CN108159419B - Application of substance for inducing IRF8 expression in preparation of medicine for treating liver cancer - Google Patents

Abstract

The invention discloses an application of a substance for inducing IRF8 expression in preparing a medicament for treating diseases caused by abnormal expression of androgen receptor. The invention finds that IRF8 can directly regulate the proliferation of prostate cancer and liver cancer cells, the expression of IRF8 can regulate the sensitivity of prostate cancer cells to AR antagonist Enz, and the knocking-down of IRF8 can cause the apoptosis tolerance of the prostate cancer cells to the Enz. Substances that induce the expression of IRF8, such as IFN α, up-regulate IRF8 expression by AR and are thus involved in the regulation of prostate cancer cell sensitivity to Enz. In clinical trials, IFN α in combination with maximal androgen blockade endocrine therapy can enhance the therapeutic efficacy of prostate cancer patients. Therefore, the substance capable of inducing the expression of IRF8 can be combined with the androgen receptor antagonist to enhance the sensitivity of a patient with low clinical IRF8 expression to the androgen receptor antagonist, improve the curative effect of the androgen receptor antagonist and delay the occurrence of drug resistance of the patient.

Description

Application of substance for inducing IRF8 expression in preparation of medicine for treating liver cancer

Technical Field

The invention belongs to the field of medicines, and relates to application of a substance for inducing IRF8 expression in preparation of a medicine for treating diseases caused by abnormal expression of androgen receptor.

Background

In European and American countries, the prostate cancer is a common male malignant tumor with the second-ranked morbidity and mortality, and the incidence of the prostate cancer in China is lower than that in the Western countries, but the prostate cancer is in a trend of remarkable increase along with the change of life style and the aging of population. It is expected that the incidence of prostate cancer will be as high as 40/10 ten thousand and 35 ten thousand new cases in the year by 2020, which is equivalent to the incidence of liver cancer.

Primary Prostate Cancer is hormone-dependent, where endocrine therapy is sensitive, symptoms of Prostate Cancer improve, disease progression is delayed, but under Castration-selective pressure, it becomes Castration-Resistant Prostate Cancer (CRPC) after typically 12-18 months. In recent years, drugs for treating CRPC have been developed, and for example, an Androgen antagonist abiraterone (abiraterone), an Androgen Receptor (AR) antagonist Bicalutamide (Bic), Enzalutamide (Enzalutamide, enzz) and the like are effective for CRPC, but all of them have a problem of easy drug resistance. The occurrence and drug resistance of CRPC are the main causes of death of prostate cancer patients, and are the main difficulties and research directions of prostate cancer treatment at present.

The androgen receptor belongs to a superfamily of nuclear receptors, functions as a transcription factor in the presence of a corresponding ligand, directs the correct distribution of prostate epithelial cells, is an essential factor in normal prostate tissue development, but is overexpressed to cause tissue proliferation and is negatively associated with the staged differentiation of prostate cancer. Animal models show that the overexpression of AR in prostate epithelial cells can cause proliferation and malignant transformation, and in prostate cancer patients, the high expression of AR is closely related to the malignant progression and the reduction of relapse-free survival rate of clinical cases. AR is widely involved in the growth, differentiation and functioning of prostate cells, as well as in the initiation and progression of prostate cancer, and is the most important signal required for prostate cancer growth. In addition, abnormal increase of AR activity is also considered as a key to development of CRPC, and CRPC is often accompanied by abnormal AR signaling pathway, and abnormal expression of AR and AR synergistic molecules, disorder of AR signaling pathway and the like are involved in the generation and development of CRPC. In addition, abnormal AR activity is also an important cause of the drug resistance of the CRPC therapeutic drug Enz targeting AR.

The treatment of prostate cancer mainly involves surgery, castration, radiation therapy and chemotherapy, depending on the age and disease course of the patient. Early stage prostate cancer is androgen dependent, and after endocrine treatment, prostate cancer symptoms improve and disease progression is delayed, but the prostate cancer generally becomes CRPC after 12-18 months. In recent years, drugs for treating CRPC are developed successively, and androgen antagonists Abiraterone, androgen receptor antagonists bicalutamide, Enzalutamide and the like are effective on CRPC, but all the drugs have the problem of easy drug resistance. The occurrence and drug resistance of CRPC are the main causes of death of prostate cancer patients, and are the main difficulties and research directions of prostate cancer treatment at present.

Enzalutamide (also called MDV3100) is a new generation of antiandrogen drug with high oral bioavailability and directly acting on androgen receptor, and mainly acts by inhibiting the combination of testosterone and androgen receptor, inhibiting the nuclear translocation of androgen receptor, inhibiting the combination of androgen receptor and DNA without agonist effect, and the like, thereby improving the overall survival and disease-free survival of CRPC patients (including patients who fail docetaxel chemotherapy). Compared with the prior antiandrogen (such as bicalutamide), the affinity of the antiandrogen with an androgen receptor can be remarkably enhanced. The enzalutamide competitively binds with a ligand binding region of AR, blocks the combination of testosterone, dihydrotestosterone and AR, inhibits the nuclear translocation of androgen receptor, inhibits the combination process of AR and target gene DNA, inhibits the proliferation of prostate cells, promotes the apoptosis of prostate cells, and further exerts the antitumor activity. Therefore, if the androgen receptor changes, including the change of the activity of AR protein, point mutation, shearing isomer and the like, the generation of enzalutamide resistance can be caused. Overexpression of AR in androgen-independent transplants can desensitize prostate cancer cells to androgen and further facilitate the transformation of prostate transplants from an androgen-dependent mode to an androgen-independent mode, underscoring the potential mechanisms of AR expansion and overexpression for developing resistance in androgen-deprivation therapy as well as second generation therapy.

IRF8, also known as Interferon consensus sequence-binding protein (ICSBP), belongs to a member of the Interferon Regulatory Factor (IRF) family. IRF is a transcription factor discovered when researching type I IFN system, the expression of the IRF is regulated by IFN, and IFN signal path is feedback regulated, and the IRF plays an important role in IFN transcription regulation, innate immunity and adaptive immunity involved by IFN, cytokine signal transduction and cell proliferation regulation. A total of 9 members of the IRF family (IRF1-9), and the functions of multiple members of the IRF family (IRF1, IRF3, IRF4, IRF5, IRF7, IRF8) in pattern recognition receptor-triggered innate immune responses have been demonstrated. Significant progress was also made in the important role of IRF family members IRF1, IRF2, IRF4, IRF8 in immune cell development. In recent years, more and more researches are focused on the functions of IRF family members IRF1, IRF2, IRF3, IRF5, IRF6 and IRF8 in the growth, survival and tumorigenesis of blood and epithelial cells, wherein the functions of IRF1, IRF2, IRF3, IRF5 and IRF6 in blood and solid tumors are proved by a large number of researchers. The lack of IRF8 expression in immunocytes can lead to the development of hematological diseases such as Acute Myelogenous Leukemia (AML) and Chronic Myelogenous Leukemia (CML), which indicates that IRF8 may be used as a leukemia inhibitory gene in hematological tumors, induce the transcription of Tumor inhibitory gene (TSG) and apoptosis gene, and inhibit the expression of oncogene and anti-apoptosis gene Bcl-xl, Bcl-2, etc., thereby exerting its Tumor inhibitory function. However, only a few papers have reported that IRF8 functions in epithelial cells, including the fact that IRF8 gene promoter hypermethylation results in low/no expression of its proteins in most tumor tissues (human protein Atlas, http:// www.proteinatlas.org). However, the expression and function of IRF8 in prostate cancer are not reported.

Disclosure of Invention

The invention aims to solve the problem of androgen receptor resistance, and provides application of a substance for inducing IRF8 expression in preparation of a medicament for assisting in treating diseases caused by abnormal androgen receptor expression.

Another object of the present invention is to provide a pharmaceutical composition for treating diseases caused by abnormal expression of androgen receptor.

The purpose of the invention can be realized by the following technical scheme:

use of a substance that induces the expression of IRF8 in the manufacture of a medicament for the adjunctive treatment of a condition caused by aberrant expression of the androgen receptor.

Preferably the use of a substance which induces the expression of IRF8 in the manufacture of a medicament for the adjuvant treatment of a disorder resulting from aberrant expression of an androgen receptor antagonist or a disorder resulting from aberrant expression of an androgen receptor resistant to an androgen receptor antagonist.

The substance inducing the expression of IRF8 is preferably a substance capable of increasing the in vivo expression level of IRF8 or the in vivo activity of IRF 8. The substance for inducing the expression of IRF8 is further preferably selected from IFN alpha, histone deacetylase inhibitor (HDACI).

The androgen receptor antagonist is preferably enzalutamide or bicalutamide.

The diseases caused by abnormal androgen expression are preferably prostate cancer and liver cancer; castration-resistant prostate cancer is further preferred.

A pharmaceutical composition for treating diseases caused by androgen abnormal expression is composed of androgen receptor antagonist in therapeutically effective amount and the substance for inducing IRF8 expression.

The androgen receptor antagonist is selected from enzalutamide and bicalutamide; the substance for inducing the expression of IRF8 is a substance capable of improving the in vivo expression quantity of IRF8 or the in vivo activity of IRF 8.

