CN115877005A - Application of Musashi-1 gene and protein as castration-resistant prostate cancer treatment target - Google Patents

Application of Musashi-1 gene and protein as castration-resistant prostate cancer treatment target Download PDF

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CN115877005A
CN115877005A CN202211132180.XA CN202211132180A CN115877005A CN 115877005 A CN115877005 A CN 115877005A CN 202211132180 A CN202211132180 A CN 202211132180A CN 115877005 A CN115877005 A CN 115877005A
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musashi
protein
castration
prostate cancer
gene
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井立佳
郭建全
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Abstract

The application provides an application of Musashi-1 gene and protein as castration-resistant prostate cancer treatment targets, and belongs to the fields of molecular biology and biomedicine. The Musashi-1 gene and the protein are used as the target for treating castration-resistant prostate cancer. The invention provides a novel therapeutic target aiming at castration-resistant prostate cancer, which can be effectively used for the development judgment, treatment scheme selection and/or prognosis evaluation of the castration-resistant prostate cancer, thereby providing a novel diagnosis agent and/or treatment agent for the castration-resistant prostate cancer for the field and having clinical application prospect.

Description

Application of Musashi-1 gene and protein as castration-resistant prostate cancer treatment target
Technical Field
The application relates to the technical field of molecular biology and biomedicine, in particular to application of a Musashi-1 gene and a Musashi-1 protein as a castration-resistant prostate cancer treatment target.
Background
Prostate Cancer (Prostate Cancer) is a common malignancy in men, and the incidence of Prostate Cancer is the first malignancy in men in western countries. The Chinese annual report on cancer 2019 shows that the incidence rate of prostate cancer in China ranks 6 th in male malignant tumors, but the incidence rate of prostate cancer is the first in nearly ten years. Clinically, androgen-driven therapy (ADT) is the primary approach to intermediate-to-late metastatic prostate cancer, but after a median period of 18 to 24 months, the vast majority of Castration-sensitive prostate cancers (CSPCs) are classified as Castration-resistant prostate cancers (CRPCs). CRPC patients have a very poor prognosis with a median survival of less than 2 years. Although various therapeutic approaches such as chemotherapy (docetaxel, cabazitaxel), radiotherapy (X-rays and brachytherapy radionuclides), new endocrine drug therapy (enzalutamide, abiraterone) and immunotherapy (Sipuleucel-T) are currently used to treat CRPC, the existing therapeutic approaches are still not completely effective in treating CRPC and, therefore, the search for new therapies for CRPC is of great importance.
Musashi-1 is an evolutionarily conserved RNA binding protein, contains 2 conserved RNA recognition domains at the N-terminus, and can regulate the translation level by specifically binding to the 3' untranslated region of the target gene. In general, musashi-1 acts as a stem cell gene to maintain stem cells in an undifferentiated state by regulating post-transcriptional translation processes. In recent years, musashi-1 is found to be related to the occurrence and development of tumors, and is overexpressed in various tumor tissues such as colorectal cancer, cervical cancer, pancreatic cancer, lung cancer, glioblastoma and the like to play a cancer promotion role, but the cancer promotion mechanisms of Musashi-1 are different, but the related discovery that Musashi-1 participates in the development of CRPC is not found at present.
Disclosure of Invention
In view of the above, the present application aims to provide an application of Musashi-1 gene and protein as a target for treating castration-resistant prostate cancer, so as to solve or partially solve the problems of the background art.
In view of the above, the present application provides, in a first aspect, a use of a Musashi-1 protein as a target for treating castration-resistant prostate cancer.
We find that the Musashi-1 protein has high expression level in CRPC tumor tissues and tumor cells for the first time and is related to the proliferation of the CRPC tumor cells. The research finds that the expression of Musashi-1 is related to the proliferation, migration and invasion capacity of CRPC tumor cells, and the inhibition of the expression of the Musashi-1 protein causes the reduction of the proliferation, migration and invasion capacity of the CRPC tumor cells, so that the Musashi-1 protein is a new target for potential castration-resistant prostate cancer treatment.
