CN108159419A

CN108159419A - Induce application of the substance of IRF8 expression in the drug for preparing treatment androgen receptor unconventionality expression associated diseases

Info

Publication number: CN108159419A
Application number: CN201711439167.8A
Authority: CN
Inventors: 杨勇; 吴红茜; 赵志立; 李岩; 高晓芳; 赵伟; 毛杰
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2017-12-27
Filing date: 2017-12-27
Publication date: 2018-06-15
Anticipated expiration: 2037-12-27
Also published as: CN108159419B

Abstract

Application in the drug for treating androgen receptor unconventionality expression associated diseases in preparation the invention discloses the substance of induction IRF8 expression.Present invention discover that IRF8 can directly adjust prostate cancer and hepatoma cell proliferation, and the adjustable prostate gland cancer cell of IRF8 expression is to the sensibility of AR antagonists Enz, and IRF8, which strikes, low causes prostate gland cancer cell to be resistant to the apoptosis caused by Enz.The substance such as IFN α of IRF8 expression is induced to raise IRF8 expression by AR, and then take part in adjusting of the prostate gland cancer cell to Enz sensibility.In clinical test, endocrine therapy endocrinotherapy can enhance patients with prostate cancer treatment curative effect to IFN α joint to greatest extent.It can be seen that the substance of induction IRF8 expression, which can combine androgen receptor antagonists enhancing clinic IRF8, expresses low patient to androgen receptor antagonists sensibility, androgen receptor antagonists curative effect is improved, its drug resistance is delayed to occur.

Description

Induce substance disease caused by treatment androgen receptor unconventionality expression is prepared of IRF8 expression Application in the drug of disease

Technical field

The invention belongs to field of medicaments, are related to inducing the substance of IRF8 expression preparing treatment androgen receptor unconventionality expression Application in the drug of associated diseases.

Background technology

In American-European countries, prostate cancer is that incidence, the death rate ranked second the common male malignancy of position, China forefront Though the incidence of gland cancer is less than western countries, become with the aging of living-pattern preservation and population in what is significantly increased Gesture.Expect the year two thousand twenty, the incidence of prostate cancer will be up to 40/,100,000, year new cases up to 350,000, the incidence with liver cancer Quite.

Initial prostate cancer has hormonal dependent, and at that time, endocrine therapy is sensitive, and the symptom of prostate cancer improves, Disease process delays, but in the case where castration selects pressure, generally can become castration-resistant prostate cancer after 12-18 months (Castration Resistant Prostate Cancer, CRPC).In recent years, the drug for having treatment CRPC successively comes out, Such as androgen antagonist abiraterone, androgen receptor (Androgen receptor, AR) antagonist Bicalutamide (Bicalutamide, Bic), the miscellaneous Shandong amine (Enzalutamide, Enz) of grace etc. is effective to CRPC, but all exists easily drug resistant Problem.The generation of CRPC and drug resistance are the underlying cause of death of patients with prostate cancer and the Major Difficulties of current prostate cancer therapy And research direction.

Androgen receptor belongs to one kind of nuclear receptor superfamily, in the presence of respective ligand, plays transcription factor, The correct distribution of prostate epithelial cell is instructed, in the development of normal prostata tissue, androgen receptor is essential Factor, but its overexpression can then lead to hyperblastosis, and negatively correlated with the differentiation by stages of prostate cancer.Animal model table Bright prostate epithelial cell AR overexpressions can cause proliferation and vicious transformation, in patients with prostate cancer, AR high expression and clinic Case malignant progression and recurrence-free survival rate reduce closely related.AR widely participates in the growth of prostatic cell, differentiation and Function simultaneously participates in the starting and development of prostate cancer, is most important signal needed for prostate cancer growth.It is in addition, different Normal AR activity increases are also considered as the key of CRPC development, and CRPC is often abnormal with AR signal paths, AR expression and AR associations The generation and development of CRPC is taken part in abnormal, AR signal paths imbalance of developed by molecule etc..In addition, it is also targeting that AR activity is abnormal The drug resistant major reasons of CRPC medicines Enz of AR.

According to the age of patient and disease process, the treatment means of prostate cancer mainly include operation, castration, radiotherapy and change It treats.Early prostate cancer has androgen-dependent, and after endocrine therapy, the symptom of prostate cancer improves, disease process prolongs It is slow, but generally can become CRPC by 12-18 months.In recent years, the drug for having treatment CRPC successively comes out, as androgen is short of money Anti-agent abiraterone, androgen receptor antagonists Bicalutamide, the miscellaneous Shandong amine of grace etc. are effective to CRPC, but all exist easily drug resistant Problem.The generation of CRPC and drug resistance are the underlying cause of death of patients with prostate cancer and the Major Difficulties of current prostate cancer therapy And research direction.

The miscellaneous Shandong amine of grace (also known as MDV3100) be the oral administration biaavailability of a new generation it is high directly act on androgen receptor The antiandrogen drug of body, mainly by the way that the combination of testosterone and androgen receptor, the core of inhibition androgen receptor is inhibited to shift, press down Androgen receptor processed is combined with DNA to play a role simultaneously without approach such as agonist effects, so as to improve CRPC patient (including mostly western He match chemotherapy failure patient) Overall survival and DFS phase.Compared with previous antiandrogen drug (such as than card Shandong Amine), its affinity with androgen receptor can be significantly increased.The miscellaneous Shandong amine of grace by the ligand binding domain of competitive binding AR, The combination of testosterone, protona and AR are blocked, while androgen receptor core is inhibited to shift, inhibits what AR was combined with target gene DNA Process inhibits the proliferation of prostatic cell, promotes its apoptosis, and then play antitumor activity.If so androgen receptor sheet Body changes, and changes including AR protein actives, point mutation, and Shear criteria etc. can lead to the miscellaneous drug resistant generation of Shandong amine of grace. AR is overexpressed in androgen independence transplantable tumor, prostate gland cancer cell can be made to reduce Androgen-sensitive, and further Prostate transplantable tumor is promoted to be transformed into androgen independence pattern from Androgen-dependent pattern, this just highlights AR amplifications and mistake In Androgen deprivation therapy and the treatment of two generations for there is drug resistant potential mechanism in expression.

IRF8, also referred to as interferon consensus sequence binding protein (Interferon consensus sequence- Binding protein, ICSBP), belong to interferon regulatory factor (Interferon regulatory factor, IRF) family Race's a member.The transcription factor that IRF has found when being a kind of research I type IFN systems, expression are regulated and controled, and feedback regulation by IFN IFN signal paths, in IFN transcriptional controls, the IFN congenital immunity participated in and adaptive immunity, cytokine signaling, thin Play an important roll in born of the same parents' multiplication regulatory.IRF families share 9 members (IRF1-9), the multiple members of IRF families (IRF1, IRF3, IRF4, IRF5, IRF7, IRF8) pattern recognition receptors triggering innate immune response in function demonstrate,proved It is real.The important function of IRF family members IRF1, IRF2, IRF4, IRF8 in immunocyte growth course also obtain significantly into Exhibition.More and more research concern IRF family members IRF1, IRF2, IRF3, IRF5, IRF6, IRF8 are in blood and epithelium in recent years Function in growth, survival and the tumour generation of cell, wherein IRF1, IRF2, IRF3, IRF5, IRF6 are in blood and solid tumor In function obtained the confirmation of a large number of researchers.IRF8 expression deletions in immunocyte can then cause acute myelocytic white Blood disease (Acute myeloid leukemia, AML) and chronic granulocytic leukemia (Chronic myelocytic Leukemia, CML) etc. blood tumors disease generation, show in blood tumor, IRF8 possibly as a kind of leukaemia inhibit base Cause, inducing tumor suppressor genes (Tumor suppressor gene, TSG) and apoptogene transcription inhibit oncogene and resist to wither The expression such as gene Bcl-xl, Bcl-2 is died so as to play its tumor suppression function.But IRF8 only has several articles at present It reports its function in epithelial cell, leads to its protein in most tumours including IRF8 promoter hypermethylations All it is low expression/do not express (human protein Atlas, http in tissue://www.proteinatlas.org).But IRF8 has not been reported in the expression of prostate cancer and function.

Invention content

The problem of the purpose of the present invention is being directed to current androgen receptor drug resistance, the substance for providing induction IRF8 expression are being made Application in the drug of standby auxiliary treatment androgen receptor unconventionality expression associated diseases.

It is a further object of the present invention to provide a kind of for treating the medicine group of androgen receptor unconventionality expression associated diseases Close object.

The purpose of the present invention can be achieved through the following technical solutions：

The substance of IRF8 expression is induced in the drug for preparing auxiliary treatment androgen receptor unconventionality expression associated diseases Using.

It is preferred that the substance of induction IRF8 expression is preparing auxiliary androgen receptor antagonists treatment androgen receptor exception table Application up in the drug of associated diseases or the drug resistant androgen receptor unconventionality expression associated diseases of androgen receptor antagonists.

Wherein, the substance of described induction IRF8 expression is preferably able to improve IRF8 expression quantity or IRF8 is in vivo in vivo The substance of activity.The substance of the induction IRF8 expression is further preferably from IFN α, histon deacetylase (HDAC) inhibitor (histone deacetylase inhibitor, HDACI).

The miscellaneous Shandong amine of the preferred grace of the androgen receptor antagonists, Bicalutamide.

The preferred prostate cancer of androgen unconventionality expression associated diseases, liver cancer；Before further preferred castration-resistant Row gland cancer.

