Use of pus1 in increasing sensitivity of enzalutamide to prostate cancer treatment

Abstract

The application relates to application of PUS1 in improving sensitivity of enzalutamide to prostate cancer treatment. Through bioinformatics analysis and a series of in-vivo and in-vitro experiments, the application first discovers and determines the key role of the expression level of PUS1 in reducing the sensitivity of enzalutamide in treating prostate cancer, and deeply studies the mechanism, and determines that the PUS1 promotes enzalutamide resistance of prostate cancer in dependence on the Psi modification activity. The application enriches the related mechanism of drug resistance generation and regulation in the process of enzalutamide in treating prostate cancer, provides sufficient scientific basis and theoretical basis for exploring new prostate cancer diagnosis, prognosis judgment and treatment molecular target, and developing new targeted drugs, and has important social value and scientific significance.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the application of PUS1 in improving the sensitivity of enzalutamide to prostate cancer treatment. Background Technology

[0002] Prostate cancer is a common malignant tumor in men, ranking first in incidence and second in mortality in Western countries. Although the incidence of prostate cancer in my country is relatively low, it has been rapidly increasing in recent years due to changes in environment and lifestyle, posing a threat to men's health. Early and mid-stage prostate cancer is mainly treated with surgery and radiotherapy, but most patients are diagnosed at an advanced stage and rely primarily on endocrine therapy. Enzalutamide, a second-generation nonsteroidal androgen receptor antagonist (ARSI) approved by the US FDA in 2012, is used to treat metastatic castration-resistant prostate cancer (CRPC). However, drug resistance in tumor cells has become a major obstacle to treatment. There have been reports of enzalutamide resistance abroad, and many patients are too old to tolerate chemotherapy, leaving them without available treatment options.

[0003] The mechanisms of enzalutamide resistance mainly include the following aspects: (1) AR gene mutation: Long-term ARSI treatment patients will have the F876L mutation in the LBD region of the AR protein ligand binding domain, which causes enzalutamide to change from an antagonist to an agonist, resulting in drug resistance in prostate cancer; (2) AR splice variant (ARV) formation: AR-V7 positive prostate cancer is unresponsive to both abiraterone and enzalutamide; (3) Neuroendocrineization: About 30-40% of mCRPC patients develop a neuroendocrine phenotype after endocrine therapy, which makes them resistant to ARSI treatment; (4) Activation of other signaling pathways: Increased expression of glucocorticoid receptors can bypass the AR signaling pathway and promote the progression of prostate cancer. Existing technologies have revealed the important role of non-mutant epigenetic reprogramming in tumor development and have incorporated it into novel tumor phenotypic markers. This study explains for the first time the important role of pseudouracil (Ψ) modification in the epigenetic regulation of prostate cancer and conducts an in-depth discussion.

[0004] Ψ is the C5-glycosidic isomer of uridine and is the earliest and most abundant modified nucleoside in RNA, often referred to as the "fifth nucleoside" in RNA. Initially discovered in non-coding RNAs such as rRNA, tRNA, and snRNA, Ψ modification sites have been found in almost all types of RNA, including mRNA, thanks to advancements in detection methods and techniques. In eukaryotes, Ψ primarily modifies RNA through two mechanisms: an RNA-independent mechanism where pseudouridine synthase directly recognizes and catalyzes the substrate; and an RNA-dependent mechanism where pseudouridine synthase relies on guide RNA and the H / ACA ribonucleoprotein complex for catalysis, primarily occurring in structural non-coding RNAs. In the Ψ structure, the typical NC bond between ribose and bases is replaced by a CC bond, creating additional hydrogen bond donors and making the RNA more spatially stable. Furthermore, Ψ participates in regulatory processes such as tRNA codon-anticodon base pairing, rRNA folding, snRNP biogenesis, pre-mRNA splicing, and mRNA coding. In 1983, Salvatore et al. pointed out that serum Ψ could serve as a tumor marker. DCK1 was the first Ψ-modifying enzyme discovered to have pro-cancer effects. It promotes the growth of colorectal carcinoma (CRC) by binding to and catalyzing ribosomal protein RPS3 mRNA, increasing its stability, and directly activating HIF-1α transcription to enhance CRC angiogenesis and cell metastasis. Furthermore, the Ψ-modifying activity of PUS7 has also been shown to be closely related to the progression of various tumors. Shi et al., through small RNA Ψ-seq detection, found that PUS7-targeted modified tRNA is crucial for codon-specific translation control of key regulatory factors in glioma stem cells, and selective inhibition of PUS7 enzyme activity can significantly inhibit glioma growth. With the increasing number of advanced prostate cancer patients receiving enzalutamide treatment, drug resistance is a significant issue. Therefore, in-depth exploration of the molecular mechanisms of enzalutamide resistance in prostate cancer is of great importance in providing new ideas and molecular targets for drug therapy. Summary of the Invention

