Application of MIIP as a regulatory molecule and drug target for prostate cancer

Abstract

The present invention discloses the application of MIIP as a regulatory molecule and drug target for prostate cancer. Specifically, the present invention discloses the expression changes of MIIP in prostate cancer patients; the regulatory effect of MIIP on the proliferation, migration and invasion of prostate cancer cells; and the regulatory effect of MIIP in the tumor formation process of prostate cancer cells. Therefore, the present invention provides the application of MIIP as a drug target for the treatment of prostate cancer, and the present invention also relates to the application of MIIP as a target molecule for screening prostate cancer treatment, prevention drugs and related molecular diagnostic markers.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to prostate cancer regulatory molecules and applications thereof. Background Art

[0002] Prostate cancer (PCa) is the most common malignant tumor in men. In Europe and the United States, it ranks first in male tumor incidence and second in mortality. In recent years, with the aging of the population and the improvement of diagnostic technology, the incidence of prostate cancer in my country has also been increasing year by year. The latest statistics show that the incidence rate in the Chinese population is 4.3 / 100,000, and about 5% of patients have already had distant metastasis when they are first diagnosed. The pathogenesis of PCa is still not fully understood. Studies have shown that 86-98% of PCa are hormone-dependent tumors.

[0003] Androgen deprivation therapy (ADT) is the main method for treating first-episode PCa, but most PCa patients, especially those with high Gleason scores that have metastasized, will progress to metastatic castration-resistant prostate cancer (CRPC). Metastatic CRPC is the final stage of PCa progression. Under existing medical conditions, the survival time is generally less than 2 years, and it is the leading cause of death in PCa patients. Therefore, clarifying the molecular mechanism of CRPC progression and discovering effective signaling molecule therapeutic targets have become current research hotspots.

[0004] The molecular mechanism of CRPC progression is very complex. Existing studies have revealed that the disorder of AR signaling pathway is the main cause of CRPC. Androgen synthesis in the tumor, upregulated expression of AR receptors, mutations and splice variants will lead to increased sensitivity and abnormal activation of the AR signaling pathway, leading to the occurrence of CRPC. At present, drugs based on AR signaling regulation pathways, such as abiraterone and enzalutamide, have been used in the treatment of clinical CRPC. Although both can alleviate the progression of CRPC and prolong the survival of patients, they cannot ultimately prevent the progression of CRPC. This shows that the progression of CRPC is subject to more and more complex molecular regulation. Only by finding key regulatory molecules as therapeutic targets can CRPC be fundamentally treated.

[0005] MIIP (migration and invasion inhibitory protein) is a protein that interacts with IGFBP2 (insulin-like growth factor binding protein 2) discovered by researchers in glioma research using yeast two-hybrid in 2003. Because its molecular weight is about 45kDa, it is also called IIp45 (invasion inhibitory protein 45). The MIIP gene is located in the chromosome 1p36.2 region, with a total length of 12595bp and a total of 10 exons. The mRNA it encodes is 2024bp, and the protein contains 388 amino acid residues. It is widely expressed in various tissues in the human body. Structural analysis of the full-length MIIP protein found that it contains 3 SEG (segments of low compositional complexity) domains and an RGD (arginine-glycine-aspartic acid peptide) motif; there are also 42 serines, 6 threonines and 1 potential threonine phosphorylation site.

[0006] Functional studies on MIIP have fully shown that the MIIP gene is a new tumor suppressor gene, based on the following: ① The chromosome region 1p36.2 where the MIIP gene is located is deleted in many types of tumors, including gliomas, breast cancer, etc. ② In glioma and lung cancer tissues, MIIP expression is significantly lower than that in normal tissues, and its expression level is negatively correlated with the pathological stage and prognosis of lung cancer. It is currently believed that the mechanism of action of MIIP includes: A. MIIP binds to and antagonizes the action of IGFBP-2, inhibiting cell migration and invasion; B. Binds to and promotes the degradation of HDAC6, inhibiting the latter's deacetylase activity, thereby affecting microtubule formation and inhibiting cell migration; C. By directly binding to Cdc20, it blocks the degradation of Cyclin B1, causing cells to stagnate in the metaphase of mitosis, thereby inhibiting cell cycle progression and cell proliferation.

