccttccc aatgaccatt ctagggtgat tctgagccaa 540
ctggatggaa ttccctgttc agactacatc aatgcttcct acatagatgg ttacaaagag 600
aagaataaat tcatagcagc tcaaggtccc aaacaggaaa cggttaacga cttctggaga 660
atggtctggg agcaaaagtc tgcgaccatc gtcatgttaa caaacttgaa agaaaggaaa 720
gaggaaaagt gccatcagta ctggcccgac caaggctgct ggacctatgg aaacatccgg 780
gtgtgcgtgg aggactgcgt ggttttggtc gactacacca tccggaagtt ctgcatacag 840
ccacagctcc ccgacggctg caaagccccc aggctggtct cacagctgca cttcaccagc 900
tggcccgact tcggagtgcc ttttaccccc attgggatgc tgaagttcct caagaaagta 960
aagacgctca accccgtgca cgctgggccc atcgtggtcc actgtagcgc gggcgtgggc 1020
cggacgggca ccttcattgt gatcgatgcc atgatggcca tgatgcacgc ggagcagaag 1080
gtggatgtgt ttgaatttgt gtctcgaatc cgtaatcagc gccctcagat ggttcaaacg 1140
gatatgcagt acacgttcat ctaccaagcc ttactcgagt actacctcta cggggacaca 1200
gagctggacg tgtcctcact ggagaagcac ctgcagacca tgcacggcac cacaacccac 1260
ttcgacaaga tcgggctgga ggaggagttc aggaaattga caaatgtccg gatcatgaag 1320
gagaacatga ggacgggcaa cttgccggca aacatgaaga aggccagggt catccagatc 1380
atcccgtatg acttcaaccg agtgatcctt tccatgaaaa ggggtcaaga atacacagac 1440
tacatcaacg catccttcat agacggctac cgacagaagg actatttcat cgccacccag 1500
gggccactgg cacacacggt tgaggacttc tggaggatga tctgggaatg gaaatcccac 1560
actatcgtga tgctgacgga ggtgcaggag agagagcagg ataaatgcta ccagtattgg 1620
ccaaccgagg gctcagttac tcatggagaa ataacgattg agataaagaa tgataccctt 1680
tcagaagcca tcagtatacg agactttctg gtcactctca atcagcccca ggcccgccag 1740
gaggagcagg tccgagtagt gcgccagttt cacttccacg gctggcctga gatcgggatt 1800
cccgccgagg gcaaaggcat gattgacctc atcgcagccg tgcagaagca gcagcagcag 1860
acaggcaacc accccatcac cgtgcactgc agtgccggag ctgggcgaac aggtacattc 1920
atagccctca gcaacatttt ggagcgagta aaagccgagg ggcttttaga tgtatttcaa 1980
gctgtgaaga gtttacgact tcagagacca catatggtgc aaaccctgga acagtatgaa 2040
ttctgctaca aagtggtaca agattttatt gatatatttt ctgattatgc taatttcaaa 2100
tga 2103
<210> 6
<211> 2103
<212> DNA
<213> 突变型PTPRE(PTPRE-Y164A)
<400> 6
atggagccct tgtgtccact cctgctggtg ggttttagct tgccgctcgc cagggctctc 60
aggggcaacg agaccactgc cgacagcaac gagacaacca cgacctcagg ccctccggac 120
ccgggcgcct cccagccgct gctggcctgg ctgctactgc cgctgctgct cctcctcctc 180
gtgctccttc tcgccgccta cttcttcagg ttcaggaagc agaggaaagc tgtggtcagc 240
accagcgaca agaagatgcc caacggaatc ttggaggagc aagagcagca aagggtgatg 300
ctgctcagca ggtcaccctc agggcccaag aagtattttc ccatccccgt ggagcacctg 360
gaggaggaga tccgtatcag atccgccgac gactgcaagc agtttcggga ggagttcaac 420
tcattgccat ctggacacat acaaggaact tttgaactgg caaataaaga agaaaacaga 480
gaaaaaaaca gagctcccaa catccttccc aatgaccatt ctagggtgat tctgagccaa 540
ctggatggaa ttccctgttc agactacatc aatgcttcct acatagatgg ttacaaagag 600
aagaataaat tcatagcagc tcaaggtccc aaacaggaaa cggttaacga cttctggaga 660
atggtctggg agcaaaagtc tgcgaccatc gtcatgttaa caaacttgaa agaaaggaaa 720
