CN107385082A - The application of MST1P9 genes and its expression product in kidney - Google Patents
The application of MST1P9 genes and its expression product in kidney Download PDFInfo
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- CN107385082A CN107385082A CN201710775687.XA CN201710775687A CN107385082A CN 107385082 A CN107385082 A CN 107385082A CN 201710775687 A CN201710775687 A CN 201710775687A CN 107385082 A CN107385082 A CN 107385082A
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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
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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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
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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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
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Abstract
The invention discloses the application of MST1P9 genes and its expression product in kidney, MST1P9 is in low expression in renal carcinoma tissue, increase MST1P9 expression can reduce cell propagation and the invasion and attack of kidney, the present invention substantially increases the Sensitivity and Specificity of Diagnosis of Renal Cell Carcinoma, while provides new target spot for the accurate treatment of kidney.
Description
Technical field
The invention belongs to biomedicine field, is related to the application of MST1P9 genes and its expression product in kidney, specifically
Be related to Diagnosis of Renal Cell Carcinoma and treatment in application.
Background technology
Clear-cell carcinoma (renal cell carcinoma, RCC) also known as kidney, are initiated by kidney essence uriniferous tubule epithelium
The malignant tumour of cell, the 80%-90% of kidney malignant cancer is accounted for, account for the 2%-3% of adult malignancies, in the world respectively
It is individual it is national, the interlocal kidney incidence of disease is different, the incidence of disease of developing country is less than developed country.According to statistics, kidney
Difference, M-F are about 1.81 to the incidence of disease of male and female:1, the city incidence of disease is compared with rural area height.To clear-cell carcinoma
Histopathology classified, clear cell carcinoma of kidney, renal papilla shape gland cancer (I types and II types), kidney chromophobe cell tumor can be divided into
And unfiled 4 partings of clear-cell carcinoma.Clear cell carcinoma of kidney (renal clear cell carcinoma, RCCC) be kidney most
For common pathological, the 60%-85% of the kidney incidence of disease is accounted for.Imageological examination is still the master of clinical diagnosis kidney at present
Will foundation, pathological examination is then the goldstandard for making a definite diagnosis kidney.Combined by stages according to AJCC in 2010 TNM stage and AJCC
(island), kidney is divided into Limitable renal cell carcinoma, Local advancement kidney and metastatic renal cell carcinoma.The preferred treatment of Limitable renal cell carcinoma at present
It is surgery excision, radical nephrectomy need to be taken according to tumor size, position, patient profiles and the experience of clinician
(radical nephrectomy, RN) or Nephron -sparing surgery (nephron sparing surgery, NSS).At present also
The therapeutic scheme do not generally acknowledged, surgery excision primary lesion are the complementary treatment means of kidney, and targeted drug treatment can
It is obviously prolonged the life cycle of patient.
At present, pathological staging is the main factor for influenceing kidney prognosis, and in addition, the prognosis of kidney may be also with swelling
Whether contain downright bad group in the histological grade of knurl, the KPS scale (such as ECOG life quality scores) of patient, tumour
Knit and the exception of some biochemical indicators to change etc. factor it is related.The invasion and attack transfer of tumour is the master for causing tumor prognosis bad
Reason is wanted, but discovery can be sensitive and specifically prompts clear cell carcinoma of kidney to occur and attack the tumor-marker of transfer yet at present
Thing.With the further investigation in canceration field and the continuous development of molecular biology, find there are a variety of bases in renal carcinoma tissue so far
Because of unconventionality expression, but its specific pathogenesis is still unclear.Therefore generation, the development machine of tumour are explored from gene variation direction
System, the gene related to tumor development is found, the early diagnosis, treatment, judging prognosis and related drugs to tumour are ground
Hair etc. all has great importance.
The content of the invention
In order to make up the deficiencies in the prior art, an object of the present invention, there is provided a kind of related to kidney occurrence and development
Gene, the expression by detecting the gene realize the diagnosis of kidney.
The second object of the present invention, there is provided a kind of therapeutic targets of kidney, the treatment of kidney is realized by targeting target.
The third object of the present invention, there is provided a kind of method for the potential material for screening treatment kidney, pass through the material energy
It is no regulation biological target expression come judge screened material whether be treat kidney potential material.
To achieve these goals, the present invention adopts the following technical scheme that：
The invention provides the purposes of a kind of MST1P9 genes and its expression product, for preparing the production of early diagnosis kidney
Product.
Further, the product includes the reagent of detection MST1P9 genes and its expression product expression.
Further, the reagent includes being examined with RT-PCR, real-time quantitative PCR, in situ hybridization, chip or immunoassay
Survey the reagent of MST1P9 genes and its expression product expression.
Wherein, the reagent that MST1P9 genes and its expression product expression are detected with RT-PCR is special including at least a pair
Property amplification MST1P9 genes primer；The reagent of MST1P9 genes and its expression product expression is detected with real-time quantitative PCR
Including at least the primer of a pair of specific amplification MST1P9 genes；It is described to detect MST1P9 genes and its expression production with situ hybridization
The reagent of thing expression includes the probe with the nucleic acid array hybridizing of MST1P9 genes；It is described to detect MST1P9 genes with chip
And its reagent of expression product expression includes and the probe of MST1P9 nucleic acid array hybridizing or special with MST1P9 albumen
Property combine antibody or part；The reagent that MST1P9 genes and its expression product expression are detected with immunoassay
Including the antibody and/or part combined with MST1P9 protein-specifics.