[0007] However, there are no reports on the expression status and role of MIIP in prostate cancer. Summary of the invention

[0008] The purpose of the present invention is to provide the application of MIIP as a regulatory molecule and drug target for prostate cancer.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] Application of migration and invasion inhibitory protein MIIP in the preparation of anti-tumor drugs for prostate cancer.

[0011] The anti-tumor drug is selected from a gene therapy drug containing a MIIP overexpression vector.

[0012] Application of agonists of migration and invasion inhibitory protein MIIP in the preparation of anti-tumor drugs for prostate cancer.

[0013] Application of migration and invasion inhibitory protein MIIP as a molecular diagnostic marker for prostate cancer.

[0014] Application of migration and invasion inhibitory protein MIIP as an agonist or inhibitor of prostate cancer progression regulatory molecules.

[0015] Application of migration and invasion inhibitory protein MIIP as a target molecule for screening of therapeutic drugs for prostate cancer.

[0016] The application of migration and invasion inhibitory protein MIIP as a detection index to evaluate the application of prostate cancer therapeutic drugs.

[0017] Application of migration and invasion inhibitory protein MIIP as a target molecule for screening molecular diagnostic markers for prostate cancer.

[0018] The beneficial effects of the present invention are embodied in:

[0019] The present invention has discovered for the first time through experiments that MIIP expression is reduced in prostate cancer and is associated with CRPC progression, revealing that MIIP can be used as a new prostate cancer regulatory factor, which not only provides a new theoretical basis for the pathogenesis of prostate cancer, but more importantly, can provide a new approach for the development of prostate cancer drugs, using MIIP as a target molecule to screen new prostate cancer therapeutic drugs, and MIIP can also be used as a detection indicator to evaluate prostate cancer therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The expression levels of MIIP in cancerous tissues and adjacent non-cancerous tissues of some prostate cancer patients (Patients) are compared; among them: ANT (adjacent non-cancerous tissues): adjacent non-cancerous tissues, CT (cancerous tissues): prostate cancer tissues.

[0021] Figure 2 The effects of MIIP on the malignant phenotype of CRPC cells; A. MIIP expression levels after transfection with MIIP overexpression plasmid or small interfering RNA (siRNA) detected by immunoblotting; B. Effect of MIIP expression regulation on cell growth; C. Effect of MIIP expression regulation on cell clone formation; D. Effect of MIIP expression regulation on cell invasion ability.

[0022] Figure 3The effect of stable knockdown of MIIP on the malignant phenotype of tumor formation in mice; A. Comparison of tumor volume; B. Comparison of tumor growth rate; C. Comparison of cell proliferation in tumor tissue of prostate cancer-bearing mouse model. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0024] The invention discloses the regulatory mechanism of migration and infiltration inhibitor protein MIIP in prostate cancer and its application as a drug target.

[0025] The present invention is conducted through the following experiments: (1) detecting MIIP in samples of prostate cancer population; (2) constructing MIIP overexpression and MIIP shRNA knockdown vectors, lentiviral packaging of MIIP cDNA and MIIP shRNA, establishing MIIP stable overexpression and MIIP shRNA stable knockdown prostate cancer cell lines, and detecting biological behavior of prostate cancer cells; (3) constructing MIIP shRNA stable knockdown prostate cancer tumor-bearing mouse model, and detecting tumor growth and tumor tissue cell proliferation in tumor-bearing mice. The results showed that in some prostate cancer patients, the expression of MIIP in cancer tissue was significantly lower than that in adjacent tissues; cell level studies confirmed that overexpression of MIIP in CRPC cell lines PC-3, DU-145, and C4-2, respectively, could significantly inhibit cell growth, clone formation, and invasion ability; while knocking down MIIP in PC-3 and DU-145 using siRNA significantly enhanced cell growth, clone formation, and invasion ability. These results indicate that MIIP can significantly inhibit the malignant phenotype of CRPC. Animal studies have found that nude mice tumor formation experiments were performed by injecting CRPC cell lines (PC-3-shMIIP and DU-145-shMIIP) with MIIP stably knocked down. Compared with the normal group, the experimental group showed malignant phenotypes such as increased tumor growth rate and enhanced invasiveness. Therefore, the present invention points out that MIIP plays an extremely important role in the progression of prostate cancer, especially in the malignant evolution of CRPC, and provides a new pathogenesis theoretical basis and therapeutic target for prostate cancer.