The substance for inducing the expression of IRF8 is selected from IFN alpha.

The diseases caused by abnormal androgen expression are selected from prostate cancer and liver cancer; castration-resistant prostate cancer is preferred.

Has the advantages that:

the invention shows that IRF8 is expressed in cytoplasm and nucleus of prostate cancer epithelial cells through prostate cancer tissue chip and prostate cancer tissue immunohistochemistry and Western blot research results, and the expression of IRF8 is positively correlated with prostate cancer pathological grade. IRF8 has no report on the fact that its expression is significantly up-regulated in clinical prostate tissue, and it acts as a tumor suppressor in solid tumors such as hematological tumors and colon cancer. This study reports for the first time the expression of IRF8 in prostate cancer, and its expression is regulated by the androgen AR signaling pathway.

The Western blot result shows that the IRF8 protein is expressed in both AR positive and AR negative prostate cancer cells. Wherein the content of the cells in the AR positive prostate cancer cell strain is higher than that of the AR negative cell strain; AR protein levels were significantly increased after IRF8 knockdown, and overexpression of IRF8 reduced AR protein expression. In vitro experiments show that the effect of prostate cancer cells over expressing IRF8 on prostate cancer cell proliferation is related to AR expression status. IRF8 inhibits the proliferation of AR positive prostate cancer cells, but has no effect on the proliferation of AR negative prostate cancer cells. Upon IRF8 knockdown, LNCaP cells proliferate faster, with the proliferation-enhancing property persisting at both low and high androgen levels. In vivo experiments show that after IRF8 is knocked down, the in vivo tumor formation speed of LNCaP cells is remarkably accelerated, and the overall survival time of tumor-bearing mice is remarkably reduced. And the tumor formation speed increasing characteristic caused by IRF8 knock-down still exists in NOD-SCID mice with complete loss of immune systems, which indicates that IRF8 can directly regulate the proliferation of prostate cancer cells.

The invention also finds that IRF8 expression can regulate the sensitivity of prostate cancer cells to AR antagonist Enz, and IRF8 knock-down can lead to prostate cancer cells resistant to apoptosis caused by Enz. IFN α up-regulates IRF8 expression by AR and is therefore involved in the regulation of sensitivity of prostate cancer cells to Enz. In clinical trials, IFN α in combination with maximal androgen blockade endocrine therapy can enhance the therapeutic efficacy of prostate cancer patients. Therefore, the substance capable of inducing the expression of IRF8 can be combined with the androgen receptor antagonist to enhance the sensitivity of a patient with low clinical IRF8 expression to the androgen receptor antagonist, improve the curative effect of the androgen receptor antagonist and delay the occurrence of drug resistance of the patient.

Drawings

FIG. 1IRF8 expression in prostate cancer tissue microarray chips.

Figure 2 bioinformatic analysis found that IRF8 was down-regulated in prostate cancer progression.

(A) Database analysis IRF8 in normal prostate and prostate cancer Tissues (TCGA); proliferative prostate tissue, local and metastatic prostate cancer tissue (GSE 3325); metastatic androgen sensitive and resistant prostate cancer tissue (GSE6099) and castration induced tumor nadir growth and castration resistant recurrent prostate cancer tissue. (B) Expression of other members of the IRF family in the TCGA database in the normal prostate and prostate cancer databases. (C) Expression of IRF family members IRF2, IRF4, IRF6, which have a high homology to IRF8, in proliferative prostate tissue, local and metastatic prostate cancer tissue (GSE 3325).

FIG. 3 upregulation of IRF8 in the PCa cell line was dependent on the DHT-AR signaling pathway.

(A) IRF8 expression was detected by immunofluorescence 24 hours after DHT treatment of LNCaP cells. (B) Dose-dependent effects of DHT on IRF8 mRNA levels were detected by real-time PCR 24 hours after DHT treatment of LNCaP cells. P <0.01, P < 0.001. (C) DHT dose-dependent effects on IRF8 protein levels were measured by WB 24 hours after DHT treatment of LNCaP cells. (D) Dose-dependent effects of DHT on IRF8 mRNA levels were examined by real-time PCR 24 hours after DHT treatment of 22RV1 cells. (E-G) time-dependent effects of IGF-1(10nM, E), EGF (10nM, F) and IL-6(10ng/mL, G) on IRF8 protein levels in LNCaP.

Figure 4ADT down-regulates the expression of IRF8 in vivo and in vitro.

Graph A-B detection of dose dependence of DHT on IRF8 mRNA level (A) and protein level (B) by real-time PCR and WB, respectively. P < 0.001. C-panel, time-dependent effect of IRF8 protein levels after 24-72 hours of treatment with C-FBS (5%) by WB, Enz (10. mu.M and Bic (10. mu.M). D-E panel, LNCaP mock castration (1 and 2 weeks) in LNCaP xenografts treated with castration and castration in combination with Enz, and prolonged culture in vitro in media containing 5% C-FBS, WB to detect IRF8 expression F-F panel, LNCaP cells seeded ventral side of surgically castrated Balb/C nude mice, and IRF8 expression in tumors 28 days after treatment with Enz (10mg/kg) and solvent control (n-6).

The expression and function of IRF8 in fig. 5 is related to the state of the AR.

(A) Western blot analysis protein levels of IRF8 in prostate cancer cell lines. (B) The absorbance at 450nm of DU145-IRF8 transiently transfected in pcDNA3.1(+) (DU145-vector) or as pcDNA3.1(+)/IRF 8(DU145-IRF 8). Values represent mean ± SEM, n ═ 6 samples per sample. (C) Absorbance at 450nm of PC 393.1 (+) transiently transfected (PC3-vector) or as PC DNA3.1(+)/IRF8 transiently transfected PC3(PC3-IRF 8). Values represent mean ± SEM, n ═ 6 samples per sample. (D) Absorbance at 450nm of LNCaP transiently transfected (LNCaP-vector) at pcDNA3.1(+) or as pcDNA3.1(+)/IRF 8(LNCaP-IRF 8). Values represent mean ± SEM, n ═ 6 samples per sample.

Figure 6IRF8 inhibited the proliferation of AR positive LNCaP.

(A) Western blot analysis (normalized to. beta. -actin) protein levels of IRF8 in LNCaP-shIRF 8. (B-D) absorbance at 450nM of LNCaP-shIRF8(shRNA1, shRNA2) cultured in FBS (B), C-FBS (C) or a medium containing 10nM R1881(D) for 24 hours to 72 hours.

FIG. 7 knockdown of IRF8 in LNCaP promotes tumorigenesis in castrated Balb/c nude mice.

(A) Tumor growth of LNCaP-shIRF8 in castrated Balb/c nude mice (n ═ 9). (B) Overall survival of LNCaP-shIRF8 in castrated Balb/c nude mice (n ═ 9). Values represent mean ± SEM; p <0.05, P < 0.01.

Figure 8 knock-down of IRF8 in LNCaP promoted its tumorigenesis in NOD-SCID mouse model.

(A) Tumor growth of LNCaP-shrrf 8 in NOD-SCID mice (n ═ 6). (B) NOD-SCID mouse xenograft tumor model final tumor weight (n ═ 6). (C) Photograph of tumor at experimental end point. Values represent mean ± SEM; p <0.05, P < 0.01. FIG. 9 shows that LNCaP-shIRF8 cells have reduced sensitivity to Enz.

(A) Cell viability of LNCaP-shNC and LNCaP-shIRF8 treated with different doses of Enz for 48 hours in 96 well culture plates. Cell viability was normalized to DMSO control. (B) Cell viability was measured after treating LNCaP-shNC and LNCaP-shIRF8 cells with different doses of Enz in 96 well culture plates for 72 h. DMSO was used as control. (C) Plate clones were formed after 5. mu.M and 10. mu.M Enz treatment of LNCaP-shNC and LNCaP-shIRF8 cells 7 d. The magnification was 10 times, and the scale bar was 200 μ M.

FIG. 10 Enz-induced caspase 3 activity modulated by IRF8 expression

(A) IRF8 knock-out in LNCaP reduced Enz-induced caspase 3 activity. (B) Ectopic overexpression of IRF8 enhanced Enz-induced caspase 3 activity. P <0.05, ns: not significant, n ═ 6.

FIG. 11 shows that the inhibition rate of cell proliferation and clone formation induced by Enz is reduced in LNCaP-shIRF 8.

(A) Representative clonogenic assays of LNCaP-shNC and LNCaP-shRNA1 and LNCaP-shRNA2 treated with 10. mu.M Enz for 10 days. (B) The number of clones treated in each group of (A). P <0.01 compared to LNCaP-shNC (treated with DMSO); #, P <0.01, as compared to LNCaP-shNC (processed with Enz). (C) (B) clone count clone formation inhibition rate,. P <0.05 compared to LNCaP-shNC. (D) Absorbance at 450nm of LNCaP-shrnc and LNCaP-shrrf 8 at 10 μ M Enz for 4 days,. P < 0.05.

FIG. 12 tumor regression after Enz treatment in LNCaP-shNC and LNCaP-shIRF8 xenograft models.