[0005] The purpose of this invention is to address the problem of enzalutamide resistance in prostate cancer patients treated with enzalutamide in existing technologies. This invention involves in-depth research into the resistance mechanism, identifying PUS1 as a key target associated with enzalutamide resistance in prostate cancer. Inhibiting PUS1 expression or inducing specific mutations in PUS1 can effectively reduce enzalutamide resistance in prostate cancer and enhance the therapeutic activity of enzalutamide. Furthermore, by detecting PUS1 expression and / or mutation levels in patients, the prognosis of enzalutamide treatment can be rationally assessed, providing a new strategy for prostate cancer treatment.

[0006] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution.

[0007] The first aspect of the present invention provides the use of the PUS1 mutant in the preparation of a drug that enhances the sensitivity of enzalutamide to prostate cancer treatment; wherein the mutation site of the PUS1 mutant is selected from one or more of R116, D118, R171, Y173, and R267.

[0008] Preferably, the mutation sites of the PUS1 mutant are R116, D118, R171, Y173, and R267.

[0009] Preferably, the sequence of the PUS1 mutant is shown in SEQ ID NO: 1.

[0010] Preferably, the prostate cancer is selected from castration-resistant prostate cancer.

[0011] The second aspect of this invention provides the use of PUS1 inhibitors in the preparation of medicaments that enhance the sensitivity of enzalutamide to prostate cancer treatment.

[0012] Preferably, the PUS1 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the PUS1 gene.

[0013] Preferably, the PUS1 inhibitor is selected from one or more of siRNA and shRNA designed based on the PUS1 gene.

[0014] Preferably, the sequence of the siRNA designed based on the PUS1 gene is selected from one or more of SEQ ID NO: 2 (5'-GCTGATTGACGACATTCTATT-3') and SEQ ID NO: 3 (5'-GGCCATTGTGAAGGGTTATTT-3'); the sequence of the shRNA designed based on the PUS1 gene is selected from one or more of SEQ ID NO: 4 (5'-CcggGAGCTTCATGATGCATC AGATCTCGAGATCTGATGCATCATGAAGCTCTTTTTg-3') and SEQ ID NO: 5 (5'-CcggTGTCGGGTCCTCACAATTCAACTCGAGTTGAATTGTGAGGACCCGACATTTTTg-3').

[0015] Preferably, the prostate cancer is selected from castration-resistant prostate cancer.

[0016] A third aspect of the present invention provides the use of a reagent for detecting PUS1 expression levels in the preparation of a product for evaluating the sensitivity of enzalutamide to prostate cancer treatment.

[0017] Preferably, the reagent for detecting PUS1 expression level includes primers for detecting PUS1 gene expression level and / or reagents for detecting PUS1 protein content.

[0018] Preferably, the primers for detecting the expression level of the PUS1 gene are selected from the following primer pairs, the upstream sequence of which is shown in SEQ ID NO: 6 (5'-GGGCGGGTTTAACTCCA AGA-3') and the downstream sequence is shown in SEQ ID NO: 7 (5'-ATTGTGGAAGTTGTGC GTGC-3').

[0019] Preferably, the reagent for detecting PUS1 protein content is selected from anti-PUS1 antibody (ab203010, abcam).

[0020] Preferably, the prostate cancer is selected from castration-resistant prostate cancer.