[0026] Unless otherwise indicated, the present invention will use chemical, biochemical and recombinant DNA techniques known to those skilled in the art. These techniques are fully explained in the literature. See, A Lehninger, Biochemistry (Worth Publishers, Inc. latest edition); Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd edition, 1989.

[0027] (1) Western Blot (WB) was used to detect the expression level of MIIP protein in cancer tissues of prostate cancer patients.

[0028] Patient tissue specimens were obtained from the Department of Urology, Xijing Hospital. Strong RIPA lysis buffer (Biyuntian) was used to lyse cells, and protease inhibitors PMSF and Cocktail (Sigma) were added before lysis. All operations were performed on ice, ultrasonically disrupted for 8 seconds, and the extracted proteins were stored at -80°C. The proteins were aliquoted to prevent repeated freezing and thawing. Before running the gel, loading buffer was added, mixed, and loaded directly. MIIP protein was separated by 10% SDS PAGE gel, and the molecular weight was about 42.8kD. GAPDH (Sigma) was used as an internal reference. For specific experimental results, see Figure 1 It can be seen that the expression of MIIP in prostate cancer tissue is significantly lower than that in adjacent tissues.

[0029] (2) Construction of MIIP shRNA stable knockdown and MIIP overexpression prostate cancer cell lines to detect the effect of MIIP on the malignant phenotype of prostate cancer cells

[0030] 1) Construction of MIIP shRNA and MIIP overexpression vectors and lentiviral packaging

[0031] First, MIIP siRNA was ordered from Shanghai Jikai Chemical Technology Co., Ltd., and two pairs of RNAi target sequences (siRNA1 and siRNA2) against MIIP were selected according to the siRNA sequences used and proved to be effective in previous experiments, and forward and reverse oligonucleotides were synthesized; after annealing, the oligonucleotide double strands were formed, and cloned into the pLKO.1-TRC vector (purchased from Addgene, catalog number: 10878) after restriction digestion and ligation, and constructed into pLKO.1-MIIP shRNA (restriction sites: AgeⅠ, EcoRⅠ), and the recombinant plasmid was co-transfected with the packaging plasmid psPAX2 and pMD2.G (Addgene) into 293T cells (purchased from: Chinese Academy of Sciences Cell Bank, catalog number: GNHu17) for virus packaging, and pLKO.1-sh MIIP virus particles were produced and secreted into the extracellular culture medium. The supernatant containing pLKO.1-sh MIIP virus particles was collected and stored at -80℃, which can be directly used for cell infection.

[0032] The MIIP overexpression system uses the pLEX-MCS lentiviral packaging system from Thermo Scientific. The pLEX-MCS-MIIP overexpression vector (restriction sites: SpeⅠ, XhoⅠ) was purchased and constructed using PCR and molecular cloning techniques. The recombinant plasmid was amplified and co-transfected with another two auxiliary / packaging plasmid mixtures (TLP4616 / TLP4617, Thermo Scientific) into 293T cells for virus packaging, producing pLEX-MCS-MIIP virus particles that were secreted into the extracellular culture medium. The supernatant containing pLEX-MCS-MIIP virus particles was collected and stored at -80℃, which can be directly used for cell infection.

