CN109750041A - The application of CYTOR and its inhibitor in anti-castration-resistant tumour - Google Patents

Abstract

The invention discloses the expression that CYTOR can regulate and control AR-V7, and the castration after prompting CYTOR to can be used for treating the hormone castration of androgen receptor related neoplasms is resisted and relapse and metastasis；The present invention discloses the antisense oligonucleotides that can regulate and control AR-V7 expression, are the treatment of tumour, and the castration especially after the hormone castration of androgen receptor related neoplasms is resisted and relapse and metastasis provides new therapy approach.

Description

The application of CYTOR and its inhibitor in anti-castration-resistant tumour

The application be for application No. is the 2018107698084, applying date be on July 13rd, 2018, denomination of invention " AR- The divisional application of application of the V7 variable sheer sequence in anti-castration-resistant tumour ".

Technical field

The invention belongs to fields of biomedicine, are related to the application of CYTOR and its inhibitor in anti-castration-resistant tumour.

Background technique

Prostate cancer is a kind of common malignant tumour, and is the second largest cause of disease for causing male tumor death.In recent years Come, for the disease incidence of China's prostate cancer in rapid increase trend year by year, 2004 are 5.8/10 ten thousand people, have been risen by 2009 For 8/,100,000 people.And it is Hong Kong that disease incidence absolute value, which rises most fast, 16.5/10 ten thousand people from 1999 rise to 28.1/10 ten thousand people in 2010.In Taiwan and Shanghai, prostate cancer has ranked male's kinds of tumor the 5th and urinary system System the 1st, tumour.Since screening of the China to prostate cancer Susceptible population falls behind relatively, advanced prostate cancer is caused to account for about The 70% of the prostate cancer of initial diagnosis；China's first visit is that the ratio of advanced prostate cancer is much higher than American-European countries (20-30%). Androgen deprivation therapy (Androgen deprivation therapy, ADT) is the main treatment side to this some patients Method.Although most of after about 18 months sensitive periods in ADT treatment initial stage prostate cancer by different degrees of control The disease of patient gradually and irreversibly progresses to castration-resistant prostate cancer.

Castration-resistant prostate cancer (Castration resistant prostate cancer, CRPC) refers to patient After ADT is treated, it is horizontal (< 50ng/dl or < 1.7nmol/L) that level of serum testosterone reaches castration, but disease is still in progress Prostate cancer.The clinical manifestation of progression of disease is the visible tumour progression of iconography and/or prostate-specific antigen (PSA) Persistent levels increase.The latter should meet the following conditions, i.e., 2ng/mL or more is reached in PSA, at interval of one week monitoring blood-serum P SA water Flat, continuous 3 blood-serum P SA are increased, and increase 50% or more than basic value.However, since CRPC pathogenesis is unknown so far, because And the clinical etiotropic accurate treatment of shortage, this is the difficult point and hot spot of current urinary surgery research.

AR signal path plays particularly important effect in each stage of prostate cancer development, numerous studies also table The continuous activation of bright AR signal path is still the key enablers of CRPC progress.Although the grace of the AR antagonist of FDA approval Miscellaneous Shandong amine and inhibit androgen levels two kinds of drug energy bands of abiraterone come survive benefit, but still have many patients CRPC from Start just insensitive to both drugs, and nearly all patient for receiving to treat finally can obtain drug resistance, and with PSA is increased, and has prompted the reactivation of AR signal path.AR shears the appearance of variant (AR splice variants, AR-Vs) Abnormal activation AR signal path, to drug resistance occur.AR-Vs is the AR that truncated mutant occurs, and lacked AR matches body end (ligand-binding domain, LBD), the still N-terminal of reservation AR and the combined area DNA, can play the function of AR, not have Activation AR signal path still is able in the presence of some androgens or the miscellaneous Shandong amine of grace, so that patients with prostate cancer is in ADT CRPC is progressed to after treatment and to the miscellaneous Shandong amine of grace and abiraterone drug resistance.AR-V7 is a member in AR-Vs, in patient CRPC Middle expression highest and the most universal.Nearest numerous studies concentrate the correlative study of report AR-V7, disclose AR-V7 and exist Promote the effect in CRPC progress and the important meaning as therapeutic choice mark molecule.Research has shown that the nuclear location of AR-V7 And gene function is not rely on the presence of androgen and AR overall length, and the high expression of AR-V7 can reverse ADT to be inhibited The downstream molecules of androgen activation and the particular molecule signal for participating in cell cycle progress, promote the growth and progress of CRPC. Therefore, specificity inhibits the targeted therapy of AR-V7 variable sheer very necessary.

Specific function site sequence about AR-V7 variable sheer has not been reported, and the present invention passes through underlying biological reality The key effect of the data research sequence in AR-V7 variable sheer is tested, achievement of the invention can support for AR-V7 positive castration Refractory prostate cancer treatment provides new target spot, restores the drug susceptibility of tumour.

Summary of the invention

In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide the lncRNA of controllable AR-V7 a kind of and Its application in cancer treatment, and antisense oligonucleotides relevant to AR-V7 and its application in cancer treatment.

To achieve the goals above, present invention employs following technical solutions:

The first aspect of the present invention provides a kind of antisense oligonucleotides, and the antisense oligonucleotides is selected from:

Specifically bind the antisense oligonucleotides of CYTOR；Or

Specifically bind the antisense oligonucleotides of the shearing site as shown in sequence SEQ ID NO.7.

In the present invention, " antisense oligonucleotides " refers to RNA or DNA points in conjunction with another RNA or DNA (target RNA, DNA) Son.For example, being interacted by means of RNA-RNA if it is RNA oligonucleotide and combining another RNA target and change target RNA Activity.Antisense oligonucleotides can raise or lower the expression and/or function of specific polynucleotides.This definition is meant including from controlling It treats, any foreign rna or DNA molecular useful for diagnosis or other viewpoints.This molecule includes, for example, antisense RNA or DNA molecular, RNA interfering (RNAi), microRNA, bait RNA molecule, siRNA, enzymatic RNA, therapeutic editor RNA and agonist with Antagonist RNA, antisense oligonucleotide compound, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, optional spliceosome, draw Object, probe and the other oligomeric compounds hybridized at least part of target nucleic acid.Therefore, these compounds can it is single-stranded, Double-strand, part be single-stranded or the form of cyclic oligomeric compound introduces.