A kind of pharmaceutical composition for being used to treat androgen unconventionality expression associated diseases, by the androgen receptor of therapeutically effective amount Body antagonist and the material composition of induction IRF8 expression.

The androgen receptor antagonists are selected from the miscellaneous Shandong amine of grace, Bicalutamide；The substance of the induction IRF8 expression For IRF8 expression quantity or IRF8 active substances in vivo in vivo can be improved.

The substance of the induction IRF8 expression is selected from IFN α.

The androgen unconventionality expression associated diseases are selected from prostate cancer, liver cancer；It is preferred that castration-resistant prostate cancer.

Advantageous effect：

The present invention is studied by the immunohistochemistry and Western blot of prostate cancer tissue chip and prostate cancer tissue The results show that IRF8 has expression, and expression and the prostate cancer of IRF8 in the endochylema and karyon of cancerous prostate epithelial cell Pathological grading positive correlation.IRF8 plays tumor suppressor effect in the solid tumors such as blood tumor and colon cancer, and expression is being faced Apparent up-regulation, this phenomenon are also never reported in bed prostata tissue.This research reports IRF8 in prostate for the first time Expression in cancer, and its expression is adjusted by androgen AR signal paths.

Western blot results of the present invention show that IRF8 albumen has in the AR positives and AR feminine gender prostate gland cancer cells Expression.Content wherein in the Prostatic cancer cell lines of the AR positives is higher than AR negative cells strains；IRF8 strikes low rear AR albumen water Dawn shows increase, and IRF8 is overexpressed the protein expression that can then reduce AR.Experiment in vitro shows that prostate gland cancer cell is overexpressed IRF8 Influence afterwards to prostate gland cancer cell proliferation is related with AR expression status.The prostate gland cancer cell that IRF8 can inhibit the AR positives increases It grows, and AR feminine genders prostate gland cancer cell is proliferated without influence.IRF8 strike it is low after, LNCaP cell Proliferations accelerate, proliferation accelerate characteristic Persistently exist in low androgen levels and excessive androgen level.Experiment in vivo shows, IRF8 strike it is low after, in LNCaP cell bodies It is dramatically speeded up into efflux velocity, tumor-bearing mice Overall survival is substantially reduced.And IRF8 strikes and low caused increases into efflux velocity Characteristic still exists in the NOD-SCID mouse that immune system lacks completely, shows that IRF8 can directly adjust prostate gland cancer cell increasing It grows.

It has also been found that the adjustable prostate gland cancer cell of IRF8 expression, to the sensibility of AR antagonists Enz, IRF8 strikes low Prostate gland cancer cell can be caused to be resistant to the apoptosis caused by Enz.IFN α raises IRF8 expression by AR, and then takes part in forefront Adjusting of the adenocarcinoma cell to Enz sensibility.In clinical test, endocrine therapy endocrinotherapy can increase IFN α joint to greatest extent Strong patients with prostate cancer treatment curative effect.It can be seen that the substance of induction IRF8 expression can combine androgen receptor antagonists enhancing clinic IRF8 expresses low patient to androgen receptor antagonists sensibility, improves androgen receptor antagonists curative effect, its drug resistance is delayed to send out It is raw.

Description of the drawings

IRF8 is expressed in Fig. 1 prostate cancer tissue micro-array chips.

Fig. 2 bioinformatic analysis finds that IRF8 expresses downward in prostate cancer progression.

(A) database analysis IRF8 (TCGA) in normal prostatic and prostate cancer tissue；Hyperplastic prostate tissue, Part and metastatic prostate cancer tissue (GSE3325)；Metastatic Androgen-sensitive and tolerance prostate cancer tissue (GSE6099) and castration castrates the expression induced in tumour growth minimum point and castration tolerance recurrent prostate cancer tissue.(B) In TCGA databases, expression of other members of IRF families in normal prostatic and prostate cancer database.(C) it is same with IRF8 Source property higher IRF family members IRF2, IRF4, IRF6 are in hyperplastic prostate tissue, part and metastatic prostate cancer group Knit the expression in (GSE3325).

The up-regulation of IRF8 depends on DHT-AR signal paths in Fig. 3 PCa cell lines.

(A) DHT handles the Immunofluorescence test IRF8 expression after 24 hours of LNCaP cells.(B) DHT handles LNCaP cells 24 Dose-dependent effects of the DHT to IRF8mRNA levels are detected by real-time PCR after hour.**P<0.01, * * * P<0.001. (C) DHT handles LNCaP cells and detects dose-dependent effects of the DHT to IRF8 protein levels by WB after 24 hours.(D)DHT It handles 22RV1 cells and dose-dependent effects of the DHT to IRF8mRNA levels is detected by real-time PCR after 24 hours.(E-G) IGF-1 (10nM, E) in LNCaP, EGF (10nM, F) and IL-6 (10ng/mL, G) imitate the Time Dependent of IRF8 protein levels It should.

Fig. 4 ADT lower the expression of IRF8 in vivo and in vitro.

A-B schemes:Agent of the DHT to IRF8mRNA horizontal (A) and protein level (B) is detected by real-time PCR and WB respectively Measure dependence.***P<0.001.C schemes:C-FBS (5%) is detected by WB, (10 μM and Bic (10 μM) are handled 24-72 hours Enz The time dependence effect of IRF8 protein levels afterwards.D-E schemes:In castration and the LNCaP heterografts of castration combination Enz treatments In knurl；And cultivate LNCaP simulation castrations (1 week and 2 weeks), WB detections in the culture medium containing 5%C-FBS for a long time in vitro IRF8 expressions.F schemes:LNCaP cell inoculations are in the Balb/c nude mice abdomen back sides of castration of performing the operation, Enz (10mg/kg) and solvent After compareing (n=6) processing 28 days, the expression of IRF8 in tumour.

The expression of Fig. 5 IRF8 and function are related with the state of AR.

(A) in Western blot analysis prostate cancer cell line IRF8 protein level.(B) in pcDNA3.1 (+) wink When transfection (DU145-vector) or the DU145 (DU145-IRF8) that transiently transfects of the pcDNA3.1 (+)/IRF8 that is used as exist Absorbance at 450nm.Numerical value represents average value ± SEM, each n=6 sample of sample.(C) instantaneously turn in pcDNA3.1 (+) Extinctions of the PC3 (PC3-IRF8) that dye (PC3-vector) or pcDNA3.1 (+)/IRF8 being used as are transiently transfected at 450nm Degree.Numerical value represents average value ± SEM, each n=6 sample of sample.(D) (LNCaP- is transiently transfected in pcDNA3.1 (+) ) or absorbances of the LNCaP (LNCaP-IRF8) that transiently transfects of the pcDNA3.1 (+)/IRF8 that is used as at 450nm vector. Numerical value represents average value ± SEM, each n=6 sample of sample.

Fig. 6 IRF8 inhibit the proliferation of AR positives LNCaP.

(A) in Western blot analysis (being standardized as β-actin) LNCaP-shIRF8 IRF8 protein level.(B- D the LNCaP- of -72 hours 24 hours) is cultivated in FBS (B), C-FBS (C) or culture medium containing 10nM R1881 (D) The absorbance of the 450nm of shIRF8 (shRNA1, shRNA2).

Fig. 7 strikes in LNCaP to be subtracted the tumour that IRF8 promotes it in the Balb/c nude mices of castrating and occurs.

(A) castrating Balb/c nude mices (n=9) in LNCaP-shIRF8 tumour growth.(B) LNCaP-shIRF8 is being castrated Total survival rate in the Balb/c nude mices (n=9) cut.Numerical value represents average value ± SEM；*P<0.05, * * P<0.01.

Fig. 8 strike that low IRF8 promotes it in NOD-SCID mouse models in LNCaP into knurl.

(A) tumour growths (n=6) of the LNCaP-shIRF8 in NOD-SCID mouse.(B) NOD-SCID murine heterotropics move Plant the final tumor weight of knurl model (n=6).(C) the tumour photo of experimental endpoints.Numerical value represents average value ± SEM；*P< 0.05, * * P<0.01.

Fig. 9 LNCaP-shIRF8 cells reduce Enz sensibility.

(A) LNCaP-shNC and LNCaP-shIRF8 of 48 hours is handled with the Enz of various dose in 96 well culture plates Cell viability.Cell viability is standardized as DMSO controls.(B) the Enz processing of various dose is used in 96 well culture plates Cell viability is detected after LNCaP-shNC and LNCaP-shIRF8 cells 72h.Using DMSO as control.(C) at 5 μM and 10 μM of Enz Manage LNCaP-shNC and LNCaP-shIRF8 cell 7d rear plate Clone formations.Amplification factor is 10 times, engineer's scale=200 μM.

3 activity of caspase of Figure 10 Enz inductions is adjusted by IRF8 expression

(A) IRF8 in LNCaP is knocked out, and reduces by 3 activity of caspase of Enz inductions.(B) dystopy of IRF8, which is overexpressed, increases 3 activity of caspase of strong Enz inductions.*P<0.05, ns：Not significantly, n=6.

The Clone formation and cell proliferation inhibition rate that Enz is induced in Figure 11 LNCaP-shIRF8 weaken.

(A) 10 μM of Enz handle the LNCaP-shNC and LNCaP-shRNA1 of 10 days and the representativeness gram of LNCaP-shRNA2 Grand formation.(B) clone's number of each group processing of (A).*, compared to LNCaP-shNC (with DMSO processing), P<0.01；##, with LNCaP-shNC is compared, P (with Enz processing)<0.01.(C) the Clone formation inhibiting rate of the colony count of (B), with LNCaP- ShNC is compared, * * P<0.05.(D) suction of the LNCaP-shNC and LNCaP-shIRF8 of 4 days at 450nm is handled in 10 μM of Enz Luminosity, * P<0.05.