gaggaaaagt gccatcagta ctggcccgac caaggctgct ggacctatgg aaacatccgg 780
gtgtgcgtgg aggactgcgt ggttttggtc gactacacca tccggaagtt ctgcatacag 840
ccacagctcc ccgacggctg caaagccccc aggctggtct cacagctgca cttcaccagc 900
tggcccgact tcggagtgcc ttttaccccc attgggatgc tgaagttcct caagaaagta 960
aagacgctca accccgtgca cgctgggccc atcgtggtcc actgtagcgc gggcgtgggc 1020
cggacgggca ccttcattgt gatcgatgcc atgatggcca tgatgcacgc ggagcagaag 1080
gtggatgtgt ttgaatttgt gtctcgaatc cgtaatcagc gccctcagat ggttcaaacg 1140
gatatgcagt acacgttcat ctaccaagcc ttactcgagt actacctcta cggggacaca 1200
gagctggacg tgtcctcact ggagaagcac ctgcagacca tgcacggcac cacaacccac 1260
ttcgacaaga tcgggctgga ggaggagttc aggaaattga caaatgtccg gatcatgaag 1320
gagaacatga ggacgggcaa cttgccggca aacatgaaga aggccagggt catccagatc 1380
atcccgtatg acttcaaccg agtgatcctt tccatgaaaa ggggtcaaga atacacagac 1440
tacatcaacg catccttcat agacggctac cgacagaagg actatttcat cgccacccag 1500
gggccactgg cacacacggt tgaggacttc tggaggatga tctgggaatg gaaatcccac 1560
actatcgtga tgctgacgga ggtgcaggag agagagcagg ataaatgcta ccagtattgg 1620
ccaaccgagg gctcagttac tcatggagaa ataacgattg agataaagaa tgataccctt 1680
tcagaagcca tcagtatacg agactttctg gtcactctca atcagcccca ggcccgccag 1740
gaggagcagg tccgagtagt gcgccagttt cacttccacg gctggcctga gatcgggatt 1800
cccgccgagg gcaaaggcat gattgacctc atcgcagccg tgcagaagca gcagcagcag 1860
acaggcaacc accccatcac cgtgcactgc agtgccggag ctgggcgaac aggtacattc 1920
atagccctca gcaacatttt ggagcgagta aaagccgagg ggcttttaga tgtatttcaa 1980
gctgtgaaga gtttacgact tcagagacca catatggtgc aaaccctgga acagtatgaa 2040
ttctgctaca aagtggtaca agattttatt gatatatttt ctgattatgc taatttcaaa 2100
tga 2103
<210> 7
<211> 2103
<212> DNA
<213> 突变型PTPRE(PTPRE-D250A)
<400> 7
atggagccct tgtgtccact cctgctggtg ggttttagct tgccgctcgc cagggctctc 60
aggggcaacg agaccactgc cgacagcaac gagacaacca cgacctcagg ccctccggac 120
ccgggcgcct cccagccgct gctggcctgg ctgctactgc cgctgctgct cctcctcctc 180
gtgctccttc tcgccgccta cttcttcagg ttcaggaagc agaggaaagc tgtggtcagc 240
accagcgaca agaagatgcc caacggaatc ttggaggagc aagagcagca aagggtgatg 300
ctgctcagca ggtcaccctc agggcccaag aagtattttc ccatccccgt ggagcacctg 360
gaggaggaga tccgtatcag atccgccgac gactgcaagc agtttcggga ggagttcaac 420
tcattgccat ctggacacat acaaggaact tttgaactgg caaataaaga agaaaacaga 480
gaaaaaaaca gatatcccaa catccttccc aatgaccatt ctagggtgat tctgagccaa 540
ctggatggaa ttccctgttc agactacatc aatgcttcct acatagatgg ttacaaagag 600
aagaataaat tcatagcagc tcaaggtccc aaacaggaaa cggttaacga cttctggaga 660
atggtctggg agcaaaagtc tgcgaccatc gtcatgttaa caaacttgaa agaaaggaaa 720
gaggaaaagt gccatcagta ctggcccgcc caaggctgct ggacctatgg aaacatccgg 780
gtgtgcgtgg aggactgcgt ggttttggtc gactacacca tccggaagtt ctgcatacag 840
ccacagctcc ccgacggctg caaagccccc aggctggtct cacagctgca cttcaccagc 900
tggcccgact tcggagtgcc ttttaccccc attgggatgc tgaagttcct caagaaagta 960