Further, in specific embodiments of the present invention, the Antisensedigonucleotsequence sequence of CYTOR is specifically bound such as Shown in SEQ ID NO.8, the sequence of the antisense oligonucleotides of the shearing site as shown in sequence SEQ ID NO.7 is specifically bound As shown in SEQ ID NO.9.

The antisense oligonucleotides includes the antisense oligonucleotides that modification obtains, and the modification includes one kind selected from the following Or a variety of modifications: the saccharide part of at least one modification, the internucleoside linkage of at least one modification, at least one modification nucleotide and A combination thereof.Wherein, one or more modifications include the saccharide part selected from following at least one modification: 2'-O- methoxyl group second Saccharide part, saccharide part, the saccharide part of 2'-O- alkyl modified, the saccharide part and its group of two rings of the modification of 2'- methoxyl group of base modification It closes；One or more modifications include the tnternucleotide linkage of at least one modification selected from the following: thiophosphate, 2'-O first Oxygroup ethyl (MOE), 2'- fluorine, alkyl phosphonates, phosphorodithioate, alkyl thio-phosphonate, phosphoramidate, amino first Acid esters, carbonic ester, phosphotriester, acetamide ester, carboxymethyl ester and combinations thereof；One or more modifications include selected from following At least one modification nucleotide: peptide nucleic acid (PNA), arabinose-nucleic acid (FANA), analog, spreads out at lock nucleic acid (LNA) Biology and combinations thereof.

Further, the antisense oligonucleotides is the antisense oligonucleotides of methylation modification.

The second aspect of the present invention provides a kind of pharmaceutical composition, and described pharmaceutical composition includes first aspect present invention The antisense oligonucleotides.

The third aspect of the present invention provides a kind of pharmaceutical composition, and described pharmaceutical composition includes closing such as sequence SEQ The reagent of shearing site shown in ID NO.7.

As a kind of selectable embodiment, the reagent is antisense oligonucleotides.

As a kind of preferred embodiment, the sequence of the antisense oligonucleotides is as shown in SEQ ID NO.9.

The fourth aspect of the present invention provides following described in any item applications:

A. the CYTOR of AR-V7 is specifically bound after the hormone castration of preparation treatment androgen receptor related neoplasms Castration resist and relapse and metastasis drug in application；

B. application of the antisense oligonucleotides described in first aspect present invention in the drug of preparation treatment tumour；

C. hormone of the antisense oligonucleotides described in first aspect present invention in preparation treatment androgen receptor related neoplasms Castration after castration is resisted and is applied in relapse and metastasis drug；

D. application of the pharmaceutical composition described in second aspect of the present invention in the product of preparation treatment tumour；

E. pharmaceutical composition described in second aspect of the present invention is gone in the hormone of preparation treatment androgen receptor related neoplasms Castration after gesture treatment is resisted and is applied in relapse and metastasis product；

F. application of the pharmaceutical composition described in third aspect present invention in the product of preparation treatment tumour；

G. pharmaceutical composition described in third aspect present invention is gone in the hormone of preparation treatment androgen receptor related neoplasms Castration after gesture treatment is resisted and is applied in relapse and metastasis product.

Further, drug described in a includes the inhibitor of CYTOR, it is preferred that the inhibitor be antisense oligonucleotides or Short hairpin RNA.

As a preferred embodiment, the sequence of the antisense oligonucleotides is as shown in SEQ ID NO.8.

Alternatively preferred embodiment, the sequence of the short hairpin RNA is as shown in SEQ ID NO.1.

Present invention firstly discovers that CYTOR and AR-V7 is specifically bound, and the table for reducing CYTOR has been experimentally confirmed it Up to the level that can reduce AR-V7, and the level for reducing CYTOR can restore the drug of castration-resistant prostate cancer cell line Sensibility.

In the present invention, the gene for encoding CYTOR is located at No. 21 areas 1 of the short arm of a chromosome and takes, the CYTOR packet in the present invention Include wild type, saltant type or its segment.In an embodiment of the present invention, a kind of nucleosides of the gene of representative coding CYTOR In acid sequence such as at present international public nucleic acid database GeneBank shown in CYTOR gene (NR_024204.2).Of the invention CYTOR nucleotide full length sequence or its segment can usually be obtained with PCR amplification method, recombination method or artificial synthesized method.

In the present invention, the tumour includes prostate cancer, lung cancer, breast cancer, carcinoma of testis, cervix cancer and colon Cancer.

The present invention also provides the methods of forecast assessment AR-V7 variable sheer functional site, and steps are as follows:

1) structure of the mRNA of analysis AR-V7mRNA and overall length AR (AR-FL), determines the peculiar structure of AR-V7mRNA；

2) using 3.1 module of HSF of Human Splicing Finder on-line prediction assessment website, shearing site is selected It analyzes (Splice site analysis), Gene Name AR is inputted, using maximum entropy prediction algorithm to the peculiar structure of AR-V7 It is analyzed；

3) specificity of the functional site sequence in AR pre-mRNA of variable sheer is verified.

Further, by AR transcript AR-FL (NM_000044.4) and AR-V7 transcript (NM_ in step 1) 001348061.1) information nucleic acid carries out mRNA Structure Comparison by Graphics module, and FASTA module carries out mRNA sequence Comparison, screens peculiar structure steganography exon 3.

Further, the method also includes further verifying obtained sequence, judge to be by closing the site The no variable sheer for influencing AR-V7 is to judge whether the sequence is functional shearing sequence.

In scope of the invention, term " oligonucleotides " refer to ribonucleic acid (RNA) or DNA (DNA) or The oligomer or polymer of its analogies.Term " oligonucleotides " further includes the linear of natural and/or modification monomer or bonding Or cyclic oligomeric object, including dezyribonucleoside, ribonucleotide, its replace form and the different capitiform formula of its α-, peptide nucleic acid (PNA), Lock nucleic acid (LNA), thiophosphate, methyl-phosphonate and analog.Oligonucleotides can be via the monomer and monomer of regular fashion Between interaction, such as to specifically bind target more for the base pairing of Watson-Crick type or inverse type base pairing or the like Nucleotide.

Oligonucleotides can be " being fitted into ", that is, including different zones.In scope of the invention, " chimeric " compound It is oligonucleotides, it includes two or more chemical regions, for example, region of DNA domain, the region RNA, the region PNA etc..Eachization School district domain is made of at least one monomer unit, and the monomer unit described in the situation of oligonucleotide compound is nucleotide. These oligonucleotides generally include at least one region, and wherein oligonucleotides is modified to show one or more desired characteristics. The desired characteristic of oligonucleotides includes but is not limited to, for example, to the increased tolerance of nuclease degradation, increased cellular uptake And/or to the increased binding affinity of target nucleic acid.Therefore, the different zones of oligonucleotides can have different characteristics.As described above, Chimeric oligonucleotide of the invention is formed as the mixed structure of two or more oligonucleotides, the oligonucleotides of modification, widow Nucleosides and/or oligonucleotide analogs.