Figure 12 Enz treated tumor regressions in LNCaP-shNC and LNCaP-shIRF8 xenograft models.

(A) (each processing group n=of the castrating male mice with LNCaP-shNC and LNCaP-shIRF8 xenograft tumors 6) tumour growth.Pass through daily gavage solvent control, Enz：10mg/kg/d is handled 28 days.(B) LNCaP xenograft tumors Final gross tumor volume.(C) relative tumour volume of each tumour changes percentage after 28 days.(D) final tumor weight.(E) at LNCaP xenograft tumours photos after dead mouse.Statistical analysis is examined with Student t and carried out, compared with the control * * p< 0.01, * * * p<0.001.

Figure 13 IFN αs 2a can be adjusted AR in prostate gland cancer cell by IRF8/AR axis and be expressed.

(A) influences of the IFN α 2a to the protein level of IRF8 and AR in LNCaP and 22RV1 cells.With various concentration IFN α 2a processing LNCaP or 22,RV1 24 hours, pass through AR, IRF8 and the β-actin that immunoblotting measures cell lysate. (B) the IFN α 2a 2000IU/ml of 22RV1 various concentrations are handled 24 hours, pass through AR pairs of DNA pull down experiments detection The recognition reaction in IRF8 promoter region ISRE-like sites.(C) the IFN α 2a 2000IU/ml processing of 22RV1 various concentrations 48 hours, protein synthesis inhibitor CHX processing 0-6h, WB detection AR protein half-lifes.(D) IFN α of 22RV1 various concentrations 2a 2000IU/ml are handled 48 hours, protease inhibitors MG132 processing 5h, WB detection AR expression.(E) 22RV1 is different dense The IFN α 2a 2000IU/ml of degree are handled 48 hours, protease inhibitors MG132 processing 5h, Immunoprecipitation detection AR's Ubiquitination.

Figure 14 is in LNCaP-shNC and LNCaP-shIRF8 heteroplastic transplantation models, IFN α joint Enz treated tumours Subside.

(A) in LNCaP and 22RV1 cells, IFN α (1000IU/ml) joint various dose Enz handles 96h and 48h respectively, 50% lethal dose IC50 is calculated according to cell viability.(B) various dose IFN α has no the proliferation of LNCaP and 22RV1 cells It significantly affects.(C) LNCaP-shNC and LNCaP-shIRF8 xenograft tumor models, daily gavage give solvent control, Enz (10mg/kg), IFN α (1.5 × 10 7IU, every group of n=6)/kg) or Enz (10mg/kg)+IFN α (1.5x10⁷IU/kg)30 My god.(D) final tumor weight and tumor suppression.(E) the single tumour relative tumour volume increased percentage of experimental endpoints.(F) at After dead mouse, the photo of xenograft tumours tissue and the percentage of recurrent tumor.Statistical analysis uses Student's t- It examines and carries out, compared with the control * p<0.05, * * p<0.01, * * * p<0.001.

The PSA of Figure 15 IFN αs joint Enz treatment patients with prostate cancer is horizontal.

MAB groups：With the 3.6mg/28 days+Bicalutamide combination therapy in 50mg/ days of goserelin acetate acetate.MAB combines IFN α group：MAB+IFNα3×10⁶IU/3d.Follow-up detection PSA is horizontal.

Figure 16 IRF8 express reduction in liver cancer (HCC) patient, and with clinical overall survival positive correlation.

(A) TCGA database analysis IRF8 is expressed in liver cancer patient.(B) clinical liver cancer patient cancer takes up IRF8 in cancer nests Expression.(C) WB detects IRF8 and AR expression in the rat liver cancer model cancerous tissue that DEN is induced.(D) TCGA database analysis IRF8 Expression height and liver cancer patient overall survival.(E) GEO databases GSE10141 analyzes IRF8 expression height and liver cancer patient totality Existence.

Figure 17 raisings IRF8 is expressed in liver cancer cells Hep1-6, inhibits tumour growth characteristic.

(A-B) after Hep1-6 is instantaneously overexpressed IRF8, inhibit its cell Proliferation.(C) after Hep1-6 is instantaneously overexpressed IRF8, Inhibit its Clone formation.(D) after Hep1-6 stablizes overexpression IRF8, inhibit its cell Proliferation.(E-F) Hep1-6, which stablizes, is overexpressed After IRF8, inhibit its cut transfer ability.(G-I) after Hep1-6 stablizes overexpression IRF8, inhibit its balling-up ability.

IFN α can inhibit AR expression by IRF8 in Figure 18 liver cancer cells.

(A-B) after liver cancer cells Bel7404 and HepG2 is overexpressed IRF8, WB detection AR expression.(C) Hep1-6 stablized After expressing IRF8 (band His labels), WB detection AR expression.(D) after various concentration IFN α handles liver cancer cells Bel7404 for 24 hours, WB detects IRF8 and AR expression.(E) after various concentration IFN α handles hepatocellular carcinoma H22 for 24 hours, WB detection IRF8 and AR expression.

Specific embodiment

1.1 experimental cells and animal

Balb/c nude mouse, NOD-SCID mouse are purchased from Shanghai Ling Chang bio tech ltd, for building LNCaP Nude Mouse Models.Mouse feeder is in SPF environment, and light and shade alt time is 12/12h round the clock, free water diet. All zooperies carry out by China Medicine University's management of laboratory animal regulations research and Ethics Committee requirement, and obtain Pharmaceutical university of state animal health and the approval using the committee.

DMEM culture solutions of the people renal epithelial cell HEK293T (hereinafter referred 293T) containing 10%FBS, prostate gland cancer cell Strain 1640 culture mediums of the LNCaP containing 10%FBS, in 5%CO₂It is carried out under the conditions of 37 DEG C of saturated humidities by 1:2 or 1:3 passages Culture.

Human prostate proliferative cell strain BPH1, Prostatic cancer cell lines LNCaP and IRF8 surely strike cell strain, and PC3 is with containing The RPMI-1640 cultures of 10%FBS, HEK293T, DU145 are cultivated with the DMEM containing 10%FBS；In 5%CO₂37 DEG C of saturations are wet Secondary culture is carried out under the conditions of degree, by 1:2 or 1:3 passages.

1.2 drugs and reagent

Dihydrotestosterone (Dihydrotestosterone, DHT, trade name allodihydrotestosterone, MB5035), testosterone (MG1636-5), Estrogen (Estrogen, E2, MB1098-S), Mei Lun companies；The miscellaneous Shandong amine (HY-70002) of grace, the Medchem Express U.S. Company；IGF-1 (100-11), EGF (AF-100-15), IL-6 (200-06), Peprotech companies；RPMI-1640 culture mediums (01-100-1ACS), DMEM culture mediums (01-052), fetal calf serum (Fetal Bovine Serum, FBS, 040011ACS), Carbon adsorption fetal calf serum (Certified Foetal Bovine Serum Charcoal Stripped, C-FBS, 04-201- 1A), Biological Industries companies；Dual anti-(10378-016), trypsase (25200-072), Gibco companies； Matrigel (354234), BD companies；Albumen pre-dyed albumen marker (26617), BCA protein quantification kit reagent box (NC13225CH), ECL-PLUS developer solutions (QB19798510), Dynabeads M-280Streptavidin biotins are affine Magnetic bead (11205D), Thermo Scientific companies；Protease Inhibitor Cocktail (05693132001), Roche companies；Small amount plasmid extraction kit (PL02), Ai Delai biotechnologies company；The a large amount of extracts kits of plasmid (740412.50), MACHEREY-NAGEL companies；AR (ab74272) antibody, IRF8 (ab28696) antibody, Abcam companies；β- Actin (bsm-33036M), Bo Aosen companies；Goat-anti rabbit-HRP secondary antibodies (sc-2030), Santa Cruz companies；DAPI (C1005), Dil cell membranes red fluorescence probe (C1036), anti-fluorescent quenching mounting liquid (P0126), lowlenthal serum (C0265), DAB horseradish peroxidase colour reagent boxes (P0203), CCK8 (C0039), WB and IP cell pyrolysis liquids (P0013), the green skies Bio tech ltd；QReal-time PCR reagents (PR047A, PR820A), Takara companies；DNA and SiRNA transfection reagents (101-10N), PolyPlus-transfection companies；Dual luciferase reporter gene detection kit (E1910), Promega companies；Nucleoprotamine DNA (P4505), Sigma companies；Calpain Activity Assay Kit Fluorogenic (QIA120), Millipore Corp.；SiRNA targeting IRF8 (siRNA1-5), Scramble siRNA (siNC), Ji Ma companies in Shanghai build；RT-PCR primer, Jin Sirui bio tech ltd structure；MG132 (M7449), PEZX-FR03-hIRF8-luc Dual-Luciferase reporter plasmids, the synthesis of Guangzhou reactivation bio tech ltd；pcDNA3.1 (+)-hAR (full length), pcDNA3.1 (+), pcDNA3.1 (+)-hIRF8 plasmids, Jin Sirui bio tech ltd Synthesis.Flag-Ub plasmids are purchased from Wuhan Miao Ling bio tech ltd；Leupeptin (Leu, L2884), Calpastatin (SCP0063), cycloheximide (Cycloheximide, CHX, C4859), actinomycin D (Actinomycin D, ActD, A9415), Sigma companies；MG101 (HY-18964), MCE companies；DiO cell membrane green fluorescence probes (C1038), the green skies Biotechnology company；Normal rabbit IgG (sc-2027), Santa Cruz company；Pierce^TMProtein A/G Magnetic Beads (88803), Thermo Fisher Scientific companies；IFN α 2a cell factors, perprotech companies；IFNα 2a parenteral solution (trade names：Recomvinated Interferon α-2a), Shenyang three lives pharmaceutical Co. Ltd；P-STAT1 (701), p-STAT1 (727), STAT1, IRF3, CST company；Bradford protein quantifications kit (P0006), green skies bio tech ltd；Other reagents are equal It is pure to analyze.

