CN107435074A

CN107435074A - Application of the CES8 genes in clear cell carcinoma of kidney diagnosis and treatment

Info

Publication number: CN107435074A
Application number: CN201710775715.8A
Authority: CN
Inventors: 任静; 马翠
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2017-08-31
Filing date: 2017-08-31
Publication date: 2017-12-05

Abstract

The invention discloses applications of the CES8 in clear cell carcinoma of kidney diagnosis and treatment, the present invention has found CES8 up-regulated expressions in renal clear cell carcinoma by high-flux sequence, the high expression of the gene and its coded the albumen conspicuousness in clear cell carcinoma of kidney patient is further demonstrated by experiment, prompts CES8 to be applied to as diagnosis marker in the clinical diagnosis of clear cell carcinoma of kidney.The expression that the present invention further demonstrates change CES8 by In vitro cell experiment can play inhibitory action to the propagation of cell, migration and invasion and attack, prompt CES8 to be applied to as possible potential target in clear cell carcinoma of kidney and its clinical treatment of transfer.

Description

Application of the CES8 genes in clear cell carcinoma of kidney diagnosis and treatment

Technical field

The invention belongs to biomedicine field, is related to application of the CES8 genes in clear cell carcinoma of kidney diagnosis and treatment.

Background technology

Clear-cell carcinoma (renal cell carcinoma, RCC) is also known as kidney, and the incidence of disease is only second to carcinoma of urinary bladder, is to secrete One of most common malignant tumour in urinary system, account for the 2%-3% of adult malignancies' incidence of disease.Every country and each The incidence of disease in area is different, and developed country is higher than developing country, and in China, in recent years, it is in increase trend year by year that it, which is fallen ill,.About The clear-cell carcinoma for having 70% is clear cell renal cell carcinoma (clear cell renal cell carcinoma, ccRCC), is risen Disease concealment, its atypical clinical manifestations, to rely on imageological examination could find, Newly diagnosed rate is not high, about 30% Patient has shifted in diagnosis, loses opportunity of operation.The main metastasis site of kidney includes lung, liver, bone and local leaching Fawn on, after shifting, the mean survival time (MST) of kidney is only 8~10 months, and transfer is the main original that clear-cell carcinoma causes death Cause.Kidney is insensitive to the kinds of tumors such as radiotherapy, chemotherapy and endocrine therapy treatment means, and effective percentage is less than 20%, treatment Mainly by radical nephrectomy.With the development of biology techniques, appearance the grinding for disease pathogenesis of high-throughput techniques Study carefully and provide more comprehensively and quick analysis means, using high throughput sequencing technologies combination bioinformatic analysis, find and The related important gene of disease development, and the biological experiment of orientation is combined, to realize that disease precision treatment provides New way.

The histological type that kidney shifts is mainly clear cell renal cell carcinoma (clear cell renal cell Carcinoma, ccRCC) and sarcoma sample clear-cell carcinoma hypotype.And ccRCC is most common hypotype in clear-cell carcinoma, 70% is accounted for ~80%.And ccRCC patient, compared with other RCC hypotype poor prognosis, 5 years disease-specific survival rates are 50%~69%, and nipple Shape clear-cell carcinoma is 67%~87%, and chromophobia clear-cell carcinoma is 78%~87%.The formation of kidney and transfer with it is other pernicious Tumour is the same, is polygenes, the factor, the coefficient result of step, it is that kidney turns that wherein kidney cancer cell, which obtains high transfer ability, The important step of shifting.This process needs to be related to signal transduction gene, adhesion molecule, matrix metalloproteinase etc..To improve kidney The diagnosis and survival rate of cell cancer, the biological marker of prediction kidney is found, turn into the focus of kidney research at present.

The content of the invention

In order to make up the deficiencies in the prior art, it is an object of the invention to provide a kind of and clear cell carcinoma of kidney occurrence and development Related biomarker, by the expression for detecting biomarker in sample, it can be determined that whether patient is saturating with kidney Clear cell carcinoma or suffer from clear cell carcinoma of kidney risk size, by targeting biomarker, change biomarker Expression or activity, so as to realize the accurate targeted therapy of clear cell carcinoma of kidney patient.

To achieve these goals, the present invention adopts the following technical scheme that：

The invention provides application of the detection CES8 reagent in the product for preparing diagnosis clear cell carcinoma of kidney.