[0021] A fourth aspect of the present invention provides a pharmaceutical composition for treating prostate cancer, comprising enzalutamide and an enzalutamide active agent; wherein the enzalutamide active agent is selected from one or more of PUS1 mutants and PUS1 inhibitors; wherein the mutation site of the PUS1 mutant is selected from one or more of R116, D118, R171, Y173, and R267.

[0022] Preferably, the mutation sites of the PUS1 mutant are R116, D118, R171, Y173, and R267.

[0023] Preferably, the sequence of the PUS1 mutant is shown in SEQ ID NO: 1.

[0024] The PUS1 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the PUS1 gene.

[0025] Preferably, the PUS1 inhibitor is selected from one or more of siRNA and shRNA designed based on the PUS1 gene.

[0026] Preferably, the sequence of the siRNA designed based on the PUS1 gene is selected from one or more of SEQ ID NO: 2 and SEQ ID NO: 3; the sequence of the shRNA designed based on the PUS1 gene is selected from one or more of SEQ ID NO: 4 and SEQ ID NO: 5.

[0027] Preferably, the prostate cancer is selected from castration-resistant prostate cancer.

[0028] Preferably, the pharmaceutical composition may optionally contain a pharmaceutically acceptable carrier.

[0029] Preferably, the pharmaceutically acceptable carrier includes one or more of fillers, disintegrants, binders, lubricants, flavoring agents, preservatives, antioxidants, and colorants.

[0030] It should be understood that, unless otherwise specified, in the context of this invention, the PUS1 inhibitor refers to a substance capable of specifically downregulating the expression level of PUS1 and / or the transcriptional level of its mature mRNA and / or the expression level or activity of the PUS1 protein. For example, methods such as antisense oligonucleotides, siRNA, shRNA, sgRNA, antagomiRs, miRNA sponges, miRNA erasers, target masking, and / or multi-target methods can be used to downregulate the expression level and / or activity of PUS1; any method that can reduce the level and / or activity of PUS1 is acceptable. The primers and / or primer pairs refer to PCR primers used to synthesize the PUS1 gene cDNA strand in PCR, thereby detecting the expression level of the PUS1 gene mRNA. In addition to the primers and / or primers listed in this invention, those skilled in the art are fully capable of designing corresponding primers, primer pairs, and antibodies based on the PUS1 gene sequence using conventional methods and techniques in the field, including but not limited to molecular biology, and screening the designed primers and / or primer pairs through conventional experimental methods, or obtaining commercially available primers, as long as they can specifically detect the PUS1 expression level; other conventional reagents and methods in the field can also be used to detect the PUS1 protein expression level.

[0031] This invention, through extensive research, has found a positive correlation between PUS1 expression and Gleason score in prostate cancer, with higher expression levels in mCRPC. KM curve analysis showed a significant shortening of ARSI-related overall survival in patients with high PUS1 expression. Furthermore, in both non-resistant and drug-resistant LNCaP and C4-2B cell lines, PUS1 expression was found to be higher in drug-resistant cell lines. Immunohistochemical analysis of clinical samples and PDX models revealed that PUS1 was highly expressed in CRPC samples compared to androgen-dependent prostate cancer, and even higher in enzalutamide-resistant tissues. These results indicate that PUS1 expression is associated with the malignancy and poor prognosis of prostate cancer. In vivo and in vitro studies showed that knockdown of PUS1 expression in C4-2B_ENZR and LNCaP_EZNR cell lines, validated by CCK-8 and plate clone assays, significantly reduced resistance to enzalutamide in both cell lines. Results from a subcutaneous xenograft model showed that in the enzalutamide treatment group, knockdown of PUS1 expression significantly slowed subcutaneous tumor growth and resulted in smaller final tumor weight. These results indicate that PUS1 is an important molecule mediating enzalutamide resistance in prostate cancer.