aagacgctca accccgtgca cgctgggccc atcgtggtcc actgtagcgc gggcgtgggc 1020
cggacgggca ccttcattgt gatcgatgcc atgatggcca tgatgcacgc ggagcagaag 1080
gtggatgtgt ttgaatttgt gtctcgaatc cgtaatcagc gccctcagat ggttcaaacg 1140
gatatgcagt acacgttcat ctaccaagcc ttactcgagt actacctcta cggggacaca 1200
gagctggacg tgtcctcact ggagaagcac ctgcagacca tgcacggcac cacaacccac 1260
ttcgacaaga tcgggctgga ggaggagttc aggaaattga caaatgtccg gatcatgaag 1320
gagaacatga ggacgggcaa cttgccggca aacatgaaga aggccagggt catccagatc 1380
atcccgtatg acttcaaccg agtgatcctt tccatgaaaa ggggtcaaga atacacagac 1440
tacatcaacg catccttcat agacggctac cgacagaagg actatttcat cgccacccag 1500
gggccactgg cacacacggt tgaggacttc tggaggatga tctgggaatg gaaatcccac 1560
actatcgtga tgctgacgga ggtgcaggag agagagcagg ataaatgcta ccagtattgg 1620
ccaaccgagg gctcagttac tcatggagaa ataacgattg agataaagaa tgataccctt 1680
tcagaagcca tcagtatacg agactttctg gtcactctca atcagcccca ggcccgccag 1740
gaggagcagg tccgagtagt gcgccagttt cacttccacg gctggcctga gatcgggatt 1800
cccgccgagg gcaaaggcat gattgacctc atcgcagccg tgcagaagca gcagcagcag 1860
acaggcaacc accccatcac cgtgcactgc agtgccggag ctgggcgaac aggtacattc 1920
atagccctca gcaacatttt ggagcgagta aaagccgagg ggcttttaga tgtatttcaa 1980
gctgtgaaga gtttacgact tcagagacca catatggtgc aaaccctgga acagtatgaa 2040
ttctgctaca aagtggtaca agattttatt gatatatttt ctgattatgc taatttcaaa 2100
tga 2103
<210> 8
<211> 2103
<212> DNA
<213> 突变型PTPRE(PTPRE-DM)
<400> 8
atggagccct tgtgtccact cctgctggtg ggttttagct tgccgctcgc cagggctctc 60
aggggcaacg agaccactgc cgacagcaac gagacaacca cgacctcagg ccctccggac 120
ccgggcgcct cccagccgct gctggcctgg ctgctactgc cgctgctgct cctcctcctc 180
gtgctccttc tcgccgccta cttcttcagg ttcaggaagc agaggaaagc tgtggtcagc 240
accagcgaca agaagatgcc caacggaatc ttggaggagc aagagcagca aagggtgatg 300
ctgctcagca ggtcaccctc agggcccaag aagtattttc ccatccccgt ggagcacctg 360
gaggaggaga tccgtatcag atccgccgac gactgcaagc agtttcggga ggagttcaac 420
tcattgccat ctggacacat acaaggaact tttgaactgg caaataaaga agaaaacaga 480
gaaaaaaaca gagctcccaa catccttccc aatgaccatt ctagggtgat tctgagccaa 540
ctggatggaa ttccctgttc agactacatc aatgcttcct acatagatgg ttacaaagag 600
aagaataaat tcatagcagc tcaaggtccc aaacaggaaa cggttaacga cttctggaga 660
atggtctggg agcaaaagtc tgcgaccatc gtcatgttaa caaacttgaa agaaaggaaa 720
gaggaaaagt gccatcagta ctggcccgcc caaggctgct ggacctatgg aaacatccgg 780
gtgtgcgtgg aggactgcgt ggttttggtc gactacacca tccggaagtt ctgcatacag 840
ccacagctcc ccgacggctg caaagccccc aggctggtct cacagctgca cttcaccagc 900
tggcccgact tcggagtgcc ttttaccccc attgggatgc tgaagttcct caagaaagta 960
aagacgctca accccgtgca cgctgggccc atcgtggtcc actgtagcgc gggcgtgggc 1020
cggacgggca ccttcattgt gatcgatgcc atgatggcca tgatgcacgc ggagcagaag 1080
gtggatgtgt ttgaatttgt gtctcgaatc cgtaatcagc gccctcagat ggttcaaacg 1140