(A) Tumor growth in castrated male mice bearing LNCaP-shrnc and LNCaP-shrrf 8 xenografts (n 6 per treatment group). By daily gavage solvent control, Enz: 10mg/kg/d, for 28 days. (B) Final tumor volume of LNCaP xenograft tumors. (C) Relative percent change in tumor volume for each tumor after 28 days. (D) Final tumor weight. (E) Photographs of LNCaP xenograft tumors after sacrifice of mice. Statistical analysis was performed using Student's t-test with p <0.01 and p <0.001 compared to controls.

Figure 13IFN alpha 2a through IRF8/AR axis regulation of prostate cancer cells in AR expression.

(A) Effect of IFN α 2a on protein levels of IRF8 and AR in LNCaP and 22RV1 cells. Cell lysates were assayed for AR, IRF8 and β -actin by immunoblotting for 124 hours with LNCaP or 22RV treated with different concentrations of IFN α 2 a. (B)22RV1 was treated with IFN alpha 2a 2000IU/ml at different concentrations for 24 hours, and the recognition effect of AR on ISRE-like sites in IRF8 promoter region was examined by DNA pull down experiment. (C)22RV1 was treated with IFN α 2a 2000IU/ml for 48 hours at different concentrations, CHX was treated for 0-6 hours as a protein synthesis inhibitor, and the half-life of AR protein was measured by WB. (D)22RV1 was treated with IFN α 2a 2000IU/ml at various concentrations for 48 hours, protease inhibitor MG132 was treated for 5 hours, and WB was used to detect AR expression. (E)22RV1 was treated with IFN alpha 2a 2000IU/ml for 48 hours at different concentrations, protease inhibitor MG132 was treated for 5 hours, and co-immunoprecipitation was used to detect ubiquitination of AR.

FIG. 14 tumor regression after IFN α in combination with Enz treatment in LNCaP-shNC and LNCaP-shIRF8 xenograft models.

(A) In LNCaP and 22RV1 cells, IFN alpha (1000IU/ml) was treated for 96h and 48h in combination with different doses of Enz, and a 50% lethal dose IC50 was calculated based on cell viability. (B) Different doses of IFN α did not have a significant effect on the proliferation of LNCaP and 22RV1 cells. (C) LNCaP-shNC and LNCaP-shIRF8 xenograft tumor models, solvent control, Enz (10mg/kg), IFN α (1.5 × 107 IU, n ═ 6/kg per group) or Enz (10mg/kg) + IFN α (1.5x 10) were administered by daily gavage⁷IU/kg) for 30 days. (D) Final tumor weight and tumor suppression. (E) Experimental end-point percentage increase in individual tumors relative to tumor volume. (F) Photographs of xenograft tumor tissues and percentage of recurrent tumors after sacrifice of mice. Statistical analysis was performed using Student's t-test, p compared to control<0.05，**p<0.01，***p<0.001。

Figure 15 PSA levels in prostate cancer patients treated with IFN α in combination with Enz.

MAB group: the treatment is carried out by combining 3.6mg/28 days of goserelin acetate and 50 mg/day of bicalutamide. MAB in combination with IFN α group: MAB + IFN alpha 3X 10⁶IU/3 d. Follow-up measures PSA levels.

Figure 16IRF8 was reduced in expression in liver cancer (HCC) patients and positively correlated with clinical overall survival.

(A) TCGA database analysis IRF8 is expressed in liver cancer patients. (B) IRF8 expression in cancer side and nest of clinical liver cancer patients. (C) WB detected the expression of IRF8 and AR in DEN-induced mouse liver cancer model cancer tissues. (D) The TCGA database analyzes the expression level of IRF8 and the overall survival of the liver cancer patients. (E) The GEO database GSE10141 analyzes the level of IRF8 expression and the overall survival of the liver cancer patients.

FIG. 17 shows that IRF8 is increased to be expressed in hepatoma cell Hep1-6, and the tumor growth characteristic is inhibited.

(A-B) after Hep1-6 transiently over-expresses IRF8, cell proliferation is inhibited. (C) After Hep1-6 transiently over-expresses IRF8, its clonogenic activity is inhibited. (D) After Hep1-6 stably overexpresses IRF8, cell proliferation is inhibited. After (E-F) Hep1-6 stably overexpresses IRF8, the scratch migration capability of the (E-F) Hep1-6 is inhibited. (G-I) Hep1-6 stably over-expresses IRF8, and then inhibits the balling capacity of the IRF 8.

FIG. 18 IFN alpha in hepatoma cells could inhibit AR expression by IRF 8.

(A-B) after the liver cancer cells Bel7404 and HepG2 overexpress IRF8, WB detects AR expression. (C) After Hep1-6 stably overexpressed IRF8 (His-tagged), WB detected AR expression. (D) After treating hepatoma cell Bel 740424 h with different concentrations of IFN alpha, WB detects IRF8 and AR expression. (E) After the liver cancer cells HepG 224 h are treated by IFN alpha with different concentrations, WB detects IRF8 and AR expression.

Detailed Description

1.1 Experimental cells and animals

Balb/c nude mice and NOD-SCID mice are purchased from Shanghai Ling Biotechnology Limited and used for constructing an LNCaP nude mouse transplantation tumor model. The mice were kept in SPF environment with alternating light and dark time of 12/12h, and were fed with water. All animal experiments were performed as required by the institutional animal care and ethics committee of the university of chinese pharmacy and were approved by the animal care and use committee of the university of chinese pharmacy.

DMEM culture solution containing 10% FBS for human kidney epithelial cells HEK293T (293T), 1640 culture solution containing 10% FBS for prostate cancer cell line LNCaP, and 5% CO₂ 37℃Subculture was carried out at 1: 2or 1:3 under saturated humidity conditions.

Human prostate hyperplasia cell line BPH1, prostate cancer cell lines LNCaP and IRF8 stable knock cell lines, PC3 with 10% FBS RPMI-1640 culture, HEK293T, DU145 with 10% FBS DMEM culture; at 5% CO₂Subculturing at 37 deg.C under saturated humidity condition, and subculturing at 1: 2or 1: 3.

1.2 drugs and reagents

Dihydrotestosterone (DHT, trade name androsaponol, MB5035), testosterone (MG1636-5), Estrogen (Estrogen, E2, MB1098-S), melem; enzalutamide (HY-70002), Medchem Express, USA corporation; IGF-1(100-11), EGF (AF-100-15), IL-6(200-06), Peprotech Corp; RPMI-1640 medium (01-100-1ACS), DMEM medium (01-052), Fetal Bovine Serum (Fetal Bovine Serum, FBS, 040011ACS), carbon-adsorbed Fetal Bovine Serum (Certified dietary Bovine Serum Charcoal Strepped, C-FBS, 04-201-1A), Biological Industries, Inc.; diabody (10378-; matrigel (354234), BD corporation; protein prestainer (26617), BCA protein quantification kit (NC13225CH), ECL-PLUS developer (QB19798510), Dynabeads M-280Streptavidin biotin affinity magnetic beads (11205D), Thermo Scientific; protease Inhibitor Cocktail (05693132001), Roche; plasmid miniprep kit (PL02), edley biotechnology; a plasmid bulk extraction kit (740412.50), MACHEREY-NAGEL; AR (ab74272) antibody, IRF8(ab28696) antibody, Abcam corporation; beta-Actin (bsm-33036M), Boosen; goat anti-rabbit-HRP secondary antibody (sc-2030), Santa Cruz; DAPI (C1005), Dil cell membrane red fluorescent probe (C1036), anti-fluorescence quenching mounting solution (P0126), goat serum (C0265), DAB horseradish peroxidase color development kit (P0203), CCK8(C0039), WB and IP cell lysate (P0013), bi yun tian biotechnology limited; qReal-time PCR reagent (PR047A, PR820A), Takara;

and siRNA transfection reagent (101-10N), PolyPlus-transfection Co;dual-luciferase reporter assay kit (E1910), Promega corporation; protamine DNA (P4505), Sigma; calpain Activity Assay Kit fluoro (QIA120), Millipore; siRNA targeting IRF8(siRNA1-5), Scamble siRNA (siNC), constructed by Shanghai Jima corporation; RT-PCR primer, constructed by Kinsley Biotechnology, Inc.; MG132(M7449), pEZX-FR03-hIRF8-luc dual-luciferase reporter plasmid, synthesized by Guangzhou bioenergy Biotech, Inc.; pcDNA3.1(+) -hAR (full length), pcDNA3.1(+), pcDNA3.1(+) -hIRF8 plasmid, available from Kinsley Biotechnology Ltd. The Flag-Ub plasmid was purchased from Wuhan vast Ling Biotech, Inc.; leuteptin (Leu, L2884), Calpastatin (SCP0063), Cycloheximide (CHX, C4859), Actinomycin D (Actinomycin D, ActD, A9415), Sigma; MG101(HY-18964), MCE; DiO cell membrane green fluorescent probe (C1038), bio-yunnan biotechnology; normal rabbit IgG (sc-2027), Santa Cruz; pierce^TMProtein A/G Magnetic Beads (88803), Thermo Fisher Scientific; IFN α 2a cytokine, perprotech; IFN alpha 2a injection (trade name: Intfin), Shenyang Sansheng pharmaceutical Co., Ltd; p-STAT1(701), p-STAT1(727), STAT1, IRF3, CST corporation; bradford protein quantification kit (P0006), Biyunnan Biotech Ltd; other reagents were analytically pure.