In the present invention, " drug " and " pharmaceutical composition " can carry out equivalent substitute.The pharmaceutical composition in addition to It further include pharmaceutically acceptable carrier including the active constituent in the present invention.The carrier includes but is not limited to: diluent, Buffer, emulsion, granule, encapsulation agents, excipient, filler, adhesive, spray, cutaneous permeable agent, moistens suspension Agent, disintegrating agent, sorbefacient, surfactant, colorant, corrigent, absorption carrier etc..

In the present invention, transfer of the exogenous nucleic acid into host cell or organism can be by directly detecting cell or biology The presence of the nucleic acid is evaluated in body.This detection can be realized by a variety of methods well known in the art.For example, exogenous nucleic acid Presence can pass through polymerase by southern blotting technique or using the primer for specifically expanding relevant to nucleic acid nucleotide sequence Chain reaction (PCR) technology detects.The expression of exogenous nucleic acid is also measured using the conventional method including gene expression analysis. For example, can detect and quantify using RNA trace and reverse transcription PCR (RT-PCR) from the mRNA that exogenous nucleic acid generates.

RNA can also be detected from the expression of exogenous nucleic acid by measurement enzymatic activity or reporter protein activity.For example, antisense Adjusting activity can measure indirect as decreasing or increasing for target nucleic acid expression, and target nucleic acid expression decreases or increases as outer Source nucleic acid is in the instruction for generating effect RNA.It, can design primer and the code area for expanding target gene based on sequence conservation Domain.Initially, the coding region of the topnotch expression from each gene can be used for forming types crt gene, appoint although can be used What coding or non-coding region.By being inserted into each coding region between reporter gene coding region and its poly (A) signal To assemble each crt gene.These plasmids by generate reporter gene gene upstream portion and possible RNAi target it is non-in 3' The mRNA of coding region.The effect of independent antisense oligonucleotides will be measured by the adjusting of reporter gene.It can be used for the present invention The reporter gene of method includes acetohydroxy acid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), β glucose Aldehydic acid enzyme (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow are glimmering Photoprotein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS) and its derivative.It assigns mould to ampicillin, bleomycin, chloramphenicol, gentamicin, tide Element, kanamycins, lincomycin, methotrexate (MTX), glufosinate, the multiple choices of puromycin and tetracyclin resistance label are can to obtain 's.The method for determining the adjusting of reporter gene is it is known in the art that including but is not limited to, fluorescent method is (for example, fluorescence spectrum , fluorescence activated cell sorting (FACS), fluorescence microscopy, antibiotic resistance determine.

In embodiments, handled using antisense oligonucleotides of the present invention sample (for example, internal or external cell or Tissue) in AR-V7 expression (for example, mRNA or albumen) by being evaluated with the AR-V7 expression in control sample.Example Such as, the expression of albumen or nucleic acid can utilize the ratio in method known to those skilled in the art and simulation process or untreated samples Compared with.Optionally, depend on desired information, can with compare antisense oligonucleotides (for example, have change or it is not homotactic Antisense oligonucleotides) processing sample be compared.In another embodiment, processing sample and AR-V7 in untreated samples The differential expression of albumen or nucleic acid (can appoint from nucleic acid different in processing sample and untreated samples including what researcher was deemed appropriate What standard, for example, house-keeping gene) differential expression compare.

The difference observed can indicate as expected, for example, in the form of ratio or score, for compare.? In embodiment, AR-V7mRNA or albumen horizontally relative to untreated in the sample that is handled with antisense oligonucleotides of the present invention Sample increases or decreases about 1.25 times to about 10 times or more with the sample that control nucleic acid is handled.AR- in embodiments The level of V7mRNA or albumen increase or decrease at least about 1.25 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times, At least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 Again, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 5.5 times, at least about 6 times, at least about 6.5 times, at least about 7 Times, at least about 7.5 times, at least about 8 times, at least about 8.5 times, at least about 9 times, at least about 9.5 times or at least about 10 times or more It is more.

In the present invention, " cultivating system " is defined as expressing or becoming competence to express appointing for AR-V7 gene product What organism, cell, cell culture or tissue.These include but is not limited to people, transgenic animals, cell, cell culture Object, tissue, xenograft, graft and combinations thereof.

As a non-limiting example, by the expression in the cell or tissue handled with one or more antisense compounds Mode belongs to according to it such as disease of checked gene compared with the control cell or tissue that are not handled by antisense compounds Sick association, signal transduction path, cellular localization, expression, size, the generated mode of structure or function analysis gene table Up to level of difference.These analyses can carry out stimulation or the cell not stimulated, and deposit in the other compounds for influencing expression pattern Or in the absence of.

The example of gene expression analysis method known in the art includes DNA array or microarray, SAGE (gene expression system Column analysis), the READS restriction enzyme of the cDNA of digestion (expand), TOGA (total gene expression analysis), protein arrays and protein Group is learned, the sequencing of the sequence label (EST) of expression, abatement RNA fingerprint technique (SuRF), abatement clone, mRNA differential display mRNA (DD), is compared Genomic hybridization, FISH (fluorescence in situ hybridization) technology and mass spectrometry method.

The specificity and sensitivity that those skilled in the art also utilize antisense are for therapeutical uses.Antisense compounds exist It is used as treatment part in the treatment of the morbid state of animal (including people).Antisense oligonucleotides drug is securely and effectively applied For people, many clinical tests are currently carried out.Therefore it has been determined that antisense compounds can be useful treatment form, It can be configured to the therapeutic scheme for the treatment of cell, tissue and animal (especially people).

For treatment, is treated and suspected with can be by adjusting AR-V7 by applying antisense compounds according to the present invention Expression come the animal of the disease or illness treated, preferably people.For example, in one non-limiting embodiment, method packet Include the step of applying the AR-V7 regulator of therapeutically effective amount to animal in need for the treatment of.AR-V7 regulator of the invention is effectively It adjusts the activity of AR-V7 or adjusts the expression of AR-V7 albumen.In one embodiment, compared with the control, AR-V7 in animal Activity or expression be suppressed about 10%.Preferably, the activity of AR-V7 or expression are suppressed about 30% in animal.It is highly preferred that The activity of AR-V7 or expression are suppressed 50% or more in animal.Therefore, compared with the control, oligomeric compounds adjust AR- The expression of V7mRNA at least 10%, at least 50%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, extremely Few 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100%.