The invention provides a kind of product for diagnosing kidney, the product includes MST1P9 genes or its volume in detection sample
The reagent of the protein expression level of code.The product includes but is not limited to chip, preparation, kit.
Further, the reagent includes specific recognition MST1P9 probe or specific amplification MST1P9 primer；Or
Specifically bind the antibody or part of the albumen of MST1P9 codings.
In the present invention, the product of the diagnosis kidney can be used for detection related to kidney more including MST1P9
The expression of individual gene and/or its expression product.Multiple gene association diagnosis, the accuracy of Diagnosis of Renal Cell Carcinoma can be increased.
The invention provides the purposes of a kind of MST1P9 genes and its expression product, for screening the potential thing for the treatment of kidney
Matter.
Further, the step of potential material of screening treatment kidney is as follows：
The system for the albumen expressed or containing MST1P9 genes or its coding is handled with candidate substances；With
Detect the expression of the albumen of MST1P9 genes or its coding or activity in the system；
Wherein, if the candidate substances can promote the expression of the albumen of MST1P9 genes or its coding or activity (preferable
Significantly rise, it is such as high by more than 20%, it is preferably high by more than 50%；It is more preferably high more than 2 times), then it is pre- to show the candidate substances
Anti- or treatment kidney potential material.
The invention provides a kind of method for the potential material for screening prevention or treatment kidney, screening step is：
The system for the albumen expressed or containing MST1P9 genes or its coding is handled with candidate substances；With
Detect the expression of the albumen of MST1P9 genes or its coding or activity in the system；
Wherein, if the candidate substances can promote the expression of the albumen of MST1P9 genes or its coding or activity (preferably aobvious
Rise is write, it is such as high by more than 20%, it is preferably high by more than 50%；It is more preferably high more than 2 times), then it is prevention to show the candidate substances
Or the potential material for the treatment of kidney.
In test group, candidate substances are added to the system for the albumen expressed or containing MST1P9 genes or its coding
In；And/or the expression of the albumen of MST1P9 genes or its coding or activity in the system of detection test group, and it is relative with control group
Than wherein described control group is expression or or the albumen containing MST1P9 genes or its coding for not adding the candidate substances
System；If the expression of the albumen of MST1P9 genes or its coding or activity are higher than control group in test group, that is, show the time
It is prevention or the potential material for treating kidney to select material.
Further, the step also includes：Further cell experiment and/or animal examination are carried out to the potential material of acquisition
Test, further to select and determine for preventing, alleviating or treating the useful material of kidney from potential material.
In the present invention, the system includes but is not limited to：Cell system, subcellular fraction system, solution system, organizer
System, organ systems or animal system.The candidate compound includes but is not limited to：For MST1P9 genes or its upstream or under
The nucleic acid for swimming gene design promotes thing, albumen accelerator, protein binding molecule.
The invention provides the purposes of a kind of MST1P9 genes and its expression product, for preparing the medicine group for the treatment of kidney
Compound.
Further, described pharmaceutical composition includes MST1P9 genes, albumen or its accelerator.The MST1P9 albumen includes
Total length MST1P9 albumen, ripe MST1P9 albumen, the MST1P9 domain combined with DNA or containing the domain
MST1P9 active fragments；The accelerator include MST1P9 gene expression products, promoted type miRNA, promoted type transcriptional control because
Son or promoted type targeting micromolecular compound.
Further, described pharmaceutical composition includes promoting the accelerator of MST1P9 functional expressions.
The invention provides a kind of pharmaceutical composition for treating kidney, described pharmaceutical composition includes：MST1P9 features
The accelerator of expression；And/or pharmaceutically acceptable carrier；The accelerator includes improving MST1P9 genes or its expression product
Stability, the expression of up-regulation MST1P9 genes or its expression product, increase MST1P9 genes or its expression product are effectively made
With the material of time.
Preferably, the accelerator is MST1P9 over-express vectors.
The pharmaceutically acceptable carrier, including but not limited to diluent, adhesive, surfactant, Humectant,
Absorption carrier, lubricant, filler, disintegrant.
Brief description of the drawings
Fig. 1 is the expression figure in renal carcinoma tissue using QPCR detection MST1P9 genes；
Fig. 2 is the expression figure in renal carcinoma tissue using Western blot detection MST1P9 albumen；
Fig. 3 is expression figures of the MST1P9 in clear cell carcinoma of kidney cell；Wherein, figure A is detection MST1P9 mRNA
Expression figure in clear cell carcinoma of kidney cell；Figure B is the table for detecting MST1P9 albumen in clear cell carcinoma of kidney cell
Up to situation map；
Fig. 4 is transfected condition figures of the MST1P9 in kidney cancer cell；Wherein, figure A is to kidney using QPCR detection transfections
The influence figure that MST1P9 mRNA are expressed in cell；Figure B is to MST1P9 in kidney cancer cell using Western blot detection transfections
The influence figure of albumen；
Fig. 5 is the influence figure bred with mtt assay detection MST1P9 gene pairs kidney cancer cell；
Fig. 6 is the influence figure migrated using cell scratch experiment detection MST1P9 to kidney cancer cell；
Fig. 7 is the influence figure attacked using Transwell cells detection MST1P9 to kidney cancer cell.
Specific embodiment
The present invention is found that in kidney that MST1P9 conspicuousnesses are lowered first by high throughput sequencing technologies.It is it is demonstrated experimentally that logical
Increase MST1P9 expression is crossed, can effectively suppress growth, the invasion and attack of kidney cancer cell, prompt MST1P9 genes can be used for
The clinical early diagnosis and therapy of kidney.
MST1P9 genes
MST1P9 is taken positioned at the area 6 of No. 1 the short arm of a chromosome of people 3, and the MST1P9 in the present invention includes wild type, saltant type
Or its fragment.MST1P9 bases in a kind of representational MST1P9 gene orders such as current international public nucleic acid database GeneBank
Because shown in (NC_000001.11).
The people's MST1P9 nucleotides full length sequence or its fragment of the present invention can generally use PCR TRAPs, recombination method or people
The method of work synthesis obtains., can be according to published relevant nucleotide sequence, especially ORFs for PCR TRAPs
Sequence designs primer, and with commercially available cDNA storehouses or the cDNA storehouses as prepared by conventional method well known by persons skilled in the art
As template, expand and obtain relevant sequence.When sequence is longer, it is often necessary to carry out twice or multiple PCR is expanded, then again will
Each time the fragment amplified is stitched together by proper order.
The present invention can utilize any method known in the art measure gene expression.Those skilled in the art should manage
Solution, the means for determining gene expression are not the importances of the present invention.The table of biomarker can be detected on transcriptional level
Up to level.
Diagnostic products
In the present invention, the product for diagnosing kidney can be any form, including but not limited to chip, preparation, reagent
Box, as long as it can detect MST1P9 genes or the expression of its expression product.
Chip in the present invention includes：Solid phase carrier；And the oligonucleotides being fixed in order on the solid phase carrier is visited
Pin, described oligonucleotide probe specifically correspond to the part or all of sequence shown in MST1P9.
Specifically, suitable probe can be designed, is fixed on solid phase carrier according to gene of the present invention, is formed
" oligonucleotide arrays ".Described " oligonucleotide arrays " refer to addressable point (i.e. with distinctive, addressablely
The position that location is characterized) array, each addressable point is containing a coupled characteristic oligonucleotides.According to need
Will, oligonucleotide arrays can be divided into multiple sub- battle arrays.
Term " probe " refers to the molecule that can be combined with the particular sequence or subsequence or other parts of another molecule.It is unless another
Point out, term " probe " is often referred to match by complementary base and another polynucleotides (often referred to as " target polynucleotide ")
With reference to polynucleotide probes.Lack sufficient sequence complementarity according to the stringency of hybridization conditions, probe energy and with the probe
Target polynucleotide combines.Probe can make direct or indirect mark, and its scope includes primer.Crossing system, including, but it is unlimited
In：Solution, solid phase, mixed phase or in situ hybridization determination method.
Heretofore described solid phase carrier can use the various common used materials in genetic chip field, such as include but is not limited to
Plastic products, microparticle, membrane carrier etc..The plastic products can be resisted by non-covalent or physical absorption mechanism with antibody or albumen
Original is combined, and the most frequently used plastic products are small test tube, globule and micro-reaction plate made of polystyrene；The microparticle is
The microballoon or particle aggregated into by high polymer monomer, its diameter are mostly micron, due to the functional group that can be combined with protein,
Chemical coupling easily is formed with antibody (antigen), binding capacity is big；The membrane carrier includes nitrocellulose filter, glass fibre element film
And the miillpore filter such as nylon membrane.
Described MST1P9 chips prepare the common manufacturing method that can use biochip known in the art.For example,
If solid phase carrier is gone here and there using modification slide or silicon chip, 5 ' ends of probe containing amido modified poly- dT, can be by few nucleosides
Acid probe is configured to solution, then using point sample instrument by its point modification slide or silicon chip on, be arranged in predetermined sequence or battle array
Row, are then fixed by standing overnight, so that it may obtain the genetic chip of the present invention.
The invention provides a kind of kit, the kit can be used for detection MST1P9 expression.Preferably, it is described
Also contain the label for labeled RNA sample, and the substrate corresponding with the label in preparation or kit.This
Outside, may also include in described kit for extracting the various reagents needed for RNA, PCR, hybridization, colour developing etc., including it is but unlimited
In：Extract, amplification liquid, hybridization solution, enzyme, comparison liquid, nitrite ion, washing lotion etc..In addition, in described kit also including the use of
Specification and/or chip image analysis software.In kit can also have kit operation instructions, wherein describe how
Detected using kit, and how tumor development to be judged using testing result, therapeutic scheme is selected.
Accelerator and pharmaceutical composition
Discovery based on inventor, the invention provides a kind of MST1P9 accelerator, the accelerator includes improving
MST1P9 genes or its expression product stability, the expression of up-regulation MST1P9 genes or its expression product, increase MST1P9
The material of gene or its expression product effective acting time.The accelerator can be MST1P9 gene expression products, promoted type
MiRNA, promoted type transcription regulatory factor or promoted type targeting micromolecular compound.
In the present invention, be various carriers known in the art, such as commercially available carrier including plasmid, clay, bacteriophage,
Virus etc..
Present invention also offers a kind of pharmaceutical composition, and it contains the described MST1P9 of effective dose accelerator, and
Pharmaceutically acceptable carrier.Described composition can be used for treating kidney.Any foregoing MST1P9 accelerator is available
In the preparation of composition.The pharmaceutical composition of the present invention can be used for the missing or deficiency for supplementing endogenic MST1P9 albumen,
By improving the expression of MST1P9 genes or albumen, so as to treat the kidney caused by MST1P9 is reduced.
The pharmaceutically acceptable carrier, including but not limited to diluent, adhesive, surfactant, Humectant,
Absorption carrier, lubricant, filler, disintegrant.
Wherein, diluent such as lactose, sodium chloride, glucose, urea, starch, water etc.；Adhesive such as starch, pregelatinated form sediment
Powder, dextrin, maltodextrin, sucrose, Arabic gum, gelatin, methylcellulose, carboxymethyl cellulose, ethyl cellulose, poly- second
Enol, polyethylene glycol, PVP, alginic acid and alginate, xanthans, hydroxypropyl cellulose and hydroxypropyl methyl
Cellulose etc.；Surfactant such as polyoxyethylene sorbitan fatty acid ester, lauryl sodium sulfate, stearic acid list glycerine
Ester, hexadecanol etc.；Humectant such as glycerine, starch etc.；Absorption carrier such as starch, lactose, bentonite, silica gel, kaolin and soap
Clay etc.；Lubricant such as zinc stearate, glycerin monostearate, polyethylene glycol, talcum powder, calcium stearate and magnesium, polyethylene glycol,
Boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyl 40 stearate, single bay sucrose acid ester, laruyl alcohol sulfuric acid
Sodium, magnesium laurylsulfate, Stepanol MG etc.；Filler such as mannitol (granular or powdery), xylitol, sorbierite, wheat
Bud sugar, erythrose, microcrystalline cellulose, polymerization sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, sea-tangle
Polysaccharide powder, agar powder, calcium carbonate and sodium acid carbonate etc.；Disintegrant such as cross-linked ethylene pyrrolidones, sodium carboxymethyl starch, low
Substitute hydroxypropyl methyl, Ac-Di-Sol, soybean polyoses etc..
Pharmaceutical composition in the present invention can also include stabilizer, bactericide, buffer, isotonic agent, chelating agent, pH controls
The additive such as preparation and surfactant.
Wherein, stabilizer includes Human serum proteins, l-amino acid, sugar and cellulose derivative.L-amino acid can be with
Including any one in glycine, cysteine and glutamic acid.Carbohydrate includes monose, such as glucose, mannose, gala
Sugar, fructose etc.；Sugar alcohol, such as mannitol, inositol, xylitol etc.；Disaccharides, such as sucrose, maltose, lactose etc.；Polysaccharide,
Such as glucan, hydroxypropul starch, vulcanization chondroitin, hyaluronic acid etc. and their derivative.Cellulose derivative includes first
Base cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, HPMC and sodium cellulose glycolate.
Surfactant includes ion or nonionic surfactant, such as polyethylene glycol oxide Arrcostab, sorbitan monoacyl ester, fat
Fatty acid glyceride.Additive buffer can include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid and corresponding salt (they
Alkali metal or alkaline rare earth metal salt, such as sodium salt, sylvite, calcium salt and magnesium salts).Isotonic agent include potassium chloride, sodium chloride, sugar and
Glycerine.Chelating agent includes sodium ethylene diamine tetracetate and citric acid.
Pharmaceutical composition Orally-administrable of the present invention, parenteral administration, by suck spray delivery, it is local to
Medicine, rectally, nasal administration, cheek administration, vagina administration are administered by the storage medicine device of implantation.It is preferred that it is administered orally or injects
Administration.Pharmaceutical composition of the present invention contains any commonly employed nontoxic pharmaceutical acceptable carrier, auxiliary material or excipient.
The mode that the medicine of the present invention imports tissue either cell can be divided into external or internal mode.Vitro formats
Including the medicine containing MST1P9 genes or the medicine containing MST1P9 protein are imported in cell, then by cell transplantation or
Feed back in vivo.Internal mode includes directly noting the medicine containing MST1P9 genes or the medicine containing MST1P9 protein
Enter in in-vivo tissue.
Over the course for the treatment of, can be adjusted according to the order of severity, the frequency of recurrence and the physiologic response of therapeutic scheme of symptom
The dosage of whole pharmaceutical composition of the present invention.
The pharmaceutical composition of the present invention can also can be with the drug combination of other treatment kidney, other therapeutic compound
It is administered simultaneously with main active component, or even is administered simultaneously in same composition.Can also with single composition or with
The different dosage form of main active component individually gives other therapeutic compounds.
In the present invention, term " sample " is used with its broadest sense.It is intended to include the sample obtained from any source
Or culture, and biology and environmental samples.Biological specimen available from animal (including people) and cover liquid, solid, tissue and
Gas.Biological specimen includes blood product, blood plasma, serum etc..However, such sample, which should not be construed as limitation, is applied to this
The sample type of invention.
In a particular embodiment of the present invention, experiment all completed according to being at least repeated 3 times, result data be all with
The mode of mean+SD represents, carries out statistical analysis using SPSS18.0 statistical softwares, paired sample is using t inspections
Test, multisample is analyzed using the variance test (ANOVA) of mean, it is believed that works as P<There is statistical significance when 0.05.
The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this
Invention rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in embodiment, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.
Embodiment 1 screens the gene marker related to kidney
1st, sample collection
Collect 6 renal clear cell carcinomas and corresponding cancer beside organism, the preoperative non-row chemicotherapy of all patients, art
Row check pathological section is clarified a diagnosis afterwards.The acquirement of tissue samples obtains the informed consent of patient, and obtains and pass through tissue
The agreement of Ethics Committee.
2nd, the preparation of RNA sample
1) liquid nitrogen grinding tissue is added to powder, adds 1ml TRIzol (Invitrogen) solution, piping and druming mixes, and makes group
Abundant cracking is knitted, stands 5min；
2) 4 DEG C of centrifugation 5min of 12000rpm, supernatant is transferred in 1.5ml RNase free EP pipes；
3) 200 μ l chloroforms are added, acutely vibration mixes 30s, aqueous phase and organic phase is fully contacted, is stored at room temperature 15min；
4) for 12000g from 15min, solution centrifugal is three layers in 4 DEG C of environment, and it is new to move to another in upper strata aqueous phase by RNA
RNase free EP are managed；
5) 0.5ml isopropanols are added, it is soft fully to mix, it is stored at room temperature 10min；
6) at 4 DEG C, 12000g centrifugation 10min, the 75% ethanol precipitation RNA isometric with RNAiso Plus is added,
4 DEG C of centrifugation 5min of 7500g, remove supernatant；
7) washed twice with 75% ethanol, super-clean bench air-dries；Add 30 μ l DEPC water dissolving precipitation.
8) quality analysis of RNA sample
The RNA concentration and purity extracted are detected using Nanodrop2000, agarose gel electrophoresis detection RNA
Integrality, Agilent2100 measure RIN values.Concentration >=200ng/ μ l, OD260/280 is between 1.8~2.2.
3rd, rRNA is removed
The rRNA in total serum IgE is removed using Ribo-Zero kits.
4th, construction cDNA library
The structure of cDNA library, specific behaviour are carried out using the Truseq RNA sample Prep Kit using Illumina
Make by specification progress.
5th, upper machine sequencing
CDNA library is sequenced using Hiseq4000 microarray datasets, concrete operations by specification is carried out.
6th, high flux transcript profile sequencing data is analyzed
Bioinformatic analysis is carried out to sequencing result, RNA-seq read positioning is carried out using TopHat v1.3.1, leads to
Cross Cufflinks v1.0.3 and RNA-seq segment numbers are standardized to the relative abundance for calculating transcript, utilize
Cuffdiff detects differential expression, works as p value<0.001, | log2 (Fold_change) normalized |>When 1, it is believed that mRNA shows
Write differential expression.
7th, result
RNA-seq results show that expression quantity of the gene M ST1P9 in renal carcinoma tissue is substantially less than the table in cancer beside organism
Up to amount.
The differential expression of the QPCR sequence verification MST1P9 genes of embodiment 2
1st, large sample QPCR checkings are carried out to MST1P9 gene differential expressions.According to the sample collection mode in embodiment 1
Select patients with renal cell carcinoma cancer beside organism and each 50 of renal carcinoma tissue.
2nd, RNA extracts specific steps as described in Example 1.
3rd, reverse transcription
3rd, reverse transcription：Using FastQuant cDNA the first chain synthetic agent box (article No.s：KR106 mRNA reversions) are carried out
Record.Comprise the following steps that：
(1) the μ l of 5 × gDNA Buffer 2.0 are added, the μ g of total serum IgE 1, add Rnase Free ddH2O makes cumulative volume to 10 μ
L, 42 DEG C of heating 3min in water-bath；
(2) 20 μ l reaction systems, 10 × Fast RT Buffer, 2.0 μ L, RT Enzyme Mix 1.0 μ l, FQ- are built
μ l, the RNase Free ddH of RT Primer Mix 2.02Add in the mixed liquor in (1) and mix after the μ l of O 5.0 mixing；
(3) 42 DEG C of heating 15min in water-bath, 95 DEG C of heating 3min, -20 DEG C store for future use.
4th, QPCR is expanded
(1) design of primers
QPCR amplifications are designed according to the coded sequence of MST1P9 genes in Genebank and house-keeping gene GAPDH genes to draw
Thing, synthesized by Bo Maide companies.The primer sequence of MST1P9 genes is as shown in SEQ ID NO.1~2, the primer of GAPDH genes
Sequence is as shown in SEQ ID NO.3~4
(2) PCR reaction systems：Forward primer and each μ of 0.6 μ l, 2 × SuperReal PreMix Plus 10 of reverse primer
L, DNA profiling 2 μ l, ddH2μ l, the 50 × ROX Reference Dye of O 7.4△2 μ l, the μ l of sterile purified water 4.8.
(3) PCR reaction conditions：95 DEG C of 15min, (95 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 32s) × 40 circulations, 95 DEG C of 15s,
60 DEG C of 60s, 95 DEG C of 15s.In the enterprising performing PCR reaction of the type quantitative real time PCR Instruments of ABI 7300, pass through melt curve analysis analysis and electrophoresis
Purpose band is determined, Δ Δ CT methods carry out relative quantification.
5th, result
As a result as shown in figure 1, compared with cancer beside organism, MST1P9 expresses downward in renal carcinoma tissue, and difference has statistics
Learn meaning (P<0.05) it is, consistent with high-flux sequence result.
The differential expression of the protein immunization imprinting of embodiment 3 experiment detection MST1P9 albumen
1st, the extraction of total protein is organized
Put it into and be placed in the glass homogenizer in ice after shredding tissue with scissors, RIPA lysates and PMSF are with 100:
1 ratio is mixed, and the RIPA lysates of the ratio addition respective amount of 100 μ l lysates, glass are added according to every 20mg tissue specimens
Glass homogenizer pulverize tissue until its fully crack, the liquid after cracking is drawn in EP pipes, at 4 DEG C 14000rpm centrifuge
5min, collect supernatant.
2nd, total protein concentration determines
The measure of protein concentration is carried out according to the specification of BCA determination of protein concentration kits.
3rd, SDS-PAGE electrophoresis
According to the separation gel of the specification preparation 8% of PAGE gel reagent preparation box and 5% concentration glue and carry out
Electrophoresis.
4th, western is detected
1) electrotransfer
Pvdf membrane is put into methanol solution and activates 5min, is put into transferring film buffer solution and balances 20min.PAGE glue is taken out to put
Enter in transferring film buffer solution, cut corresponding PAGE glue, according to be followed successively by from down to up filter paper, pvdf membrane, PAGE glue, filter paper it is suitable
Sequence is put into half-dried transferring film instrument, constant pressure 25V transferring films 1.5h；
2) immuning hybridization
Pvdf membrane is taken out, PBS is placed in 5%BSA solution after rinsing shakes closing 2h at room temperature, and pvdf membrane is put into hybridization
In bag, add primary antibody and stay overnight, wash pvdf membrane with TBST buffer solutions, add corresponding secondary antibody, be incubated 2h at room temperature, TBST delays
Fliud flushing is washed.
3) DAB develops the color
The slightly dry rear DAB nitrite ions that Fresh is added dropwise of pvdf membrane, record is scanned after pvdf membrane colour developing.Made with β-actin
For internal reference, sxemiquantitative gray analysis is carried out to band using Quantity One Labworks image acquisition and analysis softwares, experiment repeats 3
It is secondary, as a result take average gray value；
5th, result
As a result as shown in Fig. 2 expression of the MST1P9 albumen in renal carcinoma tissue is substantially less than cancer beside organism.
Differential expression of the MST1P9 genes of embodiment 4 in people's clear cell carcinoma of kidney cell
1st, cell culture
People's clear cell carcinoma of kidney cell line RLC-310,786-0, Caki-2, human embryonic kidney cells HEK293T, with containing 10% tire
Cow's serum and 1%P/S DMEM culture mediums are in 37 DEG C, 5%CO2, relative humidity be 90% incubator in cultivate.Change within 2-3 days
Liquid 1 time, cell growth is good, is grown in monolayer adherence.Passed on using the 0.25% trypsase conventional digestion containing EDTA.
2nd, the detection of MST1P9 gene mRNAs
2.1RNA extraction
The cell in exponential phase is taken, the Trizol cell lysis of respective amount is added according to cell quantity, piping and druming is mixed
Even and be transferred in the centrifuge tube of no RNase, fully homogenate, subsequent step is total with the operation to tissue specimen, extraction cell
RNA。
2.2 reverse transcriptions and QPCR expand specific steps with embodiment 2
3rd, the detection of MST1P9 albumen
The extraction of 3.1 cell proteins
The cell of the different disposal group in logarithmic phase is collected, cell is washed with the PBS of precooling.By RIPA cell pyrolysis liquids
With PMSF with 100:1 ratio is mixed, and the above-mentioned μ l of lysate 150 are added into cell, 30min is placed on ice, is scraped using cell
Knife scrapes off the cell of cracking, and the liquid after cracking is drawn in EP pipes using pipettor, and 14000rpm is centrifuged at 4 DEG C
5min.Supernatant after careful collection centrifugation.
3.2 total protein concentrations determine
The measure of protein concentration is carried out according to the specification of BCA determination of protein concentration kits.
3.3SDS-PAGE and Western detects specific steps with embodiment 3
4th, result
As a result as shown in figure 3, mRNA and protein expression level of the MST1P9 genes in clear cell carcinoma of kidney cell are notable
Less than HEK293T cells, wherein differential expression is the most notable in RLC-310 cells.
The overexpression of the MST1P9 genes of embodiment 5
1st, cell culture step is the same as embodiment 4
2nd, transfect
1) precellular processing is transfected
The day before transfection, 3~5 × 10 are planted on 6 well culture plates5Individual cells/well, one is cultivated in antibiotic-free culture medium
My god, cell density is 30~50% during transfection, changes serum free medium into before transfection.
2) structure of gene overexpression carrier
According to the special pcr amplification primer thing of the sequent synthesis of MST1P9 in GeneBank, primer sequence SEQ ID NO.5~
Shown in 6.
Two restriction enzyme sites of KpnI and XhoI are added respectively in 5 ' end primers and 3 ' end primers.With patients with renal cell carcinoma group
Knit the cDNA that extraction and reverse transcription obtain and be used as amplification template, above-mentioned cDNA sequence is through the double enzymes of restriction enzyme KpnI and XhoI
The recombinant vector that acquisition is connected in the eukaryotic expression vector pcDNA3.1 through KpnI and XhoI double digestions is inserted into after cutting
PcDNA3.1-1 is used for subsequent experimental.
3) transfect
Kidney cancer cell is divided into 3 groups, respectively control group (RLC-310), blank control group (transfection pcDNA3.1-NC),
Experimental group (transfection pcDNA3.1-1).The transfection of carrier is carried out using liposome 2000, specific transfection method is to specifications
Instruction is carried out.PcDNA3.1 empty carriers and pcDNA3.1-1 transfection concentrations are 0.5 μ g/ml.
3rd, QPCR detects the expression of MST1P9 genes
1) extraction of cell total rna
The RNA in cell is extracted using Qiagen cell RNA extracts kit, experimental implementation is to specifications
Carry out.
2) reverse transcription step is the same as embodiment 2.
3) QPCR amplification steps are the same as embodiment 2.
4th, Western blot detect the expression specific steps of MST1P9 albumen with embodiment 4
5th, result
As a result as shown in figure 4, with non-transfection group compared with transfecting empty plasmid group, the MST1P9 mistakes in MST1P9 groups are transfected
Expression, difference have statistical significance (P<0.05).
The influence of the MST1P9 gene pairs kidney cancer cell of embodiment 6 propagation
Influenceed using MTT experiment detection MST1P9 gene pairs kidney cancer cells multiplication capacity.
1st, the good cell of upgrowth situation is taken, conventional digestion counts cell, cell is diluted into conjunction into after single cell suspension
The cell suspension of suitable concentration；
2nd, in 96 well culture plates, the different disposal group cell per well after dilution is inoculated with 2000 cells, 3 is at least set and puts down
Row hole, 37 DEG C, 5%CO2Cultivate 24h；
3rd, 1 after inoculation, 2,3,4,5 days daily OD values taken out 3 hole cells and its 570nm is detected with mtt assay, counted
Number, calculate average value；
4th, abandoning supernatant before detecting, nutrient solution are washed 3 times, and MTT free serum cultures based sols (0.2mg/ml) are added per hole
100 μ l, continue in 37 DEG C of incubators to cultivate 4h；
5th, culture is terminated, careful inhale abandons supernatant, 150 μ l DMSO are added per hole, shake 10min, make crystal fully molten
Solution, with wavelength it is 570nm measure optical density (OD) values on ELIASA, using the time as transverse axis, it is thin that OD value is that the longitudinal axis is drawn
Intracellular growth curve.
6th, result
As a result as shown in figure 4, compared with the control, the propagation of experimental group cell is substantially inhibited, difference has statistics
Learn meaning (P<0.05) illustrate that MST1P9 overexpressions have the function that to suppress cancer cell multiplication.
The influence of the MST1P9 gene pairs Change of Apoptosis in Renal Cancer Cells of embodiment 7
Use the influence of flow cytomery MST1P9 gene pairs Apoptosis.
1st, cell culture step is the same as embodiment 3.
2nd, cell transfecting step is the same as embodiment 3.
3rd, step
1) cell of the different disposal group in exponential phase is broken into cell suspension through pancreatin digestion after-blow and counted.
Take 106The cell suspension of amount, 1000rpm centrifugations 5min；
2) supernatant is abandoned, 195 μ l Annexin V-FITC combination liquid is added and cell is gently resuspended；
3) 5 μ l Annexin V-FITC are added, soft to mix, lucifuge is incubated 10min at room temperature；
4) 1000rpm centrifuges 5min, abandons supernatant, and cell is gently resuspended in the Annexin V-FITC combination liquid for adding 190 μ l；
5) 10 μ l propidium iodides (PI) dyeing liquors are added, it is soft to mix, ice bath avoid light place, carry out flow cytomery
Apoptosis situation, all experiments are repeated 3 times, results averaged.
4th, result：
As a result show, for experimental group compared with control group, the apoptosis rate of cell illustrates that MST1P9 is thin to kidney without significant change
The Apoptosis of born of the same parents is little.
The cell scratch experiment of embodiment 8
1st, the μ g/ml of 1ml 50 fibronectin is added per hole into 6 orifice plates, is placed in 4 DEG C of refrigerator overnights；
2nd, remaining Fibronectin solution is discarded, is cleaned with serum free medium, by not existing together in exponential phase
Reason group cell is inoculated in 6 orifice plates for being covered with fibronectin after pancreatin digestion is resuspended, and every group of cell sets 2 multiple holes, per hole 5
×105Individual cell；
3rd, 37 DEG C, 5%CO are inserted2Overnight incubation in incubator；
4th, when cell length to about 90% fusion, an acellular thin trace is marked with 10 μ l Tip heads, PBS solution is washed
The cell to come off is removed, serum free medium is added and continues to cultivate；
5th, 0h, 48h observe the healing state at cell cut and taken pictures after cut.Experiment is repeated 3 times, and is as a result taken
Average value.