[0032] Due to limited research on the relationship between PUS1 and tumors, recent studies have revealed that PUS1's Ψ-modification activity is related to the translation efficiency of various proto-oncogenes (IRS1, MYC, etc.), but the specific mechanisms remain unclear. Previous studies have found that PUS1 is associated with alternative splicing at the 3′ end of pre-mRNA. In vitro validation revealed significant Ψ-modification sites in the exons or flanking introns of genes such as PUM2, and significant splicing differences were observed in cells with PUS1 knockout. In addition, studies have shown that PUS1 can modify the terminator of target mRNA, causing a gene readthrough effect and altering the full-length amino acid sequence of the protein. To clarify whether PUS1 regulates enzalutamide resistance in prostate cancer through Ψ-activity, overexpression plasmids were constructed by mutagenizing five sites (R116L, D118K, R171T, Y173E, R267L). CCK-8 and plate clone assays revealed that overexpression of mutant PUS1_mut cells helped reduce enzalutamide resistance in prostate cancer cells, indicating that PUS1's promotion of enzalutamide resistance in prostate cancer depends on its Ψ-modification activity.

[0033] In summary, this invention, through bioinformatics analysis and a series of in vitro and in vivo experiments, for the first time discovered and clarified the crucial role of PUS1 expression level in the decreased sensitivity of enzalutamide in prostate cancer treatment, and further investigated its mechanism of action: PUS1 promotes enzalutamide resistance in prostate cancer in a manner dependent on its Ψ-modification activity. This invention enriches our understanding of the mechanisms underlying the development and regulation of enzalutamide resistance in prostate cancer treatment, providing ample scientific evidence and theoretical foundation for exploring new molecular targets for prostate cancer diagnosis, prognosis, and treatment, and for developing new targeted drugs. It possesses significant social and scientific value. Attached Figure Description

[0034] Figure 1 This is a schematic diagram showing the relationship between PUS1 expression level and Gleason score in the TCGA-PRAD dataset.

[0035] Figure 2 This is a schematic diagram showing the differences in PUS1 expression levels in normal prostate tissue, prostate cancer in situ, and mCRPC in the GSE35988 dataset.

[0036] Figure 3 This is a schematic diagram illustrating the correlation between PUS1 expression level and ARSI-related overall survival in mCRPC patients using KM curve analysis.

[0037] Figure 4 A schematic diagram illustrating the results of Western blotting to verify changes in PUS1 protein expression in drug-resistant and non-drug-resistant prostate cancer cells.

[0038] Figure 5A schematic diagram illustrating the results of IHC validation of differential expression of PUS1 in ADPC, CRPC, and enzalutamide-resistant CRPC tissues.

[0039] Figure 6 A schematic diagram illustrating the efficiency of PUS1 knockdown in C4-2B_ENZR and LNCaP_ENZR cells as verified by Western blotting.

[0040] Figure 7 A schematic diagram showing the changes in the sensitivity of C4-2B_ENZR cells to enzalutamide after PUS1 knockdown as detected by CCK8 assay.

[0041] Figure 8 A schematic diagram showing the changes in the sensitivity of LNCaP_ENZR cells to enzalutamide after PUS1 knockdown detected by CCK8 assay.

[0042] Figure 9 This is a schematic diagram illustrating the changes in the colony-forming ability of C4-2B_ENZR cells stimulated with enzalutamide after PUS1 knockdown in a plate colony formation assay.

[0043] Figure 10 This diagram illustrates the changes in the colony-forming ability of LNCaP_ENZR cells after PUS1 knockdown under enzalutamide stimulation in a plate colony formation assay.

[0044] Figure 11 This is a schematic diagram showing the effect of PUS1 expression level on tumor growth in mice treated with enzalutamide.

[0045] Figure 12 This is a schematic diagram showing the quantitative analysis results of the effect of PUS1 expression level on tumor growth in mice treated with enzalutamide.

[0046] Figure 13 This is a schematic diagram showing the results of mutations at the active site of the PUS1 enzyme.

[0047] Figure 14 A schematic diagram showing the results of Western blotting to verify the expression efficiency of wild-type PUS1_wt and mutant PUS1_mut plasmids.

[0048] Figure 15 This is a schematic diagram showing the changes in the sensitivity of prostate cancer cells to enzalutamide after CCK8 assays for overexpression of PUS1_wt and PUS1_mut.