1 immunohistochemistry of embodiment

After the fresh prostate cancer of Jiangsu TCM Hospital's collection, hyperplastic prostate tissue, liquid nitrogen flash freezer, prepare paraffin and cut Piece, from Shanghai, Xin Chao companies buy prostate cancer tissue chip (HPro-Ade180PG-02,180 points) and entrust its immunohistochemistry IRF8 expression is detected, and senior pathologist is asked to carry out interpretation to ImmunohistochemistryResults Results：

(1) stained positive rate judges：After the observation for interlacing point being carried out first under low power lens whole visual field, then choose The different visual field of 3 staining powers carries out interpretation under high power lens, if when being positioned at karyon, remembers at random in each visual field 100 cells, then remember the percentage X1% that positive cell accounts in 100 cells, and same principle sees the positive in other 2 visuals field X2%, X3% after the percentage that cell accounts for, the final interlacing point stained positive rate take the average of X1%, X2% and X3%；Such as If fruit is positioned at endochylema or after birth, 3 different staining power visuals field are equally chosen, carry out taking after estimating its positive rate respectively Its average value.

(2) staining power is quantitative：One sample, after the observation for interlacing point being carried out first under low power lens whole visual field, Then it chooses the different visual field of 3 staining powers to take pictures under high power lens, under complete same microscopic conditions, once All samples are shot.Immunohistochemistry photo densitometry is analyzed using IPP softwares.After optical density correction is illustrated, selection Average optical density density mean and accumulation optical density degree (integrated optical density sum, IOD Sum it) is quantified, takes quantitative result of the average value in three visuals field for a sample.

The organization chip takes up cancer nests tissue samples and 5 normal prostate tissues, base comprising 64 same patient's cancers This information is as shown in table 1.Immunohistochemical location result shows that IRF8 has expression (figure in prostate gland cancer cell endochylema and karyon 1).The results show that in normal cancer beside organism's endochylema, the weak dyeing of IRF8 and moderate strength positive staining reach IRF8 positive stainings rate 40%；Strong dyeing is up to 20%, and in cancer beside organism's endochylema, IRF8 moderate strength positive stainings are reduced to 16%；In prostate cancer nests The dyeing of IRF8 strong positives is reduced to 9.37% in cell cytosol.In nucleus IRF8 by force dyeing by its in the normal tissue 20% 1.5625% be reduced in cancer nests tissue, expression and its expression in Hypertrophic cancer beside organism and normal cancer beside organism Significant difference (table 2).

By 1 64 cancers of table/cancer nests patient clinical case feature

2 IRF8 of table is normal, by cancer, the immunohistochemical staining in cancer nests prostata tissue

Embodiment 2

2.1 DHT can raise IRF8 expression

Ligand of the androgen as AR is played in the prostate carcinoma cell growth and vicious transformation that are mediated in AR to closing weight The effect wanted.Generally existing Androgen-AR signal path abnormal activations during prostate cancer occurrence and development.We have found that with The increase of prostate cancer pathological grading, the expression quantity of IRF8 rises, and IRF8 expression expresses positive correlation with AR.These researchs carry Show us, there are certain between IRF8 and AR to contact.Research shows that have in prostatic cell a group ISG albumen at the same time as Androgen stimulated gene is regulated and controled by androgen.Whether we detect the expression of IRF8 by AR signal paths in the present embodiment Adjusting.AR positive prostate cancer cells strain LNCaP pre-process rs, Zhi Houyong for 24 hours with 5% carbon adsorption change of serum C-FBS bed boards The 10nM DHT stimulations IRF8 expression of rs cellular immunofluorescences detection for 24 hours, ethyl alcohol is as solvent control group.Immunostaining results are found In the case of no DHT exists, IRF8 has faint expression (Fig. 3 A-DHT), DHT stimulation activation AR in LNCaP cell cytosols and karyon After signal path, IRF8 expression significantly up-regulation (Fig. 3 A+DHT) in endochylema karyon.Simultaneously LNCaP is stimulated with various concentration DHT Cell rs for 24 hours extracts RNA and albumen, respectively with Real-time PCR and WB detection IRF8 expression, it is found that DHT can dose-dependant Property the up-regulation mRNA level in-site (Fig. 3 B) of IRF8 and protein expression (Fig. 3 C).In another AR positive prostate cancer cells strain DHT activation AR can also raise the expression (Fig. 3 D) of IRF8 in 22RV1.During prostate cancer vicious transformation, low-level androgen In the case of, growth factor I GF-1, EGF and cell factor IL-6 etc. can alternative activation AR promotion prostate gland cancer cell androgens Dependent/non-dependent is grown.Thus we guess whether alternative activation AR can equally raise the expression of IRF8It is interesting that AR bypasses swash Factor IGF-1 (Fig. 3 E) living, EGF (Fig. 3 F) can lower the expression of IRF8, and without time dependence；IL-6 to the expression of IRF8 then It does not make significant difference (Fig. 3 G).These results prompting IRF8 may be a kind of androgen-induced gene.

2.2 castrations can lower the expression of IRF8

DHT mainly by being combined with its receptor AR, forms dimer, enters core and then play functional transcription.A upper small nodule Fruit finds that DHT can induce the expression of IRF8 in AR positive cell strains, then blocks whether AR signal paths can inhibit the table of IRF8 It reachesIn view of the androgen-dependent of initial prostate cancer, androgen deprivation therapy (Androgen deprivation Therapy, ADT), it is the main therapy of early prostate cancer including operation or medical castration.The miscellaneous Shandong amine of grace is that second generation AR is short of money Anti-agent can inhibit the core of AR to shift, inhibit AR a variety of to the combination of target gene DNA etc. by inhibiting the combination of androgen and AR Approach blocks AR signal paths.Carbon adsorption serum can remove the micro androgen in serum, and 5%C-FBS is commonly used to culture LNCaP For castration outside analogue body.Enz processing LNCaP after rs, extracts RNA and Protein Detection IRF8 expression respectively for 24 hours.As a result, it has been found that Enz can the dose dependent downward mRNA level in-site (Fig. 4 A) of IRF8 and protein level (Fig. 4 B).C-FBS, Enz and Bicalutamide (Bicalutamide, Bic) act on 24-72hrs, WB results show C-FBS can time dependence reduce IRF8 expression；It is potent Rs can lower IRF8 albumen to floor level for 24 hours for AR antagonists Enz effects, have no time dependence；Bic is short of money for first generation AR Anti-agent because it has partial excitatory activity to AR, is less than Enz (Fig. 4 C) to the downward intensity of IRF8 expression.To study long-term castration Influence to IRF8 expression, we are using the outer castration of 5%C-FBS culture medium longterm culture in vitro LNCaP analogue bodies.WB results are shown IRF8 can be lowered for 1 week to floor level by showing that C-FBS is handled, and further extend castration to 2 weeks, the expression of IRF8 is then without more into one Step reduces (Fig. 4 D, n=3).Further to study the influence that castration expresses IRF8 in PCa bodies, we are by LNCaP cell inoculations In Balb/c nude mice abdomen back sides, once tumour is grown.It is randomly divided into 3 groups, respectively sham-operation group；Surgical removal bilateral testes group (castration), internal androgen levels are reduced；Castration+Enz (10mg/kg) group further blocks AR signals.28 It wins tumor tissue after it to be homogenized, IRF8 is expressed in WB detection tumor mass.As a result display cuts off merely bilateral compared to sham-operation group Testis can slightly lower the expression of IRF8, after operation excision bilateral testes joint gastric infusion Enz treatments, IRF8 expressions into One step reduces (Fig. 4 E, n=3).The influence expressed further to detect Enz IRF8, we are performing the operation LNCaP cell inoculations The Balb/c nude mice abdomen back sides of bilateral testes are cut off, build LNCaP-CRPC Nude Mouse Models, Enz (10mg/kg) gavage Administration, wins tumor tissue after 28 days and is homogenized, and IRF8 is expressed in WB detection tumor mass.As a result after showing Enz treatments CRPC, IRF8 Expression generally reduces (Fig. 4 F, n=6), illustrates AR signal paths further to be blocked to remain unchanged in low androgen levels IRF8 expression can be lowered.

3 LNCaP cells of embodiment, which strike low IRF8 expression cells proliferation, to be accelerated

3.1IRF8 surely strikes the design and structure of cell strain LNCaP-shNC, LNCaP-shRNA1, LNCaP-shRNA2.

3.1.1Oligo design

DNA oligo use Designer3.0 (Genepharma) Software for Design, and synthetic primer is by Shanghai Ji agate pharmacy skill Art Co., Ltd completes.Loop structures in LV3-shRNA templates have selected TTCAAGAGA to avoid termination signal is formed.Just GATCC is contained at 5 ' ends of adopted chain, with the cohesive end complementation formed after BamHI digestions；AATTC is contained at 5 ' ends of antisense strand, with EcoRI The cohesive end formed after digestion is complementary.

