CN105779600A - Application of AKR1C1 protein - Google Patents

Application of AKR1C1 protein Download PDF

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Publication number
CN105779600A
CN105779600A CN201610211769.7A CN201610211769A CN105779600A CN 105779600 A CN105779600 A CN 105779600A CN 201610211769 A CN201610211769 A CN 201610211769A CN 105779600 A CN105779600 A CN 105779600A
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China
Prior art keywords
akr1c1
lung cancer
albumen
product
cancer metastasis
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CN201610211769.7A
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Chinese (zh)
Inventor
杨波
何俏军
朱虹
应美丹
常琳琳
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Zhejiang University ZJU
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Zhejiang University ZJU
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Priority to CN201610211769.7A priority Critical patent/CN105779600A/en
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    • 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
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • G01N33/57423—Specifically defined cancers of lung
    • 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
    • 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
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)

Abstract

The invention provides application of AKR1C1 protein. The product comprises a product for diagnosing lung cancer and predicting lung cancer metastasis by RT-PCR, real-time quantitative PCR, immunodetection, hybridization in situ or gene chip, and is used for preparing a product for diagnosing lung cancer and preparing a medicine for treating lung cancer. Overexpressed AKR1C1 protein can be used for remarkably improving in-vivo metastasis of lung cancer cells and remarkably enhancing in-vitro metastasis and invasion capacity of lung cancer cells, and AKR1C1 can be used for remarkably inhibiting the in-vitro metastasis and invasion capacity of lung cancer cells by RNA interference suppression. The AKR1C1 protein can be used as specific marker protein for diagnosing lung cancer, predicting lung cancer metastasis and prognosing, so that lung cancer diagnosis can be accurately and quickly performed, the accuracy for diagnosing lung cancer and predicting lung cancer metastasis can be improved, and a new treatment target and new effective medicine are provided to lung cancer prevention and treatment.