Further, the use of an inhibitor of the Musashi-1 protein for the manufacture of a medicament for the prevention and/or treatment of castration-resistant prostate cancer.
Further, the medicament comprises a pharmaceutically acceptable carrier and an effective amount of active ingredients, wherein the active ingredients are inhibitors of Musashi-1 protein.
Further, the Musashi-1 protein inhibitor is selected from an antibody of the Musashi-1 protein, a binding protein of the Musashi-1 protein, a compound for inhibiting the function of the Musashi-1 protein and/or a PROTAC medicament for targeted degradation of the Musashi-1 protein.
Wherein the PROTAC is a protein degradation Targeting complex (protein Targeting Chimeras).
Based on the same inventive concept, the application provides an application of Musashi-1 gene as a castration-resistant prostate cancer treatment target.
Further, the use of an inhibitor of the Musashi-1 gene for the preparation of a medicament for the prevention and/or treatment of castration-resistant prostate cancer.
Further, the medicine comprises a pharmaceutically acceptable carrier and an effective amount of active ingredients, wherein the active ingredients are inhibitors of Musashi-1 genes.
Furthermore, the Musashi-1 gene inhibitor is selected from one or more of Musashi-1 gene specific RNAi, musashi-1 gene specific micro RNA, musashi-1 gene specific gene editing drugs or Musashi-1 gene promoter inhibitor.
Based on the same inventive concept, the third aspect of the present application provides a medicament for preventing and/or treating castration-resistant prostate cancer, comprising a pharmaceutically acceptable carrier and effective amounts of the following active ingredients: an inhibitor of the Musashi-1 protein and/or an inhibitor of the Musashi-1 gene.
Further, the Musashi-1 protein inhibitor is selected from an antibody of the Musashi-1 protein, a binding protein of the Musashi-1 protein, a compound inhibiting the function of the Musashi-1 protein and/or a PROTAC medicament for targeted degradation of the Musashi-1 protein;
the Musashi-1 gene inhibitor is selected from one or more of Musashi-1 gene specific RNAi, musashi-1 gene specific micro RNA, musashi-1 gene specific gene editing drugs or Musashi-1 gene promoter inhibitor.
The term "effective amount" or "effective dose" refers to an amount that is functional or active in humans and/or animals and is acceptable to humans and/or animals.
The term "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
Such pharmaceutically acceptable carriers include, but are not limited to: water, saline, buffer, glycerol, ethanol, liposomes, lipids, proteins, protein-antibody conjugates, peptidic substances, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched with the mode of administration, and these are well known to those skilled in the art.
The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition of the invention can be prepared into injections, oral preparations (tablets, capsules, oral liquids), transdermal agents and sustained-release agents. For example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.
The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, etc.
From the above, it can be seen that the application of the Musashi-1 gene and protein provided by the present application as a castration-resistant prostate cancer therapeutic target provides a novel therapeutic target for castration-resistant prostate cancer, and the target can be effectively used for castration-resistant prostate cancer development judgment, treatment scheme selection and/or prognosis evaluation, so as to provide a novel castration-resistant prostate cancer diagnostic agent and/or therapeutic agent for the field, and have clinical application prospects.