Further, the reagent of the detection CES8 includes：Pass through sequencing technologies, nucleic acid hybridization technique, nucleic acid amplification technologies Or the method for immunoassays detects the expression reagent of CES8 genes in sample.

Further, the reagent is selected from：

The probe of specific recognition CES8 genes；Or

The primer of specific amplification CES8 genes；Or

Specifically bind the antibody or part of the albumen of CES8 codings.

Further, the reagent comprises at least a pair of specific amplification CES8 primer, in the specific embodiment of the present invention In, the primer sequence of the specific amplification CES8 genes is as shown in SEQ ID NO.3~4.

In the present invention, any method detection CES8 of this area expression can be used, those skilled in the art know The method of road detection CES8 expressions is to realize the means that technical solution of the present invention is realized.

The invention provides a kind of product for diagnosing clear cell carcinoma of kidney, the product, which includes CES8 in detection sample, expresses Horizontal preparation, chip or kit, its chips include genetic chip, protein chip；The genetic chip carries including solid phase Body and the oligonucleotide probe for being fixed on solid phase carrier, the oligonucleotide probe include being used to detect CES8 genetic transcription water The flat oligonucleotide probe for CES8 genes；The protein chip includes solid phase carrier and is fixed on the pin of solid phase carrier To the specific antibody of CES8 albumen；Kit includes gene detecting kit, protein detection kit.

Product of the present invention can be used for the expression of multiple genes or gene expression product of the detection including CES8 Horizontal (the multiple genes or its expression product related to clear cell carcinoma of kidney), by multiple marks of clear cell carcinoma of kidney simultaneously Detected, be greatly improved the accuracy rate of clear cell carcinoma of kidney diagnosis.

The invention provides applications of the CES8 in the candidate compound of screening prevention or treatment clear cell carcinoma of kidney.

Further, the step of screening candidate compound is as follows：

In test group, test compound is added in cultivating system, and observes the table of CES8 in the cell of the test group Up to amount and/or activity；In control group, test compound is not added in identical cultivating system, and observes the institute of control group State the expression quantity and/or activity of CES8 in cell；

Wherein, if the CES8 of cell expression quantity and/or activity are less than control group in test group, the test is indicated that Compound is expression to CES8 and/or activity have inhibitory action treating cancer candidate compound.

In the present invention, described step also includes：The candidate compound of acquisition is carried out further cell experiment and/ Or animal experiment, further to select and determine from candidate compound to have for preventing, alleviating or treat clear cell carcinoma of kidney Material.

In the present invention, the system of the candidate compound of screening prevention or treatment clear cell carcinoma of kidney is not limited to cell body System, in addition to cell system, subcellular fraction system, solution system, organizational framework, organ systems or animal system etc., the system Above-mentioned form is not limited to, as long as the system can detect test compound and can reduce CES8 expression and/activity.

The candidate compound includes but is not limited to：For CES8 genes or the albumen or its upstream or downstream base of its coding Disturbing molecule, nucleic acid inhibitor, binding molecule (such as antibody or part), the micromolecular compound of cause or protein design.

The invention provides the inhibitor of CES8 functional expressions to prepare the medicine of prevention or treatment clear cell carcinoma of kidney Application in composition.

Further, the inhibitor of the CES8 functional expressions is selected from：Nucleic acid inhibitor, protein inhibitor, proteolysis Enzyme, protein binding molecule, the preferable inhibitor is nucleic acid inhibitor siRNA.

In the present invention, the inhibitor of the CES8 can be additionally used in invasion and attack and the propagation for suppressing clear cell carcinoma of kidney cell.

In a particular embodiment of the present invention, the sequence of the siRNA is as shown in SEQ ID NO.11~12.

The invention provides a kind of pharmaceutical composition for treating clear cell carcinoma of kidney, described pharmaceutical composition includes CES8 work( The inhibitor of energy property expression.

The inhibitor is selected from：CES8 gene expressions or gene as target sequence and can be suppressed using CES8 or its transcript The disturbing molecule of transcription, including：ShRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisensenucleic acids, Or it can express or be formed the construction of the shRNA, siRNA, dsRNA, Microrna, antisensenucleic acids；Or specificity with The protein bound binding molecule (if suppressing the antibody or part of CES8 protein actives) of CES8 codings.