Positive-sense strand：5’-GATCC-(GN18)-(TTCAAGAGA)-(N18C)-TTTTTTG-3’

Antisense strand：3’-G(CN18)-(AAGTTCTCT)-(N18G)-AAAAAACTTAA-5’

The DNA Oligo that table 3 designs

3.1.2 the annealing of LV3-shDNA templates

DNA oligo are dissolved in the TE solution of pH8.0, a concentration of 100 μM.Take in previous step corresponding sense and Antisense oligo solution matches the annealing reaction system of pallet according to following ratio：

4 shDNA template annealing reaction systems of table

PCR instrument cycle of annealing is set：95℃5min；85℃5min；75℃5min；70℃5min；4 DEG C of preservations.PCR product This PCR product is diluted 50 times, makes final concentration of 200nM by as shRNA templates (10 μM).

3.1.3 the linearisation of LV3 carriers

10 μ g LV3 carriers are taken, digestion processing is carried out according to following system：

5 endonuclease reaction system of table

37 DEG C of digestion 1h, agarose electrophoresis are returned using Agarose Gel DNA Purification Kit Ver2.0 It receives, electrophoresis detection estimated concentration, diluted concentration to 50ng/ μ L.

3.1.4 the structure of LV3-shRNA carriers

The coupled reaction of carrier is carried out according to following system：

6 carrier coupled reaction system of table

22 DEG C of reaction 1h, product are used for subsequent transformation competent cell.

3.1.5 the conversion and amplification of plasmid

(1) competent cell DH5 α are taken out from -80 DEG C of refrigerators, will just melt cell suspension and dispense 500 μ L to sterile pre- In cold EP pipes, it is placed in ice bath.

(2) target DNA is added in into competent cell suspension, flicks mixing, 30min is stood in ice bath.

(3) centrifuge tube is placed in 42 DEG C of water-baths and places 90s, then quickly pipe is transferred in ice bath, make cell cold But 2min, the process not shake centrifuge tube.

(4) 500 μ l LB culture mediums (without antibiotic) are added in into every centrifuge tube, mixing is placed on 37 DEG C of shaking tables, 220rpm shaken cultivation 1h, make thalline recover.

(5) 4000rpm centrifuges 1min, abandons part supernatant, retains 150 μ l, and mixing draws the competence that 100 μ l have been converted Cell is spread evenly across on the LB solid agar mediums containing 50 μ g/mL Ampicillin.

(6) tablet is placed in room temperature until liquid is absorbed, inversion tablet, 37 DEG C are cultivated 12-16h.

3.1.6 the identification and sequencing of positive colony

(1) it from 4 independent, full bacterium colonies of picking on cultured tablet, is placed in containing 5ml (containing 50 μ g/ml Ampicillin) in the 50mL tubes of LB culture mediums；37 DEG C of shaking tables are placed in, 250rpm cultivates 16h

(2) by cultured bacterium solution, plasmid is extracted in a small amount, and gained plasmid carries out double digestion identification with Xba I and Nhe I.

(3) the correct bacterial strain conservation of sequencing, which is stayed, does follow-up test.

3.1.7 plasmid extraction

(1) it takes in the bacterium solution to 50mL centrifuge tubes being incubated overnight, 5000rpm room temperatures centrifugation 2min falls do as far as possible Take back collection thalline completely.This step is repeated, until collecting enough thalline.

(2) bacterial sediment (ensuring to have added RNase A in solution I) is resuspended with 7.5mL solution P1, vortex oscillation is to thorough It suspends.

(3) add 7.5mL solution P2, leniently spinning upside down 6-8 times makes thalline fully crack, and is stored at room temperature 4-5min.This When bacterium solution should become limpid sticky.

(4) add 7.5mL solution N3, leniently spin upside down 6-8 times immediately, abundant mixing will appear white flock and sinks at this time It forms sediment, 5000rpm low-temperature centrifugations 20min.It is careful that supernatant is taken to avoid being drawn to the white precipitate of floating to new pipe.

(5) about 10mL isopropanols are added in into supernatant, (adsorption column in adsorption column DC is transferred to several times after fully reverse mixing It is put into collecting pipe), 5000rpm centrifugation 2min outwell waste liquid in collecting pipe.

(6) 10mL rinsing liquids (having added in absolute ethyl alcohol) are added in, 5000rpm centrifugation 2min discard waste liquid.Add 10mL Rinsing liquid WB repeats to rinse primary.

(7) adsorption column DC is put back in sky collecting pipe, 5000rpm centrifugation 5min with residual ethanol on dry matrices film, are beaten Seed cell's temperature of uncapping dries 5min.

(8) adsorption column DC is being taken out, is being put into a clean centrifuge tube, 1-2mL is added to wash in the centre position of adsorbed film De- liquid EB (being preheated in 65-70 DEG C of water-bath in advance) is stored at room temperature 3min, 5000rpm centrifugations 5min.Gained liquid is as high-purity Spend plasmid；Its concentration and purity are measured with UV Absorption method, ensure to carry the A260/A280 of Plasmid DNA 1.8~2.0 it Between.

3.1.8 293T cell packaging virus

(1) it takes the logarithm growth period 293T cell, removes culture solution, cell two is washed with 1mL D-Hank ' s solution It is secondary.1mL Trypsin-EDTA solution are added in, after mixing, 37 DEG C of placement 1-3min.

(2) Trypsin-EDTA solution are sucked, add in 2mL DMEM culture solutions (containing 10%FBS) to terminate digestion, Piping and druming makes cell form single cell suspension.In inoculated and cultured ware, DMEM culture solutions of the 10mL containing 10%FBS is added in, 37 after mixing DEG C 5%CO₂Overnight incubation.

(3) 1.5mL serum-free DMEM are added in a sterile 5mL centrifuge tube, are proportionally added into the sequence containing IRF8 Shuttle plasmid and packaging plasmid (pGag/Pol, pRev, pVSV-G), mixing take another sterile 5mL centrifuge tube, add in 1.5mL serum-free DMEM, add 300 μ L RNAi-Mate, and two pipes after being placed at room temperature for 5min are mixed, are placed at room temperature for by mixing 20-25min。

(4) culture solution in (2) is absorbed, adds in the DMEM culture solutions of 5mL serum-frees.Training is added dropwise in mixture in (3) It supports in ware, soft mixing puts 37 DEG C of 5%CO₂4-6h in incubator.

(5) transfection liquid is absorbed, adds in DMEM culture solutions of the 10mL containing 10%FBS.37 DEG C of 5%CO₂Continue to cultivate 72h.

(6) supernatant for cultivating cell is drawn onto in 50mL centrifuge tubes, 4000rpm centrifugations 4min.Take out centrifuged supernatant, 0.45 μm of filter filtering.

(7) low-temperature and high-speed centrifugation 2h is carried out to filtrate.Concentrate is collected in packing to EP pipes, -80 DEG C save backup.

3.1.9 virus infection and puromycin screening IRF8 surely strike cell line

(1) the LNCaP cells in growth period of taking the logarithm are inoculated in 24 orifice plates by every 2000, hole, and culture medium added by per hole is 100μL.To ensure there is preferable transfection efficiency, the density of cell is 40%-60%, 37 DEG C of 5%CO when carrying out virus infection₂Training It supports for 24 hours, it is adherent.

(2) two groups of infection preliminary experiments, every group of virus for having various concentration gradient are designed.First group is felt under normal circumstances Dye only adds virus liquid in complete medium；The Polybrene of 5 μ g/mL is added in during second group of infection, Polybrene is in big portion Efficiency of infection can be effectively improved by dividing in cell.

(3) virus liquid is taken out from refrigerator in advance, melted on ice.It draws in 10 μ L virus liquids to new EP pipes, soft mixing. According to 10 times, 100 times of concentration gradient dilutes virus liquid.

(4) suck the culture solution in 24 orifice plates, respectively taken from the virus liquid of three different gradients 500 μ L be added to every group three In a hole, while blank control group is set up, cell, which is then put back to incubator, is incubated.Cellular morphology is observed after 8-12h.It is if thin Born of the same parents' form shows that virus liquid does not have overt toxicity to cell with being uninfected by a group no significant difference.

(5) culture sucks in 24 orifice plates afterwards for 24 hours dilutes virus liquid, and the 1640 training liquid of 500 μ l 10%FBS are added in per hole, are added Enter puromycin to 1.0 μ g/ml of final concentration, in 37 DEG C of 5%CO₂Continue to cultivate.

(6) liquid, dosing are changed daily, until when blank dosing group is all dead, expands the cell of screening, are received sample and are used for RT-PCR Poor efficiency is struck with Western blot detections IRF8, IRF8 surely strikes cell strain LNCaP-shNC, LNCaP-shRNA1, LNCaP- ShRNA2 is built successfully.

3.2 CCK8 detect cell viability

(1) 0.25% pancreatin digests logarithmic phase cell, is resuspended and blown and beaten into unicellular outstanding with the culture solution containing 10%FBS Liquid.With every hole 1 × 10⁴In 96 well culture plates, 100 μ L/ holes, cell shakes up a cell inoculation, and each time point is done three and put down Row hole.It is put into incubator, 37 DEG C of 5%CO₂Cultivate rs for 24 hours.

(2) micro- Microscopic observation cell is adherent and in normal growth form, you can is administered.According to experiment purpose and Drug to be measured is first made mother liquor, carries out gradient dilution successively by plan, the property according to drug to be measured.Old culture solution is drawn, is added Enter diluted drug culture solution culture 48-72 hours.