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In order to more clearly illustrate the technical solutions in the present application or the related art, the drawings needed to be used in the description of the embodiments or the related art will be briefly introduced below, and it is obvious that the drawings in the following description are only embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 shows the comparison of the expression levels of Musashi-1 protein in castration-sensitive prostate cancer cell line LNCaP and castration-resistant prostate cancer cell lines PC-3 and DU-145; in FIG. 1, A is a representative protein band for detecting the expression level of Musashi-1 in three cell strains of LNCaP, PC-3 and DU-145 by western blot; in FIG. 1, B is the gray scale value analysis (n = 3) of the expression band of Musashi-1 in western blot detection LNCaP, PC-3 and DU-145 cell strains;
FIG. 2 is a comparison of the expression levels of Musashi-1 protein in castration-sensitive prostate cancer tissue and castration-resistant prostate cancer tissue; FIG. 2A is a photograph showing immunohistochemical results of Musashi-1 in CSPC cancer tissue and CRPC cancer tissue of a patient (representative 2 patients); FIG. 2B is a quantitative analysis of the results of immunohistochemistry for CSPC cancer tissue and CRPC cancer tissue Musashi-1 in patients (10 patients);
FIG. 3 shows that knocking down Musashi-1 protein inhibits the proliferation of castration-resistant prostate cancer cells PC-3 and DU-145; (in FIG. 3, untreated cells are denoted as cells, cells transfected with negative control siRNA are denoted as siNC, and cells transfected with Musashi-1-specific siRNA are denoted as siRNA);
FIG. 4 is a graph of the inhibition of castration-resistant prostate cancer cell migration of PC-3 and DU-145 by knockdown of the Musashi-1 protein; FIG. 4A is a photograph showing the comparison of the number of cells spanning the Transwell basement membrane between PC-3 and DU-145 cells before and after the Musashi-1 protein knockdown; FIG. 4B is a cell count analysis of PC-3 and DU-145 cells crossing the Transwell basement membrane before and after knocking down Musashi-1 protein (n = 3);
FIG. 5 shows that knocking down Musashi-1 protein inhibits invasion of castration-resistant prostate cancer cells PC-3 and DU-145; FIG. 5A is a photograph comparing the number of PC-3 and DU-145 cells crossing the stromal gel-coated Transwell basement membrane before and after knocking down the Musashi-1 protein; in FIG. 5B is the cytometric analysis of PC-3 and DU-145 cells crossing the matrigel coated Transwell basement membrane before and after knockdown of Musashi-1 protein (n = 3).
FIG. 6 is a depiction of the knockdown of Musashi-1 protein inhibiting the growth of castration-resistant prostate cancer tumors; in FIG. 6, A is the growth of the tumor-bearing PC-3 cells on the surface of the mouse before and after the Musashi-1 protein knock-down, and B is the growth of the tumor-bearing DU-145 cells on the surface of the mouse before and after the Musashi-1 protein knock-down (n = 3).
Detailed Description
To make the objects, technical solutions and advantages of the present disclosure more apparent, the present disclosure is further described in detail below with reference to specific embodiments.
It is to be noted that, unless otherwise defined, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are all conventional methods unless otherwise specified.
Materials and methods
1. Experimental Material
(1) Castration-sensitive prostate cancer cell line LNCaP and castration-resistant prostate cancer cell lines PC-3 and DU-145 were purchased from American Type Culture Collection (ATCC, USA).
(2) Castration-sensitive prostate cancer tissue and castration-resistant prostate cancer tissue are provided by the first hospital affiliated with the university of medical science, shanxi.
(3) anti-Musashi-1 antibody (Abcam, ab 5286).
(4)Matrigel matrix(Corning，354234)。
2. Experimental methods
(1) The expression of Musashi-1 protein in castration-sensitive prostate cancer cell strain LNCaP and castration-resistant prostate cancer cell strains PC-3 and DU-145 is detected by WesternBlotting.
The specific steps of WesternBlotting are as follows:
cell lysis: cells in a 6-well plate were collected in a 1.5mL EP tube using a cell scraper, washed with PBS, and added with RIPA (Strong) lysis solution (CW 2333S, century Biotechnology Co., ltd.), wherein protease inhibitor mixture (CW 2200S, century Biotechnology Co., ltd.) was added to RIPA lysis solution, lysed 30min on ice, centrifuged at 14000g and 10min at 4 ℃, and the supernatant was transferred to a new 1.5mL EP tube.