Example 1 immunohistochemistry

Collecting fresh prostate cancer and prostatic hyperplasia tissues from the traditional Chinese medicine institute of Jiangsu province, quickly freezing by liquid nitrogen, preparing paraffin sections, purchasing a prostate cancer tissue chip (HPro-Ade180PG-02, point 180) from Shanghai core super company, entrusting immunohistochemical detection of IRF8 expression, and asking a senior pathological specialist to interpret the immunohistochemical result:

(1) and (3) judging the dyeing positive rate: firstly, observing a tissue point in a whole visual field under a low-power microscope, then selecting 3 visual fields with different staining intensities, judging under a high-power microscope, randomly recording 100 cells in each visual field if the tissue point is positioned in a nucleus, then recording the percentage X1% of positive cells in 100 cells, and finally taking the average of X1%, X2% and X3% of the staining positive rate of the tissue point after the percentage X2% and X3% of the positive cells in the other 2 visual fields are also viewed on the same principle; if located in the cytoplasm or the cell membrane, 3 different fields of staining intensity are also selected, and the positive rate is estimated and averaged.

(2) Quantification of staining intensity: a sample is obtained by observing a tissue point in a whole visual field under a low power microscope, then selecting 3 visual fields with different staining intensities to take a picture under a high power microscope, and taking all samples at one time under the same microscope condition. Immunohistochemical optical density measurements were analyzed using IPP software. After the optical density correction is performed as described, the average optical density mean and the integrated optical density degree (IOD sum) are selected for quantification, and the average value of three fields is taken as the quantification result of one sample.

The tissue chip comprises 64 samples of paracancer and cancer-nidus tissues of the same patient and 5 samples of normal prostate tissues, and the basic information is shown in table 1. Immunohistochemical localization results showed that IRF8 was expressed in both the cytoplasm and nucleus of prostate cancer cells (fig. 1). The result of the positive staining rate of IRF8 shows that the weak staining and the medium-intensity positive staining of IRF8 in the cytoplasm of normal paracarcinoma tissues reach 40 percent; strong staining reaches 20%, and moderate positive staining in IRF8 in cytoplasm of para-carcinoma tissue is reduced to 16%; the strong positive staining of IRF8 decreased to 9.37% in the cytoplasm of prostate cancer nests. The strong staining of IRF8 in the nucleus was reduced from 20% in normal tissues to 1.5625% in cancer niche tissues, with significant differences in its expression from that in both proliferative and normal paracancerous tissues (table 2).

Table 164 clinical case characteristics of Paracarcinoma/cancer nest patients

TABLE 2 immunohistochemical staining of IRF8 in Normal, paracancerous, cancer-nestled prostate tissue

Normal, paracarcinoma, cancero, χ 2 test

Example 2

2.1DHT can upregulate IRF8 expression

Androgens, as ligands for AR, play a crucial role in the growth and malignant transformation of prostate cancer cells mediated by AR. Abnormal activation of the android-AR signaling pathway is prevalent in the development of prostate cancer. We found that with the increase of the pathological grade of prostate cancer, the expression level of IRF8 increased, and IRF8 expression was positively correlated with AR expression. These studies suggest that there is some connection between IRF8 and AR. Research shows that a group of ISG proteins in prostate cells can be simultaneously used as androgen stimulating genes to be regulated by androgen. In this example we tested whether expression of IRF8 is regulated by the AR signaling pathway. AR positive prostate cancer cell line LNCaP was pretreated with 5% carbon adsorbed serum C-FBS plating for 24hrs, followed by 24hrs cell immunofluorescence detection of IRF8 expression with 10nM DHT, ethanol as solvent control. The immunostaining results show that IRF8 has weak expression in cytoplasm and nucleus of LNCaP cells under the condition of no DHT (figure 3A-DHT), and IRF8 expression in cytoplasm and nucleus is obviously up-regulated after the AR signal pathway is activated by DHT stimulation (figure 3A + DHT). While different concentrations of DHT were used to stimulate LNCaP cells for 24hrs, RNA and protein were extracted, and IRF8 expression was detected by Real-time PCR and WB, respectively, and DHT was found to dose-dependently up-regulate the mRNA level (FIG. 3B) and protein level expression (FIG. 3C) of IRF 8. DHT-activated AR also up-regulated IRF8 expression in another AR-positive prostate cancer cell line 22RV1 (fig. 3D). In the malignant transformation process of the prostate cancer, under the condition of low level of androgen, growth factors IGF-1, EGF, cytokine IL-6 and the like can bypass activation of AR to promote androgen-independent growth of prostate cancer cells. Thus we guess if the expression of IRF8 could be up-regulated by the bypass-activated AR as well? Interestingly, the AR shunt activator IGF-1 (fig. 3E), EGF (fig. 3F) down-regulated IRF8 expression without time-dependency; IL-6 had no significant effect on IRF8 expression (FIG. 3G). These results suggest that IRF8 may be an androgen inducible gene.

2.2 castration downregulates the expression of IRF8

DHT mainly binds to its receptor AR to form a dimer, which enters the nucleus and then exerts a transcription function. The results of the previous subsection found that DHT can induce the expression of IRF8 in AR positive cell lines, and then whether blocking the AR signaling pathway can inhibit the expression of IRF 8? In view of the Androgen dependence of primary prostate cancer, Androgen Deprivation Therapy (ADT), including surgery or drug castration, is the primary treatment for early stage prostate cancer. Enzalutamide is a second generation of AR antagonist, and can block an AR signal pathway through various ways such as inhibiting the combination of androgen and AR, inhibiting the nuclear translocation of AR, inhibiting the combination of AR to target gene DNA and the like. Carbon-adsorbed serum removed trace androgen from serum, and 5% C-FBS was commonly used to culture LNCaP for mimicking in vitro castration. After LNCaP is processed by Enz for 24hrs, RNA and protein are respectively extracted and IRF8 expression is detected. As a result, it was found that Enz could dose-dependently down-regulate the mRNA level (fig. 4A) and protein level (fig. 4B) of IRF 8. C-FBS, Enz and Bicalutamide (Bic) act for 24-72hrs, and WB results show that C-FBS can reduce the expression of IRF8 in a time-dependent manner; the potent AR antagonist Enz can down-regulate IRF8 protein to the lowest level by 24hrs without time dependence; bic, the first generation AR antagonist, was less down-regulated in IRF8 expression than Enz due to partial AR agonistic activity (fig. 4C). To study the effect of long-term castration on IRF8 expression, we simulated in vitro castration by long-term culture of LNCaP in 5% C-FBS culture medium. WB results showed that C-FBS treatment reduced IRF8 down to the lowest level for 1 week, further prolonged castration to 2 weeks, and no further reduction in IRF8 expression (fig. 4D, n ═ 3). To further investigate the effect of PCa castration in vivo on IRF8 expression, we seeded LNCaP cells on the dorsal ventral side of Balb/c nude mice once tumors grew. Randomly dividing the operation into 3 groups which are respectively a false operation group; surgically removing bilateral testicular groups (trapping) to reduce androgen levels in vivo; the casting + Enz (10mg/kg) group, further blocked the AR signal. Tumor tissues are picked up for homogenate after 28 days, and WB detects the expression of IRF8 in tumor blocks. The results showed that the expression of IRF8 was slightly down-regulated by simple resection of bilateral testes compared to the sham group, and that the expression level of IRF8 was further reduced after combined surgical resection of bilateral testes with intragastric administration of Enz (fig. 4E, n-3). To further detect the influence of Enz on the expression of IRF8, LNCaP cells are inoculated on the dorsal ventral side of Balb/c nude mice with bilateral testicles removed through an operation to construct an LNCaP-CRPC nude mouse transplantation tumor model, Enz (10mg/kg) is intragastrically administered, tumor tissues are picked up after 28 days for homogenization, and WB detects the expression of IRF8 in tumor blocks. The results show a general decrease in IRF8 expression levels following Enz treatment of CRPC (fig. 4F, n ═ 6), suggesting that further blockade of the AR signaling pathway in the presence of low androgen levels may still down-regulate IRF8 expression.

Example 3 knockdown of IRF8 expressing cells by LNCaP cells proliferation accelerated

3.1 design and construction of stable knock-out cell strains LNCaP-shNC, LNCaP-shRNA1 and LNCaP-shRNA2 of IRF 8.

3.1.1 design of Oligo

The DNA oligo was designed using Designer3.0 (Genephrma) software, and the synthesis of primers was carried out by Shanghai Jima pharmaceutical technology, Inc. TTCAAGAGAGA is selected as the loop structure in the LV3-shRNA template to avoid the formation of a termination signal. The 5' end of the sense chain contains GATCC, and is complementary with a sticky end formed after BamHI enzyme digestion; the 5' end of the antisense strand contains AATTC, which is complementary to the sticky end formed by EcoRI cleavage.

Sense strand: 5 '-GATCC- (GN18) - (TTCAAGAGAGA) - (N18C) -TTTTTTG-3'

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

DNA Oligo designed in Table 3

3.1.2 annealing of LV3-shDNA template

The DNA oligo was dissolved in TE solution at pH8.0 at a concentration of 100. mu.M. Taking the corresponding sense and antisense oligo solutions in the previous step, and preparing the annealing reaction system of the template according to the following proportion:

TABLE 4 annealing reaction System for shDNA template

Setting a PCR instrument annealing program: 5min at 95 ℃; 5min at 85 ℃; 5min at 75 ℃; 5min at 70 ℃; storing at 4 ℃. The PCR product was the shRNA template (10. mu.M), and this PCR product was diluted 50-fold to a final concentration of 200 nM.