[0049] Figure 16 This is a schematic diagram illustrating the changes in colony-forming ability of prostate cancer cells after overexpression of PUS1_wt and PUS1_mut in a plate colony-forming assay under enzalutamide treatment. Detailed Implementation

[0050] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0051] Unless otherwise specified, all cell lines listed in this invention, including LNCaP and C4-2B, were purchased from ATCC and cultured according to ATCC guidelines. All cell lines were identified by short tandem repeat analysis at the China Center for Type Culture Collection (Wuhan), and the presence of mycoplasma contamination was verified using a PCR detection kit (Shanghai Biothrive Sci). All cell lines were cryopreserved in liquid nitrogen for subsequent experiments. All reagents, consumables, and equipment used in this invention were commercially available. The upstream primer sequence for detecting PUS1 mRNA expression levels is shown in SEQ ID NO: 6, 5'-GGGCGGGTTTAACTCCAAGA-3', and the downstream primer sequence is shown in SEQ ID NO: 7, 5'-ATTGTGGAAGTTGTGCGTGC-3'.

[0052] In this invention, informed consent was obtained from patients for all clinical samples used; the pathology and histological morphology of all tissue samples were confirmed by the pathology department. The procedures and methods of this invention have been approved by the Institutional Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University, complying with medical ethics requirements and Good Clinical Practice (GCP) guidelines for drug clinical trials. All experimental procedures adhered to the Declaration of Helsinki. The experimental methods used in this invention, such as bioinformatics analysis, molecular biology experiments, cell biology experiments, and immunohistochemistry, are all conventional methods and techniques in the field. Representative results from repeated biological experiments are presented in the accompanying figures, and data are displayed as mean ± SD and mean ± SEM as specified in the figures. All in vitro experiments were repeated at least three times, and animal experiments were repeated twice. Data were analyzed using GraphPad Prism 8.0 software. Conventional medical statistical methods such as t-tests, chi-square tests, and analysis of variance were used to compare the differences in means between two or more groups. *p < 0.05 was considered a significant difference.

[0053] Example 1

[0054] Firstly, analysis of the TCGA-PRAD dataset revealed a positive correlation between PUS1 expression and Gleason score in prostate cancer (see [link]). Figure 1Subsequently, the expression of PUS1 in normal prostate tissue, primary prostate cancer, and metastatic castration-resistant prostate cancer (mCRPC) was analyzed in the GSE35988 dataset. The analysis revealed significantly increased PUS1 expression in the tumor tissue of mCRPC patients (see [link to study]). Figure 2 Kaplan-Meier curve analysis revealed a significant correlation between PUS1 expression and ARSI treatment-related overall survival in mCRPC patients; specifically, patients with high PUS1 expression had significantly shorter ARSI treatment-related overall survival (see [link to Kaplan-Meier curve analysis]). Figure 3 ).

[0055] Furthermore, non-drug-resistant (WT) and enzalutamide-resistant (ENZR) prostate cancer cell lines (LNCaP and C4-2B) were collected, and the expression of PUS1 was detected by Western blot. The specific steps are as follows:

[0056] (1) After taking prostate cancer cells in the logarithmic growth phase, digest them and collect them into 1.5 mL EP tubes. Wash them twice with PBS, add cell lysis buffer containing 1× protease inhibitor and phosphatase inhibitor, and lyse them on ice for 30 min. After cell lysis, collect them into 1.5 mL EP tubes and centrifuge at 12000 rpm and 4℃ for 10 min.

[0057] (2) After protein quantification, add 5× protein loading buffer according to the sample volume and mix well. Incubate at 95℃ for 5 min. Perform protein electrophoresis on the obtained protein sample. Set the initial voltage to 80V. When the protein marker bands are clearly separated, adjust the voltage to 120V.

[0058] (3) After protein electrophoresis, the transfer can be performed. First, place the protein gel in the transfer solution to equilibrate, then add an appropriate amount of transfer solution to the container, and place the sponge, filter paper, PVD F membrane (activated with methanol), gel, filter paper, and sponge in sequence. Remove the gas, and place the transfer tank in ice water. The transfer conditions are 300mA constant current for 1 hour.