gatatgcagt acacgttcat ctaccaagcc ttactcgagt actacctcta cggggacaca 1200
gagctggacg tgtcctcact ggagaagcac ctgcagacca tgcacggcac cacaacccac 1260
ttcgacaaga tcgggctgga ggaggagttc aggaaattga caaatgtccg gatcatgaag 1320
gagaacatga ggacgggcaa cttgccggca aacatgaaga aggccagggt catccagatc 1380
atcccgtatg acttcaaccg agtgatcctt tccatgaaaa ggggtcaaga atacacagac 1440
tacatcaacg catccttcat agacggctac cgacagaagg actatttcat cgccacccag 1500
gggccactgg cacacacggt tgaggacttc tggaggatga tctgggaatg gaaatcccac 1560
actatcgtga tgctgacgga ggtgcaggag agagagcagg ataaatgcta ccagtattgg 1620
ccaaccgagg gctcagttac tcatggagaa ataacgattg agataaagaa tgataccctt 1680
tcagaagcca tcagtatacg agactttctg gtcactctca atcagcccca ggcccgccag 1740
gaggagcagg tccgagtagt gcgccagttt cacttccacg gctggcctga gatcgggatt 1800
cccgccgagg gcaaaggcat gattgacctc atcgcagccg tgcagaagca gcagcagcag 1860
acaggcaacc accccatcac cgtgcactgc agtgccggag ctgggcgaac aggtacattc 1920
atagccctca gcaacatttt ggagcgagta aaagccgagg ggcttttaga tgtatttcaa 1980
gctgtgaaga gtttacgact tcagagacca catatggtgc aaaccctgga acagtatgaa 2040
ttctgctaca aagtggtaca agattttatt gatatatttt ctgattatgc taatttcaaa 2100
tga 2103
Claims (8)
1.靶向PTPRE基因的shRNA在制备抗肝癌药物中的用途。
2.如权利要求1所述的用途,其特征在于,所述抗肝癌药物包括抑制肝癌细胞的增殖、侵袭与转移的药物。
3.如权利要求1所述的用途,其特征在于,所述抗肝癌药物的作用机理包括:抑制PTPRE基因的表达或者抑制PTPRE蛋白的酶活从而抑制其对TGF-β/SMAD3、SRC信号通路的调节。
4.如权利要求1所述的用途,其特征在于,所述shRNA的序列如SEQIDNO.1-3任一所示。
5.如权利要求1所述的用途,其特征在于,所述抗肝癌药物还包括可诱导shRNA在体内表达的诱导剂。
6.如权利要求1所述的用途,其特征在于,所述抗肝癌药物的有效成分为含有干扰PTPRE基因的慢病毒载体,所述慢病毒载体的表达区序列如SEQNO.1-3任一所示。
7.如权利要求6所述的用途,其特征在于,所述抗肝癌药物为慢病毒药物,其有效成分含有PTPRE基因干扰慢病毒,且所述PTPRE基因干扰慢病毒由慢病毒载体在慢病毒包装质粒、细胞系的辅助下,经过病毒包装而成。
8.PTPRE作为分子标志物在制备肝癌细胞的增殖、侵袭与转移的检测试剂盒中的用途。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911327688.3A CN110923319B (zh) | 2019-12-20 | 2019-12-20 | Ptpre作为靶标在制备或筛选抗肝癌药物中的用途及其相关药物 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911327688.3A CN110923319B (zh) | 2019-12-20 | 2019-12-20 | Ptpre作为靶标在制备或筛选抗肝癌药物中的用途及其相关药物 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110923319A CN110923319A (zh) | 2020-03-27 |
CN110923319B true CN110923319B (zh) | 2023-09-12 |
Family
ID=69863419
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201911327688.3A Active CN110923319B (zh) | 2019-12-20 | 2019-12-20 | Ptpre作为靶标在制备或筛选抗肝癌药物中的用途及其相关药物 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110923319B (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011004049A1 (es) * | 2009-07-08 | 2011-01-13 | Centro Nacional De Investigaciones Cardiovasculares (Cnic) | Método de identificación de compuestos inmunomoduladores para la piel |
WO2016201365A2 (en) * | 2015-06-12 | 2016-12-15 | Visani Giuseppe | Methods for treating cancers |
CN109381701A (zh) * | 2017-08-11 | 2019-02-26 | 复旦大学 | 促癌基因jcad及其作为靶点在制备治疗肝癌的药物中的用途 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100681763B1 (ko) * | 2005-02-28 | 2007-02-15 | 재단법인 목암생명공학연구소 | 인간 리포칼린 2를 유효성분으로 포함하는 암 전이 억제용약학적 조성물, 이를 이용한 암 전이 억제 방법 |