Oligonucleotides of the invention includes activity, the cell distribution that the oligonucleotides chemistry is connected to enhancing oligonucleotides Or one or more parts or the conjugate of cellular uptake.These parts or conjugate may include covalent bond functional group such as primary The conjugate group of hydroxyl or secondary hydroxy group.Conjugate group of the invention includes intercalator, report molecule, polyamine, polyamides Amine, polyethylene glycol, polyethers, the group for enhancing oligomer pharmacokinetic properties, the group for enhancing oligomer pharmacodynamic profiles.Allusion quotation Type conjugate group includes cholesterol, lipid, phosphatide, biotin, azophenlyene, folic acid, phenanthridines, anthraquinone, acridine, fluorescein, Luo Dan Bright, cumarin and dyestuff.In scope, the group for enhancing pharmacodynamic profiles includes improving intake, enhancing to degradation Tolerance and/or the group of reinforcement and target nucleic acid sequence specific hybrid.In scope, enhance pharmacokinetic properties Group include improve the compounds of this invention intake, distribution, metabolism or excretion group.Conjugate fraction includes but unlimited In lipid part such as cholesterol moiety, cholic acid, thioether such as hexyl -5- trityl mercaptan, sulphur cholesterol, aliphatic chain Such as dodecanediol or undecyl residues, phosphatide such as two-cetyl-rac- glycerol or bis--O- cetyl of 1,2-- Rac- glycerol -3-H phosphonic acids triethyl ammonium, polyamine or polyglycol chain or adamantane acetic acid, palmityl part or octadecylamine or Hexylamino-carbonyl-oxycholesterol part.Oligonucleotides of the invention can also be conjugated in active drug substance, for example, A Si Woods, warfarin, bute, brufen, suprofen, fenbufen, Ketoprofen, (S)-(+)-pranoprofen, Carprofen, red flesh Propylhomoserin, 2,3,5 triiodobenzoic acid, flufenamic acid, folinic acid, benzothiadiazine, chlorothiazide, diazacyclo heptantriene, indoles beauty Pungent, barbiturate, cephalosporin, sulfonamide, antidiabetic, antimicrobial or antibiotic.

In order to assist intake, distribution and/or absorb, the compound of the present invention can also be with other molecules, molecular structure or change Close the mixture mixing of object, encapsulating, conjugation or be associated in other ways, for example, liposome, receptor target molecule, oral, rectum, Local or other preparations.

Although antisense oligonucleotides need not be applied to adjust target expression and/or function, this hair with the background of carrier Bright embodiment includes the expression vector constructs for antisence oligonucleotides, including promoter, heterozygote promoter Gene order, and have the promoter activity that strong constitutive promoter is active or can be induced in desired situation.

In embodiments, invention practice includes at least one above-mentioned antisense widow core of nucleic acid delivery systems appropriate application Thuja acid.In one embodiment, which includes the non-virus carrier for being operably connected to polynucleotides.It is this non-viral The example of carrier includes the combination of independent oligonucleotides or oligonucleotides and albumen appropriate, polysaccharide or lipid formulations.

In addition nucleic acid delivery systems appropriate include viral vectors, and usual sequence is from least one below: adenovirus, Adeno-associated virus (AAV) (AAV), helper virus dependent form adenovirus, retrovirus or hemagglutinating virus of Japan-liposome (HVJ) Complex.Preferably, viral vectors includes the strong eukaryotic promoter for being operably connected to polynucleotides, such as giant cell disease Malicious (CMV) promoter.

In addition preferred carrier includes viral vectors, fusion protein and chemically conjugated object.Retroviral vector includes not Lip river Nissl murine leukemia virus and the virus based on HIV.A kind of viral vectors being preferably based on HIV includes at least two carrying Body, wherein gag and pol gene is from HIV genome and env gene comes from another virus.DNA viral vector is preferred.This A little carriers include poxvirus vector such as vaccinia subgroup virus or fowl pox virus vectors, herpesvirus vector such as I type herpe simplex disease Malicious (HSV) carrier, adenovirus vector and gland relevant viral vector.

Pharmaceutical composition of the invention covers any pharmaceutically acceptable salt, the salt of ester or this ester or when being applied to Any other compound or its residue of (direct or indirect) bioactive metabolites are capable of providing when animal (including people).

The invention also includes pharmaceutical compositions and preparation comprising antisense oligonucleotides of the present invention.Also depending on expectation part It is systematic treating and region to be treated, pharmaceutical composition of the invention can be applied in many ways.Application can be local (including eyes and to mucous membrane, including vagina and rectal delivery), lung, such as by sucking or spraying into powder or aerosol, wrap It includes and passes through sprayer；Intratracheally, intranasally, epidermis and percutaneous), it is oral or extra-parenteral.Parenteral administration includes intravenous, artery In interior, subcutaneous, peritonaeum or intramuscular injection or infusion；Or encephalic, such as intrathecal or intraventricular application.

Composition of the invention can be formulated as any many possible dosage forms, such as, but not limited to, tablet, capsule, gel Capsule, liquid syrups, soft gel, suppository and enema.Composition of the invention can be also formulated as aqueous, not aqueous or mixed Close the suspending agent in medium.Aqueous suspension agent can further include the substance for increasing suspending agent viscosity, including for example, carboxymethyl is fine Tie up plain sodium, sorbierite and/or glucan.Suspending agent also may include stabilizer.

Pharmaceutical composition of the invention includes but is not limited to solution, lotion, foaming agent and pharmaceutical preparations containing liposomes.The present invention Pharmaceutical composition and preparation may include one or more penetration enhancers, carrier, excipient or other activity or inactive match Material.

