CN109371130A - RIPOR3 is in preparation for the application in the biological products of breast cancer detection and treatment - Google Patents
RIPOR3 is in preparation for the application in the biological products of breast cancer detection and treatment Download PDFInfo
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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
- G01N33/57415—Specifically defined cancers of breast
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- 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
Abstract
The invention discloses breast cancer molecular marker RIPOR3 in preparation for the application in the biological products of breast cancer detection and treatment, the inventors discovered that expression of the RIPOR3 in breast cancer tissue or breast cancer cell is lowered.The invention also discloses the kit for breast cancer detection or prognosis, the kit includes the primer sequence of specific amplification RIPOR3.The auxiliary diagnosis and prognosis evaluation that breast cancer is carried out using kit of the invention have directive significance to follow-up clinical treatment.In addition, the RIPOR3 pharmaceutical composition for being used to prepare treatment breast cancer can also be used for the clinical applications such as the gene therapy of breast cancer, drug therapy.
Description
Technical field
The present invention relates to oncomolecularbiology technical fields, and in particular to RIPOR3 preparation for breast cancer detection and
Application in the biological products for the treatment of.
Background technique
Breast cancer has become the highest tumour of disease incidence in women in the world, seriously affects the life and health of women.Although
The development of screening scheme and complementary therapy achieves success, but significant percentage of patient with breast cancer dies of cancer metastasis.With it
His most countries are the same, and breast cancer also becomes the most common cancer of Chinese women；Annual Chinese Breast Cancer is newly sent out quantity and is accounted for
The 12.2% of the whole world, The dead quantity account for the 9.6% of the whole world.Since the nineties, Chinese breast cancer incidence growth rate is complete
More than twice of ball, urban area is especially pronounced.The key of breast cancer diagnosis and treatment is early discovery, early diagnosis and early treatment.Oncogene
Activation or the inactivation of tumor suppressor gene play key effect during tumor development.As extensive, high pass measures
The popularization and application of sequence technology, finding new oncogene and tumor suppressor gene not is only that the diagnosis of tumour and prognosis provide and have much value
Standard, while also laying the foundation to find new tumor biotherapy target spot.
FAM65 protein family includes member FAM65A (also referred to as Rho family interaction polarization regulatory protein 1),
(also referred to as Rho family interacts by FAM65B (also referred to as Rho family interaction polarization regulatory protein 2) and FAM65C
Polarization regulatory protein 3, i.e. Rho family-interacting cell polarization regulator 3,
RIPOR3).FAM65A has been displayed and is positioned at sertoli cell main process and nephrocyte body.FAM65B early stage during myocyte breaks up
It lowers, and with the generation of two kinds long and short of isotype, the differential expression in different tissues.FAM65C (RIPOR3) seems
Occur in two kinds of isotypes, is generated by alternative splicing.
RIPOR3 gene is located at the position NC_000020.11 of chromosome 20, and the reference of RIPOR3 includes NCBI
(GenBank) 1 (NP_ of shorter protein isoform of reference sequences transcriptional variants 1 (NM_080829.3) and its coding
543019.2)；And NCBI (GenBank) reference sequences transcriptional variants 2 (NM_001290268.1) and its coding have more
The longer protein isoform 2 (NP_001277197.1) of the long end N-, compared with variant 1 variant 2 it is different in 5'UTR and
Use the upstream AUG of substitution.
Currently, unclear about the mechanism of action of RIPOR3 gene or albumen in breast cancer, function and expression become
The clinical meaning of change not yet has been reported that.Therefore, the relationship of RIPOR3 gene and breast cancer occurrence and development is analyzed, exploration is directed to
The oncotherapy of RIPOR3 approach and prediction policy are most important.
Summary of the invention
To solve the above problems, a purpose of the invention is to provide breast cancer molecular marker RIPOR3 in preparation for cream
Gland cancer detection, prognosis or treatment biological products in application, wherein the molecular marked compound is RIPOR3 gene or albumen,
Gene I/D with ncbi database: shown in the NM_001290268.1 or NP_001277197.1 of 140876 leted others have a look at RIPOR3
Sequence.