Further, described pharmaceutical composition also includes pharmaceutically acceptable carrier.The carrier includes but is not limited to Diluent, excipient, adhesive, wetting agent, sorbefacient, surfactant, Humectant, absorption carrier, lubricant, buffering Agent, stabilizer, bacteriostatic agent, isotonic agent, chelating agent, pH controlling agents.

Brief description of the drawings

Fig. 1 is the expression figure in renal clear cell carcinoma using QPCR detection CES8 genes；

Fig. 2 is the expression figure in renal clear cell carcinoma using western blot detection CES8 albumen；

Fig. 3 is the expression figure for detecting CES8 in cell；Wherein figure A is saturating in kidney using QPCR detections CES8mRNA Expression figure in clear cell carcinoma cell；Figure B is thin in clear cell carcinoma of kidney using Western blot detection CES8 albumen Expression figure in born of the same parents；

Fig. 4 is to detect the interference effect figure that siRNA is expressed CES8 in the transparent cancer cell of kidney；Wherein figure A is detection siRNA To the interference effect figure of CES8mRNA in cell；Figure B is to detect interference effect figures of the siRNA to CES8 albumen in cell；

Fig. 5 is the influence figure bred with mtt assay detection CES8 gene pairs clear cell carcinoma of kidney cell；

Fig. 6 is to utilize influence figures of the cell scratch experiment detection CES8 to clear cell carcinoma of kidney cell migration；

Fig. 7 is to utilize influence figures of the Transwell cells detection CES8 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 CES8 conspicuousnesses raise in clear cell carcinoma of kidney.It is demonstrated experimentally that pass through reduction It CES8 expression, can effectively suppress the growth and invasion and attack of clear cell carcinoma of kidney cell, prompt detection CES8 genes Expression can turn into one of auxiliary diagnostic index of clear cell carcinoma of kidney early diagnosis, and interference CES8 gene expressions can turn into pre- Anti- or treatment clear cell carcinoma of kidney or the new way of clear cell carcinoma of kidney transfer.

Mark

Mark (is used alone or such as clear cell carcinoma of kidney mark, kidney hyaline cell is combined with other qualitative terms Cancer Specific marker, control mark, external source mark, endogenous mark) refer to it is measurable, can calculate or can be otherwise Obtain, it is related to any molecule or molecular combinations, it can be used as the parameter of the indicant of biology and/or chemical state.In this hair In bright, " mark " refers to the parameter related to one or more biomolecule (i.e. " biomarker ") and for example naturally or manually closed Into caused nucleic acid (i.e. genes of individuals, and coding and noncoding DNA and RNA) and albumen (such as peptide, polypeptide).In the present invention " mark " also include referring to can be by considering that expression data from two or more unlike signal things calculate or with its other party The single parameter that formula obtains.

It is transparent carefully that clear cell carcinoma of kidney mark refers to and may be used as and (combine individually or with other marks) kidney in subject The certain types of mark of born of the same parents' cancer indicant, in specific embodiments of the present invention, clear cell carcinoma of kidney mark can use In the mark for providing and (individually or with other marks combining) clear cell carcinoma of kidney clinical assessment in subject.

CES8 genes

CES8 is taken positioned at the area 2 of No. 16 chromosome long arms of people 2, the present invention in CES8 include wild type, saltant type or Its fragment.Nucleotide sequence and amino acid sequence shown in a kind of representational CES8 genes of SEQ ID NO.1 and SEQ ID NO.2 Row.People's CES8 nucleotides full length sequence of the present invention or its fragment can generally use PCR TRAPs, recombination method or artificial synthesized Method obtains.

Detection technique/method

The gene of the present invention is detected using a variety of detection techniques known to persons of ordinary skill in the art, these technologies Including but not limited to：Nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technologies, immunoassay technology.

The exemplary, non-limitative example of Nucleic acid sequencing techniques includes but is not limited to chain terminator (Sanger) sequencing and dye Expect terminator sequencing.One of ordinary skill in the art it will be recognized that due to RNA in cell less stable and in an experiment Be more vulnerable to nuclease attack, thus before sequencing generally by RNA reverse transcriptions into DNA.