3.1.3 linearization of LV3 vector

Taking 10 mu g of LV3 vector, and carrying out enzyme digestion treatment according to the following system:

TABLE 5 digestion system

The enzyme was digested at 37 ℃ for 1h, subjected to Agarose electrophoresis, recovered using Agarose Gel DNA Purification Kit Ver2.0, subjected to electrophoresis to determine the estimated concentration, and diluted to 50 ng/. mu.L.

3.1.4 construction of LV3-shRNA vector

The ligation of the support was carried out as follows:

TABLE 6 Carrier ligation reaction System

The reaction is carried out for 1h at 22 ℃, and the product is used for subsequent transformation of competent cells.

3.1.5 transformation and amplification of plasmids

(1) The competent cells DH5 α were removed from the-80 ℃ freezer and the freshly thawed cell suspension was aliquoted in 500 μ L aliquots into sterile, pre-chilled EP tubes and placed in an ice bath.

(2) Adding the target DNA into the competent cell suspension, gently and uniformly mixing, and standing in an ice bath for 30 min.

(3) The centrifuge tube was placed in a 42 ℃ water bath for 90s, and then the tube was quickly transferred to an ice bath to cool the cells for 2min without shaking the centrifuge tube.

(4) Adding 500 μ l LB culture medium (without antibiotic) into each centrifuge tube, mixing, placing in 37 deg.C shaking table, shaking at 220rpm, and culturing for 1h to recover thallus.

(5) The mixture was centrifuged at 4000rpm for 1min, a portion of the supernatant was discarded, 150. mu.l of the supernatant was retained, mixed well, and 100. mu.l of the transformed competent cells were pipetted and spread well on LB solid agar medium containing 50. mu.g/mL Ampicillin.

(6) The plate was left at room temperature until the liquid was absorbed, inverted and incubated at 37 ℃ for 12-16 h.

3.1.6 identification and sequencing of Positive clones

(1) 4 individual, filled colonies were picked from the cultured plates and placed in 50mL vials containing 5mL (50. mu.g/mL Ampicillin) of LB medium; placing in a shaker at 37 deg.C, rotating at 250rpm, and incubating for 16h

(2) And extracting plasmids from the cultured bacterial solution in a small amount, and carrying out double enzyme digestion identification on the obtained plasmids by using Xba I and Nhe I.

(3) The strains with the correct sequencing were kept for subsequent testing.

3.1.7 plasmid extraction

(1) And (3) putting the overnight cultured bacterial liquid into a 50mL centrifugal tube, centrifuging at the room temperature of 5000rpm for 2min, and pouring out the supernatant as much as possible to collect the bacterial. This step was repeated until enough biomass was collected.

(2) Resuspend pellet with 7.5mL of solution P1 (ensure that RNase A was added to solution I) and vortex until suspension was complete.

(3) Adding 7.5mL of solution P2, gently turning over for 6-8 times to fully crack the thallus, and standing at room temperature for 4-5 min. At this time, the bacterial liquid becomes clear and viscous.

(4) Adding 7.5mL of solution N3, immediately turning gently up and down for 6-8 times, mixing well until white flocculent precipitate appears, and centrifuging at 5000rpm for 20 min. The supernatant was carefully removed to a new tube to avoid aspiration of a floating white precipitate.

(5) Adding 10mL of isopropanol into the supernatant, fully reversing, uniformly mixing, transferring into an adsorption column DC (the adsorption column is placed into a collection tube) for multiple times, centrifuging at 5000rpm for 2min, and pouring waste liquid in the collection tube.

(6) 10mL of the rinse solution (to which absolute ethanol had been added) was added, centrifuged at 5000rpm for 2min, and the waste solution was discarded. Then, 10mL of the rinsing solution WB was added and the rinsing was repeated once.

(7) And (4) putting the adsorption column DC back into the empty collection tube, centrifuging at 5000rpm for 5min to dry residual ethanol on the matrix membrane, opening the cover and airing at room temperature for 5 min.

(8) Taking out the adsorption column DC, placing into a clean centrifuge tube, adding 1-2mL eluent EB (preheated in water bath at 65-70 deg.C in advance) at the middle position of the adsorption membrane, standing at room temperature for 3min, and centrifuging at 5000rpm for 5 min. The obtained liquid is high-purity plasmid; the concentration and purity of the DNA are measured by an ultraviolet absorption method, and the A260/A280 of the DNA of the improved grains is ensured to be between 1.8 and 2.0.

3.1.8293T cell packaging virus

(1) 293T cells in the logarithmic growth phase were taken, the culture medium was removed, and the cells were washed twice with 1mL of D-Hank's solution. Adding 1mL of Trypsin-EDTA solution, mixing uniformly, and standing at 37 ℃ for 1-3 min.

(2) Trypsin-EDTA solution was aspirated, 2mL of DMEM medium (containing 10% FBS) was added to stop digestion, and the cells were blown to form a single cell suspension. Inoculating into a culture dish, adding 10mL DMEM culture solution containing 10% FBS, mixing well, and then 5% CO at 37 ℃₂The culture was carried out overnight.

(3) Adding 1.5mL serum-free DMEM into one sterile 5mL centrifuge tube, proportionally adding shuttle plasmid and packaging plasmid (pGag/Pol, pRev and pVSV-G) containing IRF8 sequence, mixing, adding 1.5mL serum-free DMEM into the other sterile 5mL centrifuge tube, adding 300 μ L RNAi-Mate, mixing, standing at room temperature for 5min, mixing the two tubes, and standing at room temperature for 20-25 min.

(4) The medium in (2) was aspirated, and 5mL of serum-free DMEM medium was added. Dropwise adding the mixture obtained in the step (3) into a culture dish, gently mixing the mixture uniformly, and placing the mixture at 37 ℃ in 5% CO₂The incubator lasts for 4-6 h.

(5) The transfection solution was aspirated, and 10mL of DMEM medium containing 10% FBS was added. 5% CO at 37 ℃₂The culture was continued for 72 h.

(6) The supernatant of the cultured cells was pipetted into a 50mL centrifuge tube and centrifuged at 4000rpm for 4 min. The supernatant was removed and filtered through a 0.45 μm filter.

(7) The filtrate was centrifuged at low temperature and high speed for 2 h. Collecting the concentrated solution, subpackaging into EP tubes, and storing at-80 deg.C for use.

3.1.9 Virus infection and puromycin screening IRF8 Stable knockdown cell line

(1) LNCaP cells in logarithmic growth phase were seeded in 24-well plates at 2000 cells per well with 100. mu.L of medium added per well. To ensure good transfection efficiency, the cell density at 37 ℃ with 5% CO was 40% -60% when viral infection was performed₂Culturing for 24h, and adhering to the wall.

(2) Two infection pilot experiments were designed, each with a different concentration gradient of virus. The first group was normally infected by adding only virus solution to the complete medium; polybrene was added at 5. mu.g/mL in the second group of infection, and it was found that Polybrene was effective in increasing the infection efficiency in most cells.

(3) The virus solution was taken out of the refrigerator in advance and melted on ice. Aspirate 10. mu.L of virus into a new EP tube and mix gently. The virus solution was diluted according to a 10-fold, 100-fold concentration gradient.

(4) The culture medium in the 24-well plate was aspirated, 500. mu.L each of the virus solutions from the three different gradients was added to three wells of each group, a blank was set up simultaneously, and the cells were returned to the incubator for incubation. Cell morphology was observed after 8-12 h. If the cell morphology is not significantly different from that of the uninfected group, it indicates that the virus fluid has no significant toxicity to the cells.

(5) After 24h of culture, the virus solution diluted in a 24-well plate is aspirated, 500. mu.l of 1640 culture solution of 10% FBS is added to each well, puromycin is added to a final concentration of 1.0. mu.g/ml, and 5% CO is added at 37 DEG C₂And (5) continuing culturing.

(6) And (3) changing and adding medicine every day until all the blank medicine adding groups die, amplifying the screened cells, collecting samples for RT-PCR and Western blot detection to detect the IRF8 knocking efficiency, and stably knocking cell strains LNCaP-shNC, LNCaP-shRNA1 and LNCaP-shRNA2 by IRF8 to successfully construct.

3.2CCK8 detection of cell viability

(1) The log phase cells were digested with 0.25% trypsin, resuspended in culture medium containing 10% FBS and blown into a single cell suspension. At a rate of 1X 10 per hole⁴Individual cells were plated in 96-well plates at 100. mu.L/well and shaken well, with three parallel wells being made at each time point. Placing into incubator, and culturing at 37 deg.C with 5% CO₂Culturing for 24 hrs.

(2) The administration can be carried out by observing the attached cells and the cells in a normal growth state under a microscope. According to the purpose and plan of the experiment, the drug to be tested is firstly prepared into mother liquor according to the property of the drug to be tested, and then the gradient dilution is carried out in sequence. Sucking the old culture solution, adding diluted medicinal culture solution, and culturing for 48-72 hr.