(3) experimental endpoints add in CCK8 solution 20 μ L, 37 DEG C of incubation 4hrs per hole.

(4) 450nm wavelength is selected, each hole absorbance value is measured on multi-function microplate reader, experimental result is recorded, as a result adopts With 6.0 mapping analysis of GraphPad prism.

3.3 IRF8 are related to AR expression status to the influence that prostate gland cancer cell is proliferated

It is cloudy to people source AR using Western blot methods first for expression of the detection IRF8 in Prostatic cancer cell lines Property cell strain hyperplastic prostate epithelial cell BPH1, Prostatic cancer cell lines PC3, DU145 and AR positive prostate cancers are thin The expression of IRF8 albumen is detected in born of the same parents' strain LNCaP, using immunocyte Raw264.7 as IRF8WB positive controls. As a result it shows that IRF8 has expression in 4 kinds of prostate epithelial cells, and highest (figure is expressed in AR positive LNCaP cells 5A), expression intensity is about 2 times of AR feminine gender prostatic cells.For different expression feelings of the IRF8 in Prostatic cancer cell lines Condition, respectively by low expression IRF8 cell strains DU145 and PC3 transiently transfect pcDNA3.1 (+)-hIRF8 plasmids (IRF8 plasmids) with Height expression IRF8 (DU145-IRF8, PC3-IRF8), IRF8 with respect to overexpression cell line LNCaP transient transfection IRF8 plasmids with into One step dystopy height expression IRF8 (LNCaP-IRF8), the influence that CCK8 methods detection IRF8 is proliferated prostate gland cancer cell.As a result it shows Show compared to unloaded control group, AR feminine gender Prostatic cancer cell lines DU145 is overexpressed IRF8, and it is proliferated and (Fig. 5 B- that do not make significant difference C), then proliferation slowed down (Fig. 5 D), prompting IRF8 are proliferated prostate gland cancer cell to AR positive cells strain LNCaP dystopys height expression IRF8 Influence may be related with the expression status of prostate gland cancer cell AR.

3.4 LNCaP, which strike low IRF8 expression cells proliferation, to be accelerated

In view of the unstability to gene expression is transiently transfected, in early-stage study, to explore IRF8 in prostate cancer Function, we are struck using shRNA viral steadies subtracts in Prostatic cancer cell lines LNCap IRF8 and expresses, obtain stablize strike subtracts it is thin Born of the same parents' strain has carried out a series of inside and outside experiments.CCK8 is detected and immunofluorescence observation finds that LNCaP-shIRF8 cells increase Quickening is grown, violet staining shows that LNCaP-shIRF8 Clone formations increase, and the culture LNCaP-shIRF8 balling-up abilities that suspend are shown Enhancing is write, apoptosis caused by hydrogen peroxide is resistant to, prompts IRF8 that may participate in prostate cancer vicious transformation and to apoptosis susceptibility Regulation and control.But prostate gland cancer cell proliferation has androgen-dependent, occurrence and development and the Androgen-AR signal paths of CRPC It is abnormal closely related.Thus, whether we wonder the IRF8 adjusted by Androgen-AR signal paths to Androgen-AR Signal path generates regulation and controlWe use Western blot methods, further verify IRF8 in LNCaP-shIRF8 cell strains It expresses (Fig. 6 A).In the case where IRF8 low expression level CCK8 methods detect androgen presence or absence, IRF8 is to forefront The influence of adenocarcinoma cell proliferation.As a result show that LNCaP strikes cell Proliferation quickening (Fig. 6 B) after low IRF8, proliferation is accelerated characteristic and gone Except (Fig. 6 D, 10nM R1881) persistently exists in the case of androgen (Fig. 6 C) and low-level androgen.

Embodiment 4 strikes one-tenth knurl ability in low IRF8 enhancings LNCaP cell bodies

4.1 animal model

4.1.1 LNCaP-shIRF8 castrates Nude Mouse Model

(1) by 18 male Balb/c nude mices of 4-6 weeks or so, operation cuts off bilateral testes and epididymis, in SPF animal houses It adapts to one week.

(2) 18 nude mices are randomly divided into 2 groups, every group 9.

(3) 0.25% pancreatin digest the shNC cells and shRNA2 of exponential phase, and 1640 culture mediums containing 5%FBS are light Soft piping and druming makes into single cell suspension.PBS is washed three times, is centrifuged 5 minutes with 1000rpm.Serum-free RPMI1640 is resuspended, and 0.4% Trypan blue detects cell survival rate (survival rate>90%) it is 5 × 10 that, matrigel, which adjusts cell concentration,⁷A/mL (matrigel： RPMI 1640=1：1).Under aseptic condition, nude mice dorsal sc injection 0.2mL cell suspensions.

(4) shNC (inoculation shNC cells) is named as according to inoculating cell difference, shIRF8 (inoculation IRF8 silence efficiencies compared with High shRNA2 cells).

(5) situations such as the weight of animals, diet are observed, monitor tumour variation daily.Tumour is measured every other day with vernier caliper Long and short diameter, calculating knurl product, (knurl accumulates=1/2 × major diameter × minor axis²), and draw tumor growth curve.

(6) in tumour length to 10% weight, stop surveying knurl product, draw tumor growth curve.

(7) continue to observe mouse growth situation, record the tumor-bearing mice death time, draw overall survival curve.

4.1.2 LNCaP-shIRF8NOD-SCID mice-transplanted tumor models

(1) it by 18 male NOD-SCID mouse of 4-6 weeks or so, is adapted to one week in SPF animal houses.

(2) 18 nude mices are randomly divided into 2 groups, every group 9.

(6) in tumour length to 10% weight, stop surveying knurl product, above-mentioned nude mice is put to death, tumor mass is taken out in dissection, measures Its final volume is simultaneously weighed, and draws tumor growth curve and knurl weight block diagram.

4.2 strike low IRF8 enhancing LNCaP in Nude mouse body into knurl, reduce its existence

In early-stage study, it has been found that LNCaP stabilization strike low IRF8 cell strains LNCaP-shIRF8 in nude mouse into Efflux velocity is accelerated, and experimental endpoints knurl weight significantly increases, proliferating cell nuclear antigen (Proliferating cell in tumor tissue Nuclear antigen, PCNA) immunostaining is remarkably reinforced, show IRF8 can inhibit prostate gland cancer cell proliferation and One-tenth knurl ability in nude mouse.Above we have found that external IRF8 inhibits prostate gland cancer cell proliferative capacity and the expression status of AR It is related, then we, which think further to investigate, reduces whether androgen levels in Mice Body still can maintain to strike the cell strain of low IRF8 One-tenth knurl ability in nude mouse.4-6 week old mouse operation excision mouse bilateral testes and epididymis reduce internal androgen levels, Again in castrating mouse abdomen back side inoculation 1 × 10⁷LNCaP-shIRF8 cells and its negative control LNCaP-shNC, monitor its into Efflux velocity.The results show that under the conditions of castration, after being inoculated with 10 days, two groups of tumor mass are grown, and it is apparent to strike low IRF8 compositions efflux velocity Accelerate, inoculation strikes knurl product in low IRF8 groups and reaches 1368 ± 188.1mm after 3 weeks³, n=9, and negative control group knurl product is then For 688 ± 90.45mm³, n=9, only strike low group 1/2, two groups of tumour growths, that is, significant difference (Fig. 7 A).Its postscript Two groups of tumor-bearing mice survival states are recorded, observe the difference of two groups of tumor-bearing mice Overall survivals, IRF8 strikes low rear tumor-bearing mice Survival condition is deteriorated, and Overall survival is substantially less than negative control group (Fig. 7 B, P=0.0326).The above result shows that it strikes low One-tenth knurl ability enhancing is unrelated with internal androgen levels in prostate gland cancer cell body caused by IRF8 expression.

4.3 strike low IRF8 enhancing LNCaP in NOD-SCID Mice Bodies into knurl

Literature research shows that IRF8 is mainly expressed in immunocyte and influenced the function of immunocyte, and enhancing macrophage is thin The immune functions such as born of the same parents, DC cells.Balb/c nude mices are thymus gland deficient mice, T cell malfunction, but still have the DC thin in vivo The expression such as born of the same parents, NK cells, and non-fully immunodeficient mouse strike for verification LNCaP and subtract tumour cell caused by IRF8 into knurl Speed is accelerated non-to reduce and then influence caused by immune function reduces, therefore we are in immune system because tumour cell IRF8 is expressed More the NOD-SCID mouse abdomens back side inoculation shNC cells of defect and shIRF8 cells, tumor growth speed.As a result it sends out Existing LNCaP cells strike subtract IRF8 after, accelerate (Fig. 8 A) into efflux velocity, experimental endpoints knurl weight has significant difference (Fig. 8 B), into The characteristic of knurl ability enhancing still exists in the Mice Body of complete immune deficiency.The above result shows that IRF8 can directly inhibit The proliferation of tumour cell and internal one-tenth knurl ability.

In conclusion it is all can induce IRF8 expression substance can directly inhibit prostate tumor cells proliferation and in vivo into Knurl ability.

LNCaP-shIRF8 reduces Enz sensibility in 5 in vitro test of embodiment

5.1 LNCaP-shIRF8 reduce various concentration Enz sensibility

The miscellaneous Shandong amine of grace blocks testosterone, protona to combine, while inhibit androgen receptor consideration convey by competitive binding LBD Record, the process combined with DNA.So if AR activity changes, including protein expression and activity change etc., it can lead to grace The miscellaneous drug resistant generation of Shandong amine.Whether the present embodiment research IRF8 can influence sensibility of the prostate gland cancer cell to Enz.