Certain embodiments of the present invention are provided comprising one or more oligomeric compounds and are acted as by non-antisense mechanism The pharmaceutical composition of one or more other chemotherapeutics.The example of this chemotherapeutics includes but is not limited to cancer chemotherapy medicine Object for example daunorubicin, daunomycin, D actinomycin D, Doxorubicin, epirubicin, idarubicin, esorubicin, it is rich come it is mould Element, Mafosfamide, ifosfamide, cytarabine, double chloroethene nitroso ureas, busulfan, mitomycin C, actinomycin D, light Refreshing mycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, Dacarbazine, procarbazine, hemel, pentamethylmelamine, rice support anthracene Quinone, amsacrine, Chlorambucil, methyl cyclohexane nitroso ureas, mustargen, melphalan, cyclophosphamide, 6-MP, 6- sulphur bird are fast Purine, cytarabine, 5-azacitidine, hydroxycarbamide, deoxycoformycin, 4- hydroxyl peroxide cyclophosphamide, 5 FU 5 fluorouracil (5- FU), floxuridine (5-FUdR), methotrexate (MTX) (MTX), colchicin, taxol, vincristine, vincaleukoblastinum, Etoposide (VP-16), Trimetrexate, Irinotecan, Hycamtin, gemcitabine, Teniposide, cis-platinum and diethylstilbestrol (DES).When with When the compound of the present invention is used together, (for example, 5-FU and oligonucleotides) can be used alone in this chemotherapeutics, sequentially made With (for example, 5-FU and oligonucleotides continue for some time, being followed by MTX and oligonucleotides) or with it is one or more other this Chemotherapeutic agent combination uses (for example, 5-FU, MTX and oligonucleotides or 5-FU, radiotherapy and oligonucleotides).It is including but not limited to non- The anti-inflammatory drug of steroidal anti-inflammatory medicines and cortin and including but not limited to Ribavirin, arabinosy ladenosine, acyclovir and The antiviral drugs of Ganciclovir also can be combined in composition of the invention.The group of antisense compounds and other non-antisense drugs It closes also within the scope of this invention.The compound of two or more combinations together or can be used sequentially.

Think therapeutic composition preparation and its subsequent application (administration) those skilled in the art limit of power It is interior.Administration depend on morbid state to be treated severity and responsiveness, the period last from days for the treatment of to the several months, or until Realize the mitigation cured or realize morbid state.Best drug dosage schedule can be calculated from the measurement of patient's body drug accumulation.This Field those of ordinary skill can easily determine optimal dose, medication and repetitive rate.Optimal dose can be according to independent few core The relative effectivenes of thuja acid and change, be generally based on the effective EC in effective in vitro and in vivo animal model₅₀To estimate.

The advantages of the present invention:

Present invention firstly discovers that the variable sheer specific function site of AR-V7, can be inhibited by closing the site AR-V7, so that the hormone castration for the tumour for inhibiting AR-V7 to mediate is resisted, restores the medicine of tumour without influencing the expression of AR-FL Object sensibility.

Present invention firstly discovers that lncRNA CYTOR can regulate and control the expression of AR-V7, the expression water of CYTOR is reduced It is flat to restore tumour and tumour cell to the sensibility of drug.

Present invention firstly discovers that the variable sheer specific function site of lncRNA CYTOR specific binding AR-V7, Regulate and control the expression of AR-V7, so that the treatment for AR-V7 positive castration-resistant tumour provides new target spot, restores tumour Drug susceptibility.

Detailed description of the invention

Fig. 1 is the cellular morphology figure of androgen-independent prostate cancer cell lines in vitro system LNCaP-AI establishment process.

Fig. 2 is Cell growth ability figure in androgen-independent prostate cancer cell lines in vitro system LNCaP-AI establishment process.

Fig. 3 is androgen-independent prostate cancer cell lines in vitro system LNCaP-AI to protona (DHT) and the miscellaneous Shandong amine of grace, ratio The response diagram of card Shandong drug amine.

Fig. 4 is AR-V7 expression figure in androgen-independent prostate cancer cell lines in vitro system LNCaP-AI establishment process.

Fig. 5 is influence diagram of the shCYTOR to CYTOR and AR-V7 expression, wherein figure A is the shadow to CYTOR mRNA Ring figure；Figure B is the influence diagram to AR-V7 albumen.

Fig. 6 targets AR-V7 variable sheer specific function site sequence and designs antisense oligonucleotides ASOAR-V7 schematic diagram.

Fig. 7 is transfection ASO^AR-V7Influence diagram of the different time to AR-V7mRNA level in different cell lines；Wherein, figure A is 5h, figure B are 10h.

Fig. 8 is transfection ASO^CYTORInfluence diagram of the different time to AR-V7mRNA level in different cell lines；Wherein, figure A is LNCaP-AI cell, figure B are 22Rv1 cell.

Fig. 9 is MTT detection after striking low CYTOR, sensibility of the LNCaP-AI and 22Rv1 cell to the miscellaneous Shandong drug amine of grace Figure.

Figure 10 is the response diagram of mouse drug susceptibility, and figure A is mouse tumor size figure；Scheming B is tumor statistical chart.

Specific embodiment

The present invention is described in further detail with reference to the accompanying drawings and examples.Therefore, width and model of the invention It encloses and should not necessarily be limited by any of above embodiment.Following embodiment is merely to illustrate the present invention rather than limits model of the invention It encloses.Test method without specific conditions in embodiment, usually according to normal condition, or according to item proposed by manufacturer Part.

The building and verifying of the positive castration-resistant prostate cancer cell line LNCaP-AI of embodiment 1

1, cell line goes androgen culture

The prostate cancer cell line LNCaP of Androgen-dependent is incubated at 10% active carbon and removes androgen fetal calf serum In RPMI-1640 culture medium.Cell is cultivated in cell incubator, and condition of culture is 37 DEG C, 5%CO₂。

2, the proliferation activity of mtt assay detection cell

2 × 10 will be diluted to after cell count to be detected⁴/ ml is inoculated in 96 orifice plates, and the dilution of 100 μ l cells is added in every hole Liquid, about 2 × 10³Cells/well, 4-5 repeating hole of every group of experimental setup is to reduce error.37 DEG C are placed in, 5%CO₂Cell incubator Middle culture takes out 96 orifice plates 1 after inoculation, after 2,3 days, carefully sucks cell culture fluid and every hole adds 100 μ l in gaging hole 1 × MTT solution, 37 DEG C of incubators are incubated for 4h, supernatant are carefully sucked out, every hole adds 150 μ l DMSO to keep formazan molten in gaging hole Solution, is shaken up with plate shaker and solution to be tested is placed in microplate reader, and optical density is detected at 570nm wavelength.Record each test Optical density (opticaldelnsity, the OD) value in hole.

3, reaction of the mtt assay detection castration-resistant prostate cancer cell line to chemotherapeutics

Using mtt assay detection castration-resistant prostate cancer cell line LNCaP-AI to protona (DHT), the miscellaneous Shandong amine of grace (MDV3100) and the reaction of Bicalutamide (bicalutamide), concrete operations are referring to step 2.