(3) At the end of the experiment, 20. mu.L of CCK8 solution was added to each well and incubated at 37 ℃ for 4 hrs.

(4) Selecting a wavelength of 450nm, measuring the absorbance value of each hole on a multifunctional microplate reader, recording the experimental result, and carrying out mapping analysis on the result by using GraphPad prism 6.0.

3.3 Effect of IRF8 on prostate cancer cell proliferation correlates with AR expression status

In order to detect the expression of IRF8 in prostate cancer cell strains, the expression level of IRF8 protein in human AR negative cell strains, proliferative prostate epithelial cells BPH1, prostate cancer cell strains PC3, DU145 and AR positive prostate cancer cell strains LNCaP is firstly detected by a Western blot method, and immune cells Raw264.7 are used as IRF8 WB positive control. The results showed that IRF8 was expressed in all 4 prostate epithelial cells and was highest in AR positive LNCaP cells (fig. 5A), with an expression intensity of approximately 2-fold higher than that of AR negative prostate cells. Aiming at different expression conditions of IRF8 in prostate cancer cell lines, pcDNA3.1(+) -hIRF8 plasmids (IRF8 plasmids) of low-expression IRF8 cell lines DU145 and PC3 are transiently transfected to highly express IRF8(DU145-IRF8, PC3-IRF8) and IRF8 is transiently transfected to IRF8 plasmids of relatively high-expression cell lines LNCaP to further ectopically highly express IRF8(LNCaP-IRF8), and the CCK8 method is used for detecting the influence of IRF8 on the proliferation of prostate cancer cells. The results show that compared with the unloaded control group, the proliferation of the AR negative prostate cancer cell strain DU145 over-expressing IRF8 has no significant effect (fig. 5B-C), the proliferation of the AR positive cell strain LNCaP ectopically high-expressing IRF8 is slowed down (fig. 5D), and the influence of IRF8 on the proliferation of prostate cancer cells is probably related to the expression state of the prostate cancer cells AR.

3.4 proliferation of cells with knockdown of IRF8 expression by LNCaP

In view of the instability of transient transfection on gene expression, in the early stage research, in order to explore the function of IRF8 in prostate cancer, the IRF8 expression in a prostate cancer cell strain LNCap is stably knocked down by shRNA virus to obtain a stably knocked-down cell strain, and a series of in vitro and in vivo experiments are carried out. CCK8 detection and immunofluorescence observation show that LNCaP-shIRF8 cell proliferation is accelerated, crystal violet staining shows that LNCaP-shIRF8 clone formation is increased, the balling capacity of suspension culture LNCaP-shIRF8 is obviously enhanced, and apoptosis tolerance caused by hydrogen peroxide suggests that IRF8 may participate in malignant transformation of prostate cancer and regulation and control of apoptosis sensitivity. However, the prostate cancer cell proliferation is Androgen-dependent, and the generation and development of CRPC are closely related to the abnormality of the Androgen-AR signaling pathway. Thus, we want to know if IRF8 regulated by the android-AR signaling pathway produces regulation of the android-AR signaling pathway? We further verified the expression of IRF8 in the LNCaP-shIRF8 cell line by Western blot method (FIG. 6A). Under the condition of low IRF8 expression level, the CCK8 method detects the influence of IRF8 on the proliferation of prostate cancer cells under the condition of existence or nonexistence of androgen. The results show that cell proliferation is accelerated after knockdown of IRF8 by LNCaP (fig. 6B), and the proliferation-accelerating property persists with androgen ablation (fig. 6C) and low levels of androgen (fig. 6D, 10nM R1881).

Example 4 knock-down of IRF8 enhances the tumorigenicity of LNCaP cells in vivo

4.1 animal models

4.1.1LNCaP-shIRF8 nude mouse graft tumor model

(1) 18 male Balb/c nude mice of about 4-6 weeks were surgically excised from bilateral testis and epididymis and acclimatized in SPF animal room for one week.

(2) 18 nude mice were randomly divided into 2 groups of 9 mice each.

(3) shNC cells and sh in 0.25% pancreatin-digested logarithmic growth phaseRNA2, 5% FBS in 1640 medium was gently pipetted into a single cell suspension. PBS was washed three times and centrifuged at 1000rpm for 5 minutes. Serum-free RPMI1640 resuspension, cell viability (viability) by 0.4% trypan blue assay>90%) matrigel adjusted to a cell concentration of 5X10⁷one/mL (matrigel: RPMI1640 ═ 1: 1). Nude mice were injected subcutaneously in the back with 0.2mL of cell suspension under sterile conditions.

(4) According to different inoculated cells, the cell is named shNC (inoculated shNC cell) and shIRF8 (inoculated IRF8 shRNA2 cell with higher silencing efficiency).

(5) The animals were observed for body weight, diet, etc. and tumor changes were monitored daily. The tumor length and tumor length were measured with a vernier caliper every other day, and the tumor volume was calculated (1/2 × long diameter × short diameter)²) And drawing a tumor growth curve.

(6) Tumor volume was stopped when tumors grew to 10% of body weight and tumor growth curves were plotted.

(7) And continuously observing the growth condition of the mouse, recording the death time of the tumor-bearing mouse, and drawing an overall survival curve.

4.1.2LNCaP-shIRF8 NOD-SCID mouse transplantation tumor model

(1) 18 male NOD-SCID mice, approximately 4-6 weeks old, were acclimated in SPF animal houses for one week.

(2) 18 nude mice were randomly divided into 2 groups of 9 mice each.

(3) shNC cells and shRNA2 in logarithmic growth phase were digested with 0.25% trypsin, and gently shaken in 1640 medium containing 5% FBS to form a single cell suspension. PBS was washed three times and centrifuged at 1000rpm for 5 minutes. Serum-free RPMI1640 resuspension, cell viability (viability) by 0.4% trypan blue assay>90%) matrigel adjusted to a cell concentration of 5X10⁷one/mL (matrigel: RPMI1640 ═ 1: 1). Nude mice were injected subcutaneously in the back with 0.2mL of cell suspension under sterile conditions.

(4) According to different inoculated cells, the cell is named shNC (inoculated shNC cell) and shIRF8 (inoculated IRF8 shRNA2 cell with higher silencing efficiency).

(5) The animals were observed for body weight, diet, etc. and tumor changes were monitored daily. Measuring the length and the short diameter of the tumor every other day by using a vernier caliper, and calculating the tumor volume (tumor body)Product of 1/2 × long diameter × short diameter²) And drawing a tumor growth curve.

(6) When the tumor grows to 10% of the body weight, the tumor volume is stopped, the nude mice are sacrificed, tumor masses are dissected out, the final volume is measured and weighed, and a tumor growth curve and a tumor weight column chart are drawn.

4.2 knock-down of IRF8 enhances the tumorigenesis of LNCaP in castrated nude mice and decreases their survival

In the previous research, the LNCaP stably knockdown IRF8 cell strain LNCaP-shIRF8 is found to have an increased tumor forming speed in a nude mouse, the tumor weight at the end of the experiment is obviously increased, and the immunostaining of Proliferating Cell Nuclear Antigen (PCNA) in tumor tissues is obviously enhanced, which indicates that IRF8 can inhibit the proliferation of prostate cancer cells and the tumor forming capability in the nude mouse. Before we found that the ability of IRF8 to inhibit prostate cancer cell proliferation in vitro correlates with the expression state of AR, we wanted to further investigate whether reducing androgen levels in mice would still maintain the tumorigenic capacity of cell lines knocking down IRF8 in nude mice. The 4-6 week old mice are surgically removed bilateral testis and epididymis to reduce androgen level in vivo, and then the dorsal ventral side of the castrated mice is inoculated with 1 × 10⁷LNCaP-shrrf 8 cells and their negative control, LNCaP-shrnc, were monitored for tumor formation rates. The results show that under castration conditions, after 10 days of inoculation, the two groups of tumor masses grow up, the speed of knocking down IRF8 to form the tumor is obviously increased, and the tumor volume in the IRF8 group which is knocked down after 3 weeks of inoculation reaches 1368 +/-188.1 mm³N is 9, while the tumor volume of the negative control group is 688 +/-90.45 mm³When n is 9, 1/2 in the knockdown group only, there was a significant difference in tumor growth in both groups (fig. 7A). Thereafter, the survival status of two groups of tumor-bearing mice was recorded, and the difference of the overall survival time of the two groups of tumor-bearing mice was observed, and the survival status of the tumor-bearing mice became worse after IRF8 was knocked down, and the overall survival time was significantly lower than that of the negative control group (fig. 7B, P ═ 0.0326). The above results indicate that the enhancement of tumorigenicity in prostate cancer cells in vivo due to knockdown of IRF8 expression is not related to androgen levels in vivo. 4.3 knockdown of IRF8 enhances the tumorigenesis of LNCaP in NOD-SCID mice

The literature research shows that IRF8 is mainly expressed in immune cells and influences the functions of the immune cells, and enhances the immune functions of macrophages, DC cells and the like. Balb/c nude mice are thymus-deficient mice and have T cell dysfunction, but are not completely immunodeficient mice expressed in vivo by old DC cells, NK cells and the like, and in order to verify that the tumor cell tumorigenic speed caused by LNCaP knock-down of IRF8 is increased and is not caused by the reduction of the expression of tumor cell IRF8 and further the reduction of immune function, shNC cells and shIRF8 cells are inoculated on the ventral side of NOD-SCID mice with more deficient immune system, and the tumor growth speed is observed. As a result, it was found that after IRF8 was knocked down by LNCaP cells, the tumor formation rate was increased (fig. 8A), the tumor weights at the end of the experiment were significantly different (fig. 8B), and the characteristic of enhanced tumor formation ability remained in the fully immunodeficient mice. The results show that IRF8 can directly inhibit the proliferation and in vivo tumor forming ability of tumor cells.