Protein concentration determination: the BSA standard was diluted with PBS according to the instructions in the BCA kit (kang century biotechnology ltd, CW 0014S), and then the solutions a and B in the BCA kit were mixed according to 50:1, respectively adding 25 mu L of diluted A-G BSA standard substance and protein samples to be detected into a marked 96-well plate, making 2-3 multiple wells for each sample to be detected, adding 200 mu L of BCA working solution into each well, fully and uniformly mixing, covering a 96-well plate cover, incubating at 37 ℃ for 30min, measuring the light absorption value of each sample and the BSA standard substance at 562nm by using an enzyme labeling instrument, drawing a standard curve, and calculating the protein concentration in the sample.
Protein denaturation: mu.g of each sample was denatured for 5min in a metal bath at 100 ℃ by adding 10. Mu.g of protein to 1/4 volume of 5 XSDS-PAGE protein loading buffer (well known as Centrio Biotechnology Co., ltd., CW 0027).
10% SDS-PAGE, sequentially adding 10. Mu.g of denatured protein sample to the wells of the gel column, concentrating the sample through the gel column with 18mA of current in the electrophoresis chamber, and then adding 25mA of current to separate the sample protein into the gel column.
And (3) membrane conversion, soaking the filter paper, the sponge and the PVDF membrane activated by the methanol and the separation gel after electrophoresis in an electric conversion buffer solution for 30min in advance, then clamping the filter paper, the sponge, the filter paper, the PVDF membrane, the separation gel, the filter paper, the sponge and the black clamping surface of the membrane in sequence, placing the membrane in a membrane converter, and pouring the electric conversion buffer solution into an ice bath to operate for 90min at constant pressure of 80V.
And (3) sealing, taking down the PVDF membrane after the membrane conversion is finished, placing the PVDF membrane in 5% skimmed milk powder prepared by 1XTBST, and sealing for 1h at room temperature.
Once anti-incubation, musashi-1 antibody (Abcam, ab52865, dilution ratio 1: 1000) and Tubulin antibody (parent biological research center, inc. # AF7011, dilution ratio 1: 3000) were diluted with 5% nonfat dry milk, shaken overnight at 4 ℃ and washed 4 times with 1X TBST, 15min each.
The secondary antibody was incubated, and horseradish peroxidase (HRP) -conjugated secondary antibody (SoC. # S0001, dilution ratio 1: 10000) was diluted with 5% skim milk powder, followed by shaking incubation at room temperature for 2h, and washing with 1X TBST for 15min each time.
Chemiluminescence imaging, eECL-A and eECL-B (kang century Biotechnology Ltd., CW 0049S) as follows 1:1, uniformly dripping the mixture on a PVDF membrane in a dark place, and carrying out exposure and imaging by using a chemiluminescence imager.
(2) The expression level of Musashi-1 in castration-sensitive prostate cancer tissues and castration-resistant prostate cancer tissues is detected through immunohistochemistry, and the cancer tissues are divided into a high expression group and a low expression group according to the degree and the area of staining. The method comprises the following specific steps:
(1) tissues were deparaffinized with xylene 2d,100%, 95%, 85%, 75% ethanol gradient hydration for 2min each, pbs hydration for 10min.
(2) And (3) performing antigen retrieval (p H6.0 citric acid retrieval solution) for 30min by microwave, and cooling to room temperature.
(3) The goat serum (Beijing Zhongxiu jin Qiao Biotech Co., ltd.) was sealed for 30min.
(4) The primary antibody was incubated at a dilution of 1: 100 of anti-MTH 2 antibody overnight at 4 ℃ and washed 3 times with PBS for 5min each.
(5) The secondary antibody was incubated with PV-6001 goat anti-rabbit Ig G/HRP polymer for 20min at room temperature, and washed 3 times with PBS for 5min each.
(6) DAB (Chinese shirt gold bridge) color development.
(7) Hematoxylin counterstaining, gradient dehydration and transparent mounting.
(3) Detection of knockdown Musashi-1 protein for inhibiting castration-resistant prostate cancer cell proliferation
Firstly, musashi-1 siRNA screened in a laboratory is used for knocking down the expression level of Musashi-1 mRNA in a PC3 cell strain and a DU145 cell strain respectively, then Western Blotting is carried out to verify the knocking-down effect, and finally, the influence of the instantaneous knocking-down of the Musashi-1 in the PC3 cell strain and the DU145 cell strain on the cell activity is detected by a CCK8 method. The method comprises the following specific steps:
(1) cell transfection: PC3 cells and DU145 cells were plated in 96-well plates at 1X104 cells per well and incubated for 24h, then 50nM siMusashi-1 and siControl were transfected per well using RFect small nucleic acid transfection reagent (hundred Biotechnology, inc.), 6 replicates per well.
(2) And (3) detecting the cell viability: after incubation at 37 ℃ for 1h in a 5% CO2 incubator with 10. Mu.L CCK8 (DOJINDO, japan Dojindo Chemicals) added to each well 24h, 48h and 72h after transfection, respectively, the absorbance was measured at 450nm using a microplate reader.
(4) Detection of capability of knocking down Musashi-1 protein to inhibit migration of castration-resistant prostate cancer cells
(1) Cell transfection: PC3 and DU145 cells were plated into 6-well plates with 8X105 cells per well. After 24h incubation, 50nM siMusashi-1 and siControl were transfected per well using RFect small nucleic acid transfection reagent (hundred Biotech Co., ltd.) with 3 replicates per group.
(2) Inoculating cells: after 72 hours of cell transfection, the cells in the 6-well plate were collected, the cells were resuspended in a serum-free medium and counted, 2000 cell suspensions per well were added to a transwell cell (Corning 3422,8.0 μm Polycarbon Membrane), the medium containing 10% FBS was added to the lower chamber, gently mixed, and left to stand at 37 ℃ for 24 hours in a 5-part CO2 incubator.
(3) Cell fixation: the medium in the upper and lower chambers was discarded, and 4% paraformaldehyde was added and fixed at room temperature for 15min.
(4) Cell staining: the fixation solution was discarded and crystal violet staining solution (Biyuntian Biotech Co., ltd., C0121) was added thereto, and the staining was carried out at room temperature for 10min.
(5) Cleaning: discard the staining solution, add deionized water to wash the chamber 2-3 times, and lightly wipe the chamber with a moist cotton swab.
(6) And (4) observation: and (5) observing under a microscope at 10X.
(7) Counting: the Image J software was used to count and analyze the observed cell images.
(5) Detection of inhibition of invasion ability of castration-resistant prostate cancer cells by knocking-down Musashi-1 protein
(1) Coating of basement membrane: matrigel (Corning 354234) was mixed with basal medium according to a ratio of 1:8 vol% dilution, mixing on ice, 45. Mu.L per well in a transwell upper chamber (Corning 3422,8.0 μm Polycarbonate Membrane), mixing crosswise, placing at 37 ℃ and 5% CO2 incubator overnight to polymerize the gel.
(2) Cell transfection: PC3 and DU145 cells were plated into 6-well plates with 8X105 cells per well. After 24h incubation, 50nM siMusashi-1 and siControl were transfected per well using RFect small nucleic acid transfection reagent (hundred Biotech, inc.), 3 replicates per group.
(3) Inoculating cells: after 72h of cell transfection, the cells in 6-well plates were collected, the cells were resuspended in serum-free medium and counted, 2000 cell suspensions per well were added to the transwell chamber previously coated with matrigel, medium containing 10% FBS was added to the lower chamber, gently mixed, placed at 37 ℃ and incubated in a CO2 incubator at 5% for 24h.
(4) Cell fixation: the medium in the upper and lower chambers was discarded, and 4% paraformaldehyde was added and fixed at room temperature for 15min.
(5) Cell staining: the fixation solution was discarded and 1% crystal violet staining solution was added to the solution, and the solution was stained at room temperature for 20min.
(6) Cleaning: discard staining solution add deionized water to wash the chamber 2-3 times and lightly wipe the chamber with a moist cotton swab.
