Application of Musashi-1 gene and protein as therapeutic targets for castration-resistant prostate cancer

Abstract

The present application provides an application of Musashi-1 gene and protein as a therapeutic target for castration-resistant prostate cancer, which belongs to the field of molecular biology and biomedicine. Application of Musashi-1 gene and protein as a therapeutic target for castration-resistant prostate cancer. The present invention provides a new therapeutic target for castration-resistant prostate cancer, which can be effectively used for the development judgment, treatment regimen selection and / or prognosis evaluation of castration-resistant prostate cancer, thereby providing a novel diagnostic agent and / or therapeutic agent for castration-resistant prostate cancer in the art, which has clinical application prospects.

Description

Technical Field

[0001] The present application relates to the fields of molecular biology and biomedicine technology, and in particular to the application of a Musashi-1 gene and protein as a therapeutic target for castration-resistant prostate cancer. Background Art

[0002] Prostate cancer is a common malignant tumor in men. In Western countries, the incidence of prostate cancer ranks first among male malignant tumors. According to the 2019 China Cancer Annual Report, the incidence of prostate cancer in my country ranks sixth among male malignant tumors, but its incidence rate has increased the fastest in the past decade. Clinically, androgen deprive therapy (ADT) is the main treatment for advanced metastatic prostate cancer, but after a median of 18 to 24 months, the vast majority of castration-sensitive prostate cancer (CSPC) will transform into castration-resistant prostate cancer (CRPC). The prognosis of CRPC patients is extremely poor, with a median survival of less than 2 years. Although there are currently multiple treatment options for CRPC, such as chemotherapy (docetaxel, cabazitaxel), radiotherapy (X-rays and brachytherapy), new endocrine drug therapy (enzalutamide, abiraterone), and immunotherapy (sipuleucel-T), existing treatments are still not completely effective in treating CRPC. Therefore, it is of great significance to explore new therapies for CRPC.

[0003] Musashi-1 is an evolutionarily conserved RNA-binding protein with two conserved RNA recognition domains at its N-terminus. It can regulate the translation level of target genes by specifically binding to their 3' untranslated regions. Generally, Musashi-1, as a stem cell gene, maintains stem cells in an undifferentiated state by regulating the post-transcriptional translation process. Recent studies have found that Musashi-1 is associated with the occurrence and development of tumors. Overexpression in various tumor tissues, including colorectal cancer, cervical cancer, pancreatic cancer, lung cancer, and glioblastoma, plays a pro-oncogenic role, but the mechanisms of its promotion vary. Currently, no findings have shown that Musashi-1 is involved in the progression of CRPC. Summary of the Invention

[0004] In view of this, the purpose of this application is to propose an application of Musashi-1 gene and protein as a therapeutic target for castration-resistant prostate cancer, so as to solve or partially solve the problems raised in the background technology.

[0005] Based on the above objectives, the first aspect of the present application provides an application of Musashi-1 protein as a therapeutic target for castration-resistant prostate cancer.

[0006] We have discovered for the first time that Musashi-1 protein expression is elevated in CRPC tumor tissues and cells and correlates with CRPC cell proliferation. Our study found that Musashi-1 expression is associated with the proliferation, migration, and invasion of CRPC tumor cells. Inhibiting Musashi-1 expression reduces the proliferation, migration, and invasion of CRPC tumor cells. Therefore, Musashi-1 protein is a potential new target for the treatment of castration-resistant prostate cancer.

[0007] Furthermore, the invention relates to use of an inhibitor of Musashi-1 protein in the preparation of a drug for preventing and / or treating castration-resistant prostate cancer.

[0008] Furthermore, the drug comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is an inhibitor of Musashi-1 protein.

[0009] Furthermore, the inhibitor of Musashi-1 protein is selected from antibodies to Musashi-1 protein, binding proteins to Musashi-1 protein, compounds that inhibit the function of Musashi-1 protein and / or PROTAC drugs that target and degrade Musashi-1 protein.

[0010] Among them, the PROTAC is a protein degradation targeting consortium (Proteolysis Targeting Chimeras).