6th, result
As a result as shown in figure 5, experimental group is compared to for control group, the migration distance after cells in vitro cut substantially reduces,
And illustrate that MST1P9 is overexpressed the migration that can suppress kidney cancer cell without significant difference between control group.
The cell invasion of embodiment 9 is tested
1st, prepared by Transwell cells
By 50mg/L Matrigel glue with the serum free medium of 4 DEG C of precoolings with 1:8 dilution proportion, mix, coating
The upper chamber face of the bottom film of Transwell cells, 4 DEG C air-dry.Take the Matrigel glue (3.9 μ g/ μ l) of the μ l of 60 μ l~80 dilutions
It is placed on the polycarbonate membrane for the Transwell upper chambers that aperture is 8 μm, all micropores on film is covered by Matrigel
Lid, being placed in 37 DEG C of 30min makes Matrigel aggregate into gel.
2nd, cell suspension is configured
The cell of different disposal group in exponential phase is digested through pancreatin, after serum free medium is resuspended, adjustment
Cell concentration is 5 × 104Individual/ml.
3rd, cell is inoculated with
2ml cell suspension is added in Transwell upper chambers, lower room adds the 1ml complete training containing 10% hyclone
Base is supported, is positioned on 6 supporting orifice plates, 37 DEG C, 5%CO2Under the conditions of cultivate 20-24h；Transwell cells are taken out, cotton swab is wiped
The Matrigel glue in most upper chamber face and the cell for not penetrating film.
4th, dye
After cell culture terminates, Transwell cells are taken out, cotton swab wipes the Matrigel glue in upper chamber face to the greatest extent and do not penetrate film
Cell, lower room face is with after 95% alcohol fixation 15min, haematoxylin dyeing 2min, and 5 high power lenses are taken at random under inverted microscope
Visual field observation, count and take pictures.The cell number for counting cell Xia Shi faces is to penetrate the cell number of Matrigel glue, is taken the mean
As experimental result, and the invasiveness of tumour cell is represented with the cell number, experiment is repeated 3 times, and every group of cell sets 3 multiple holes.
5th, result
As a result as shown in fig. 7, compared with control group, the cell of experimental group passes through Transwell cells polycarbonate membrane
Cell number significantly reduces, and no significant difference between control group, as a result illustrates that MST1P9 is related to the invasion and attack of cancer cell.
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention
And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
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Claims (10)
1. the purposes of a kind of MST1P9 genes and its expression product, it is characterised in that for preparing the product of early diagnosis kidney.
2. purposes according to claim 1, it is characterised in that the product includes detection MST1P9 genes and its expression production
The reagent of thing expression.
3. the purposes according to right wants 2, it is characterised in that the reagent is included with RT-PCR, real-time quantitative PCR, original position
Hybridization, chip or immunoassay detection MST1P9 genes and its expression product expression reagent.
4. a kind of product for diagnosing kidney, it is characterised in that the product includes MST1P9 genes or its coding in detection sample
Protein expression level reagent.
5. product according to claim 4, it is characterised in that the probe of the reagent including specific recognition MST1P9 or
Specific amplification MST1P9 primer；Or the antibody or part of the albumen of specific binding MST1P9 codings.
6. the purposes of a kind of MST1P9 genes and its expression product, it is characterised in that for screening the potential material for the treatment of kidney.
A kind of 7. method for the potential material for screening prevention or treatment kidney, it is characterised in that screening step is：
The system for the albumen expressed or containing MST1P9 genes or its coding is handled with candidate substances；With
Detect the expression of the albumen of MST1P9 genes or its coding or activity in the system；
Wherein, if candidate substances can increase MST1P9 expression or activity, it is prevention or treatment lung gland to show the candidate substances
The potential material of cancer.
8. the purposes of a kind of MST1P9 genes and its expression product, it is characterised in that for preparing the drug regimen for the treatment of kidney
Thing.
9. purposes according to claim 8, it is characterised in that described pharmaceutical composition include MST1P9 genes, albumen or
Its accelerator.
10. a kind of pharmaceutical composition for treating kidney, it is characterised in that described pharmaceutical composition includes：MST1P9 feature tables
The accelerator reached, and/or pharmaceutically acceptable carrier；The preferable accelerator is MST1P9 over-express vectors.
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Citations (4)
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|---|---|---|---|---|
| US20020142981A1 (en) * | 2000-06-14 | 2002-10-03 | Horne Darci T. | Gene expression profiles in liver cancer |
| US20050130193A1 (en) * | 2003-09-10 | 2005-06-16 | Luxon Bruce A. | Methods for detecting, diagnosing and treating human renal cell carcinoma |
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| US20020142981A1 (en) * | 2000-06-14 | 2002-10-03 | Horne Darci T. | Gene expression profiles in liver cancer |
| US20050130193A1 (en) * | 2003-09-10 | 2005-06-16 | Luxon Bruce A. | Methods for detecting, diagnosing and treating human renal cell carcinoma |
| CN104718455A (en) * | 2012-09-07 | 2015-06-17 | 基诺麦因有限公司 | Method and kit for detecting renal cancer blood biomarkers |
| CN107083428A (en) * | 2017-04-10 | 2017-08-22 | 徐州医科大学 | Applications of the PAK5 in cancer diagnosis prognosis treatment and drug screening |
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