As used in the present invention, term RIPOR3 (the polarization regulatory protein 3 of Rho family interaction) refers to people
RIPOR3 gene or albumen or its homologue or the variant form with its bioactivity.
The present invention has detected expression of the RIPOR3 gene in human breast carcinoma tissue and cancer beside organism using RT-PCR method,
And it demonstrates RIPOR3 gene and expresses downward in breast cancer tissue or breast cancer cell.
As used in the present invention, term " expression is lowered " refers to that the sequence measurement of expressed gene proves, by cancer
Normal galactophore tissue or Healthy People breast tissue, the gene expression amount measured in patient breast cancer tissue detected have extremely
Few 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more expression reduces, such as should in breast cancer tissue
Gene expression amount is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or lower in normal galactophore tissue.
One aspect of the invention provides reagent of the molecular marked compound RIPOR3 in preparation for breast cancer detection or prognosis
Application in box, in some embodiments, the RIPOR3 are people RIPOR3.
In some embodiments of the present invention, the detection or prognosis is according in the biological samples of institute's test object
RIPOR3 expression judges the disease condition of breast cancer in object, carries out curative effect evaluation or transfer and relapse monitoring, preferred institute
Stating biological sample is breast cancer tissue or breast cancer cell.
Another aspect of the invention provides the kit for breast cancer detection or prognosis, and it includes specific amplification people
The primer pair of RIPOR3 gene, wherein forward primer is the sequence as shown in SEQ ID NO.1, and reverse primer is such as SEQ ID
Sequence shown in NO.2；In addition, for expand internal reference GAPDH primer pair be the forward primer such as SEQ ID NO.3 shown in
The reverse primer as shown in SEQ ID NO.4.Further, the kit further includes that RNA extracts reagent, Reverse Transcription,
And PCR reacts common agents such as reverse transcriptase, buffer, dNTPs, MgCl2, DEPC water and Taq enzyme etc., mark can also be contained
Quasi- product and/or reference substance.
Another aspect of the present invention provides RIPOR3 or its analog or agonist in preparation prevention or treatment breast cancer
Pharmaceutical composition in application, wherein the analog or agonist are its variant form with RIPOR3 bioactivity.
In some embodiments of the present invention, described pharmaceutical composition include a effective amount of RIPOR3 or its analog or
Agonist and pharmaceutically acceptable carrier, further described pharmaceutical composition further includes other for preventing or treating breast cancer
Medicament.
As used herein, described " effective quantity " refers to can generate function or active and can quilt to mammal (preferably people)
The amount that mammal (preferably people) is received." pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, packet
Include various excipient and diluent；Described carrier itself is not necessary active constituent, and does not have excessive toxicity after applying.
The suitable carrier is well known to those of ordinary skill in the art.Pharmaceutically acceptable load in described pharmaceutical composition
Body can contain liquid such as water, salt water, buffer, and there is likely to be auxiliary substances for example filler, lubricant, glidant,
Wetting agent or emulsifier, pH buffer substance etc..Cell (such as host cell) transfection reagent can also be contained in the carrier.
The present invention can using a variety of methods well known in the art by RIPOR3 albumen, its analog or agonist or
Its encoding gene or its pharmaceutical composition are applied to patient such as mammal, preferably people.Including but not limited to: subcutaneous injection, flesh
Meat injection, for percutaneous administration of, administer locally to, be implanted into, be sustained and give；Preferably, the administration mode is that non-bowel is given.
Also may be selected using gene therapy means carry out breast cancer treatment, such as directly by RIPOR3, its analog or
Agonist is applied to patient by such as the methods of injection；Alternatively, RIPOR3, its analog can will be carried by certain approach
Or the ceneme of agonist such as expression vector or virus are delivered on target spot, and be allowed to express active RIPOR3,
Its analog or agonist, concrete condition need to be depending on the pharmacy types, these are well known to those skilled in the art
's.