[0011] Based on the same inventive concept, the second aspect of the present application provides an application of the Musashi-1 gene as a therapeutic target for castration-resistant prostate cancer.

[0012] Furthermore, the invention relates to use of an inhibitor of the Musashi-1 gene in the preparation of a drug for preventing and / or treating castration-resistant prostate cancer.

[0013] Furthermore, the drug comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is an inhibitor of the Musashi-1 gene.

[0014] Furthermore, the inhibitor of the Musashi-1 gene 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 inhibitors that inhibit the Musashi-1 gene promoter.

[0015] Based on the same inventive concept, the third aspect of the present application provides a drug for preventing and / or treating castration-resistant prostate cancer, comprising a pharmaceutically acceptable carrier and an effective amount of the following active ingredients: an inhibitor of Musashi-1 protein and / or an inhibitor of Musashi-1 gene.

[0016] Furthermore, the inhibitor of Musashi-1 protein is selected from antibodies to Musashi-1 protein, binding proteins to Musashi-1 protein, compounds that inhibit the function of Musashi-1 protein and / or PROTAC drugs that target and degrade Musashi-1 protein;

[0017] The inhibitor of the Musashi-1 gene 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 inhibitors that inhibit the Musashi-1 gene promoter.

[0018] The term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0019] The term "pharmaceutically acceptable" refers to a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents.

[0020] The pharmaceutically acceptable carrier includes, but is not limited to, water, saline, buffer, glycerol, ethanol, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be compatible with the mode of administration, as is well known to those skilled in the art.

[0021] 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. Generally, the pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared as an injection, oral preparation (tablet, capsule, oral solution), transdermal preparation, or sustained-release formulation. For example, the pharmaceutical composition can be prepared using conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

[0022] 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. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration.

[0023] From the above, it can be seen that the application of the Musashi-1 gene and protein provided in this application as a therapeutic target for castration-resistant prostate cancer provides a new therapeutic target for castration-resistant prostate cancer. This target can be effectively used for the judgment of the development of castration-resistant prostate cancer, the selection of treatment options and / or the prognosis evaluation, thereby providing a novel diagnostic agent and / or therapeutic agent for castration-resistant prostate cancer in the field, which has clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in this application or related technologies, the following briefly introduces the drawings required for use in the embodiments or related technical descriptions. Obviously, the drawings described below are merely embodiments of this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0025] Figure 1 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; Figure 1 Middle A is the representative protein band of Musashi-1 expression level in three cell lines, LNCaP, PC-3 and DU-145, detected by western blot; Figure 1 Middle B is the gray value analysis of Musashi-1 expression bands in LNCaP, PC-3 and DU-145 cell lines detected by western blot (n=3);

[0026] Figure 2 Comparison of Musashi-1 protein expression levels in castration-sensitive prostate cancer tissues and castration-resistant prostate cancer tissues; Figure 2 Middle A is the immunohistochemical results of Musashi-1 in CSPC cancer tissue and CRPC cancer tissue of patients (representative 2 patients); Figure 2 Middle B is a quantitative analysis of the Musashi-1 immunohistochemistry results of CSPC cancer tissues and CRPC cancer tissues of patients (10 patients);

[0027] Figure 3Knockdown of Musashi-1 protein inhibits the proliferation of castration-resistant prostate cancer cells PC-3 and DU-145; ( Figure 3 In the figure, untreated cells are denoted as Cell, cells transfected with negative control siRNA are denoted as siNC, and cells transfected with Musashi-1-specific siRNA are denoted as siRNA);

[0028] Figure 4 Knockdown of Musashi-1 protein inhibits the migration of castration-resistant prostate cancer cells PC-3 and DU-145; Figure 4 Middle A is a comparison of the number of PC-3 and DU-145 cells crossing the Transwell basement membrane before and after knockdown of Musashi-1 protein; Figure 4 Middle B is the cell counting analysis of PC-3 and DU-145 cells crossing the Transwell basement membrane before and after knockdown of Musashi-1 protein (n=3);

[0029] Figure 5 Knockdown of Musashi-1 protein inhibits the invasion of castration-resistant prostate cancer cells PC-3 and DU-145; Figure 5 Middle A is a comparison of the number of PC-3 and DU-145 cells crossing the Matrigel-coated Transwell basement membrane before and after knockdown of Musashi-1 protein; Figure 5 Middle B is the cell counting analysis of PC-3 and DU-145 cells crossing the Transwell basement membrane coated with matrigel before and after knockdown of Musashi-1 protein (n=3).