EP2531859A4 (en) * | 2010-02-02 | 2013-07-31 | Jolla Inst Allergy Immunolog | COMPOSITIONS AND METHOD FOR MODULATING RECEPTOR PROTEIN TYROSINE PHOSPHATASES |
US9274117B2 (en) * | 2013-12-21 | 2016-03-01 | Catholic University Industry Academic | Use of SIRT7 as novel cancer therapy target and method for treating cancer using the same |
-
2019
- 2019-12-20 CN CN201911327688.3A patent/CN110923319B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011004049A1 (es) * | 2009-07-08 | 2011-01-13 | Centro Nacional De Investigaciones Cardiovasculares (Cnic) | Método de identificación de compuestos inmunomoduladores para la piel |
WO2016201365A2 (en) * | 2015-06-12 | 2016-12-15 | Visani Giuseppe | Methods for treating cancers |
CN109381701A (zh) * | 2017-08-11 | 2019-02-26 | 复旦大学 | 促癌基因jcad及其作为靶点在制备治疗肝癌的药物中的用途 |
Non-Patent Citations (1)
Title |
---|
肝癌患者血清磷酸酪氨酸蛋白含量;王天然;马布仁;陈莉;陈建中;栗群英;曾祥元;;世界华人消化杂志(12);1467-1468 * |
Also Published As
Publication number | Publication date |
---|---|
CN110923319A (zh) | 2020-03-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Li et al. | Expression of the chemokine receptor CXCR4 in human hepatocellular carcinoma and its role in portal vein tumor thrombus | |
CN115335064B (zh) | 修饰的红细胞及其用于递送药剂的用途 | |
US20230399616A1 (en) | Modified Red Blood Cells and Uses Thereof for Delivering Agents | |
US10961533B2 (en) | Stem cell with suppressed SOCS and improved immunosuppressive ability and use thereof | |
US10155808B2 (en) | Monoclonal antibody against human PrPc and use thereof | |
CN117730143A (zh) | 通过缀合的n-末端甘氨酸修饰的细胞及其用途 | |
US20100074894A1 (en) | Pharmaceutical composition containing antibodies directed against the herv-w envelope | |
CN110923319B (zh) | Ptpre作为靶标在制备或筛选抗肝癌药物中的用途及其相关药物 | |
JP6986263B2 (ja) | 抗ウイルス薬 | |
CN117159748A (zh) | Tmprss12基因在制备预防或治疗新型冠状病毒感染药物的应用 | |
CN103966334A (zh) | Csf2rb基因在前列腺癌骨转移中的应用 | |
CN114703190B (zh) | 一种靶向抑制KIAA1429基因表达的ShRNA在慢性髓细胞白血病中的应用 | |
CN114917325B (zh) | 一种人可溶性tim-4蛋白在制备抗肿瘤药物中的应用 | |
KR20070029199A (ko) | B형 간염에 대한 치료제, 예방제 및 진단제 | |
CN114672460A (zh) | 一种靶向cd44的异质型cic细胞模型的制备方法及应用 | |
Suzuki et al. | Reduction of caveolin-1 expression in tumorigenic human cell hybrids | |
JP7033143B2 (ja) | Dctn1タンパク質とretタンパク質との融合タンパク質 | |
CN103204921A (zh) | 具有趋化活性的分泌蛋白及其编码序列和用途 | |
CN114149500B (zh) | 抗人emc10的单克隆抗体在制备治疗和/或预防脂肪肝的产品中的应用 | |
CN114149498B (zh) | 抗人emc10的单克隆抗体在防治2型糖尿病中的应用 | |
CN110859967A (zh) | 人cdc37l1基因及其编码的蛋白质在制备治疗胃癌的药物中的应用 | |
CN113827607B (zh) | 乌苯苷在制备下调肿瘤细胞pd-l1水平的制剂中的应用 | |
CN114149499B (zh) | 抗人emc10的单克隆抗体及其在治疗和/或预防肥胖症中的应用 | |
CN114592051B (zh) | 一种用于心肌损伤辅助诊断的生物标志物及试剂盒 | |
CN116549480B (zh) | 针对HIF1α的shRNA在制备治疗肿瘤的药物中的应用 |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |