The diagnosis marker and its therapeutic targets of a kind of clear cell carcinoma of kidney
Technical field
The invention belongs to biomedicine field, it is related to the diagnosis marker and its therapeutic targets of a kind of clear cell carcinoma of kidney,
The specific diagnosis marker and its therapeutic targets are MRO.
Background technology
Clear-cell carcinoma (renal cell carcinoma, RCC), also known as Grawitz's tumor, referred to as kidney, it is small to be initiated by kidney
The malignant tumour of pipe epithelial cell, its incidence of disease occupy the second of urological cancer.In recent years, the kidney incidence of disease is on obvious
The trend of liter.Clear cell carcinoma of kidney (clear cell renal cell carcinoma, ccRCC) is the most common in kidney
Tissue typing, account for 80-85%.The treatment most important method of kidney is exactly Radical Nephrectomy at present, is confined to for lump
Kidney in perirenal fascia, it is the most definite the effect of surgery excision, but for the patient of infiltration metastasis has occurred, then curative effect is very
It is limited.In addition, about recurrence or transfer occurs in 20-40% patient in 3 years after surgery after radical nephrectomy.Because kidney is for putting
Chemotherapy is insensitive, still lacks highly effective treatment means at present for advanced renal cell cancer and kidney postoperative recurrence transfer patient,
Although prognosis of the development of molecular targeted agents treatment to such patient has certain improvement, the overall treatment of patients with renal cell carcinoma
Effect is still difficult satisfactory.Therefore, it has been very urgent for kidney to seek new effective diagnosis and treatment means.
The research of the past shows that Multiple factors promote ccRCC occurrence and development, such as activation, the suppression cancer of oncogene
The inactivation of gene or the unconventionality expression of growth factor etc..Domestic and foreign scholars have carried out numerous studies in the field, find in succession
VHL, VEGF etc. are a series of with kidney occurrence and development closely related gene or signal path.Nevertheless, we are to ccRCC
The specific mechanism of occurrence and development is still imperfectly understood, it is necessary to carry out more Basic Experiment Studies.
The research for developing into disease pathogenesis of high throughput sequencing technologies provides more analyzes hand comprehensively and quickly
Section, high throughput sequencing technologies are widely used because it produces digitized signal, high sensitivity and Whole genome analysis.
By carrying out high-flux sequence to the sample of patient, the gene of the differential expression in disease is found, and arrange by further experiment
Except false positive, the gene directly related with disease development is found, so as to the research for pathogenic mechanism, and the prediction of disease
Research foundation is provided with drug development.
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 diagnosis mark of clear cell carcinoma of kidney
Will thing, judge patient whether with clear cell carcinoma of kidney or to suffer from kidney transparent by detecting the expression of mark in sample
The risk of cell cancer.
The second object of the present invention, there is provided a kind of therapeutic targets of clear cell carcinoma of kidney, by changing target in patient
Expression, so as to reach the purpose for the treatment of clear cell carcinoma of kidney.
The third object of the present invention, there is provided a kind of method for the potential material for screening prevention or treatment clear cell carcinoma of kidney,
Can the expression of biological target be adjusted by the material to judge whether screened material is that prevention or treatment kidney are saturating
The potential material of clear cell carcinoma.
To achieve these goals, the present invention adopts the following technical scheme that:
The invention provides the reagent of detection MRO expressions answering in the product for preparing diagnosis clear cell carcinoma of kidney
With.
Further, the product includes chip, preparation or kit.Wherein, the product can by RT-PCR, in real time
The detection techniques such as quantitative PCR, immune detection, in situ hybridization or chip technology detect MRO expression.
The invention provides the albumen of MRO genes or its coding in a kind of chip, preparation or kit, including detection sample
The reagent of expression.
Further, the reagent includes specific recognition MRO probe or specific amplification MRO primer;Or specificity
With reference to the antibody or part of the MRO albumen encoded.
Further, the reagent comprises at least a pair of specific amplification MRO primer, the preferable specific amplification
The sequence of MRO primer is as shown in SEQ ID NO.1~2.
In the present invention, the chip, preparation or kit can be used for detecting including MRO and clear cell carcinoma of kidney
The expression of related multiple genes and/or its expression product.Multiple gene association diagnosis, can increase clear cell carcinoma of kidney
The accuracy of diagnosis.
The invention provides applications of the MRO in the potential material of screening prevention or treatment clear cell carcinoma of kidney.
Further, the potential material is the protein expression level or activity that can promote MRO genes and/or its coding
Material.
Further, the step of screening potential material is as follows:
The system for the albumen expressed or containing MRO genes or its coding is handled with candidate substances;With
Detect the expression of the albumen of MRO genes or its coding or activity in the system;
Wherein, if the candidate substances can promote the expression of the albumen of MRO genes or its coding or activity (preferably significantly to rise
Height, 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 show that the candidate substances are preventions or controlled
Treat the potential material of clear cell carcinoma of kidney.
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 clear cell carcinoma 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 MRO genes or its upstream or downstream
The nucleic acid of gene design promotes thing, albumen accelerator, protein binding molecule.
The invention provides the accelerator of MRO functional expressions in the pharmaceutical composition for preparing treatment clear cell carcinoma of kidney
Application.
Further, the accelerator includes improving MRO genes or its expression product stability, up-regulation MRO genes or its table
Expression, increase MRO genes or the material of its expression product effective acting time up to product.
The invention provides a kind of pharmaceutical composition for treating clear cell carcinoma of kidney, described pharmaceutical composition includes MRO work(
Can property expression accelerator, and/or with other medicine classes and pharmaceutically acceptable carrier of the accelerator compatibility and/or auxiliary
Material.The accelerator includes the expression for improving MRO genes or its expression product stability, up-regulation MRO genes or its expression product
Horizontal, increase MRO genes or the material of its expression product effective acting time.
Further, the accelerator is MRO over-express vectors.The pharmaceutically acceptable carrier and/or auxiliary material, 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 clear cell carcinoma using QPCR detection MRO genes;
Fig. 2 is the expression figure in renal clear cell carcinoma using Western blot detection MRO albumen;
Fig. 3 is expression figures of the MRO in clear cell carcinoma of kidney cell;Wherein, figure A is that detection MRO mRNA are saturating in kidney
Expression figure in clear cell carcinoma cell;Figure B is the expression figure for detecting MRO albumen in clear cell carcinoma of kidney cell;
Fig. 4 is transfected condition figures of the MRO in clear cell carcinoma of kidney cell;Wherein, figure A is to utilize QPCR detection transfections pair
The influence figure that MRO mRNA are expressed in clear cell carcinoma of kidney cell;Figure B is transparent to kidney using Western blot detection transfections
The influence figure of MRO albumen in cell carcinoma;
Fig. 5 is the influence figure bred with mtt assay detection MRO gene pairs clear cell carcinoma of kidney cell;
Fig. 6 is the influence figure with flow cytomery MRO gene pairs clear cell carcinoma of kidney Apoptosis;
Fig. 7 is to utilize influence figures of the cell scratch experiment detection MRO to clear cell carcinoma of kidney cell migration;
Fig. 8 is to utilize influence figures of the Transwell cells detection MRO to clear cell carcinoma of kidney cell invasion.
Specific embodiment
The present invention, by high-flux sequence method, is detected in clear cell carcinoma of kidney sample by in-depth study extensively
Gene tumor tissues and cancer beside organism expression, find wherein have obvious differential expression gene, it is transparent with kidney to inquire into it
Relation between the generation of cell cancer, so as to the early detection for clear cell carcinoma of kidney and targeted therapy find more preferable approach and
Method.By screening, present invention firstly discovers that MRO conspicuousnesses are lowered in clear cell carcinoma of kidney.It is demonstrated experimentally that pass through increase
It MRO expression, can effectively suppress growth, apoptosis and the invasion and attack of clear cell carcinoma of kidney cell, prompt detection MRO genes
Expression can turn into one of auxiliary diagnostic index of clear cell carcinoma of kidney early diagnosis, up-regulation MRO gene expressions can turn into
Prevention or treatment clear cell carcinoma of kidney or the new way of clear cell carcinoma of kidney transfer.
MRO genes
MRO is taken positioned at the area 1 of No. 18 chromosome long arms of people 2, the present invention in MRO include wild type, saltant type or its
Fragment.MRO genes (NC_ in a kind of representational MRO gene orders such as current international public nucleic acid database GeneBank
000018.10) shown in.
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.
Chip, preparation, kit
In the present invention, " chip ", " microarray ", " array " can be included but is not limited to equivalent substitute:The micro- battle arrays of DNA
Row (for example, cDNA microarrays and oligonucleotide microarray), protein microarray, micro-array tissue, transfection or cell microarray,
Chemical compound microarray and Antibody microarray.The DNA microarray of commonly referred to as genetic chip, DNA chip or biochip is
The set of microcosmic DNA points, these points are connected on the surface of solids (for example, glass, plastics or silicon), formed for thousands of
Kind gene carries out expression pattern analysis or the array of expression monitoring simultaneously.Fixed DNA fragmentation is referred to as probe, its it is thousands of can
For in single DNA microarray.Microarray can be used for identifying disease by comparing the gene expression in disease and normal cell
Gene or transcript (for example, ncRNA).Multiple technologies can be used to be manufactured for microarray, and include but is not limited to:Printed with apicule needle
Brush on slide, carry out photoetching using prefabricated mask, carry out photoetching, ink jet printing or microelectrode using dynamic micro mirror element
Electrochemical method on array.
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.
In the present invention for MRO genes oligonucleotide probe can be DNA, RNA, DNA-RNA chimera, PNA or its
Its derivative.The length of the probe does not limit, as long as completing specific hybrid, being tied with purpose nucleotide sequence specificity
Close, any length can.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the probe
Length can grow to 60,80,100,150,300 base-pairs or longer, or even whole gene.Due to different probe lengths pair
Hybridization efficiency, signal specificity have different influences, and the length of the probe is typically at least 14 base-pairs, most it is long it is general not
More than 30 base-pairs, complementary length is optimal with 15-25 base-pair with purpose nucleotide sequence.The probe self-complementary
Sequence is most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
The invention provides a kind of kit, the kit can be used for detection MRO expression.Preferably, described system
Also contain the label for labeled RNA sample, and the substrate corresponding with the label in agent or kit.In addition,
It may also include for extracting the various reagents needed for RNA, PCR, hybridization, colour developing etc., include but is not limited in described kit:
Extract, amplification liquid, hybridization solution, enzyme, comparison liquid, nitrite ion, washing lotion etc..In addition, also including the use of saying in described kit
Bright book and/or chip image analysis software.
Can also have the operation instructions of kit in the kit of the present invention, how be entered wherein describing using kit
Row detection, and how tumor development to be judged using testing result, therapeutic scheme is selected.
In the present invention, the antibody or part with MRO specific bindings include monoclonal antibody, polyclonal antibody;This hair
It is bright not only to include complete antibody molecule, also any fragment including antibody or modification, for example, chimeric antibody, scFv, Fab, F
(ab ') 2, Fv etc..As long as the fragment can retain the binding ability with MRO albumen.Antibody for protein level
Preparation when well known to a person skilled in the art and the present invention can prepare the antibody using any method
Accelerator and pharmaceutical composition
Discovery based on inventor, the invention provides a kind of MRO accelerator, the accelerator includes improving MRO
Gene or its expression product stability, the expression of up-regulation MRO genes or its expression product, increase MRO genes or its expression
The material of product effective acting time.
Generally, these accelerator can be formulated in nontoxic, inert and pharmaceutically acceptable aqueous carrier medium,
Wherein pH ordinarily be about 5-8, and preferably pH is about 6-8, although pH value can be with the property and disease to be treated for being formulated material
Disease and be varied from.The pharmaceutical composition prepared can be administered by conventional route, including (but being not limited to):
Knurl is interior, intramuscular, intraperitoneal, intravenous, subcutaneous, intracutaneous or part are administered.
As a kind of preferred embodiment of the present invention, described MRO accelerator is a kind of MRO expression vector.Described
Expression vector is generally also containing promoter, replication orgin and/or marker gene etc..
Method well-known to those having ordinary skill in the art can be used to build the required expression vector of the present invention.These methods include
Recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc..Described expression vector is preferably comprising one or more
Selected marker, to provide the phenotypic character for the host cell for being used to select conversion, such as kalamycin, gentamicin, tide
Mycin, amicillin resistance.
In the present invention, be various carriers known in the art, such as commercially available carrier including plasmid, clay, bacteriophage,
Virus etc..Importing of the expression vector into host cell can use electroporation, calcium phosphate method, liposome method, DEAE Portugals to gather
The known methods such as sugared method, microinjection, viral infection, the combination of liposome transfection and cell-membrane permeable peptide.
In the present invention, " host cell " born of the same parents can be prokaryotic, such as bacterial cell;Or low eukaryotic, such as
Yeast cells;Or higher eucaryotic cells, such as mammalian cell.Representative example has:Escherichia coli, the bacterium of streptomyces
Cell;Fungal cell's such as yeast;Plant cell;Drosophila S2 or Sf9 insect cell;The animal of CHO, COS or 293 cells is thin
Born of the same parents etc..
It can be carried out with recombinant DNA conversion host cell with routine techniques well known to those skilled in the art.When host is original
When core biology is such as Escherichia coli, can absorb DNA competent cell can harvest after exponential phase of growth, use CaCl2Method processing, institute
With the step of it is generally well-known in the art.Another method is to use MgCl2.If desired, conversion can also use the side of electroporation
Method is carried out.When host is eucaryote, following DNA transfection methods can be selected:Calcium phosphate precipitation, conventional mechanical methods are such as
Microinjection, electroporation, liposome packaging etc..
Present invention also offers a kind of pharmaceutical composition, and it contains the described MRO of effective dose accelerator, and pharmacy
Upper acceptable carrier.Described composition can be used for treating clear cell carcinoma of kidney.Any foregoing MRO accelerator is available
In the preparation of composition.The pharmaceutically acceptable carrier and/or auxiliary material, including but not limited to diluent, adhesive, table
Face activating agent, 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.
As used herein, described " effective dose " refer to that people and/or animal can be produced function or activity and can by people and/
Or the amount that animal is received.The effective dose of accelerator can become with the pattern of administration and the order of severity etc. of disease to be treated
Change.The selection of preferable effective dose can be determined (such as to pass through clinic by those of ordinary skill in the art according to various factors
Experiment).Described factor includes but is not limited to:The pharmacokinetic parameter of the accelerator of described MRO genes such as biology profit
With rate, metabolism, half-life period etc.;The order of severity of the disease to be treated of patient, the body weight of patient, patient immune state, to
Approach of medicine etc..
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 pharmaceutical composition of the present invention can also be with the drug combination of other treatment clear cell carcinoma of kidney, other therapeuticization
Compound can be administered simultaneously with main active component, or even be administered simultaneously in same composition.Can also be with single group
Compound or the dosage form different from main active component individually give other therapeutic compounds.
Preferably, the means of gene therapy can be used to carry out.Such as can be directly by MRO accelerator by such as injecting
The methods of deliver medicine to subject;Or ceneme (such as the table that MRO promotion is adjusted can will be carried by certain approach
Up to carrier or virus etc.) it is delivered on target spot, concrete condition need to be depending on the type of described accelerator, and these are this areas
Known to technical staff.
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 represented, statistical analysis is carried out using SPSS18.0 statistical softwares, cancerous tissue with by cancer
The paired comparisons of tissue are examined using t, and paired sample is examined using t, and multisample is carried out using the variance test (ANOVA) of mean
Analysis, it is believed that work 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 clear cell carcinoma of 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 RO in renal clear cell carcinoma is substantially less than in cancer beside organism
Expression quantity.
The differential expression of the QPCR sequence verification MRO genes of embodiment 2
1st, large sample QPCR checkings are carried out to MRO gene differential expressions.Selected according to the sample collection mode in embodiment 1
Clear cell carcinoma of kidney patient cancer beside organism and each 50 of renal clear cell carcinoma.
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 amplimers are designed according to the coded sequence of MRO genes in Genebank and house-keeping gene GAPDH genes, by
Bo Maide companies synthesize.
The primer sequence of MRO genes:
Forward primer sequence is 5 '-TGCTGGTGTATGGACTGTAT-3 ' (SEQ ID NO.1);
Reverse primer sequences are 5 '-CAGAACGACGGTCAGAGT-3 ' (SEQ ID NO.2).
The primer sequence of GAPDH genes:
Forward primer sequence is 5 '-GGAGCGAGATCCCTCCAAAAT-3 ' (SEQ ID NO.3);
Reverse primer sequences are 5 '-GGCTGTTGTCATACTTCTCATGG-3 ' (SEQ ID NO.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 clear cell carcinoma of kidney cancer beside organism, MRO is expressed in renal clear cell carcinoma
Lower, difference has statistical significance (P<0.05) it is, consistent with high-flux sequence result.
The differential expression of the protein immunization imprinting of embodiment 3 experiment detection MRO 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 MRO albumen in renal clear cell carcinoma is substantially less than cancer beside organism.
Differential expression of the MRO 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 MRO gene mRNAs
2.1RNA extraction
Take and be in exponential phase cell, the Trizol cell lysis of respective amount is added according to cell quantity, piping and druming mixes
And be transferred in the centrifuge tube of no RNase, fully homogenate, subsequent step extracts cell total rna with the operation to tissue specimen.
2.2 reverse transcriptions and QPCR amplifications
Specific steps are the same as embodiment 2
3rd, the detection of MRO 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 MRO genes in clear cell carcinoma of kidney cell are substantially less than
Differential expression is the most notable in HEK293T cells, wherein RLC-310 cells.
The overexpression of the MRO 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
It is as follows according to the special pcr amplification primer thing of the sequent synthesis of MRO in GeneBank, primer sequence:
Forward primer:5’-CCGAAGCTTGCCACCATGGCGTGGGGAAGCAAAGG-3’(SEQ ID NO.5)
Reverse primer:5’-CGGCTCGAGCAGAATTTTATTTGCGTAGAAGAAC-3’(SEQ ID NO.6)
Two restriction enzyme sites of HindIII and XhoI are added respectively in 5 ' end primers and 3 ' end primers.Suffered from kidney
CDNA that person's tissue extraction and reverse transcription obtain as amplification template, above-mentioned cDNA sequence through restriction enzyme HindIII and
It is inserted into after XhoI double digestions in the eukaryotic expression vector pcDNA3.1 through HindIII and XhoI double digestions, connection obtains
Recombinant vector pcDNA3.1-1 be 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 MRO 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 MRO albumen with embodiment 4
5th, result
As a result such as Fig. 4 is shown, with non-transfection group compared with transfecting empty plasmid group, the MRO transfected in MRO groups is overexpressed,
Difference has statistical significance (P<0.05).
The influence of the MRO gene pairs clear cell carcinoma of kidney cell of embodiment 6 propagation
Influenceed using MTT experiment detection MRO gene pairs clear cell carcinoma of kidney ability of cell proliferation.
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;Abandoning supernatant before detection, nutrient solution are washed 3 times, and MTT free serum culture based sols (0.2mg/ is added per hole
Ml) 100 μ l, continue in 37 DEG C of incubators to cultivate 4h;
4th, 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.
5th, result
As a result as shown in figure 5, compared with the control, the propagation of experimental group cell is substantially inhibited, difference has statistics
Learn meaning (P<0.05), illustrate that MRO has the function that to suppress cell propagation.
The influence of the MRO gene pairs clear cell carcinoma of kidney Apoptosis of embodiment 7
Use the influence of flow cytomery MRO 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 as shown in fig. 6, experimental group is compared with control group, the apoptosis rate of cell significantly raises, and illustrates MRO overexpression
Promote the apoptosis (P of clear cell carcinoma of kidney cell<0.05).
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;Insert 37 DEG C, 5%CO2Overnight incubation in incubator;
3rd, 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;Respectively at the healing after cut at 0h, 48h observation cell cut
Situation is simultaneously taken pictures.Experiment is repeated 3 times, results averaged.
4th, result
As a result as shown in fig. 7, experimental group is compared to for control group, the healing rate after cells in vitro cut substantially reduces, and
Without significant difference between control group, illustrate that MRO is relevant with the migration of kidney cancer cell.
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 figure 8, 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 the expression of MRO genes and the invasion and attack phase of kidney cancer cell
Close.
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.
Sequence table
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