4, Western blot detects the protein level of AR-V7 in castration-resistant prostate cancer cell line

1) extraction of total protein of cell

The cell for being in logarithmic phase is collected, washs cell with the PBS of pre-cooling.By RIPA cell pyrolysis liquid and PMSF with 100: 1 ratio mixes, and above-mentioned 150 μ l of lysate is added into cell, places 30min on ice, using pipettor by the liquid after cracking Body is drawn in EP pipe, and 14000rpm is centrifuged 5min, the supernatant after careful collection centrifugation at 4 DEG C.

2) total protein concentration measures

The measurement of protein concentration is carried out according to the specification of BSA determination of protein concentration kit.

3) SDS-PAGE electrophoresis

8% separation gel and 5% concentration glue are prepared according to the specification of PAGE gel reagent preparation box and are carried out Electrophoresis.

4) western is detected

A. electrotransfer

Pvdf membrane is put into methanol solution and activates 5min, is put into transferring film buffer and balances 20min.PAGE glue is taken out to put Enter in transferring film buffer, cut corresponding PAGE glue, according to be followed successively by from bottom to top filter paper, pvdf membrane, PAGE glue, filter paper it is suitable Sequence is put into half-dried transferring film instrument, constant pressure 25V transferring film 1.5h；

B. immuning hybridization

Pvdf membrane is taken out, PBS flushing is placed in 5% milk is incubated for 1h at room temperature, and pvdf membrane is put into hybridization bag, is added Enter primary antibody to stay overnight, washs pvdf membrane with TBST buffer, add corresponding secondary antibody, be incubated for 1h at room temperature, TBST buffer is washed It washs.

C.DAB colour developing

The slightly dry rear DAB developing solution that Fresh is added dropwise of pvdf membrane, scans record after pvdf membrane colour developing.With β-actin work For internal reference, taken pictures using Bio-Rad Labworks image acquisition and analysis software to pvdf membrane.

5, statistical analysis

For statistical analysis to experimental data using SPSS18.0 statistical software, difference between the two is examined using t, is recognized To have statistical significance as P < 0.05.

6, result

1) cellular morphology of androgen-independent prostate cancer cell lines in vitro system LNCaP-AI establishment process is as shown in Figure 1.When When LNCaP cell grew into for 10 generation under the conditions of no androgen (incubation time about 1 month), cell growth slows down gradually, greatly The cell death of amount, only remaining a small amount of cell survival, and cell state is poor, the androgen removal processing for illustrating that experiment carries out has Effect can simulate the effect of ADT treatment in vitro.Cell stretches out more elongated feeler sample pseudopodium structure in incubation, pseudo- Length ratio is larger between sufficient structure and cytoplasm, and part cell pseudopodium structure is interweaved, the webbed nerve cell sample knot of shape Structure prompts cell gradually to be converted to neuroendocrine direction.With the extension of incubation time, cell death persistently aggravates, Only remaining cell survival fragmentarily, neuroendocrine like cell disappear, and the cell of survival is distributed in colony, cellular morphology For ellipticity, cell state gradually restores, and the cell in this period is known as LNCaP-AD cell (Androgen Dependent LNCaP).The preferable LNCaP-AD cell colony of growth conditions is carefully digested with pancreatin, is transferred in 96 orifice plates and continues Culture.When cell grew into for 40 generation (incubation time about 3 months), cell activity is gradually restored, and growth is gradually accelerated, and prompts Its growing environment for gradually having adapted to androgen deprivation, it is gradually excessive to androgen independent direction.When cell grows into the 60th For when (incubation time about 6 months), it is in good condition it can be observed that vitro growth rates are very fast, can be in the item of no androgen Fast breeding under part prompts it to come into androgen independent state, and this character is hereafter respectively kept for cell, will be this The LNCaP cell subsets of androgen independence is referred to as LNCaP-AI (Androgen Independent)；

2) cell-proliferation activity in LNCaP-AI establishment process compared with mother is for LNCaP cell as shown in Fig. 2, will go The cell of hero culture early stage, vitro growth rates are slow, and as the emasculation time extends, cell growth is slower and slower.Work as emasculation When entering mid-term, i.e. 40 generation, cell growth status increased compared with earlier generations, it is prompted gradually to adapt to androgen deprivation Growing environment, it is gradually excessive to the direction CRPC, and finally establish resulting LNCaP-AI cell line, then the speed of growth always compared with Fastly, can under the conditions of androgen independent fast breeding, prompt it to come into castration resistance state.

3) LNCaP-AI cell is to protona (DHT), the miscellaneous Shandong amine (MDV3100) of grace and Bicalutamide (bicalutamide) reaction as shown in figure 3, the cell Proliferation between control group (DMSO) and medicine group there was no significant difference, Illustrate tolerance of the LNCaP-AI cell to androgen antagonist drug；

4) AR-V7 in LNCaP-AI cell protein level as shown in figure 4, with the castration time extension, AR-V7 table It is stepped up up to amount, prompts long-term emasculation processing that can cause gradually to express AR-V7 in LNCaP-AI cell.

Influence of 2 CYTOR of embodiment to AR-V7 expression in castration-resistant prostate cancer cell line

1, cell culture

External six orifice plates culture LNCaP-AI and 22Rv1 cell, plating cells transfected cell after 24 hours.

2, it transfects

1) design of shRNA

For the short hairpin RNA of the binding sequence design synthesis targeting CYTOR of long-chain non-coding RNA CYTOR and AR-V7 (shCYTOR) and (shSCR) is compareed.

ShCYTOR sequence is as follows:

5’-TCTATGTGTCTTAATCCCTTGTCCT-3’(SEQ ID NO.1)

The sequence of shSCR:

5’-TTCTCCGAACGTGTCACGTTT-3’(SEQ ID NO.2)

2) liposome transfection

The short hairpin RNA synthesized in vitro and liposome transfection are tried using 2000 kit of Lipofectamine of Roche Agent mixing transfection prostate gland cancer cell, specific steps are detailed in specification.

3, QPCR detects the mRNA level in-site of the CYTOR in cell

1) extraction of cell total rna

After cell transfecting 72h, RNA is extracted using the cell RNA extracts kit of Tiangen company, specific steps are detailed in Specification.

2) reverse transcription

Use Thermo RevertAid First Strand cDNA Synthesis Kit by total serum IgE reverse transcription for CDNA, concrete operations are detailed in kit specification.