(7) And (4) observation: and (4) observing under a microscope 10X.
(8) Counting: the Image J software was used to count and analyze the observed cell images.
(6) Detection of knockdown Musashi-1 protein for inhibiting growth of castration-resistant prostate cancer tumor
(1) Cell line selection: two CRPC cell lines, PC-3 and DU145, were used in the study and were normally subcultured.
(2) Tumor inoculation: the CRPC tumor cells of the mice in the logarithmic growth phase are digested, counted and prepared into 1 multiplied by 10 7 Selecting BALB/c male nude mice as model animals, injecting 0.2mL subcutaneously at the right anterior scapula of the mice, feeding for 10-15 days, measuring the size of the tumor every two days, and carrying out related experiments when the tumor grows to a proper volume.
(3) And (3) drug treatment: siRNA (Musashi-1 siRNA or negative control siRNA) was transfected into tumors using a small RFect transfection reagent (hundred Biotech, inc.), each of which was transfected with 200 nM siRNA, for three consecutive days from day one to day three. Untreated tumors were used as controls.
(4) And (3) tumor inhibition effect detection: the size of the tumor in each treatment group was measured, and the growth of the tumor was recorded to investigate the in vivo therapeutic effect.
(5) Tumor volume calculation: measuring the major diameter (a) and the minor diameter (b) of the tumor with a vernier caliper,recorded and expressed as follows (V = a × b) 2 And/2) converting tumor bodies.
3. MTH2 protein expression high-low scoring standard in the tissue chip:
(1) Dyeing strength: 0 (none); 1 (weak); 2 (middle); 3 (strong).
(2) Dyeing area: 0 (0%); 1 (1-25%); 2 (26-50%); 3 (51-75%); 4 (76-100%).
(3) The final score is the staining intensity X stained area: low expression (0-6); high expression (7-12).
4. Statistical method
Data analysis and charting were performed using software such as GraphPad Prism 6.0 and SPSS statistics version 19, and Student's t-test was used to compare the two sets of means.
Second, experimental results
1. Increased expression of Musashi-1 in castration-resistant prostate cancer cells
The expression level of Musashi-1 protein in castration-sensitive prostate cancer cell strains LNCaP and castration-resistant prostate cancer cell strains DU-145 and PC-3 is detected by Western blotting. In FIG. 1, A is a representative protein band for western blotting detection of expression levels of Musashi-1 in three cell strains including LNCaP, PC-3 and DU-145, and from the gray level of the band, it can be clearly seen that the expression level of Musashi-1 in the cell strains including PC-3 and DU-145 is higher than that of LNCaP. Then, 3 times of western blotting repeated detection are carried out on the Musashi-1 expression level in three cell strains of LNCaP, PC-3 and DU-145, and the Musashi-1 protein band gray value is quantitatively analyzed, so that the protein expression level is quantified. As shown at B in fig. 1, by analyzing the gray values we gave the following results: compared with the castration-sensitive prostate cancer cell strain LNCaP, the expression levels of Musashi-1 protein in the castration-resistant prostate cancer cell strains DU-145 and PC-3 are respectively up-regulated by about 4 times and 3 times, and the up-regulation is extremely obvious (the difference is that P is less than 0.05).
2. Increased expression of Musashi-1 in castration-resistant prostate cancer tissue
We obtained 10 castration-sensitive prostate cancer tissues and 10 castration-resistant prostate cancer tissues, and compared the expression level of Musashi-1 protein by immunohistochemistry. In FIG. 2, A is the results of immunohistochemistry for Musashi-1 protein in castration-sensitive prostate cancer tissue and castration-resistant prostate cancer tissue in two patients, and it can be observed that the staining intensity of Musashi-1 protein in castration-resistant prostate cancer tissue is higher than that in castration-sensitive prostate cancer tissue. We then performed immunohistochemical analysis of Musashi-1 protein and comparative analysis of Musashi-1 protein levels on 10 castration-sensitive prostate cancer tissues and 10 castration-resistant prostate cancer tissues. As shown in B of FIG. 2, the expression of Musashi-1 protein was significantly up-regulated in castration-resistant prostate cancer tissue compared to castration-sensitive prostate cancer tissue (P < 0.05 is a significant difference).