Pharmaceutical composition of the invention can further include one or more anticancer agents.In specific embodiments,
Pharmaceutical composition includes at least one compound for promoting RIPOR3 gene expression and at least one chemotherapeutics.For of the invention
Chemotherapeutics in pharmaceutical composition, including but not limited to: DNA- alkylating agent, antitumor antibiotics agent, antimetabolite, tubulin
Stabilizer, tubulin destabilizing agent, hormone antagonist, topoisomerase enzyme inhibitor, kinases inhibitor, HMG-COA suppression
Preparation, CDK inhibitor, cyclin inhibitors, caspase inhibitors, metal protease inhibitors, antisense nucleic acid,
Triple helix DNA, the virus of aptamer and molecular modification, bacterium and exotoxin reagent.
In the present invention, " prognosis " refers to cancer patient by suppressions such as operation, chemotherapy, drug therapy or combinations thereof processing
System alleviates process or result after tumour growth.Prognosis, which can be to handle by operation, chemotherapy, drug therapy or combinations thereof, to be pressed down
Life state when 1,2,3,4,5,6,7,8,9,10,15,20 year or more long after system or alleviation growth of breast cancers.Prognosis can lead to
Detection marker is crossed to assess, the marker is one or more genes.Prognosis evaluation can be performed such that according to marker
With or without or being raised and lowered, determine whether the prognosis of patient good, or determine the general of good prognosis or poor prognosis
Rate.
Pharmaceutical composition of the invention can also can be with the drug combination of other treatment breast cancer, other therapeutic compound
It is administered simultaneously with main active, or even is administered simultaneously in same composition.
Pharmaceutical composition of the invention can also be with individual composition or the dosage form different from main active
Individually give other therapeutic compounds.The Fractional of main active can be given simultaneously with other therapeutic compounds
Medicine, and other dosage can be administered alone.Over the course for the treatment of, it according to the severity of symptom, the frequency of recurrence and can control
The physiologic response for the treatment of scheme adjusts the dosage of pharmaceutical composition of the present invention.
The utility model has the advantages that
The inventors discovered that molecular marked compound RIPOR3 is in the breast cancer tissue of patient relative to the table in cancer beside organism
It is lowered up to obvious, therefore RIPOR3 can be used as to the marker of Computer-aided Diagnosis of Breast Cancer and prognosis, and preparation is used for mammary gland
The biological products of cancer detection and/or treatment.Provided by the present invention for detecting the kit of breast cancer, it can be used for breast cancer
Auxiliary diagnosis and prognosis evaluation；In addition the pharmaceutical composition for the treatment of breast cancer provided by the invention can also be used for the gene of breast cancer
The clinical applications such as treatment, drug therapy.
Detailed description of the invention
Fig. 1 show relative expression quantity of the RIPOR3 in breast cancer tissue and cancer beside organism and compares, wherein relative to just
Normal cancer beside organism, expression of the RIPOR3 in breast cancer tissue are obviously lowered.
Fig. 2 show relative expression quantity of the RIPOR3 in breast cancer cell line MCF-7, MDA-MB-453 significantly lower than it
Relative expression quantity in normal breast epithelium cell system MCF-10A.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means.
Embodiment 1
This example demonstrates that content of the RIPOR3 in patient breast cancer tissue sample is significantly lower than it in cancer beside organism's sample
Content in this.
1, it samples
Take in October, 2013 to during in December, 2017 in BeiJing University ShenZhen Hospital's surgical operation obtain breast cancer cancer
Side tissue and each 50 of breast cancer tissue, all samples are confirmed through pathological examination, and -80 DEG C of low temperature refrigerators of number postposition are protected
It deposits.RNA is extracted to be used to do real-time fluorescence quantitative PCR.The written informing of all patients, sample collection are cured through Peking University's biology
Institutional Review Board is learned to agree to.
2, Total RNAs extraction is carried out to tissue samples
UsingReagent (invitrogen, article No. 15596-018) carries out sample rna extraction, experiment behaviour
Make to carry out by product description, concrete operations are as follows:
It freezes to be put into the mortar being pre-chilled tissue after liquid nitrogen, taking-up after collection sample and be ground, sample to be organized
This is at powdered rear:
1. Trizol is added, room temperature preservation 5 minutes；
2. chlorination imitates 0.2mL, with forced oscillation centrifuge tube, mix well, places 5-10 minutes at room temperature；
3. drawing upper strata aqueous phase (inhaling 70%) after 12000rpm high speed centrifugation 15 minutes into another new centrifuge tube pipe, pay attention to
The protein substance that be not drawn between two layers of water phase.New pipe is moved into, the pre- cold isopropanol of isometric -20 DEG C is added, it is sufficiently reverse
It mixes, is placed in 10 minutes on ice；
4. 12000rpm high speed from 15 minutes after carefully discard supernatant, in 1mL/mL Trizol ratio be added 75%
DEPC ethanol washing precipitating (4 DEG C of preservations), washing precipitate, oscillation mixes, 12000rpm high speed centrifugation 5 minutes at 4 DEG C；
5. discarding ethanol liquid, 5 minutes are placed at room temperature sufficiently to dry precipitating, it is heavy that the processed water dissolution of DEPC is added
It forms sediment；
6. measuring RNA purity and concentration with Nanodrop2000 ultraviolet specrophotometer, freeze in -80 DEG C.RNA mass is sentenced
Calibration is quasi-: the OD260/OD280 value of RNA sample is between 1.7-2.2；Total serum IgE electrophorogram has clearly 28S, 18S band；
70 DEG C of water-baths keep the temperature 1 hour after electrophorogram and the map no significant difference before water-bath heat preservation.
3, reverse transcription synthesizes cDNA
UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044)
CDNA reverse transcription is carried out, experimental implementation is carried out by product description, and concrete operations are as follows:
Using Reverse Transcriptase kit, converse record is carried out to l μ g total serum IgE with RT Buffer and synthesizes cDNA.Using 25 μ L
Reaction system, each sample take 1 μ g total serum IgE as template ribonucleic acid.It is spare that -20 DEG C of refrigerators are put in the cDNA preservation of acquisition.
4、RT-PCR
With 7500 type fluorescence quantitative PCR instrument of ABI, using 2-ΔΔCtMethod carries out data relative quantitative assay.
Using online primer-design software, gene order is referring to NCBI:RIPOR3 (gene I/D: 140876, NM_
001290268.1), interior participation in the election GAPDH is synthesized by invitrogen company after design of primers.Specific primer sequence is as follows:
Table 1: primer sequence
Operating process is as follows:
Table 2:RT-PCR reaction system
Reaction system: Power is usedGreen PCR Master Mix (invitrogen, article No. 4367659)
It is expanded, experimental implementation is carried out by product description.
Amplification program are as follows: 95 DEG C of initial denaturation 5min, (95 DEG C of denaturation 15sec, Tm annealing 45sec, 72 DEG C of extension 35sec) ×
40 circulations, specific Tm is referring to table 1.
Sample RT-PCR detection: 2 μ L will be taken to make template after 10 times of each sample cDNA dilutions, uses target gene primer respectively
It is expanded with reference gene primer.Simultaneously in 60-95 DEG C of progress solubility curve analysis.
Experimental result shows that real-time quantitative PCR amplification curve inflection point understands, amplification curve entirety collimation is good, shows each
The amplification efficiency of reaction tube is close, and the limit is put down without the presence that raises up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；
Sample amplified production solubility curve be all it is unimodal, illustrate that amplified production only has one, be specific amplification；According to qRT-PCR's
Relative quantification formula: 2-ΔΔCt× 100%, expression of the icp gene in breast cancer tissue and cancer beside organism.As a result it shows
Show: qRT-PCR stable amplification result, as shown in Figure 1, relative expression quantity of the RIPOR3 gene in breast cancer tissue is significantly lower than
Its relative expression quantity in cancer beside organism, and the difference has statistical significance (P < 0.05)；This shows that RIPOR3 is suffering from
The expression in breast of person is lowered.
Expression of the embodiment 2RIPOR3 in breast cancer cell line
Firstly, culture human breast carcinoma cell lines MCF-7, MDA-MB-453 and normal breast epithelium cell system MCF-10A,
In, MDA-MB-453 is incubated in the L15 culture medium of 10% fetal calf serum, MCF-7 and normal breast epithelial MCF-10A
It is incubated in the DMEM culture medium containing 10% fetal calf serum.1%P/S (Pen .- Strep mixed solution) is added in culture solution.
It is cultivated in 37 DEG C, 5%CO2, the incubator that relative humidity is 90%.It changes within 2-3 days liquid 1 time, pancreas of the use 0.25% containing EDTA
The passage of protease conventional digestion.
Then, the total serum IgE in cell is extracted using the cell RNA extracts kit of QIAGEN and measure its concentration, specifically
The specific steps are the same as those in embodiment 1 with qPCR for operation.Experiment is completed according to being repeated 3 times, and result data is all with average value ± standard
The mode of difference indicates, using SPSS18.0 statistical software come for statistical analysis, breast cancer cell line and normal breast cell
The paired comparisons of system are examined using t, it is believed that have statistical significance as P < 0.05.
It is as shown in Figure 2 the result shows that, RIPOR3 gene is opposite in breast cancer cell line MCF-7, MDA-MB-453
Expression quantity is significantly lower than its relative expression quantity in normal breast epithelium cell system MCF-10A, which anticipates with statistics
Adopted (P < 0.05).
Overexpression of the embodiment 3RIPOR3 gene in breast cancer cell
1, cell culture
Human breast cancer cell line MDA-MB-453, with the L15 culture medium containing 10%FBS and 1%P/S 37 DEG C, 5%CO2,
It is cultivated in the incubator that relative humidity is 90%.It changes within 2-3 days liquid 1 time, trypsase conventional digestion of the use 0.25% containing EDTA
Passage.
Cell in culture bottle is digested with pancreatin and is seeded in 6 orifice plates, guarantee cell number is 2-8 × 105A/
L15 cell culture medium is added in hole.Overnight, second day observation cell density, cell density can be transfected for 70% or more.
2, the building of gene overexpression carrier
Specific PCR amplimer is synthesized according to the cDNA sequence of people's RIPOR3 gene, in 5 ' end primers and 3 ' end primers
Two restriction enzyme sites of HindIII and XhoI are added respectively.It is obtained with the extraction of patient with breast cancer's blood and reverse transcription
CDNA as amplification template, above-mentioned cDNA sequence be inserted into after restriction enzyme HindIII and XhoI double digestion through
In the eukaryotic expression vector pcDNA3.1 of HindIII and XhoI double digestion, the recombinant vector pcDNA3.1-1 of acquisition is connected
For subsequent experimental.
3, it transfects
Experiment is divided into three groups: control group (MDA-MB-453), negative control group (pcDNA3.1-NC) and experimental group
(pcDNA3.1-1), the transfection of carrier is carried out using liposome 3000, the instruction of specific transfection method to specifications carries out.
4, qPCR detects the level of RIPOR3 gene
1) extraction of cell total rna
The total serum IgE in cell is extracted using QIAGEN cell extraction kit, concrete operations are detailed in specification.
2) the specific steps are the same as those in embodiment 1 for qPCR amplification.
5, statistical method
Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD,
Using SPSS18.0 statistical software come for statistical analysis, the difference between RIPOR3 Gene Experiments group and control group uses t
It examines, it is believed that there is statistical significance as P < 0.05.
6, result
As a result compared with the control group, the RIPOR3 gene expression dose of MDA-MB-453 cell dramatically increases in experimental group,
And the difference has statistical significance (P < 0.05).
The influence of embodiment 4RIPOR3 gene pairs Cells Proliferation of Human Breast Cancer
Detection RIPOR3 gene pairs Cells Proliferation of Human Breast Cancer capacity is tested using CCK-8.
1, with embodiment 3,6h changes liquid after transfection, places cell incubator mistake for MDA-MB-453 cell culture and transfection procedure
Night.
2, trypsin digestion cell, the mixing of L15 cell culture medium, which is added, makes cell suspend, and carries out cell count.
3, it is inoculated in 96 orifice plates, 3000/hole, is inoculated with 8 multiple holes.Altogether spread 4 piece of 96 orifice plate be respectively used to for 24 hours,
4 detection time points of 48h, 72h and 96h.
4, after for 24 hours, first piece of 96 orifice plate is taken out, the CCK-8 detection liquid of 10 μ l is added in every hole, 96 orifice plates are continued to put
Enter and be incubated for 4h or so in cell incubator, detect absorbance value of each hole at 450nm wavelength with microplate reader and records data.
5, the operation in 4 is respectively repeated steps after 48h, 72h, 96h, finally counts the absorbance value at each time point,
Make growth curve chart.
6, statistical analysis
Experiment is completed according to being repeated 3 times, using SPSS18.0 statistical software come for statistical analysis, the two it
Between difference using t examine, it is believed that as P < 0.05 have statistical significance.
7, result
As a result compared with the control, after transfecting pcDNA3.1-1, the proliferation of cell is obviously inhibited experimental group, and
The difference has statistical significance (P < 0.05), it is related to the cell Proliferation of breast cancer to illustrate RIPOR3, and RIPOR3 crosses table
Up to the proliferation that can inhibit breast cancer cell.
5 breast cancer detection of embodiment or prognosis evaluation reagent kit
Based on the primer sets of table 1 in embodiment 1, the kit of the present invention for being used to detect breast cancer is assembled, it is described
Kit includes primer pair (forward primer shown in SEQ ID NO:1 and the SEQ ID NO:2 of specific amplified people's RIPOR3 gene
Shown in reverse primer) and specific amplified house-keeping gene (GAPDH) the primer pair (forward primer and SEQ of SEQ ID NO:3
Reverse primer shown in ID NO:4)；It further include SYBR Green polymerase chain reaction system, such as PCR buffer, SYBR
Green fluorescent dye, dNTPs.The ingredient of the PCR buffer is 25mM KCl, 2.5mM MgCl2, 200mM (NH4)2SO4；
It further include normal breast tissue cDNA: as negative control and the common quantitative PCR detection of sample cDNA to be detected, each reactant
It is use and detection sample cDNA equal amount.It is preferred that PCR reaction system is as shown in table 3:
Table 3PCR reaction system
| Component | Additional amount |
| SYBR Green polymerase chain reaction system | 12.5μL |
| Upstream primer (10 μM) | 0.5μL |
| Downstream primer (10 μM) | 0.5μL |
| Template cDNA | 2.0μL |
| Sterile purified water is added | To 25 μ L |
Optimum reaction condition are as follows: 95 DEG C of initial denaturation 5min, (95 DEG C of denaturation 15sec, 60 DEG C of annealing 45sec, 72 DEG C extend
35sec) × 40 circulation, 72 DEG C of extension 15min.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>BeiJing University ShenZhen Hospital's (Peking University's Shenzhen Clinical Medical Institute)
<120>RIPOR3 is in preparation for the application in the biological products of breast cancer detection and treatment
<130> P18033
<160> 4
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gacactgaca agcaatcggc 20
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<213>artificial sequence (Artificial Sequence)
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tgataatgca gccgggactc 20
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aatgggcagc cgttaggaaa 20
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Claims (10)
1. breast cancer molecular marker RIPOR3 answering in the biological products that preparation is used for breast cancer detection, prognosis or treatment
It is RIPOR3 gene or albumen with, which is characterized in that the molecular marked compound, the gene I/D with ncbi database:
Sequence shown in the NM_001290268.1 or NP_001277197.1 of 140876 leted others have a look at RIPOR3.