[0033] 2) Establishment of MIIP shRNA and MIIP overexpression stable cell lines

[0034] ① Cell culture

[0035] The cells used in this experiment include hormone-independent / CRPC cell lines (C4-2, DU-145, PC3) and human embryonic kidney cells (293T cells), which were routinely cultured. Among them, C4-2 was obtained by transplanting LNCaP (derived from lymph node metastases of PCa patients) into tumor mice and screening by castration, and is AR positive; DU-145 and PC3 are derived from brain and bone metastases of CRPC patients, respectively, and are both AR negative. Cell lines DU-145, PC3, and 293T were purchased from the Chinese Academy of Sciences Cell Bank (Catalog Numbers: TCHu222, SCSP-532, and GNHu17), and C4-2 originated from the Urology Department of Xijing Hospital, Fourth Military Medical University. (Reference: [1] Murphy G, editor. Models for prostate cancer. 37. New York: Liss; 1980, pp. 115-132. [2] Gibas Z, et al. A high-resolution study of chromosome changes in a human prostatic carcinoma cell line (LNCaP). Cancer Genet. Cytogenet. 11: 399-404, 1984. [3] Papsidero LD,et al.Prostate antigen: a marker for human prostate epithelialcells.J.Natl.Cancer Inst.66:37-42,1981.[4]Stone KR,et al.Isolation of a humanprostate carcinoma cell line(DU 145).Int.J.Cancer 21:274-281,1978.[5]KaighnME,et al.Establishment and characterization of a human prostatic carcinoma cell line (PC-3). Invest. Urol. 17: 16-23, 1979. [6] Chen TR. Chromosome identity of human prostate cancer cell lines, PC-3 and PPC-1. Cytogenet. Cell Genet. 62: 183-184, 1993.[7]El Etreby MF,et,al,Antitumor activity of mifepristone in the human LNCaP,LNCaP-C4,and LNCaP-C4-2 prostate cancer models in nudemice.Prostate.2000 Feb 1;42(2):99-106.[8]Xie BX,et,al.The radiation response of androgen-refractory prostate cancer cell line C4-2 derived from androgen-sensitive cell line LNCaP.Asian J Androl.2010 May;12(3):405-14.doi:10.1038 / aja.2009.91.).

[0036] ②Screening of drug concentration

[0037] The drugs used for cell screening after transfection are mainly bleomycin and puromycin. Before establishing a stable cell line, it is necessary to determine the optimal drug concentration that can kill cells in 7-10 days. The method is as follows: Inoculate cells in a 24-well plate. When the cell density reaches 50%-70% fusion, add the drug to the culture wells at increasing concentrations. Observe cell death, change the medium every other day, and determine the optimal concentration for killing cells for subsequent screening of stable cell lines.

[0038] ③ Virus infection and establishment of stable cell lines

[0039] Overexpression or knockdown of MIIP in CRPC cell lines C4-2, DU-145, and PC3: Cells were cultured overnight and used for viral transfection when they reached about 70% confluence. Prepared viral supernatant containing pLEX-MCS-MIIP or pLKO.1-shMIIP was added to the cells in the logarithmic growth phase at a volume ratio of 1:1. After 24 hours, the optimal concentration of drug was added for screening to establish stable cell lines with MIIP overexpression (MIIP group) and MIIP knockdown (MIIP siRNA1 group, MIIP siRNA2 group); At the same time, cells infected and screened with pLEX-MCS (Vector) or pLKO.1-scramble shRNA (NC) were used as the corresponding control groups, see Figure 2 A.

[0040] (3) Detection of biological behavior of prostate cancer cells

[0041] 1) MTT assay to detect cell growth

[0042] The cells in the logarithmic phase were collected and the concentration of the cell suspension was adjusted and added to a 96-well plate (2×10 3 / well), incubate the cells at 5% CO2 and 37°C for 24h, add 20μL of freshly prepared MTT (0.5mg / mL) to each well, continue incubation at 37°C for 4h, discard the MTT solution, and place on a shaker for 10min at a low speed to fully dissolve the crystals. Measure the absorbance of each well at 490nm on an ELISA reader. Set up 6 replicates for each group, measure continuously for 5-7 days, and draw a cell growth curve. Figure 2 B, It can be seen that overexpression of MIIP inhibits cell growth, while knockdown of MIIP promotes cell growth.

[0043] 2) Clone formation experiment

[0044] Take cells in the logarithmic growth phase, digest them with 0.25% trypsin and blow them into single cells, suspend the cells in the culture medium for later use. Dilute the cell suspension in gradient multiples, and take 100-200 cells and evenly inoculate them in a 60mm culture dish. Incubate at 37°C and 5% CO2 for 2-3 weeks. When clones visible to the naked eye appear, terminate the culture. Discard the supernatant and carefully wash twice with PBS. Fix with methanol for 15 minutes, stain with an appropriate amount of Giemsa staining solution for 10-30 minutes, and air dry. Invert the plate and superimpose a transparent film with a grid, count the number of clones with the naked eye or calculate the clone formation rate, see Figure 2 C, it can be seen that overexpression of MIIP inhibits cell clone formation, while knockdown of MIIP promotes cell clone formation.