[0030] Figure 6 Knockdown of Musashi-1 protein inhibits castration-resistant prostate cancer tumor growth; Figure 6 A in the middle shows the growth of PC-3 cell tumors on the surface of mice before and after knockdown of Musashi-1 protein. Figure 6 Middle B shows the growth of DU-145 cell-bearing tumors on the surface of mice before and after knockdown of Musashi-1 protein (n=3). DETAILED DESCRIPTION

[0031] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the present disclosure is further described in detail below with reference to specific embodiments.

[0032] It should be noted that, unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods described, unless otherwise specified, are all conventional methods.

[0033] 1. Materials and Methods

[0034] 1. Experimental Materials

[0035] (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).

[0036] (2) Castration-sensitive prostate cancer tissue and castration-resistant prostate cancer tissue were provided by the First Affiliated Hospital of Shanxi Medical University.

[0037] (3) Anti-Musashi-1 antibody (Abcam, ab5286).

[0038] (4) Matrigel matrix (Corning, 354234).

[0039] 2. Experimental Methods

[0040] (1) Western Blotting was used to detect the expression of Musashi-1 protein in castration-sensitive prostate cancer cell line LNCaP and castration-resistant prostate cancer cell lines PC-3 and DU-145.

[0041] The specific steps of Western Blotting are as follows:

[0042] Cell lysis: Cells in a 6-well plate were collected into a 1.5 mL EP tube using a cell scraper. After washing with PBS, cells were added to RIPA (Strong) lysis buffer (Congwei Century Biotechnology Co., Ltd., CW2333S) supplemented with a protease inhibitor cocktail (Congwei Century Biotechnology Co., Ltd., CW2200S). The cells were lysed on ice for 30 min, centrifuged at 14,000 g for 10 min at 4°C, and the supernatant was transferred to a new 1.5 mL EP tube.

[0043] Protein concentration determination: According to the instructions of the BCA kit (Kangwei Century Biotechnology Co., Ltd., CW0014S), the BSA standard was diluted with PBS, and then the A solution and B solution in the BCA kit were prepared at a ratio of 50:1 to prepare the BCA working solution. 25 μL of the diluted AG BSA standard and the protein sample to be tested were added to a labeled 96-well plate. Each sample to be tested was prepared in 2-3 replicates, and 200 μL of BCA working solution was added to each well. The plates were thoroughly mixed, and the plates were covered with a 96-well lid. The plates were incubated at 37°C for 30 min. The absorbance of each sample and the BSA standard was measured at 562 nm using a microplate reader. A standard curve was drawn to calculate the protein concentration in the samples.

[0044] Protein denaturation: 10 μg of protein was taken from each sample and added into 1 / 4 volume of 5× SDS-PAGE protein loading buffer (CW0027, Kangwei Century Biotechnology Co., Ltd.), and denatured in a metal bath at 100°C for 5 min.

[0045] 10% SDS-PAGE electrophoresis, add 10 μg of denatured protein sample into the wells of the stacking gel in sequence, first use 18 mA current in the electrophoresis tank to make the sample pass through the stacking gel for concentration, then increase the current to 25 mA to allow the sample protein to enter the separation gel for separation.

[0046] For membrane transfer, soak the filter paper, sponge, methanol-activated PVDF membrane and the separation gel after electrophoresis in electrotransfer buffer for 30 minutes in advance. Then clamp the membrane in the order of the white side of the transfer clamp, sponge, filter paper, PVDF membrane, separation gel, filter paper, sponge and the black side of the transfer clamp, place it in the transfer instrument, pour in the electrotransfer buffer and run at a constant voltage of 80V in an ice bath for 90 minutes.