3. Knocking down proliferation capacity of Musashi-1 protein for inhibiting castration-resistant prostate cancer cell strain
The proliferation capacity changes of DU-145 and PC-3 castration-resistant prostate cancer cells after 24h, 48h and 72h of Musashi-1 protein knock-down are respectively detected by using a CCK8 method, the Musashi-1 protein in DU-145 and PC-3 cell strains is knocked down by transfecting Musashi-1 specific siRNA in the experimental process, and untreated cells and cells transfected with negative control siRNA are used as controls together. As shown in FIG. 3, after Musashi-1 protein is knocked down in DU-145 and PC-3 cell lines for 24h, 48h and 72h, the OD value in the cell well plate of the treated group is obviously reduced compared with that of the two control groups, which reflects that the number of cells is reduced, and indicates that the proliferation capacity of the cells is obviously reduced (P < 0.05 is an obvious difference).
4. Knockdown migration capacity of Musashi-1 protein for inhibiting castration-resistant prostate cancer cell strain
The change of the migration capacity of castration-resistant prostate cancer cell strains DU-145 and PC-3 after the Musashi-1 protein is knocked down is detected by using a transwell method, and the migration capacity of the cells is judged according to the number of the cells crossing a transwell basement membrane in the research. In FIG. 4, A is the visualized change of the migration ability of DU-145 and PC-3 cells after the Musashi-1 protein is knocked down by siRNA, and cells transfected with negative control siRNA are used as comparison. It was found that DU-145 and PC-3 cells cross the transwell basement membrane decreased in number after knocking down the Musashi-1 protein. In fig. 4, B is statistics of the number of DU-145 and PC-3 cells crossing the transwell basement membrane after the reduction of the Musashi-1 protein (n = 3), and the results show that the number of DU-145 and PC-3 cells crossing the transwell basement membrane after the reduction of the Musashi-1 protein is only one fourth of that of the control group (P < 0.05 is a significant difference), indicating that the cell migration ability is decreased.
5. Knockdown Musashi-1 protein inhibition castration-resistant prostate cancer cell line invasion capacity
We have detected the invasion ability change of castration-resistant prostate cancer cell strains DU-145 and PC-3 after the Musashi-1 protein is knocked down by using a transwell method, and the invasion ability of the cells is judged according to the number of the cells crossing a transwell basement membrane in the research. Unlike the migration ability test, matrigel is coated on the outer side of the basement membrane of the transwell when the invasion ability of cells is tested. In FIG. 5, A is the visual change of the invasion ability of DU-145 and PC-3 cells after the Musashi-1 protein is knocked down by siRNA, and cells transfected with negative control siRNA are used as comparison. It was found that DU-145 and PC-3 cells crossed the matrigel-coated transwell basement membrane in decreased numbers after knockdown of the Musashi-1 protein. In fig. 5, B is the statistics of the number of DU-145 and PC-3 cells crossing the stroma-coated transwell basement membrane after the knockdown of the Musashi-1 protein (n = 3), and the results show that the number of DU-145 and PC-3 cells crossing the transwell basement membrane after the knockdown of the Musashi-1 protein is only one third of that of the control group (P < 0.05 is a significant difference), indicating that the invasion capacity of the cells is reduced.