The another exemplary non-limiting examples of Nucleic acid sequencing techniques include sequencing of future generation, and (deep sequencing/high pass measures Sequence), high throughput sequencing technologies be it is a kind of based on unimolecule cluster in synthesis sequencing technologies, based on proprietary reversible termination chemistry Reaction principle.The DNA of genome random fragment is attached to optically transparent glass surface during sequencing, these DNA fragmentations warp After crossing extension and bridge amplification, hundreds of millions of clusters is formed in glass surface, each cluster is the list for having thousands of parts of same templates Molecular cluster, four kinds of special deoxyribonucleotides with fluorophor are then utilized, skill is sequenced in synthesis by reversible Template DNA to be measured is sequenced art.

The exemplary, non-limitative example of nucleic acid hybridization technique include but is not limited in situ hybridization (ISH), microarray and Southern or Northern traces.In situ hybridization (ISH) be it is a kind of use mark complementary DNA or RNA chains as probe with A position tissue part or section (original position) are the spy in whole tissue (full organization embedding ISH) if tissue is sufficiently small Different in nature DNA or RNA sequence hybridization.DNA ISH can be used for the structure for determining chromosome.RNA ISH are used to measuring and positioning group Knit the mRNA and other transcripts (for example, ncRNA) in section or full organization embedding.At generally to sample cell and tissue Reason increases the entrance of probe with fixation in situ target transcript.Probe hybridizes with target sequence at high temperature, then by unnecessary spy Pin is washed off.Autoradiograph, fluorescence microscopy or immunohistochemistry are used respectively, to using radiation, fluorescence or antigen in tissue The probe of the kilobase marker of mark is positioned and quantified.Two or more can also be used by radioactivity by ISH or other are non- The probe of radioactive label substance markers, to detect two or more transcripts simultaneously.

The present invention can simultaneously expand before detection or with detection to nucleic acid (for example, ncRNA).Nucleic acid amplification technologies Exemplary, non-limitative example include but is not limited to：PCR (PCR), reverse transcriptase polymerase chain reaction (RT- PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and based on nucleotide sequence Expand (NASBA).One of ordinary skill in the art will be it will be recognized that some amplification techniques (for example, PCR) needs will before amplification RNA reverse transcriptions are into DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).

Commonly referred to as PCR PCR uses annealing and the primer extend of denaturation, primer pair and opposite strand Multiple circulations, exponentially increase target nucleic acid sequence copy number；The amplification of TMA transcriptive intermediate is (in substantial constant Temperature, multiple copies of target nucleic acid sequence are autocatalytically synthesized under conditions of ionic strength and pH, wherein target sequence is more Individual RNA copies autocatalytically generate other copy；LCR ligase chain reaction uses miscellaneous with the adjacent area of target nucleic acid The two groups of complementary DNA oligonucleotides handed over；Other amplification methods are included for example：The commonly referred to as NASBA expansion based on nucleotide sequence Increase；Use rna replicon enzyme (the commonly referred to as Q β replicase) amplification of amplification probe molecule in itself；Amplification method based on transcription； And the sequence amplification of self―sustaining.

The nucleic acid of non-amplification or amplification can be detected by any conventional means in the present invention.

Chip, kit

In the present invention, " chip ", " microarray ", " array " can be included but is not limited to equivalent substitute：DNA microarray (for example, cDNA microarrays and oligonucleotide microarray), protein microarray, micro-array tissue, transfection or cell microarray, change Chemical combination thing microarray and Antibody microarray.The DNA microarray of commonly referred to as genetic chip, DNA chip or biochip is micro- The set of DNA points is seen, these points are connected on the surface of solids (for example, glass, plastics or silicon), are formed for thousands of kinds Gene carries out expression pattern analysis or the array of expression monitoring simultaneously.Fixed DNA fragmentation is referred to as probe, and its is thousands of available In single DNA microarray.Microarray can be used for identifying disease base by comparing the gene expression in disease and normal cell Cause 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 Photoetching is carried out on to slide, using prefabricated mask, carries out photoetching, ink jet printing or microelectrode battle array using dynamic micro mirror element Electrochemical method on row.

Kit in the present invention can be used for detection CES8 expression, it is preferred that it is effective that described kit includes detection The reagent of the detection CES8 genes of amount, the one or more materials being selected from the group：Container, operation instructions, positive control, the moon Property tester, buffer, auxiliary agent or solvent.Such as being suspended or fixing the solution of cell, detectable label or tag, make Nucleic acid is easy to the solution of hybridization, for the solution of cell lysis, or the solution for nucleic acid purification.