2. application as described in claim 1, which is characterized in that breast cancer tissue of the molecular marked compound RIPOR3 in patient
Or it expresses and lowers in breast cancer cell.
3. application as claimed in claim 1 or 2, which is characterized in that the biological products are for breast cancer detection or prognosis
Kit.
4. application as claimed in claim 3, which is characterized in that the detection or prognosis is according to the biological samples of institute's test object
RIPOR3 expression in this judges the disease condition of breast cancer in object, carries out curative effect evaluation or transfer and relapse monitoring.
5. application as claimed in claim 4, which is characterized in that the biological sample is that breast cancer tissue or breast cancer are thin
Born of the same parents.
6. the kit for breast cancer detection or prognosis, which is characterized in that include drawing for specific amplification people's RIPOR3 gene
Object pair, wherein forward primer is the sequence as shown in SEQ ID NO.1, and reverse primer is the sequence as shown in SEQ ID NO.2.
7. kit as claimed in claim 6, which is characterized in that the kit further includes that RNA extracts reagent, reverse transcription examination
Agent, quantitative PCR reagent.
The application of 8.RIPOR3 or its analog or agonist in the pharmaceutical composition of preparation prevention or treatment breast cancer, it is special
Sign is that the analog or agonist are its variant form with RIPOR3 bioactivity.
9. application as claimed in claim 8, which is characterized in that described pharmaceutical composition includes a effective amount of RIPOR3 or its class
Like object or agonist and pharmaceutically acceptable carrier.
10. application as claimed in claim 9, which is characterized in that described pharmaceutical composition further includes prevention or treatment breast cancer
Other medicaments.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811373112.6A CN109371130B (en) | 2018-11-19 | 2018-11-19 | Application of RIPOR3 in preparation of biological products for breast cancer detection and treatment |
Applications Claiming Priority (1)
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| CN110946872A (en) * | 2019-12-30 | 2020-04-03 | 北京大学深圳医院 | Application of miR-4491 in preparation of medicine for treating breast cancer |
| CN111269981A (en) * | 2020-02-17 | 2020-06-12 | 中国医科大学附属盛京医院 | Application of TROAP in preparing prognosis product for detecting breast cancer patient treated by endocrine |
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| CN101493464A (en) * | 2009-03-09 | 2009-07-29 | 昆明理工大学 | Molecular marker for breast carcinoma diagnosis |
| CN107267606A (en) * | 2017-04-20 | 2017-10-20 | 浙江理工大学 | A kind of lncRNA and its application in lung cancer detection label or prognosis recurrence label is merged as breast cancer |
| CN108277277A (en) * | 2018-01-04 | 2018-07-13 | 北京大学深圳医院（北京大学深圳临床医学院） | It is a kind of assessment familial breast cancer risk marker and its application |
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| EP1682679A1 (en) * | 2003-11-10 | 2006-07-26 | Randox Laboratories Ltd. | Molecular marker |
| CN101493464A (en) * | 2009-03-09 | 2009-07-29 | 昆明理工大学 | Molecular marker for breast carcinoma diagnosis |
| CN107267606A (en) * | 2017-04-20 | 2017-10-20 | 浙江理工大学 | A kind of lncRNA and its application in lung cancer detection label or prognosis recurrence label is merged as breast cancer |
| CN108277277A (en) * | 2018-01-04 | 2018-07-13 | 北京大学深圳医院（北京大学深圳临床医学院） | It is a kind of assessment familial breast cancer risk marker and its application |
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| CN110946872B (en) * | 2019-12-30 | 2022-11-04 | 北京大学深圳医院 | Application of miR-4491 in preparation of medicine for treating breast cancer |
| CN111269981A (en) * | 2020-02-17 | 2020-06-12 | 中国医科大学附属盛京医院 | Application of TROAP in preparing prognosis product for detecting breast cancer patient treated by endocrine |
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