[0047] After blocking, the PVDF membrane was removed and placed in 5% skim milk powder prepared in 1XTBST and blocked at room temperature for 1 hour.

[0048] For primary antibody incubation, Musashi-1 antibody (Abcam, ab52865, dilution ratio 1:1000) and Tubulin antibody (Probiotics Research Center Co., Ltd., #AF7011, dilution ratio 1:3000) were diluted with 5% skim milk powder, incubated on a shaker at 4°C overnight, and washed 4 times with 1XTBST, each time for 15 minutes.

[0049] For secondary antibody incubation, horseradish peroxidase (HRP)-conjugated secondary antibody (ProBiological Research Center Co., Ltd., #S0001, dilution ratio 1:10000) was diluted with 5% skim milk powder and incubated on a shaker at room temperature for 2 h. The sections were then washed four times with 1X TBST for 15 min each time.

[0050] For chemiluminescence imaging, eECL-A and eECL-B (CW0049S, Kangwei Century Biotechnology Co., Ltd.) were mixed in equal volumes at a ratio of 1:1, and evenly dropped onto the PVDF membrane in the dark. The membrane was then exposed and imaged using a chemiluminescence imager.

[0051] (2) The expression of Musashi-1 in castration-sensitive prostate cancer tissue and castration-resistant prostate cancer tissue was detected by immunohistochemistry. The cancer tissues were divided into high expression group and low expression group according to the degree and area of staining. The specific steps are as follows:

[0052] ① The tissue was dewaxed in xylene for 2 days, hydrated in 100%, 95%, 85%, and 75% ethanol for 2 minutes each, and hydrated in PBS for 10 minutes.

[0053] ② Antigen retrieval (pH 6.0 citric acid retrieval solution) was performed by microwave retrieval for 30 min and then cooled to room temperature.

[0054] ③ Block with goat serum (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) for 30 min.

[0055] ④ Primary antibody incubation: anti-MTH2 antibody was diluted at a ratio of 1:100, incubated at 4°C overnight, and washed 3 times with PBS, each time for 5 min.

[0056] ⑤ Secondary antibody incubation: use PV-6001 goat anti-rabbit IgG / HRP polymer, incubate at room temperature for 20 minutes, and wash with PBS three times, 5 minutes each time.

[0057] ⑥DAB (Zhongshan Jinqiao) color development.

[0058] ⑦Hematoxylin counterstaining, gradient dehydration, and transparent sealing.

[0059] (3) Detection of the inhibition of castration-resistant prostate cancer cell proliferation by knocking down Musashi-1 protein

[0060] First, we used the Musashi-1 siRNA screened in the laboratory to knock down the expression level of Musashi-1 mRNA in PC3 and DU145 cell lines, respectively. Then, we performed Western blotting to verify the knockdown effect. Finally, we used the CCK8 assay to detect the effect of transient knockdown of Musashi-1 on cell viability in PC3 and DU145 cell lines. The specific steps are as follows:

[0061] ① Cell transfection: PC3 cells and DU145 cells were plated in 96-well plates at 1×10 4 cells per well. After incubation for 24 h, 50 nM siMusashi-1 and siControl were transfected per well using RFect small nucleic acid transfection reagent (Bio-Tech Co., Ltd.), with 6 replicate wells per group.

[0062] ② Cell viability detection: 24h, 48h and 72h after transfection, 10μL CCK8 (DOJINDO, Japan Dojindo Chemical) was added to each well and incubated in a dark incubator at 37°C and 5% CO2 for 1h. The absorbance was then measured at 450nm using a microplate reader.

[0063] (4) Detection of the ability of knockdown of Musashi-1 protein to inhibit the migration of castration-resistant prostate cancer cells

[0064] ① Cell transfection: PC3 and DU145 cells were plated into 6-well plates at 8 × 105 cells per well. After 24 h of incubation, 50 nM siMusashi-1 and siControl were transfected per well using RFect small nucleic acid transfection reagent (Bio-Biotech Co., Ltd.), with triplicate wells per group.