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.

Using the kit of the present invention, CES8 can be detected by the various methods (including but is not limited to) being selected from the group：It is real When Quantitative Reverse Transcription PCR, biochip test method, southern blotting technique method or RNA blottings or hybridization in situ.The common skill in this area Art personnel can be according to physical condition and needing to be adjusted detection mode and change.

Inhibitor and pharmaceutical composition

Discovery based on inventor, it is saturating for preparing suppression kidney the invention provides a kind of purposes of CES8 inhibitor The pharmaceutical composition of clear cell carcinoma.As used herein, described CES8 inhibitor include but is not limited to inhibitor, antagonist, Retarding agent, blocking agent, nucleic acid inhibitor etc..

Described CES8 genes or the inhibitor of albumen refer to any activity for reducing CES8 albumen, reduce CES8 genes Or the stability of albumen, the expression for lowering CES8 albumen, reduction CES8 albumen effective acting times or turn for suppressing CES8 genes Record and the material of translation, these materials are used equally for the present invention, as the material useful for lowering CES8, so as to for pre- Anti- or treatment clear cell carcinoma of kidney.For example, described inhibitor is：Nucleic acid inhibitor, protein inhibitor, antibody, part, albumen Hydrolase, protein binding molecule, as long as it can lower CES8 albumen or the table of its encoding gene on albumen or gene level Reach.

As a kind of selection mode of the present invention, described CES8 inhibitor is resisting of being combined with CES8 of a species specificity Body.The specific antibody includes monoclonal antibody, polyclonal antibody；The present invention not only includes complete antibody molecule, also wraps Any fragment or the modification of antibody are included, for example, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as the fragment can protect Stay the binding ability with CES8 albumen.During preparation for the antibody of protein level well known to a person skilled in the art, And the present invention can prepare the antibody using any method

As a kind of preferred embodiment of the present invention, the inhibitor of the CES8 is a kind of specific siRNAs of CES8 Molecule.As used herein, described " siRNA " refers to a kind of short-movie section double stranded rna molecule, can be with homologous complementary sequence MRNA degraded specific mRNA for target, this process is exactly RNA interference (RNA interference) processes.Small interference RNA can be prepared into the form of double-strandednucleic acid, and it contains a positive-sense strand and an antisense strand, and this two chains are only in the bar of hybridization Double-strand is formed under part.One double-stranded RNA compound can be prepared by the positive-sense strand that is separated from each other and antisense strand.Therefore, illustrate For, complementary positive-sense strand and antisense strand are chemical syntheses, thereafter can be by anneal, and the double-stranded RNA for producing synthesis is answered Compound.

When screening effective siRNA sequence, the present inventor is optimal effective so as to find out by largely comparing analysis Fragment.The present inventor's design has synthesized a variety of siRNA sequences, and they are transfected into clear cell carcinoma of kidney by transfection reagent respectively Cell line is verified, selects the optimal siRNA of interference effect, and they have SEQ ID NO.11, SEQ ID NO.12 respectively Shown sequence, further tested in cellular level, as a result prove that suppression efficiency is very high for test cell line.

The nucleic acid inhibitor such as siRNA of the present invention can be with chemical synthesis, can also be by a recombinant nucleic acid structure Expression cassette is prepared after being transcribed into single stranded RNA.The nucleic acid inhibitors such as siRNA, can be by using appropriate transfection reagent quilt It is transported into the cell, or can be also transported into the cell using multiple technologies known in the art.

As a kind of optional mode of the present invention, described CES8 inhibitor can also be a kind of " children purpura nephritis (Small hairpin RNA, shRNA) ", it is the non-coding small RNA molecular that can form hairpin structure, children purpura nephritis energy Enough by RNA interference channels come the expression of suppressor.As described above, shRNA can be expressed by double-stranded DNA template.Double-stranded DNA Template is inserted into a carrier, such as plasmid or viral vector, is then connected to a promoter carry out table in vitro or in vivo Reach.ShRNA in the presence of DICER enzymes, can be cut into siRNA molecule in eukaryotic, hence into RNAi approach. " shRNA expression vectors " refers to plasmid of some this areas conventionally used for building shRNA structures, exist on the usual plasmid " Every sequence " and multiple cloning sites positioned at " intervening sequence " both sides or for replacing sequence, so as to people can by shRNA (or Analog) corresponding DNA sequence dna inserted by way of forward and reverse multiple cloning sites or replace thereon for replacing sequence, RNA after DNA sequence dna transcription can form shRNA (Short Hairpin) structure.Described " shRNA expression vectors " is current It can be bought and obtained by commercially available approach completely, such as some viral vectors.