Description

A kind of application of AKR1C1 albumen
Technical field
The invention belongs to technical field of bioengineering, relate to the application of an albumen, particularly relate to the application of a kind of AKR1C1 albumen.
Background technology
The sickness rate of pulmonary carcinoma increases year by year, within 2012, rises and has become as China's prevalence and the highest malignant tumor of mortality rate.According to U.S. State's National Cancer Center statistics, is in II, III, IV phase state of an illness transfer occur when the patients with lung cancer of 79% is gone to a doctor；Pulmonary carcinoma The death toll of patient accounts for the 26.8% of all cancer patient death tolls, and 5 annual survival rates of 2005-2011 are only 17.4%. The transfer case of this explanation lung cancer patient is closely related with prognosis survival condition.These data illustrate, the clinical research of pulmonary carcinoma, special It not transfer correlational study, have a great deal of practical meanings and clinical demand.
People's AKR1C1 albumen is to be encoded by AKR1C1 gene (Genebank No.NM 001353.5), is aldehyde ketone reductase AKR One of superfamily protein member.The function of this albumen is not yet verified completely at present, only understands that AKR1C1 albumen can utilize Aldehyde ketone, as coenzyme, is converted to corresponding alcohols by NADH/NADPH.Document is reported, AKR1C1 is abnormal expression in tumor tissues, Such as raise at Expressions in Lung Cancer.AKR1C1 albumen and tumor drug resistance have certain relation, but there is no and turning about AKR1C1 albumen Research report in shifting, the especially research report in lung cancer metastasis.
Summary of the invention
An object of the present invention is to provide a kind of AKR1C1 albumen in the product preparing diagnosing and prediction lung cancer metastasis Application.Described product includes: with RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or gene chip diagnosis lung Cancer and the product of prediction lung cancer metastasis.The sequence of described AKR1C1 albumen is (Aldo-keto reductase as shown in SEQ ID NO.1 Family 1 member C1 aldehyde ketone reductase, gene ID 1645, by 323 aa codings).
The product of described RT-PCR diagnosing and prediction lung cancer metastasis at least includes a pair specific amplification AKR1C1 gene Primer: AKR1C1 forward primer: 5'-agt aaa gct tta gag gcc ac-3'；Reverse primer: 5'-cac cca tgg Ttc ttc tcg g-3', amplified production is 591bp.
The product of described real-time quantitative PCR diagnosing and prediction lung cancer metastasis at least includes a pair specific amplification AKR1C1 base The primer of cause: AKR1C1 forward primer: 5'-gta aag ctt tag agg cca c-3'；Reverse primer: 5'-gag gtc Aac ata atc caa ttg-3', amplified production is 245bp.
The product of described immune detection diagnosing and prediction lung cancer metastasis includes: the antibody being combined with AKR1C1 protein-specific.
The product of described in situ hybridization diagnosing and prediction lung cancer metastasis includes: with the nucleic acid array hybridizing of AKR1C1 gene Probe.
The product of described gene chip diagnosis pulmonary carcinoma and prediction lung cancer metastasis includes: with the nucleic acid array hybridizing of AKR1C1 gene Probe.
AKR1C1 albumen of the present invention can improve pulmonary cancer diagnosis and the standard of prediction lung cancer metastasis as the label of pulmonary cancer diagnosis Really property.
In the present invention, it is possible to use a series of methods known in the art prepare the antibody special for AKR1C1 albumen.Example As, people's AKR1C1 albumen of purification or its antigen fragment are injected in animal body to produce polyclonal antibody.Equally, express The cell of people's AKR1C1 albumen or its antigen fragment may also be used for animal being caused immunity and producing antibody.Produced according to the present invention Antibody can also be monoclonal antibody, these monoclonal antibodies can be prepared by hybridoma technology.The antibody of the present invention includes permissible Prevent the antibody of AKR1C1 function, it is also possible to be the antibody not affecting people's AKR1C1 protein function.Each antibody-like can lead to Cross the segment to people's AKR1C1 albumen or functional domain causes immunity and produces, and people's AKR1C1 protein product and fragment thereof can be with weights Prescription method produces or synthesizes with Peptide synthesizer.The protein bound antibody with the AKR1C1 of non-modified form, it is possible to use The gene outcome produced in prokaryotic cell such as E.coli is carried out immune animal and is obtained.With the such as glycosylation of post translational modification form or The antibody that phosphorylation AKR1C1 albumen or polypeptide combine, it is possible to use the gene produced in eukaryotic cell such as yeast or insect cell Product carrys out immune animal and obtains.
In the present invention, described probe can be DNA, RNA, DNA-RNA chimera, PNA or other derivants.Described spy The length of pin does not limit, as long as it is specific binding with purpose nucleotide sequence to complete specific hybrid, any length is all Permissible.The length of described probe may be as little to 25,20,15 or 10 bases longs.Equally, the length of described probe can be grown To 60,80,100,150,300 base pairs or longer, the most whole gene.Owing to different probe length is to hybridization effect Rate, signal specificity have different impacts, the length of described probe to be typically at least 14 base pairs, the longest are usually no more than 30 base pairs, optimal with 15-25 base pair with the length of purpose nucleotide sequence complementary.Described probe self-complementary sequences Most preferably less than 4 base pairs, in order to avoid affecting hybridization efficiency.