[0045] 3) Transwell assay to detect cell migration and invasion ability

[0046] Matrigel was mixed with serum-free DMEM culture medium at a ratio of 1:6, plated in a Transwell chamber, and placed in an incubator at 37°C for 2 h to allow it to solidify. Cells in each group were digested with trypsin, and the cells were counted to prepare 2×10 4 / mL single cell suspension, inoculate 100μL in each Transwell chamber; add fresh DMEM culture medium containing serum to the 24-well plate, put the Transwell chamber into the 24-well plate, and continue to culture in a 37℃ incubator for 24h; wipe off the cells and Matrigel on the upper layer that have not penetrated the membrane, fix with ethanol, and stain with Giemsa. Randomly select 10 fields of view under an inverted microscope and count them, and take the average value, see Figure 2 D, It can be seen that overexpression of MIIP inhibits cell invasion.

[0047] According to the above experiments, it can be concluded that overexpression of MIIP in CRPC cell lines can significantly inhibit the growth, cloning and invasion of cancer cells; while knocking down MIIP by siRNA promotes cell growth, cloning and invasion. These results show the regulatory effect of MIIP on the proliferation, migration and invasion of prostate cancer cells, and the malignant phenotype of CRPC can be significantly inhibited by regulating the expression of MIIP.

[0048] (4) Verification of the in vivo function of MIIP in a prostate cancer-bearing mouse model

[0049] 1) Establishment of prostate cancer tumor-bearing mouse model

[0050] Male nude mice aged 5-7 weeks and weighing 18-22 g were selected as experimental subjects (10 mice / group) and CRPC cell lines with stable knockdown of MIIP (PC-3-shMIIP and DU-145-shMIIP) were subcutaneously inoculated to establish a prostate cancer tumor-bearing mouse model. 7 The inoculation was performed subcutaneously at the proximal hind limb of nude mice.

[0051] 2) Tumor growth and specimen collection in tumor-bearing mice

[0052] a) Tumor growth curve

[0053] The diet, behavior and tumor formation of tumor-bearing mice were routinely monitored daily. The size of the tumor was measured with a caliper every other day to monitor the change in its volume V, V = d × D 2 ×0.5 (d: long diameter of tumor, D: short diameter of tumor). Mice were killed after 5 weeks, tumor tissues were weighed, and tumor growth curves were drawn based on the measurement results. Statistical analysis was performed using t-tests between different groups. Figure 3 A and B, it can be seen that in the MIIP shRNA stable knockdown prostate cancer mouse model (shRNA1 group was knocked down by siRNA1, and shRNA2 group was knocked down by siRNA2), the tumor weight increased compared with the control group Scramble, and the tumor growth rate accelerated.

[0054] b) Ki-67 staining to detect cell proliferation

[0055] Specimen collection: When the mice were killed, some tumor tissues were collected to make paraffin sections. Routine treatment and fixation were performed, and the sections were blocked with 10% BSA / PBS at room temperature for 1 hour. Anti-Ki-67 antibody was added and incubated for 1 hour. After washing, biotin-labeled secondary antibody was added dropwise at room temperature for 1 hour. Peroxidase-coupled avidin was added dropwise, and then 0.1% DAB+0.05% H2O2 was added. The sections were treated for 10-30 minutes, counterstained with hematoxylin, and developed. Observe under a microscope and count the proportion of positive cells. See Figure 3 C, Ki-67 staining revealed that in the prostate cancer mouse model with stable knockdown of MIIP shRNA, cell proliferation was accelerated compared with the control group Scramble.

[0056] The above experiments revealed the regulatory role of MIIP in the tumorigenesis of prostate cancer cells. After stable knockdown in the CRPC cell line, the malignant phenotype of tumor formation in mice was significantly enhanced.

[0057] In summary, the present invention found that the expression of MIIP in tumor tissue samples of prostate cancer patients was significantly reduced, and cytological experiments and tumor-bearing nude mouse experiments confirmed that MIIP has the function of inhibiting the progression of prostate cancer. It is revealed that MIIP can be used as a key regulatory molecule to regulate the progression of prostate cancer and (or) the evolution of CRPC. MIIP can be used as a targeting molecule to screen prostate cancer treatment, prevention drugs and related molecular diagnostic markers.

Claims

1. The use of migration and infiltration inhibitory protein MIIP in the preparation of anti-tumor drugs for prostate cancer, characterized in that: The prostate cancer is castration-resistant prostate cancer.

2. The use according to claim 1, characterized in that: The anti-tumor drug is selected from a gene therapy drug containing a MIIP overexpression vector.

Images