Present invention also offers a kind of pharmaceutical composition, and it contains the described CES8 of effective dose inhibitor, and medicine Acceptable carrier on.Described composition can be used for suppressing clear cell carcinoma of kidney.Any foregoing CES8 inhibitor is equal Preparation available for composition.The carrier includes but is not limited to diluent, excipient, adhesive, disintegrant, absorption enhancement Agent, surfactant, Humectant, absorption carrier, lubricant, buffer, stabilizer, bacteriostatic agent, isotonic agent, chelating agent, pH controls Preparation.

Wherein, diluent such as lactose, sodium chloride, glucose, urea, starch, water etc.；Excipient such as lactose, sodium chloride, Portugal Grape sugar, urea, starch, water etc.；Adhesive such as simple syrup, glucose solution, starch solution, cellulose derivative, alginates, bright Glue and polyvinylpyrrolidone；Disintegrant such as dried starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and carbonic acid Hydrogen sodium；Sorbefacient quaternary ammonium compound, lauryl sodium sulfate etc.；Surfactant such as polyoxyethylene sorbitan fat Acid esters, lauryl sodium sulfate, glyceryl monostearate, hexadecanol etc.；Humectant such as glycerine, starch etc.；Absorption carrier is such as Starch, lactose, bentonite, silica gel, kaolin and soap clay etc.；Lubricant such as talcum powder, calcium stearate and magnesium, polyethylene glycol, Boric acid powder etc.；Buffer can include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid and corresponding salt (their alkali metal Or alkaline rare earth metal salt, such as sodium salt, sylvite, calcium salt and magnesium salts)；Stabilizer includes Human serum proteins, l-amino acid, sugar And cellulose derivative；Bacteriostatic agent include but is not limited to valid density (such as<Benzylalcohol, phenol, metacresol, neoprene 1%w/v) Alcohol, methyl p-hydroxybenzoate and/or propylparaben；Isotonic agent includes potassium chloride, sodium chloride, sugar and glycerine；Chela Mixture includes sodium ethylene diamine tetracetate and citric acid；

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 inhibitor 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 inhibitor of described CES8 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 CES8 inhibitor by such as injecting The methods of deliver medicine to subject；Or the ceneme for the inhibitor for carrying CES8 (for example can be expressed by certain approach Carrier or virus etc., or siRNA or shRNA) it is delivered on target spot, and it is allowed to the CES8 inhibitor of expression activity, concrete condition Need to be depending on the type of described inhibitor, these are well-known to those skilled in the art.

In the present invention, term " sample " is used with its broadest sense.It is intended to include any people for being derived from work or death Tissue or material, its may include the present invention mark.In a particular embodiment of the present invention, sample can be tumour or lung Tumor tissues, and may include for example containing the cell related to tissue or any tissue or material of mark from it.

Statistical method

In the present invention, experiment is all completed according to being at least repeated 3 times, and result data is all with average value ± standard The mode of difference represents, carries out statistical analysis using SPSS18.0 statistical softwares, paired sample is adopted using t inspections, multisample Analyzed with the variance test (ANOVA) of mean, 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 is mixed, made Tissue fully cracking, 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 C ES8 in renal clear cell carcinoma is significantly higher than cancer beside organism In expression quantity.

The differential expression of the QPCR sequence verification CES8 genes of embodiment 2

1st, large sample QPCR checkings are carried out to CES8 gene differential expressions.Selected according to the sample collection mode in embodiment 1 Select the normal cancer beside organism of clear cell carcinoma of kidney patient and each 50 of renal clear cell carcinoma.

2nd, RNA extracts specific steps as described in Example 1.

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 CES8 genes in Genebank and house-keeping gene GAPDH genes, Synthesized by Bo Maide companies.