Second object of the present invention is to provide the application in the medicine of preparation treatment pulmonary carcinoma of a kind of AKR1C1 albumen.
The medicine of described treatment pulmonary carcinoma includes: by the double stranded RNA of RNA AF panel AKR1C1 gene expression, or be used for The protein of suppression AKR1C1 protein active, or for suppressing crude extract or the micromolecular compound of AKR1C1 protein function.
The present invention tests the internal transfer proving that process LAN AKR1C1 albumen can dramatically increase lung carcinoma cell；Process LAN AKR1C1 egg External transfer and the invasive ability of lung carcinoma cell can be dramatically increased in vain；Pulmonary carcinoma can be significantly inhibited thin by RNA AF panel AKR1C1 The external transfer of born of the same parents and invasive ability；The rush transfer ability of AKR1C1 albumen is by transcriptional control metastasis related gene MMP2/Twist/Sox2.Therefore, AKR1C1 albumen can make pulmonary carcinoma examine as diagnosis and the specificity marker protein of prediction transfer Disconnected more accurate, quickly.AKR1C1 albumen of the present invention is to prevent and treat pulmonary carcinoma to provide new therapeutic targets and effective new drug.
Accompanying drawing explanation
Fig. 1 is that the present invention passes through process LAN AKR1C1 albumen and can dramatically increase hepatic metastases in the nude mouse of lung carcinoma cell.
Fig. 2 is that the present invention passes through process LAN AKR1C1 albumen and can dramatically increase external transfer and the invasive ability of lung carcinoma cell.
Fig. 3 is that the present invention passes through RNA AF panel AKR1C1 and can significantly inhibit external transfer and the invasive ability of lung carcinoma cell.
By RT-PCR technology, Fig. 4 is the present invention proves that the rush transfer ability of AKR1C1 albumen is by transcriptional control metastasis related gene MMP2/Twist/Sox2。
Fig. 5 is that after the present invention passes through the exogenous transfection of explanation of qRT-PCR technology or knocks out AKR1C1 albumen, the level of its mRNA is sent out Raw corresponding change.
Detailed description of the invention
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.
Following example are merely to illustrate the present invention rather than limit the scope of the present invention.Unreceipted actual conditions in embodiment Experimental technique, generally according to normal condition, or according to the condition proposed by manufacturer.
Embodiment 1:
Take female 4～6 week old nude mouses for testing.It is stable that non-small cell lung cancer cell strain NCI-H1299 is collected in EDTA digestion The group of high expressed AKR1C1 and the cell of matched group, after training liquid washes twice, for nude mouse tail vein injection.Every naked Mus injection 2,000,000 cells, 200 μ L volumes.After 60 days, cervical dislocation is put to death, and dissects, and observes Organ relative weight situation, Take pictures.As it is shown in figure 1, the group of high expressed AKR1C1, the hepatic metastases in nude mouse is apparently higher than comparison group.
Embodiment 2:
At the lung carcinoma cell NCI-H1650 of Secondary Culture, NCI-H1299 is respectively adopted plasmid transfection AKR1C1 gene (AKR1C1 Group), and blank (pcmv6 group) is set.After 24 hours to be transfected, use Transwell method, detection transfectional cell Vitro invasion ability.As in figure 2 it is shown, after process LAN AKR1C1 gene, the body of lung carcinoma cell NCI-H1650, NCI-H1299 Outer invasive ability dramatically increases.
Embodiment 3:
At the lung cell A549 of Secondary Culture, PC-9 is respectively adopted RNA AF panel AKR1C1 and expresses (AKR1C1 group), And blank (NC group) is set.After disturbing 24 hours, use Transwell method, the vitro invasion of detection transfectional cell Ability.As it is shown on figure 3, after AF panel AKR1C1 expresses, lung cell A549, the vitro invasion ability of PC-9 significantly drops Low.
Embodiment 4:
Use plasmid transfection AKR1C1 gene (AKR1C1 group) at the lung carcinoma cell NCI-H1650 of Secondary Culture, and blank is set Comparison (vector group).In the lung carcinoma cell PC-9 of Secondary Culture, it is respectively adopted RNA AF panel AKR1C1 expresses (AKR1C1 SiRNA group), and blank (NC group) is set.After transfecting 24 hours, Real-Time Fluorescent Quantitative PCR Technique, detection is used to turn Dye cell promotees the level change of the mRNA of the gene of transfer.As shown in Figure 4, after high expressed AKR1C1, lung carcinoma cell NCI-H1650 Transcription has raised rush metastasis related gene MMP2/Twist/Sox2；After RNA AF panel AKR1C1 expresses, lung carcinoma cell PC-9 Transcription has lowered rush metastasis related gene MMP2/Twist/Sox2.
Embodiment 5:
Plasmid transfection AKR1C1 gene (AKR1C1 group) is used at lung carcinoma cell NCI-H1650, NCI-H1299 of Secondary Culture, And blank (vector group) is set.In lung carcinoma cell PC-9 and A549 of Secondary Culture, it is respectively adopted RNA interference press down AKR1C1 processed expresses (siAKR1C1 group), and arranges blank (NC group).After transfecting 24 hours, use real-time fluorescence fixed Amount round pcr, the expression of AKR1C1 in detection transfectional cell.As it is shown in figure 5, after high expressed AKR1C1, AKR1C1 MRNA level in-site significantly raise, and RNA AF panel AKR1C1 express after, the mRNA level in-site of AKR1C1 is remarkably decreased.

Claims (9)

1. an AKR1C1 albumen is preparing diagnosing and the application in the product of prediction lung cancer metastasis, it is characterised in that described Product include: with RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or gene chip diagnosis pulmonary carcinoma and prediction lung The product of cancerometastasis, the sequence of described AKR1C1 albumen is as shown in SEQ ID NO.1.
A kind of AKR1C1 albumen the most according to claim 1 answering in the product preparing diagnosing and prediction lung cancer metastasis With, it is characterised in that the product of described RT-PCR diagnosing and prediction lung cancer metastasis at least includes a pair specific amplification The primer of AKR1C1 gene: AKR1C1 forward primer: 5'-agt aaa gct tta gag gcc ac-3'；Reverse primer: 5'-cac Cca tgg ttc ttc tcg g-3', amplified production is 591bp.
A kind of AKR1C1 albumen the most according to claim 1 is in the product preparing diagnosing and prediction lung cancer metastasis Application, it is characterised in that described real-time quantitative PCR diagnosing and prediction lung cancer metastasis product at least include a pair special Property amplification AKR1C1 gene primer: AKR1C1 forward primer: 5'-gta aag ctt tag agg cca c-3'；Reversely Primer: 5'-gag gtc aac ata atc caa ttg-3', amplified production is 245bp.
A kind of AKR1C1 albumen the most according to claim 1 is in the product preparing diagnosing and prediction lung cancer metastasis Application, it is characterised in that the product of described immune detection diagnosing and prediction lung cancer metastasis includes: special with AKR1C1 albumen Anisogamy antibody.
A kind of AKR1C1 albumen the most according to claim 1 is in the product preparing diagnosing and prediction lung cancer metastasis Application, it is characterised in that the product of described in situ hybridization diagnosing and prediction lung cancer metastasis includes: with AKR1C1 gene The probe of nucleic acid array hybridizing.
A kind of AKR1C1 albumen the most according to claim 1 is in the product preparing diagnosing and prediction lung cancer metastasis Application, it is characterised in that the product of described gene chip diagnosis pulmonary carcinoma and prediction lung cancer metastasis includes: with AKR1C1 gene The probe of nucleic acid array hybridizing.
A kind of AKR1C1 albumen the most according to claim 1 is in the product preparing diagnosing and prediction lung cancer metastasis Application, it is characterised in that AKR1C1 albumen of the present invention can improve pulmonary cancer diagnosis with pre-as the label of pulmonary cancer diagnosis Survey the accuracy of lung cancer metastasis.
A kind of AKR1C1 albumen the most according to claim 1 application in the medicine of preparation treatment pulmonary carcinoma, its feature exists In, the sequence of described AKR1C1 albumen is as shown in SEQ ID NO.1.
Application the most according to claim 8, it is characterised in that the medicine of described treatment pulmonary carcinoma includes: disturbed by RNA The double stranded RNA of suppression AKR1C1 gene expression, or for suppressing the protein of AKR1C1 protein active, or be used for suppressing The crude extract of AKR1C1 protein function or micromolecular compound.
CN201610211769.7A 2016-04-06 2016-04-06 Application of AKR1C1 protein Pending CN105779600A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106478822A (en) * 2016-12-14 2017-03-08 长江大学 A kind of preparation method of Monopterus albus (Zuiew) aldehyde ketone reductase polyclonal antibody
CN109498810A (en) * 2018-11-23 2019-03-22 中国科学院昆明动物研究所 The purposes of AKR1C1 protein inhibitor
CN111139299A (en) * 2020-01-08 2020-05-12 浙江大学 Application of JOSD2 protein in preparing medicine for treating malignant tumor
CN113881774A (en) * 2021-10-26 2022-01-04 上海市第一人民医院 Detection kit with AKR1C1 as detection target and use method thereof

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Publication number Priority date Publication date Assignee Title
WO2004111197A2 (en) * 2003-06-10 2004-12-23 Trustees Of Boston University Gene expression signatures, methods and compositions for diagnosing disorders of the lung
CN105431548A (en) * 2013-03-15 2016-03-23 生物医学研究机构基金会 Method for the diagnosis, prognosis and treatment of cancer metastasis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004111197A2 (en) * 2003-06-10 2004-12-23 Trustees Of Boston University Gene expression signatures, methods and compositions for diagnosing disorders of the lung
CN105431548A (en) * 2013-03-15 2016-03-23 生物医学研究机构基金会 Method for the diagnosis, prognosis and treatment of cancer metastasis

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CHIA-WEN CHIEN: "Induction of neoplastic transformation by ectopic expression of human aldo-keto reductase 1C isoforms in NIH3T3 cells", 《CARCINOGENESIS》 *
NCBI: "GeneBank:NP_001344.2", 《NCBI BLAST》 *
QING JI: "Selective reduction of AKRIC2 in prostate Cancer and Its Role in DHT Metabolism", 《THE PROSTATE》 *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106478822A (en) * 2016-12-14 2017-03-08 长江大学 A kind of preparation method of Monopterus albus (Zuiew) aldehyde ketone reductase polyclonal antibody
CN109498810A (en) * 2018-11-23 2019-03-22 中国科学院昆明动物研究所 The purposes of AKR1C1 protein inhibitor
CN111139299A (en) * 2020-01-08 2020-05-12 浙江大学 Application of JOSD2 protein in preparing medicine for treating malignant tumor
CN111139299B (en) * 2020-01-08 2021-12-17 浙江大学 Application of JOSD2 protein in preparing medicine for treating malignant tumor
CN113881774A (en) * 2021-10-26 2022-01-04 上海市第一人民医院 Detection kit with AKR1C1 as detection target and use method thereof

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