In summary, the substance capable of inducing IRF8 expression can directly inhibit the proliferation and in vivo tumorigenic ability of prostate tumor cells.

Example 5 in vitro assay LNCaP-shIRF8 has reduced sensitivity to Enz

5.1LNCaP-shIRF8 has reduced sensitivity to different concentrations of Enz

Enzalutamide blocks the binding of testosterone and dihydrotestosterone by competitive binding to LBD, and inhibits the process of androgen receptor nuclear transcription and binding to DNA. Therefore, if the AR activity changes, including protein expression and activity changes, the generation of enzalutamide drug resistance can be caused. This example investigates whether IRF8 can affect prostate cancer cell sensitivity to Enz.

Firstly, LNCaP-shIRF8(IRF8 has high knocking efficiency) is stably knocked out by adopting IRF8, different concentrations of Enz are given for 48-72 hrs, and CCK8 is used for detecting cell viability. The results show that IRF8 knockdown cell viability was significantly higher than the unloaded control group LNCaP-shNC at the same concentration (fig. 9A-B). Similarly, increasing the cell inoculum size, 6-well plate seeded cells were exposed to 5 μ M and 10 μ M Enz for 72hrs, and cell survival was directly observed under microscope, with LNCaP-shNC at 5 μ M Enz concentration, almost no adherent cells were present on the plates, while the group LNCaP-shIRF8 at higher concentration Enz (10 μ M) showed good cell spreading in the plates, high light transmittance, almost no dead cells, and slightly lower cell density than the DMSO control (FIG. 9C).

5.2IRF8 modulation of Enz-induced caspase 3 Activity

Enzalutamide can bind with DNA to induce apoptosis^[7]. The above shows that when 5 μ M Enz acts for 48hrs, the cell viability is about 80%, the cell density is reduced, and only the cell proliferation is inhibited; after 72hrs, the cell activity was about 70%, and dead cells appeared. Thus, 5. mu.M Enz was used to treat prostate cancer cells and caspase 3 activity was detected after 72 hrs. FIG. 10A shows that Enz upregulated caspase 3 activity, and that caspase 3 activity was unchanged by Enz treatment compared to the solvent control following IRF8 knockdown. Similarly, LNCaP overexpresses IRF8 for 48hrs followed by treatment with 5 μ M Enz for 48 hrs. FIG. 10B shows that IRF8 overexpression alone did not affect caspase 3 activity, that IRF8 group caspase 3 activity was significantly upregulated under Enz treatment, and that vector group caspase 3 did not change significantly.

5.3LNCaP-shIRF8 antagonizes the inhibition of clonogenic and proliferative responses to Enz

In this example, the effect of IRF8 knockdown on cell proliferation under the long-term action of Enz was studied, and experimental basis was provided for the therapeutic effect of long-term drug administration in vivo. LNCaP-shNC cells and two IRF8 knock-down cell strains are used, and crystal violet staining is carried out to calculate the clone proliferation inhibition rate. Under 10 μ M Enz treatment, the clone formation in the LNCaP-shNC group was significantly reduced, and the clone formation in the LNCaP-shIRF8 group was also reduced to some extent (FIG. 11A). IPP calculation clone number, solvent control group and IRF8 knock-down can obviously increase clone formation of LNCaP cells; the clonogenic increasing property due to IRF8 knockdown was still present with Enz treatment (fig. 11B). Under the treatment of Enz, the clone formation inhibition rate of the LNCaP-shNC group is 48%, while the clone formation inhibition rates of the two cell strains of the IRF8 knock-down group are 33.5% and 22.5%, respectively, and have significant difference with the LNCaP-shNC group (FIG. 11C). Similarly, cell proliferation of each group of cells under 10 μ M Enz treatment was examined using 96-well plates. In agreement with the previous results, LNCaP-shrnc cell proliferation was significantly inhibited after 3 days under Enz treatment, whereas LNCaP-shrrf 8 group had no significant effect on cell proliferation compared to the solvent control group (fig. 11D). These results are all similar to those of the short-term Enz treatment, indicating that knock-down IRF8 also functions against Enz under the long-term Enz treatment as well as under the Enz short-term treatment.

Example 6 reduction of Enz sensitivity by LNCaP-shIRF8 in vivo experiments

LNCaP is inoculated on the abdominal backs of nude mice with surgically excised bilateral testicles and epididymis Balb/c, and an LNCaP-CRPC nude mouse model is constructed. The effect of IRF8 knockdown on Enz sensitivity was further validated in vivo. The results show that the tumor growth curve of the Enz treatment group is significantly different from that of the solvent control group after the LNCaP-shNC group acts for 25 days at 10mg/kg/d Enz; after IRF8 knockdown, until the end of the experiment, the tumor growth curve of the Enz-treated group was not significantly different from that of the solvent control group (fig. 12A). At the end of the experiment, the final tumor volume of the Enz-treated group and the solvent control group in the LNCaP-shrnc model was significantly different, the growth rate of the single tumor volume under the Enz treatment was significantly reduced, and the final tumor volume of the LNCaP-shrrf 8 group and the growth rate of the single tumor volume under the Enz treatment were not significantly changed from the solvent control group (fig. 12B-C). Similarly, fig. 12D shows that the end-point LNCaP-shrnc model showed significant difference in final tumor weight between the Enz-treated group and the solvent control group, with a tumor inhibition rate of about 41.1%, whereas the LNCaP-shrrf 8 group had no effect.

Example 7IFN alpha enhanced Enz therapeutic efficacy

7.1 animal models

7.1.1 detection of Enz sensitivity by LNCaP-CRPC Balb/c nude mouse model

(1) 24 male Balb/c nude mice of about 4-6 weeks were surgically excised from bilateral testis and epididymis and acclimatized in SPF animal room for one week.

(2) 24 nude mice were randomly divided into 2 groups of 12 mice each.

(3) shNC cells and shRNA2 in logarithmic growth phase were digested with 0.25% trypsin and gently shaken in 1640 medium containing 5% FBS to form a single cell suspension. PBS was washed three times and centrifuged at 1000rpm for 5 minutes. Serum-free RPMI1640 resuspension, cell viability (viability) by 0.4% trypan blue assay>90%) matrigel adjusted to a cell concentration of 5X10⁷one/mL (matrigel: RPMI1640 ═ 1: 1). Nude mice were injected subcutaneously in the back with 0.2mL of cell suspension under sterile conditions. According to different inoculated cells, the shNC (inoculated shNC cells) and shIRF8 (inoculated IRF8 shRNA2 with higher silencing efficiencyCell)

(4) Once the tumor had grown, LNCaP-shrnc and LNCaP-shrrf 8 were randomized into two groups, respectively, with the Enz group gavage 10mg/kg (n 6) per day and the Vehicle group gavage the same volume of CMCNa per day as the solvent control group (n 6).

(5) The animals were observed for body weight, diet, etc. and tumor changes were monitored daily. The tumor length and tumor length were measured with a vernier caliper every other day, and the tumor volume was calculated (1/2 × long diameter × short diameter)²) And drawing a tumor growth curve.

(6) When the tumor grows to 10% of the body weight, stopping measuring the tumor volume, killing the tumor-bearing mice, dissecting out tumor masses, measuring the final volume and weighing, drawing tumor growth curves and tumor weight histograms, and calculating the tumor growth inhibition rate (the final tumor weight of a solvent control group-the final tumor weight of an administration group)/the final tumor weight of the solvent control group as 100.

7.1.2LNCaP-CRPC NOD-SCID mouse model detects modulation of IFN alpha sensitivity to Enz

(1) 76 male NOD-SCID mice of about 4-6 weeks were surgically excised from bilateral testis and epididymis and acclimatized in SPF animal room for one week.

(2) 76 NOD-SCID mice were randomly divided into 2 groups.

(3) shNC cells and shRNA2 in logarithmic growth phase were digested with 0.25% trypsin and gently shaken in 1640 medium containing 5% FBS to form a single cell suspension. PBS was washed three times and centrifuged at 1000rpm for 5 minutes. Serum-free RPMI1640 resuspension, cell viability (viability) by 0.4% trypan blue assay>90%) matrigel adjusted to a cell concentration of 5X10⁷one/mL (matrigel: RPMI-1640 ═ 1: 1). Nude mice were injected subcutaneously in the back with 0.2mL of cell suspension under sterile conditions. According to different inoculated cells, the cells are named shNC (inoculated shNC cells), shIRF8 (inoculated IRF8 shRNA2 cells with higher silencing efficiency)

(4) LNCaP-shrnc or LNCaP-shrrf 8 were randomly divided into 4 groups: IFN alpha group was subcutaneously injected with 1.5X10 IFN alpha 2a injection every day⁷IU/kg, gavage CMCNa daily (n 12or 8); in the Enz group, the stomach of Enzalutamide is irrigated with 10mg/kg of Enzalutamide every day, and normal saline (n is 12or 8) is injected subcutaneously; IFN alpha + Enz group was administered 10mg/kg of enzalutamide per day by subcutaneous injection of IFN alpha 2aInjection of 1.5X10⁷IU/kg (n ═ 12or 8); CMCNa was gavaged daily in the Vehicle group and injected subcutaneously with normal saline (n ═ 8).