The primer sequence of CES8 genes：

Forward primer sequence is 5 '-ATGGACATAGTTCAAGAT-3 ' (SEQ ID NO.3)；

Reverse primer sequences are 5 '-AATTCATACAGGTAGACA-3 ' (SEQ ID NO.4).

The primer sequence of GAPDH genes：

Forward primer sequence is 5 '-GGAGCGAGATCCCTCCAAAAT-3 ' (SEQ ID NO.5)；

Reverse primer sequences are 5 '-GGCTGTTGTCATACTTCTCATGG-3 ' (SEQ ID NO.6).

(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, CES8 is expressed in renal clear cell carcinoma Up-regulation, difference have 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 CES8 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.Using GAPDH as Internal reference, sxemiquantitative gray analysis being carried out to band using Quantity One Labworks image acquisition and analysis softwares, experiment is repeated 3 times, As a result average gray value is taken；

5th, result

As a result as shown in Fig. 2 expression of the CES8 albumen in renal clear cell carcinoma is significantly higher than cancer beside organism.

Differential expression of the CES8 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 cultivated in 37 DEG C, 5%CO2, the incubator that relative humidity is 90%.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 CES8 gene mRNAs

2.1 RNA 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 CES8 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

3.3 SDS-PAGE and Western detections

Specific steps are the same as embodiment 3

4th, result

As a result as shown in figure 3, mRNA and protein expression level of the CES8 genes in clear cell carcinoma of kidney cell are significantly high In HEK293T cells, the expression highest in RLC-310 cell lines.

The silence of the CES8 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) siRNA design

Negative control siRNA sequence (siRNA-NC)：

Positive-sense strand is 5 '-UUCUCCGAACGUGUCACGU-3 ' (SEQ ID NO.7)

Antisense strand is 5 '-ACGUGACACGUUCGGAGAA-3 ' (SEQ ID NO.8)

siRNA1：

Positive-sense strand is 5 '-ACUUUCAGUGGGUUACUAGUG-3 ' (SEQ ID NO.9)

Antisense strand is 5 '-CUAGUAACCCACUGAAAGUGG-3 ' (SEQ ID NO.10)

siRNA2：

Positive-sense strand is 5 '-UUAGUGAUGGUUUCCUUUCUC-3 ' (SEQ ID NO.11)

Antisense strand is 5 '-GAAAGGAAACCAUCACUAAGA-3 ' (SEQ ID NO.12)

siRNA3：

Positive-sense strand is 5 '-UUUUCAUCCUUGUUGUAGCGU-3 ' (SEQ ID NO.13)

Antisense strand is 5 '-GCUACAACAAGGAUGAAAAGU-3 ' (SEQ ID NO.14)

Experiment is divided into three groups：Control group (RLC-310), negative control group (siRNA-NC) and experimental group (siRNA1, SiRNA2, siRNA3), the sequence of wherein negative control group siRNA and CES8 genes is without homology.

3) transfect

A. take the μ l of siRNA 3 that concentration is 50pmol to add 47 μ l serum free medium, gently mix, be incubated at room temperature 5min；

B. 1 μ l Lipofectamine 2000 are taken to add 49 μ l serum free mediums.Gently mix, be incubated at room temperature 5min；

C. above two mixture is mixed into (μ l of cumulative volume 100), gently mixes, be incubated 25min at room temperature, so that compound Body is formed；

D. 100 μ l compound and appropriate culture medium are added per hole in 6 orifice plates, is gently mixed；

E. the silence effect of gene is observed after 48~96h of incubation.

5th, QPCR detects the transcriptional level of CES8 genes

The extraction of 5.1 cell total rnas

The RNA in cell is extracted using Qiagen cell RNA extracts kit, experimental implementation is to specifications Carry out.

5.2 reverse transcription steps are the same as embodiment 2.

5.3 QPCR amplification steps are the same as embodiment 2.

6th, Western blot detect the expression of CES8 albumen

Specific steps are the same as embodiment 4

7th, result

As a result as shown in figure 4, with non-transfection group compared with transfecting siRNA-NC groups, experimental group siRNA2 interference effect is most Good, difference has statistical significance (P<0.05).

The influence of the CES8 gene pairs clear cell carcinoma of kidney cell of embodiment 6 propagation

Influenceed using MTT experiment detection CES8 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；

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 5, compared with the control, for experimental group after siRNA2 is transfected, the propagation of cell substantially receives suppression System, difference have statistical significance (P<0.05).