6. Knocking down Musashi-1 protein to inhibit growth capacity of castration-resistant prostate cancer tumor
We used castration-resistant prostate cancer cell lines DU-145 and PC-3 to inoculate tumors on the surface of nude mice respectively to study the growth inhibition of two tumors after knocking down Musashi-1 protein, and used transfected Musashi-1 siRNA as treatment, transfected negative control siRNA and no treatment as control, and siRNA transfection was continuously transfected for three days from the first day to the third day. FIG. 6A shows the growth inhibition of the tumor-bearing PC-3 cells after the Musashi-1 protein is knocked down by siRNA, and it can be found that the growth of the tumor-bearing PC-3 cells after the Musashi-1 protein is knocked down is significantly inhibited compared with the tumor transfected with negative control siRNA and the tumor not treated. B in FIG. 6 is the growth inhibition of DU-145 cell tumor-bearing tumor after the Musashi-1 protein is knocked down by siRNA, and it can be found that DU-145 tumor growth is significantly inhibited after the Musashi-1 protein is knocked down compared with the tumor transfected with negative control siRNA and the tumor without any treatment.
Third, the conclusion of the experiment
We find that the Musashi-1 protein has high expression level in castration-resistant prostate cancer tissues and cell strains for the first time, and is related to the proliferation, migration and invasion capacity of castration-resistant prostate cancer cells. The inhibition of the expression of the Musashi-1 protein leads to the reduction of the proliferation, migration and invasion abilities of castration-resistant prostate cancer cells, so that the Musashi-1 protein can be used as a novel target for potential castration-resistant prostate cancer treatment.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the concept of the present disclosure, also technical features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in detail for the sake of brevity.
The disclosed embodiments are intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalents, improvements, and the like that may be made within the spirit and principles of the embodiments of the disclosure are intended to be included within the scope of the disclosure.

Claims (10)

  1. Use of the Musashi-1 protein as a target for treating castration-resistant prostate cancer.
  2. Use of an inhibitor of the Musashi-1 protein for the preparation of a medicament for the prevention and/or treatment of castration-resistant prostate cancer.
  3. 3. Use according to claim 2, characterized in that: the medicament comprises a pharmaceutically acceptable carrier and an effective amount of active ingredients, wherein the active ingredients are inhibitors of Musashi-1 protein.
  4. 4. Use according to claim 3, characterized in that: the Musashi-1 protein inhibitor is selected from an antibody of the Musashi-1 protein, a binding protein of the Musashi-1 protein, a compound for inhibiting the function of the Musashi-1 protein and/or a PROTAC medicament for targeted degradation of the Musashi-1 protein.
  5. Application of Musashi-1 gene as a castration-resistant prostate cancer treatment target.
  6. Use of an inhibitor of the musashi-1 gene for the preparation of a medicament for the prevention and/or treatment of castration-resistant prostate cancer.
  7. 7. Use according to claim 6, characterized in that: the medicine comprises a pharmaceutically acceptable carrier and an effective amount of active ingredients, wherein the active ingredients are inhibitors of Musashi-1 genes.
  8. 8. Use according to claim 7, characterized in that: the Musashi-1 gene inhibitor is selected from one or more of Musashi-1 gene specific RNAi, musashi-1 gene specific micro RNA, musashi-1 gene specific gene editing drugs or Musashi-1 gene promoter inhibitor.
  9. 9. A medicament for the prevention and/or treatment of castration-resistant prostate cancer, comprising a pharmaceutically acceptable carrier and an effective amount of the following active ingredients: an inhibitor of the Musashi-1 protein and/or an inhibitor of the Musashi-1 gene.
  10. 10. The medicament of claim 9, wherein: the inhibitor of the Musashi-1 protein is selected from an antibody of the Musashi-1 protein, a binding protein of the Musashi-1 protein, a compound for inhibiting the function of the Musashi-1 protein and/or a PROTAC medicament for targeted degradation of the Musashi-1 protein;
    the Musashi-1 gene inhibitor is selected from one or more of Musashi-1 gene specific RNAi, musashi-1 gene specific micro RNA, musashi-1 gene specific gene editing drugs or Musashi-1 gene promoter inhibitor.
CN202211132180.XA 2022-08-23 2022-09-16 Application of Musashi-1 gene and protein as castration-resistant prostate cancer treatment target Pending CN115877005A (en)

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