3) PCR reacts

Design of primers: for the sequence design synthetic primer of CYTOR and GAPDH, primer sequence is as follows:

The primer sequence of CYTOR:

Forward primer: 5 '-ACAGGAAGCTCTATGACACACTTG-3 ' (SEQ ID NO.3)

Reverse primer: 5 '-CTGCATTCCGGCTGTGAT-3 ' (SEQ ID NO.4)；

The primer sequence of GAPDH:

Forward primer: 5 '-CCATCACCATCTTCCAGGAGCGA-3 ' (SEQ ID NO.5)

Reverse primer: 5 '-GGTGGTGAAGACGCCAGTGGA-3 ' (SEQ ID NO.6)；

Using GAPDH as internal reference, QPCR reaction is carried out, using SYBR Green as fluorescent marker, in Light Cycler PCR reaction is carried out on fluorescence quantitative PCR instrument, determines that purpose band, Δ Δ CT method carry out phase by melt curve analysis analysis and electrophoresis To quantitative.

4, Western blot detects the level of AR-V7 albumen in cell

After cell transfecting 72h, the total protein in cell is collected, and carry out to the protein expression level of AR-V7 and AR-FL Western detection, specific steps are referring to embodiment 1.

5, result

As a result as shown in figure 5, figure A shows that shCYTOR can significantly reduce the mRNA level in-site of the CYTOR in cell, difference With statistical significance (P < 0.05), the protein level of the AR-V7 in cell can be significantly reduced by scheming B shCYTOR, and AR (AR-FL) albumen in the case where illustrating that long-chain non-coding RNA CYTOR can not influence AR-FL expression, is adjusted then without significant changes Control the variable sheer of AR-V7.

3 Bioinformatics Prediction of embodiment assesses AR-V7 variable sheer functional site

1, the determination of the peculiar structure of AR-V7

1) AR gene (androgen receptor [Homo sapiens is chosen using the gene module in ncbi database (hTman)] overall length transcript), that is, AR-FL (NM_000044.4) and transcript 3 are AR-V7 (NM_001348061.1) Information nucleic acid；

2) using the mRNA structure of Graphics module comparison AR-FL and AR-V7, the peculiar structure of AR-V7 is analyzed；

3) mRNA sequence is compared using FASTA module, analyzes the signal sequence of shearing, determines the sequence of AR-V7 variable sheer Column.

2, the analysis of variable sheer functional site

Using 3.1 module of HSF of HTman Splicing Finder on-line prediction assessment website, shearing site point is selected It analyses (Splice site analysis), inputs Gene Name AR, continue to select identified transcript in step 1, using most Big entropy prediction algorithm is analyzed.

3, the verifying of variable sheer functional site specificity

The shearing site sequence that analysis obtains is matched in AR pre-mRNA sequence, finding can existing for sequence The position of energy, determines the specific location of the sequence.

4, result and analysis

By the analysis to AR-V7 structure and mRNA sequence, determine that AR-V7mRNA lacks the exon of AR-FL mRNA 4-8.The structure of AR-V7mRNA is one connected in AR pre-mRNA introne 3 behind the exons 1-3 of AR-FL mRNA Sub-sequence hides exon 3.It is found when analyzing the RNA sequence for hiding exon 3, at the 3 ' ends for hiding exon 3 containing height Conservative 5 '-AATAAA-3 ' the cutoff signal sequence of degree, in AR-V7mRNA process, the multienzyme for participating in RNA shearing is compound Body can identify these cutoff signal sequences, cutting and carry out Polyadenylation, form adenylate (poly A) stern construction.? Close to 3 ' montage positions of introne 3, there are AG sequences at 5 ' ends of hiding exon 3.There are vertebrates to cut at 3 ' the montage position Connect shared sequence signal feature: the extension of 10 pyrimidine 5 '-TCTCTCTTTC-3 ' followed by any base, then meets C, most After terminate at AG, these sequences are all required to montage.The introne 3 sequence before exon 3 is hidden in AR pre-mRNA It is removed in the form of " lasso trick " structure, while exon 3 is engaged with hiding exon 3, forms mature AR-V7mRNA.Cause This determines that the distinctive hiding exon 3 of AR-V7mRNA is the result of AR Pre-mRNA (pre-mRNA) variable sheer.

It is assessed by on-line prediction, obtains AR-V7 hides exon 3 (i.e. the exon 4 of ENST00000504326) 5 ' It holds functional sequence CGGGTTGGC (SEQ ID NO.7).The sequence is subjected to specific the matching analysis, it is found that the sequence is located at AR- V7 hides at 5 ' the 9th nucleotide in end of exon 3, and with the long-chain non-coding RNA-CYTOR's of adjusting AR-V7 variable sheer Partial sequence complementarity determines that the sequence is to regulate and control the sequence of the variable sheer of AR-V7.

Embodiment 4 detects the influence that AR-V7 is expressed in shearing function site

1, cell culture

External six orifice plates culture LNCaP-AI and 22Rv1 cell, plating cells transfect cell afterwards for 24 hours.

2, it transfects

1) design of antisense oligonucleotides

According to the antisense oligonucleotides ASO of specific cleavage functional site sequence design AR-V7^AR-V7(design diagram is such as Shown in Fig. 6) and CYTOR antisense oligonucleotides ASO^CYTOR, sequence is as follows:

ASO^CYTORSequence:

5’-GTGGGCGGTTGGAACCAG-3’(SEQ ID NO.8)

ASO^AR-V7Sequence:

5’-CAATTGCCAACCCGGAAT-3’(SEQ ID NO.9)

2) liposome transfection

By the antisense oligonucleotides ASO of methylation modification^AR-V7And ASO^CYTORIt is uniformly mixed respectively with lipofectamine It is incubated at room temperature 20min, mixture is uniformly added into culture medium.So that ASO^AR-V7And ASO^CYTORFinal concentration in the medium Respectively 0nM, 100nM, 200nM.

3, PCR detects the mRNA level in-site of the CYTOR in cell

1) extraction of cell total rna

After transfecting 5h and 10h, RNA is extracted using the cell RNA extracts kit of Tiangen company, specific steps are detailed in Specification.

2) reverse transcription

Use Thermo RevertAid First Strand cDNA Synthesis Kit by total serum IgE reverse transcription for CDNA, concrete operations are detailed in kit specification.