[0065] ② Inoculation of cells: 72 hours after cell transfection, collect cells from 6-well plates, resuspend the cells in serum-free medium and count them. Add 2000 cells per well of the suspension into a transwell chamber (Corning 3422, 8.0 μm Polycarbonate Membrane). Add medium containing 10% FBS to the lower chamber, mix gently, and incubate in a 37°C, 5% CO2 incubator for 24 hours.

[0066] ③Cell fixation: discard the culture medium in the upper and lower chambers, add 4% paraformaldehyde, and fix at room temperature for 15 minutes.

[0067] ④ Cell staining: discard the fixative and add crystal violet staining solution (Biyuntian Biotechnology Co., Ltd., C0121) and stain at room temperature for 10 minutes.

[0068] ⑤ Cleaning: Discard the staining solution and add deionized water to clean the chamber 2-3 times, and gently wipe the chamber with a wet cotton swab.

[0069] ⑥Observation: Observe under a microscope at 10X.

[0070] ⑦ Counting: Use Image J software to count and analyze the observed cell images.

[0071] (5) Detection of the ability of Musashi-1 protein knockdown to inhibit the invasion of castration-resistant prostate cancer cells

[0072] ① Coating with basement membrane: Dilute Matrigel (Corning 354234) and basal culture medium at a volume ratio of 1:8, mix well on ice, and spread 45 μL per well in the upper chamber of a transwell (Corning 3422, 8.0 μm Polycarbonate Membrane). Mix well and place in a 37°C, 5% CO2 incubator overnight to polymerize the gel.

[0073] ② Cell transfection: PC3 and DU145 cells were plated into 6-well plates at 8 × 105 cells per well. After 24 h of incubation, 50 nM siMusashi-1 and siControl were transfected per well using RFect small nucleic acid transfection reagent (Bio-Biotech Co., Ltd.), with triplicate wells per group.

[0074] ③ Inoculation of cells: 72 hours after cell transfection, collect the cells in the 6-well plate, resuspend the cells in serum-free medium and count them. Add 2000 cells per well of the suspension into the transwell chamber pre-coated with matrix gel, add culture medium containing 10% FBS to the lower chamber, mix gently, and incubate in a 37°C, 5% CO2 incubator for 24 hours.

[0075] ④Cell fixation: discard the culture medium in the upper and lower chambers, add 4% paraformaldehyde, and fix at room temperature for 15 minutes.

[0076] ⑤Cell staining: discard the fixative and add 1% crystal violet staining solution, and stain at room temperature for 20 minutes.

[0077] ⑥ Cleaning: Discard the staining solution and add deionized water to clean the chamber 2-3 times, and gently wipe the chamber with a wet cotton swab.

[0078] ⑦Observation: Observe under a microscope at 10X.

[0079] ⑧Counting: Use Image J software to count and analyze the observed cell images.

[0080] (6) Detection of the inhibition of castration-resistant prostate cancer tumor growth by knocking down Musashi-1 protein

[0081] ① Cell line selection: Two CRPC cell lines, PC-3 and DU145, were used in this study and cultured normally.

[0082] ② Tumor inoculation: Mouse CRPC tumor cells in the logarithmic growth phase were digested, counted and prepared into 1×10 7 BALB / c male nude mice were selected as model animals. 0.2 mL was injected subcutaneously into the scapula of the right forelimb of the mice. The mice were raised for 10-15 days. The tumor size was measured every two days during this period. Relevant experiments were performed when the tumor grew to an appropriate size.

[0083] ③ Drug treatment: siRNA (Musashi-1 siRNA or negative control siRNA) was transfected into the tumor using RFect small nucleic acid transfection reagent (EMI Biotechnology Co., Ltd.) at a concentration of 200 nM per tumor for three consecutive days from day 1 to day 3. Untreated tumors served as controls.

[0084] ④ Tumor inhibition effect detection: Measure the size of the tumor in each treatment group and record the growth of the tumor to examine its in vivo therapeutic effect.