(5) The weight, diet and other conditions of the animals are observed, and the growth and change of the tumor are monitored every day. The tumor length and tumor length were measured with a vernier caliper every other day, and the tumor volume was calculated (1/2 × long diameter × short diameter)²) And drawing a tumor growth curve.

(6) When the tumor grows to 10% of the body weight, stopping measuring the tumor volume, killing the tumor-bearing mice, dissecting out tumor masses, measuring the final volume and weighing, drawing tumor growth curves and tumor weight histograms, and calculating the tumor growth inhibition rate (the final tumor weight of a solvent control group-the final tumor weight of an administration group)/the final tumor weight of a solvent control group of 100 (wherein each of LNCaP-shNC Vehicle and Enz groups is lethal to a gastric lavage operation, and withdrawing experimental data statistics).

7.2 clinical study

We recruited 3 high-risk prostate cancer patients, cohort conditions: primary diagnosis PSA >150ng/Ml, Gleason ≧ 9(4+5, or 5+4), multiple bone metastasis or metastatic potential. Under the conditions of knowing the conditions and meeting the ethics, maximum androgen androparation blocking endocrine therapy (MAB) or IFN alpha combined maximum androgen androparation blocking endocrine therapy (IFN alpha + MAB) is respectively carried out, and the specific scheme is as follows:

(1) maximum androgen blockade endocrine therapy (MAB): goserelin acetate sustained release implant (norrex) 3.6mg (astrikon corporation) 1 time per 28 days, bicalutamide (combatad) 50mg 1 time per day.

(2) Maximum androgen blockade endocrine therapy + IFN α 2a injection (trade name: intefin): the endocrine therapy is the same as above, and IFN alpha 2a 300 ten thousand IU is taken 10 times per month.

Treatment was continued for more than 6 months, PSA levels were measured monthly with PSA >0.2ng/mL indicating biochemical recurrence. Chemotherapy or other treatment is given to patients who require withdrawal at the time of biochemical relapse.

7.3IFN alpha Up-regulating IRF8 expression, Down-regulating AR expression

Different concentrations of IFN alpha treatment AR positive prostate cancer cells LNCaP and 22RV1, WB detection IRF8 and AR protein level after 24hrs, verification of IRF8 under IFN alpha treatment and AR protein expression correlation. Figure 13A shows that IFN α can dose-dependently up-regulate the expression of IRF8 in prostate cancer cells LNCaP and 22RV1, while down-regulating the protein level of AR. FIG. 13B shows that AR positive androgen insensitive prostate cancer cell line 22RV1 can promote the binding of AR to ISRE-like site of IRF8 promoter region under IFN alpha treatment, thereby promoting the expression of IRF 8. Given different concentrations of IFN α simultaneously, the AR half-life was significantly reduced (fig. 13C) and could be blocked by the protease inhibitor MG132 (fig. 13D) and enhance ubiquitination of AR. These results indicate that IFN α in prostate cancer cells can modulate IRF8, promoting ubiquitination degradation of AR. IFN α can be used as an intervention to delay the reduction of IRF8 expression in prostate cancer cells under castration conditions.

7.4IFN alpha enhances ADT efficacy

In vitro assay, LNCaP and 22RV1 were treated with (0, 5, 10, 20, 40, 80, 100) μm ENZ in combination with 1000IU/ml IFN α or different concentrations of IFN α (0, 100, 500, 1000, 2000, 5000, 10000IU/ml) for 96h and 48h, respectively, and cell viability was determined by CCK8 (see 3.2CCK 8). The results show that IFN α can significantly enhance the sensitivity of prostate cancer cells LNCaP and 22RV1 to Enz (fig. 14A), and that IFN α alone has no significant cytotoxic effect (fig. 14B).

To eliminate the effect of IFN α on the immune system, LNCaP-CRPC models were constructed in the complete immunodeficient mouse NOD-SCID using LNCaP-shNC and LNCaP-shIRF8 cell lines (see 7.1.2 for methods). Respectively given to Enz (10mg/kg), IFN alpha (1.5X 10)⁷IU/kg) or Enz (10mg/kg) in combination with IFN α (1.5X 10)⁷IU/kg), detecting the recurrence and growth curve of the tumor. The results show that the growth of the tumor of the Enz and IFN alpha combined group is remarkably reduced (fig. 14C), the tumor weight of the Enz and IFN alpha combined group at the experimental end point is remarkably different from that of the Enz used alone, and the tumor inhibition rate is 86.3 and is higher than 62.1 percent of that of the Enz used alone; and the recurrence rate of IFN alpha combined with Enz group is 58.3%, which is far lower than 90.9% of that of Enz group alone (FIG. 14D), while the tumor recurrence rate of each treatment group with knockdown IRF8 is 100%, the tumor growth and tumor weight are not different (FIGS. 14C-F), and the synergistic effect disappears. These results indicate that induction of IRF8 expression by prostate cancer in combination with IFN α during castration enhances the sensitivity of Enz.

Based on the above results, 3 high-risk prostate cancer patients were recruited to perform maximum androgen deprivation endocrine therapy and maximum androgen deprivation endocrine therapy with IFN α in combination, respectively, under ethical conditions. The specific scheme is shown in the chapter 7.2. The results show (fig. 15): after 2 patients block endocrine therapy by androgen to the maximum extent, the PSA level is continuously higher than 0.2ng/mL, the curative effect is poor, and the PSA level rebounds obviously after 6 months; the 2 patients were withdrawn from the experiment after 6 months with other therapies. Two patients are recruited in the IFN alpha combined endocrine therapy group, one patient keeps using the IFN alpha combined endocrine therapy during the treatment period of 11 months, the PSA level is continuously kept below the biochemical recurrence line (0.2ng/mL), and the effect is obvious. Another patient had a gradual decrease in PSA after 3 months of continued treatment. These results indicate that IFN alpha can be combined with endocrine therapy to obviously enhance the clinical treatment efficacy of the prostatic cancer.

Example 8IRF8 is reduced in expression in clinical liver cancer patients; increasing IRF8 expression inhibits its tumorigenic properties; IFN α induces IRF8 expression, inhibiting AR expression.

TCGA database analysis IRF8 was significantly less expressed in liver cancer patients than in normal liver tissue (fig. 16A). Immunohistochemical results showed that IRF8 expression was significantly lower in clinical liver cancer patients than in paraneoplastic tissues (fig. 16B). Epidemiological studies have shown that men have high incidence of liver cancer, possibly associated with abnormal elevation of AR in androgen receptor. The diethyl nitrosamine can cause mouse liver cancer, and WB detection results show that the expression of IRF8 is reduced and AR is increased in DEN-induced mouse liver cancer model cancer tissues (figure 16C). Analysis of the TCGA and GEO databases showed that IRF8 expression was high in liver cancer patients, and the overall survival time of liver cancer patients was significantly higher than that of liver cancer patients with low IRF8 expression (fig. 16D-E). Therefore we hypothesized whether increasing IRF8 expression in liver cancer patients could reduce its tumorigenic properties?

After the mouse liver cancer cell Hep1-6 transiently over-expresses IRF8, cell proliferation and clone formation are obviously inhibited (FIGS. 17A-C). Meanwhile, after Hep1-6 stably over-expresses IRF8 by using lentivirus transfection, the cell proliferation, scratch migration and balling capacity of the liver cancer cell are all inhibited, and further, the fact that the expression of IRF8 is increased is proved to inhibit the tumorigenic characteristics of the liver cancer cell (fig. 17D-F). After the hepatoma cells Bel7404 and HepG2 over-expressed IRF8, WB detection showed gradient-dependent decrease of AR expression (FIGS. 18A-B). After the murine hepatoma carcinoma cell Hep1-6 stably over-expresses IRF8 (with His tag), AR expression is remarkably reduced (FIG. 18C), and IRF8 in the hepatoma carcinoma cell is further proved to reduce AR expression. In clinical treatment, IFN alpha is mainly used for resisting hepatitis B virus infection of liver cancer patients. Our study results showed that IRF8 gradient dependence was increased and AR gradient dependence was decreased after different concentrations of IFN α treatment of hepatoma cells Bel7404 and HepG 224 h (FIGS. 18D-E). Therefore, in the liver cancer patient, IFN alpha can up-regulate IRF8 expression through AR/IRF8 axis, reduce the malignancy degree of the liver cancer cell, reduce AR expression at the same time, and obviously enhance the prognosis of the liver cancer patient.

Acronyms

Claims (1)

1. The application of the substance for improving the expression level of IRF8 in preparing the medicine for treating liver cancer is disclosed, wherein the substance for improving the expression level of IRF8 is a lentivirus over-expressing IRF 8.

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