3) PCR reacts

For the sequence design synthetic primer of AR-V7 and AR-FL, primer sequence is as follows:

The primer sequence of AR-V7:

Forward primer: 5 '-CCATCTTGTCGTCTTCGGAAATGTTA-3 ' (SEQ ID NO.10)

Reverse primer: 5 '-TTTGAATGAGGCAAGTCAGCCTTTCT-3 ' (SEQ ID NO.11)；

The primer sequence of AR-FL:

Forward primer: 5 '-CAGCCTATTGCGAGAGAGCTG-3 ' (SEQ ID NO.12)

Reverse primer: 5 '-GAAAGGATCTTGGGCACTTGC-3 ' (SEQ ID NO.13)

Using CWBIO 2 × Taq MasterMix, AR-V7 primer, AR-FL primer and Sangon Biotech GAPDH Internal control primer carries out PCR reaction.

4, result

As a result as shown in FIG. 7 and 8, ASO is transfected^AR-V7And ASO^CYTOR5h afterwards, in LNCaP-AI and 22Rv1 cell The reduction of AR-V7 expression is seen, and with ASO^AR-V7The increase of concentration, AR-V7 expression reduce more obvious；Transfection 10h afterwards can equally see the decline of AR-V7 expression, and relative to 5h after transfection, AR-V7mRNA level reduces more.Turn Dye 5h and 10h is showed no AR-FL mRNA level in-site and changes, and illustrates ASO^AR-V7And ASO^CYTORAR-FL expression is not influenced In the case of, regulate and control the variable sheer transfection ASO of AR-V7^AR-V7Group is relative to transfection ASO^CYTORGroup AR-V7mRNA level reduces more It is obvious.

Effect of 5 AR-V7 of embodiment to drug susceptibility is restored

1, the reaction of vitro detection castration-resistant prostate cancer drug susceptibility

Castration-resistant prostate cancer cell line LNCaP-AI and 22Rv1 are to grace after striking low CYTOR using mtt assay detection The reaction of miscellaneous Shandong amine (10 μM), concrete operations are referring to embodiment 1.

2, the reaction of vivo detection castration-resistant prostate cancer drug susceptibility

1) 22Rv1 transfection is struck to the control group (shSCR, CYTOR level are unchanged) and experimental group of low CYTOR (reduction of shCYTOR, CYTOR level) cell each 6 × 10⁶, it is mixed according to cell volume with Matrigel colloid product 3:1, Every group of mixture 1.2ml.

2) cellular control unit mixture is given to 66 week old nude mice subcutaneous kind of tumors of left upper extremity armpit, every 200 μ of mouse respectively The subcutaneous injection of l equivalent.Experimental group cell mixture gives other 66 week old nude mice same method kind tumors respectively.

3) after planting tumor 2.5 weeks, the visible subcutaneous tumor formation of left upper extremity armpit of two groups of nude mices of observation.At random by 3 in control group Mouse mouthful raises the miscellaneous Shandong drug amine of 25mg/kg grace, 3 mouse mouthful in experimental group is raised the miscellaneous Shandong drug amine of 25mg/kg grace at random, and mouth is raised The mouse of drug assigns to new mouse cage, continues raising and takes out subcutaneous tumor after a week, tumor weight of weighing.

3, result

After vitro detection result is as shown in figure 9, strike low CYTOR, androgen-independent prostate cancer cell lines in vitro system LNCaP-AI and 22Rv1 restores the sensibility to drug, and the results are shown in Figure 10 for vivo detection, and CYTOR level can after reducing Inhibit the growth of tumour, i.e., is less than without medication control group mice tumour, after CYTOR level reduces without medication experimental mice tumour Enhance the sensibility of prostate cancer Shandong drug amine miscellaneous for grace, i.e., experimental group mouth raise drug mouse tumor relative to experimental group without The inhibiting rate of medication mouse tumor is higher than control group mouth and raises suppression of the drug mouse tumor relative to control group without medication mouse tumor Rate processed.

The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.

Sequence table

<110>Tianjin Urological Surgical Department Inst.

<120>application of CYTOR and its inhibitor in anti-castration-resistant tumour

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

tctatgtgtc ttaatccctt gtcct 25

<210> 2

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

ttctccgaac gtgtcacgtt t 21

<210> 3

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

acaggaagct ctatgacaca cttg 24

<210> 4

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

ctgcattccg gctgtgat 18

<210> 5

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

ccatcaccat cttccaggag cga 23

<210> 6

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

ggtggtgaag acgccagtgg a 21

<210> 7

<211> 9

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

cgggttggc 9

<210> 8

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

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gtgggcggtt ggaaccag 18

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caattgccaa cccggaat 18

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<213>artificial sequence (Artificial Sequence)

<400> 10

ccatcttgtc gtcttcggaa atgtta 26

<210> 11

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

tttgaatgag gcaagtcagc ctttct 26

<210> 12

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

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cagcctattg cgagagagct g 21

<210> 13

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

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gaaaggatct tgggcacttg c 21

Claims (10)

1. a kind of antisense oligonucleotides, which is characterized in that the antisense oligonucleotides specifically binds CYTOR.

2. antisense oligonucleotides according to claim 1, which is characterized in that specifically bind the antisense oligonucleotides of CYTOR Acid sequence is as shown in SEQ ID NO.8.

3. antisense oligonucleotides according to claim 2, which is characterized in that the nucleic acid of the antisense oligonucleotides is methyl Change the nucleic acid of modification.

4. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes the described in any item antisenses of claim 1-3 Oligonucleotides.

5. following described in any item applications:

A. the CYTOR for specifically binding AR-V7 is gone after the hormone castration of preparation treatment androgen receptor related neoplasms Gesture is resisted and the application in relapse and metastasis drug；

B. application of the described in any item antisense oligonucleotides of claim 1-3 in the drug of preparation treatment tumour；

C. the described in any item antisense oligonucleotides of claim 1-3 are gone in the hormone of preparation treatment androgen receptor related neoplasms Castration after gesture treatment is resisted and is applied in relapse and metastasis drug；

D. application of the pharmaceutical composition as claimed in claim 4 in the product of preparation treatment tumour；

E. pharmaceutical composition as claimed in claim 4 is after the hormone castration of preparation treatment androgen receptor related neoplasms Castration is resisted and is applied in relapse and metastasis product.

6. application according to claim 5, which is characterized in that drug described in a includes the inhibitor of CYTOR.

7. application according to claim 6, which is characterized in that the inhibitor is the reagent for reducing CYTOR expression.

8. application according to claim 7, which is characterized in that the inhibitor is short hairpin RNA or antisense oligonucleotides.

9. application according to claim 8, which is characterized in that the sequence of the short hairpin RNA such as SEQ ID NO.1 institute Show.

10. application according to claim 8, which is characterized in that the sequence of the antisense oligonucleotides such as SEQ ID NO.8 It is shown.