[0085] ⑤ Tumor volume calculation: Use a vernier caliper to measure the long diameter (a) and short diameter (b) of the tumor, record and calculate the volume according to the following formula (V = a × b 2 / 2)Convert to tumor volume.

[0086] 3. Scoring criteria for MTH2 protein expression in tissue microarrays:

[0087] (1) Staining intensity: 0 (none); 1 (weak); 2 (moderate); 3 (strong).

[0088] (2) Staining area: 0 (0%); 1 (1-25%); 2 (26-50%); 3 (51-75%); 4 (76-100%).

[0089] (3) The final score is the staining intensity × staining area: low expression (0-6); high expression (7-12).

[0090] 4. Statistical methods

[0091] GraphPad Prism 6.0 and SPSS statistics version 19 were used for data analysis and graph preparation, and Student's t-test was used to compare the means of the two groups.

[0092] 2. Experimental results

[0093] 1. Increased expression of Musashi-1 in castration-resistant prostate cancer cells

[0094] We detected the expression level of Musashi-1 protein in castration-sensitive prostate cancer cell line LNCaP and castration-resistant prostate cancer cell lines DU-145 and PC-3 by Western blotting. Figure 1 Figure A shows representative protein bands detected by western blotting for Musashi-1 expression in LNCaP, PC-3, and DU-145 cell lines. The grayscale intensity of the bands clearly indicates that Musashi-1 expression levels in PC-3 and DU-145 are higher than in LNCaP. Subsequently, we performed three western blotting replicates to detect Musashi-1 expression in LNCaP, PC-3, and DU-145 cell lines and quantitatively analyzed the grayscale values of the Musashi-1 protein bands to quantify protein expression levels. Figure 1 As shown in Figure B, by analyzing the grayscale values, we obtained the following results: compared with the castration-sensitive prostate cancer cell line LNCaP, the expression levels of Musashi-1 protein in the castration-resistant prostate cancer cell lines DU-145 and PC-3 were increased by approximately 4 times and 3 times, respectively, and the increase was extremely significant (P < 0.05 was considered a significant difference).

[0095] 2. Increased expression of Musashi-1 in castration-resistant prostate cancer tissue

[0096] We obtained 10 castration-sensitive prostate cancer tissues and 10 castration-resistant prostate cancer tissues and compared the expression levels of Musashi-1 protein by immunohistochemistry. Figure 2 Figure A shows the immunohistochemical results of Musashi-1 protein in castration-sensitive prostate cancer tissue and castration-resistant prostate cancer tissue of two patients. 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. Subsequently, we performed immunohistochemical analysis of Musashi-1 protein in 10 cases of castration-sensitive prostate cancer tissue and 10 cases of castration-resistant prostate cancer tissue and compared the levels of Musashi-1 protein. Figure 2 As shown in Figure B, compared with castration-sensitive prostate cancer tissue, the expression of Musashi-1 protein in castration-resistant prostate cancer tissue was significantly upregulated (P < 0.05 was considered a significant difference).

[0097] 3. Knockdown of Musashi-1 protein inhibits the proliferation of castration-resistant prostate cancer cell lines

[0098] We used CCK8 assay to detect the changes in the proliferation ability of two castration-resistant prostate cancer cells, DU-145 and PC-3, after knocking down Musashi-1 protein for 24h, 48h and 72h. During the experiment, we knocked down Musashi-1 protein in DU-145 and PC-3 cell lines by transfecting Musashi-1 specific siRNA, and used untreated cells and cells transfected with negative control siRNA as controls. Figure 3 As shown in the figure, after knocking down Musashi-1 protein in DU-145 and PC-3 cell lines for 24h, 48h and 72h, the OD value in the cell well plate of the treatment group showed a significant decrease compared with the two control groups, reflecting a decrease in cell number, indicating that the cell proliferation ability was significantly decreased (P < 0.05 was a significant difference).