Stablized first using IRF8 and knock out cell LNCaP-shIRF8 (IRF8 strikes inefficient height), give Enz various concentrations Act on 48hrs-72hrs, CCK8 detection cell viabilities.The results show that under same concentrations, IRF8 strikes low cell viability and is significantly higher than Unloaded control group LNCaP-shNC (Fig. 9 A-B).Equally, increase cell inoculation amount, 6 orifice plate inoculating cells give 5 μM and 10 μM Enz acts on 72hrs, directly observes cell survival under microscope, LNCaP-shNC is several on culture plate under 5 μM of Enz concentration Without attached cell, and LNCaP-shIRF8 groups are under (10 μM) effects of higher concentration Enz, and cytochrome oxidase isozymes are good in culture plate, Light transmittance is high, and almost without dead cell, cell density is slightly below DMSO control groups (Fig. 9 C).

5.2 IRF8 adjust 3 activity of caspase caused by Enz

The miscellaneous Shandong amine of grace can be combined with DNA, inducing cell apoptosis^[7].When showing 5 μM of Enz effect 48hrs above, cell is lived Power 80% or so, cell density reduce, only cell inhibitory effect；When acting on 72hrs, cell viability 70% or so has occurred Dead cell.Prostate gland cancer cell thus is handled using 5 μM of Enz, detection caspase 3 is active after 72hrs.Figure 10 A show, Enz Can raise the activity of caspase 3, IRF8 strike it is low after, compared to solvent control group, under Enz processing, the activity of caspase 3, which has no, to be changed Become.Equally, 48hrs are handled with 5 μM of Enz after LNCaP overexpressions IRF8,48hrs.When Figure 10 B are shown, when only IRF8 is overexpressed 3 activity of caspase is had no effect on, 3 activity of IRF8 groups caspase significantly up-regulation under Enz processing, and vector groups caspase 3 without significant changes.

Clone formation and Proliferation Ability caused by 5.3 LNCaP-shIRF8 antagonisms Enz

The present embodiment research IRF8 strikes the influence of cell Proliferation under the low long term to Enz, is internal long-term administration curative effect Test basis is provided.Low cell strain is struck with LNCaP-shNC cells and two kinds of IRF8, violet staining calculates clonal expansion and inhibits Rate.Under 10 μM of Enz processing, LNCaP-shNC group Clone formations significantly reduce, and LNCaP-shIRF8 groups Clone formation also has centainly The reduction (Figure 11 A) of degree.IPP calculates clone's number, solvent control group, and IRF8 strikes the low clone for dramatically increasing LNCaP cells It is formed；Under Enz processing, IRF8, which strikes low caused Clone formation, increases characteristic still in the presence of (Figure 11 B).Under Enz processing, LNCaP-shNC group Clone formations inhibiting rate is 48%, and the Clone formation inhibiting rate that IRF8 strikes low group of two cell strains is respectively 33.5%, 22.5%, there is significant difference (Figure 11 C) with LNCaP-shNC groups.Equally, each group cell is detected using 96 orifice plates Cell Proliferation in the case where 10 μM of Enz are handled.Consistent with result above, under Enz processing, after 3 days, LNCaP-shNC cells increase It grows and is significantly inhibited, and LNCaP-shIRF8 groups, compared to solvent control group, Enz processing has no significantly the proliferation of cell Property influence (Figure 11 D).These results are similar with the result of short-term Enz processing, illustrate to strike low IRF8, under long-term Enz processing With handled in short term in Enz under, also play resist Enz function.

LNCaP-shIRF8 reduces Enz sensibility in 6 experiment in vivo of embodiment

Operation excision bilateral testes and epididymis Balb/c nude mice abdomen back sides are seeded in using LNCaP, build LNCaP-CRPC Nude mice model.Further verification IRF8 strikes the low influence to Enz sensibility in vivo.The results show that LNCaP-shNC groups, After 10mg/kg/d Enz are acted on 25 days, Enz processing groups tumor growth curve has significant difference with solvent control group； IRF8 strike it is low after, until experimental endpoints, Enz processing groups tumor growth curve i.e. with solvent control group there was no significant difference (figure 12A).Experimental endpoints, Enz processing group knurl product final with solvent control group has significant difference in LNCaP-shNC models, Single gross tumor volume growth rate significantly reduces under Enz processing, and single under the final knurl product of LNCaP-shIRF8 groups and Enz processing Gross tumor volume growth rate does not have significant changes (Figure 12 B-C) with solvent control group.Equally, Figure 12 D show experimental endpoints Enz processing group final knurl weight similary with solvent control group also has significant difference in LNCaP-shNC models, and tumour inhibiting rate is about 41.1%, and LNCaP-shIRF8 groups are then without influence.

Embodiment 7IFN α enhance Enz curative effects

7.1 animal model

7.1.1 LNCaP-CRPC Balb/c nude mice models detection Enz sensibility

(1) by 24 male Balb/c nude mices of 4-6 weeks or so, operation cuts off bilateral testes and epididymis, in SPF animal houses It adapts to one week.

(2) 24 nude mices are randomly divided into 2 groups, every group 12.

(3) 0.25% pancreatin digest the shNC cells and shRNA2 of exponential phase, with 1640 culture mediums containing 5%FBS Soft piping and druming makes into single cell suspension.PBS is washed three times, is centrifuged 5 minutes with 1000rpm.Serum-free RPMI1640 is resuspended, 0.4% trypan blue detects cell survival rate (survival rate>90%) it is 5 × 10 that, matrigel, which adjusts cell concentration,⁷A/mL (matrigel：RPMI 1640=1：1).Under aseptic condition, nude mice dorsal sc injection 0.2mL cell suspensions.According to inoculation Cell difference is named as shNC (inoculation shNC cells), shIRF8 (the higher shRNA2 cells of inoculation IRF8 silence efficiencies)

(4) once tumour is grown, LNCaP-shNC and LNCaP-shIRF8 is randomly divided into two groups respectively, Enz groups are every The miscellaneous Shandong amine 10mg/kg (n=6) of its gavage grace, the similary volume CMCNa of the daily gavage of Vehicle groups is as solvent control group (n= 6)。

(6) in tumour length to 10% weight, stopping surveying knurl product, above-mentioned tumor-bearing mice is put to death, tumor mass is taken out in dissection, It measures its final volume and weighs, draw tumor growth curve and knurl weight block diagram, calculate inhibition rate of tumor growth=(solvent pair According to the final final knurl weight of knurl weight-administration group of group) the final knurl weight * 100 of/solvent control group.

7.1.2 adjusting of the LNCaP-CRPC NOD-SCID mouse models detection IFN α to Enz sensibility

(1) by 76 male NOD-SCID mouse of 4-6 weeks or so, operation cuts off bilateral testes and epididymis, in SPF animals Room adapts to one week.

(2) 76 NOD-SCID mouse are randomly divided into 2 groups.

(3) 0.25% pancreatin digest the shNC cells and shRNA2 of exponential phase, with 1640 culture mediums containing 5%FBS Soft piping and druming makes into single cell suspension.PBS is washed three times, is centrifuged 5 minutes with 1000rpm.Serum-free RPMI1640 is resuspended, 0.4% trypan blue detects cell survival rate (survival rate>90%) it is 5 × 10 that, matrigel, which adjusts cell concentration,⁷A/mL (matrigel：RPMI-1640=1：1).Under aseptic condition, nude mice dorsal sc injection 0.2mL cell suspensions.According to inoculation Cell difference is named as shNC (inoculation shNC cells), shIRF8 (the higher shRNA2 cells of inoculation IRF8 silence efficiencies)

(4) LNCaP-shNC or LNCaP-shIRF8 are randomly divided into 4 groups：IFN α 2a injections are subcutaneously injected in IFN α group daily Liquid 1.5 × 10⁷IU/kg, daily gavage CMCNa (n=12or 8)；Enz groups are the miscellaneous Shandong amine 10mg/kg of daily gavage grace, subcutaneously Injecting normal saline (n=12or 8)；IFN α+Enz organizes the daily miscellaneous Shandong amine 10mg/kg of gavage grace, and IFN α 2a injections are subcutaneously injected Liquid 1.5 × 10⁷IU/kg (n=12or 8)；Physiological saline (n=8) is subcutaneously injected in the daily gavage CMCNa of Vehicle groups.

(5) situations such as the weight of animals, diet are observed, tumour is monitored daily and has grown situation and variation.With vernier caliper every other day The long and short diameter of tumour is measured, calculating knurl product, (knurl accumulates=1/2 × major diameter × minor axis²), and draw tumor growth curve.

(6) in tumour length to 10% weight, stopping surveying knurl product, above-mentioned tumor-bearing mice is put to death, tumor mass is taken out in dissection, It measures its final volume and weighs, draw tumor growth curve and knurl weight block diagram, calculate inhibition rate of tumor growth=(solvent pair According to organizing the final final knurl weight of knurl weight-administration group) the final knurl weight * 100 of/solvent control group (wherein LNCaP-shNC Vehicle and Enz groups respectively have a gavage operation lethal, withdraw from experimental data statistics).7.2 clinical research

We have recruited 3 high-risk patients with prostate cancer, enter a group condition：First visit PSA>150ng/Ml, Gleason >=9 (4+5 or 5+4), multiple Bone tumour or transfer may.Knowing and meeting ethics, carrying out respectively male sharp to greatest extent Element blocks endocrine therapy (maximal androgen blokade, MAB) or IFN α to combine in endocrine therapy to greatest extent Secretion treatment (IFN α+MAB), concrete scheme is：

(1) endocrine therapy endocrine therapy (MAB) to greatest extent：Goserelin acetate sustained-release implant (Nuo Lei get) 3.6mg (Astrazeneca AB), every 1 time on the 28th, Bicalutamide (Kang Shi get) 50mg, one time a day.