The influence of the CES8 gene pairs clear cell carcinoma of kidney Apoptosis of embodiment 7

Use the influence of flow cytomery CES8 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, experimental group is compared with control group, and the apoptosis rate of cell is without significant change (P<0.05) CES8 pairs, is illustrated It is little in the influence of Change of Apoptosis in Renal Cancer Cells.

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 fig. 6, the cell of transfection siRNA2 groups is compared to for control group, healing rate is bright after cells in vitro cut It is aobvious to reduce, and illustrate that CES8 is overexpressed the migration that can promote clear cell carcinoma of kidney 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 is significantly reduced, and no significant difference between control group, and clear cell carcinoma of kidney can be promoted by as a result illustrating that CES8 is overexpressed Invasion and attack.

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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Asp Pro Gly Asn Val Thr Leu Phe Gly Gln Ser Ala Gly Ala Met Ser

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Phe Gln Arg Asp Pro Glu Glu Ile Ile Trp Ser Met Ser Pro Val Val

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Asp Asn Val Asn Glu His Asp Trp Lys Met Leu Arg Asn Arg Met Met

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Asp Ile Val Gln Asp Ala Thr Phe Val Tyr Ala Thr Leu Gln Thr Ala

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His Tyr His Arg Asp Ala Gly Leu Pro Val Tyr Leu Tyr Glu Phe Glu

340 345 350

His His Ala Arg Gly Ile Ile Val Lys Pro Arg Thr Asp Gly Ala Asp

355 360 365

His Gly Asp Glu Met Tyr Phe Leu Phe Gly Gly Pro Phe Ala Thr Gly

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Leu Ser Met Gly Lys Glu Lys Ala Leu Ser Leu Gln Met Met Lys Tyr

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Trp Ala Asn Phe Ala Arg Thr Gly Asn Pro Asn Asp Gly Asn Leu Pro

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Cys Trp Pro Arg Tyr Asn Lys Asp Glu Lys Tyr Leu Gln Leu Asp Phe

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Claims

1. detect application of the CES8 reagent in the product for preparing diagnosis clear cell carcinoma of kidney.

2. application according to claim 1, it is characterised in that the reagent of the detection CES8 includes：By sequencing technologies, The expression reagent of CES8 genes in nucleic acid hybridization technique, nucleic acid amplification technologies or the method for immunoassays detection sample.

3. application according to claim 2, it is characterised in that the reagent is selected from：

The probe of specific recognition CES8 genes；Or

The primer of specific amplification CES8 genes；Or

Specifically bind the antibody or part of the albumen of CES8 codings.

4. application according to claim 3, it is characterised in that the reagent comprises at least a pair of specific amplification CES8's Primer, the sequence of the primer is as shown in SEQ ID NO.3~4.

5. a kind of product for diagnosing clear cell carcinoma of kidney, it is characterised in that the product includes CES8 expression water in detection sample Flat preparation, chip or kit, its chips include genetic chip, protein chip；Kit include gene detecting kit, Protein detection kit.

Applications of the 6.CES8 in the candidate compound of screening prevention or treatment clear cell carcinoma of kidney.

7. application according to claim 6, it is characterised in that the step of screening candidate compound is as follows：

In test group, test compound is added in cultivating system, and observes the expression quantity of CES8 in the cell of the test group And/or activity；In control group, test compound is not added in identical cultivating system, and observes the described thin of control group CES8 expression quantity and/or activity in born of the same parents；

Wherein, if the CES8 of cell expression quantity and/or activity are less than control group in test group, the test compound is indicated that Be expression to CES8 and/or activity have inhibitory action treating cancer candidate compound.

Application of the inhibitor of 8.CES8 functional expressions in the pharmaceutical composition for preparing prevention or treatment clear cell carcinoma of kidney.

9. application according to claim 8, it is characterised in that the inhibitor of the CES8 functional expressions is selected from：Nucleic acid Mortifier, protein inhibitor, proteolytic enzyme, protein binding molecule；The preferable inhibitor is nucleic acid inhibitor siRNA.

10. a kind of pharmaceutical composition for treating clear cell carcinoma of kidney, it is characterised in that described pharmaceutical composition includes CES8 functions Property expression inhibitor.