[0099] 4. Knockdown of Musashi-1 protein inhibits the migration ability of castration-resistant prostate cancer cell lines

[0100] We used the transwell assay to detect changes in the migration ability of castration-resistant prostate cancer cell lines DU-145 and PC-3 after knockdown of Musashi-1 protein. In this study, the number of cells crossing the transwell basement membrane was used to evaluate the migration ability of cells. Figure 4Center A shows the changes in the migration ability of DU-145 and PC-3 cells after knockdown of Musashi-1 protein using siRNA, compared with cells transfected with negative control siRNA. It can be seen that the number of DU-145 and PC-3 cells crossing the transwell basement membrane decreased after knockdown of Musashi-1 protein. Figure 4 Middle B is the statistics of the number of DU-145 and PC-3 cells crossing the transwell basement membrane after knocking down Musashi-1 protein (n=3). The results showed that after knocking down Musashi-1 protein, the number of DU-145 and PC-3 cells crossing the transwell basement membrane was only one-fourth of that in the control group (P<0.05 was a significant difference), indicating that the cell migration ability was reduced.

[0101] 5. Knockdown of Musashi-1 protein inhibits the invasive ability of castration-resistant prostate cancer cell lines

[0102] We used a transwell assay to examine the invasiveness of castration-resistant prostate cancer cell lines DU-145 and PC-3 following knockdown of Musashi-1 protein. In this study, invasiveness was assessed by the number of cells crossing the transwell basement membrane. Unlike migration assays, the outer side of the transwell basement membrane was coated with Matrigel when assessing invasiveness. Figure 5 Center A shows the changes in the invasive ability of DU-145 and PC-3 cells after knockdown of Musashi-1 protein using siRNA, compared with cells transfected with negative control siRNA. It can be seen that knockdown of Musashi-1 protein reduced the number of DU-145 and PC-3 cells that crossed the basement membrane of the Matrigel-coated transwell. Figure 5 Middle B is the statistics of the number of DU-145 and PC-3 cells crossing the matrix gel-coated transwell basement membrane after knocking down Musashi-1 protein (n=3). The results showed that after knocking down Musashi-1 protein, the number of DU-145 and PC-3 cells crossing the transwell basement membrane was only one-third of that in the control group (P<0.05 was a significant difference), indicating that the cell invasion ability was reduced.

[0103] 6. Knockdown of Musashi-1 protein inhibits tumor growth in castration-resistant prostate cancer

[0104] We used castration-resistant prostate cancer cell lines DU-145 and PC-3 to inoculate tumors on the surface of nude mice to study the growth inhibition of the two tumors after knocking down the Musashi-1 protein. Transfection with Musashi-1 siRNA was used as treatment, while transfection with negative control siRNA and no treatment were used as controls. siRNA transfection was continuous for three days from the first to the third day. Figure 6 Figure A shows the growth inhibition of PC-3 cell-bearing tumors after knocking down Musashi-1 protein using siRNA. It can be found that compared with tumors transfected with negative control siRNA and tumors without any treatment, the growth of PC-3 tumors was significantly inhibited after knocking down Musashi-1 protein. Figure 6 Figure B shows the growth inhibition of DU-145 cell-bearing tumors after knocking down Musashi-1 protein using siRNA. It can be found that compared with tumors transfected with negative control siRNA and tumors without any treatment, the growth of DU-145 tumors was significantly inhibited after knocking down Musashi-1 protein.

[0105] 3. Experimental Conclusion

[0106] We have discovered for the first time that Musashi-1 protein expression is elevated in castration-resistant prostate cancer tissues and cell lines and correlates with the proliferation, migration, and invasion of castration-resistant prostate cancer cells. Inhibiting Musashi-1 protein expression reduces the proliferation, migration, and invasion of castration-resistant prostate cancer cells. Therefore, Musashi-1 protein may serve as a potential new therapeutic target for castration-resistant prostate cancer.

[0107] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples. Within the scope of the present disclosure, the technical features in the above embodiments or different embodiments may be combined, the 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 simplicity.

[0108] The embodiments of the present disclosure are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present disclosure should be included in the scope of protection of the present disclosure.

Claims

1. Use of a Musashi-1 gene inhibitor in the preparation of a drug for preventing and / or treating castration-resistant prostate cancer, wherein the Musashi-1 gene inhibitor is a Musashi-1 gene-specific siRNA.

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