(2) endocrine therapy endocrine therapy+IFN α 2a parenteral solution (trade names to greatest extent：Recomvinated Interferon α-2a)：Endocrine is treated Method is same as above, 3,000,000 IU of IFN α 2a, monthly 10 times.

Continued treatment 6 months or more, it is horizontal monthly to detect PSA, PSA>0.2ng/mL shows biochemical recurrence.During biochemistry recurrence Group progress chemotherapy or an other treatment are moved back according to patient requests.

The up-regulation IRF8 expression of 7.3 IFN αs, lowers AR expression

Various concentration IFN α handles AR positive prostate cancer cells LNCaP and 22RV1, and WB detects IRF8 and AR after rs for 24 hours Protein level, the correlation of the lower IRF8 and AR protein expressions of verification IFN α processing.Figure 13 A show, IFN α can be on dose dependent The expression of IRF8 in prostate gland cancer cell LNCaP and 22RV1 is adjusted, while lowers the protein level of AR.Figure 13 B show that AR is positive male The insensitive Prostatic cancer cell lines 22RV1 of hormone can promote AR to IRF8 promoter region ISRE-like sites under IFN α processing Combination, and then promote IRF8 expression.Various concentration IFN α is awarded simultaneously, AR half-life period significantly reduces (Figure 13 C), and can quilt Protease inhibitors MG132 blocks (Figure 13 D), and enhances the ubiquitination of AR.These results illustrate IFN α in prostate gland cancer cell Adjustable IRF8, promotes the ubiquitination of AR to degrade.IFN α delays prostate gland cancer cell IRF8 expression drops under the conditions of being used as castration Low intervening measure.

7.4 IFN αs enhance ADT curative effects

In in vitro test, LNCaP and 22RV1 are respectively with (0,5,10,20,40,80,100) μm ENZ joints 1000IU/ml After IFN α or various concentration IFN α (0,100,500,1000,2000,5000,10000IU/ml) handle 96h and 48h respectively, profit Cell viability is detected with CCK8 methods (see 3.2CCK8 methods).The results show that IFN α can significantly increase prostate gland cancer cell LNCaP and 22RV1 is applied alone IFN α to have no significant cytotoxicity (Figure 14 B) sensibility (Figure 14 A) of Enz.

To exclude influence of the IFN α to immune system, exempted from completely using LNCaP-shNC and LNCaP-shIRF8 cell strains LNCaP-CRPC models are built in epidemic disease deficient mice NOD-SCID (method is shown in 7.1.2).Enz (10mg/kg), IFN α are awarded respectively (1.5×10⁷IU/kg) or Enz (10mg/kg) combines IFN α (1.5 × 10⁷IU/kg), tumor recurrence and growth curve are detected.Knot Fruit shows that Enz joint IFN α group tumour growths significantly reduce (Figure 14 C), and experimental endpoints Enz combines IFN α group knurl weight with being applied alone Enz has significant difference, tumor control rate 86.3, higher than being applied alone the 62.1% of Enz groups；And IFN α joint Enz group tumours Recurrence rate is 58.3%, far below 90.9% (Figure 14 D) of Enz groups is applied alone, and strikes low each treatment group tumors recurrences of IRF8 and reaches 100%, tumour growth and knurl weight and indifference (Figure 14 C-F), synergistic effect disappear.These results indicate that during castration, connection IFN α is closed, induction prostate cancer expression IRF8 can enhance the sensibility of Enz.

According to the above results, we have recruited 3 high-risk patients with prostate cancer, in informed ethics, respectively into Endocrine therapy endocrine therapy and IFN α combine endocrine therapy endocrine therapy to greatest extent to row to greatest extent.Concrete scheme See 7.2 methods of this chapter.As a result (Figure 15) is shown：For 2 patients to greatest extent after endocrine therapy endocrine therapy, PSA is horizontal Persistently higher than 0.2ng/mL, curative effect is poor, and PSA levels significantly rebound after 6 months；2 patients are after 6 months using other treatments Method exits experiment.IFN α joint endocrinotherapy group recruits two patients altogether, and an example patient is during the treatment up to 11 months Adhere to combining endocrinotherapy using IFN α, PSA persistent levels are maintained at biochemical recurrence line (0.2ng/mL) hereinafter, effect is shown It writes.After 3 months, PSA is gradually reduced another patient's continued treatment.These results indicate that IFN α can combine endocrinotherapy, show Write enhancing prostate cancer clinical treatment curative effect.

8 IRF8 of embodiment expresses reduction in clinical liver cancer patient；Increase IRF8 expression and can inhibit it into knurl characteristic；IFN α induction IRF8 expression, inhibits AR expression.

TCGA database analysis IRF8 is expressed in liver cancer patient is substantially less than its expression (figure in normal liver tissue 16A).IRF8 expression is substantially less than its expression (figure in cancer beside organism in Showed by immune group result clinic liver cancer patient 16B).Epidemiological study shows male's liver cancer occurred frequently, may be related with the abnormal raisings of androgen receptor AR.Diethylnitrosamine Rat liver cancer can be caused, WB testing results show that IRF8 expression reduces in the rat liver cancer model cancerous tissue that DEN is induced, AR raisings (Figure 16 C).TCGA and GEO database analysis shows that IRF8 expression is high in liver cancer patient, and liver cancer patient Overall survival is notable Low liver cancer patient Overall survival (Figure 16 D-E) is expressed higher than IRF8.Thus we guess, IRF8 is in liver cancer patient for raising In expression, if it can be reduced into knurl characteristic

After murine hepatocarcinoma cell Hep1-6 is instantaneously overexpressed IRF8, cell Proliferation, Clone formation is significantly suppressed (figure 17A-C).Simultaneously using slow-virus transfection, after Hep1-6 stablizes overexpression IRF8, cell Proliferation, cut migrates and balling-up energy Power is suppressed, and further confirms raising IRF8 expression, can inhibit liver cancer cells into knurl characteristic (Figure 17 D-F).And liver cancer After cell Bel7404 and HepG2 are overexpressed IRF8, WB detections show that AR expression gradient dependences reduce (Figure 18 A-B).Mouse source liver After cancer cell Hep1-6 stablizes overexpression IRF8 (band His labels), AR expression significantly reduces (Figure 18 C), further confirms liver cancer IRF8 can reduce AR expression in cell.In clinical treatment, IFN α is mainly used for the infection of liver cancer patient anti-hepatitis virus.Ours grinds Study carefully the results show that various concentration IFN α processing liver cancer cells Bel7404 and HepG2 for 24 hours after, the raising of IRF8 gradients dependence, and AR gradients dependence reduces (Figure 18 D-E).Thus, in liver cancer patient, IFN α can raise IRF8 expression by AR/IRF8 axis, Liver cancer cells grade malignancy is reduced, while reduces AR expression, significantly increases liver cancer patient prognosis.

Initialism

Claims

1. induce the substance of IRF8 expression answering in the drug for preparing auxiliary treatment androgen receptor unconventionality expression associated diseases With.

2. application according to claim 1, it is characterised in that the substance of induction IRF8 expression is preparing auxiliary androgen receptor Body antagonist for treating androgen receptor unconventionality expression associated diseases or the drug resistant androgen receptor of androgen receptor antagonists are abnormal Express the application in the drug of associated diseases.

3. application according to claim 1, it is characterised in that the substance of the induction IRF8 expression is can improve IRF8 expression quantity or IRF8 active substances in vivo in vivo.

4. application according to claim 2, it is characterised in that the substance of the induction IRF8 expression is selected from IFN α, HDACI。

5. application according to claim 2, it is characterised in that the androgen receptor antagonists are selected from the miscellaneous Shandong amine of grace, ratio Card Shandong amine.

6. application according to any one of claims 1-5, it is characterised in that the androgen receptor unconventionality expression institute Disease is caused to be selected from prostate cancer, liver cancer；It is preferred that castration-resistant prostate cancer.

7. a kind of pharmaceutical composition for being used to treat androgen receptor unconventionality expression associated diseases, it is characterised in that effective by treating The androgen receptor antagonists of amount and the material composition of induction IRF8 expression.

8. composition according to claim 7, it is characterised in that the androgen receptor antagonists be selected from the miscellaneous Shandong amine of grace, Bicalutamide；The substance of described induction IRF8 expression be that can improve IRF8 expression quantity or IRF8 activity in vivo in vivo Substance.

9. composition according to claim 8, it is characterised in that the substance of the induction IRF8 expression is selected from IFN α, HDACI。

10. composition according to claim 8, it is characterised in that before the androgen unconventionality expression associated diseases are selected from Row gland cancer, liver cancer；It is preferred that castration-resistant prostate cancer.

11. induce application of the substance of IRF8 expression in the drug for preparing treatment androgen receptor unconventionality expression associated diseases.

12. application according to claim 11, it is characterised in that the substance of the induction IRF8 expression is can improve IRF8 expression quantity or IRF8 active substances in vivo in vivo.

13. the application according to claim 11 or 12, it is characterised in that the substance of the induction IRF8 expression is selected from IFN α, HDACI.

14. application according to claim 11, it is characterised in that the androgen receptor unconventionality expression associated diseases choosing From prostate cancer, liver cancer；It is preferred that castration-resistant prostate cancer.