[0059] (4) After transfer, place the membrane in 5% milk and block it on a shaker at room temperature for 1 hour. After blocking, wash the membrane once with 1×TBST buffer, cut the desired band, add the corresponding primary antibody, and incubate overnight on a shaker at 4°C. Recover the primary antibody and wash the membrane three times with 1×TBST buffer for 10 minutes each time. Incubate the secondary antibody at room temperature for 1 hour, and then wash the membrane three times with TBST buffer for 10 minutes each time.

[0060] (5) After washing the membrane, chemiluminescence can be performed. Prepare the ECL luminescent solution (A:B = 1:1) and use a Biorad chemiluminescence analyzer for luminescence development. The detection results are as follows: Figure 4As shown in the figure. The results showed that PUS1 expression was significantly higher in enzalutamide-resistant prostate cancer cell lines.

[0061] Furthermore, immunohistochemical analysis was performed on samples from androgen-dependent prostate cancer (ADPC), castration-resistant prostate cancer (CRPC, enzalutamide-resistant or non-resistant), and PDX model. The specific steps are as follows:

[0062] (1) The sample tissue was embedded in paraffin for preservation. The paraffin specimen was stored at room temperature, and the cut white slides were stored in a refrigerator at 4°C. During the experiment, the paraffin slides were taken out and baked in an oven at 60°C for about 2 hours to make the slides adhere to the glass slides.

[0063] (2) Dewax the sections in two portions of xylene for 10 minutes each time. Then hydrate the sections in a gradient of alcohols (100%, 100%, 95%, 85%, 70%) for 5-7 minutes each time, and then wash them three times with PBS for 3 minutes each time.

[0064] (3) Immerse the slides in citrate antigen retrieval solution and heat in a microwave oven on high for 20 minutes for antigen retrieval. After heating, allow the slides to cool naturally to room temperature in the antigen retrieval solution and wash with PBS three times for 3 minutes each time.

[0065] (4) Place the sections in a light-proof, humidified box, cover the tissue with 3% hydrogen peroxide, and incubate at room temperature for 30 minutes to thoroughly remove endogenous peroxidase. Wash three times with PBS, 3 minutes each time. Add blocking buffer and block at room temperature for 30 minutes to reduce non-specific binding of the primary antibody.

[0066] (5) Remove the blocking solution, add the primary antibody directly to cover the tissue without washing, and block overnight at 4°C in a humidified chamber. The next day, incubate at room temperature for 1 hour, then wash with PBS for 5 minutes, repeating 3 times. Add the reaction sensitizing solution and incubate at room temperature for 20 minutes, then wash with PBS three times; add the biotin-labeled secondary antibody, incubate at room temperature for 30 minutes, then wash with PBS for 5 minutes, repeating 3 times.

[0067] (6) Add DAB for color development, and after color development is complete, place the slide in water to stop the reaction. Add hematoxylin for staining for 25-30 seconds for counterstaining, and place the slide in water to stop staining; then rinse in running water for 30-45 minutes until the color of the slide is stable for inversion treatment. Gradient alcohol dehydration: Soak the slide in 70%, 85%, 95%, 100%, and 100% alcohol, and xylene for 5 minutes each time, and finally mount the slide.

[0068] Two independent researchers analyzed the patient's clinicopathological immunohistochemical sections separately, without knowing the patient's information. The test results are as follows: Figure 5As shown in the figure. The results showed that, compared with ADPC, PUS1 expression was significantly increased in CRPC samples, and even higher in enzalutamide-resistant tissues. These results indicate that PUS1 expression is associated with the malignancy and poor prognosis of prostate cancer.

[0069] Example 2

[0070] The foregoing examples clearly demonstrated the adverse effects of PUS1 overexpression on enzalutamide's resistance to prostate cancer. To further investigate the influence of PUS1 on the therapeutic activity of enzalutamide in prostate cancer, a series of in vivo and in vitro experiments were conducted.

[0071] First, siRNA and shRNA were designed to reduce intracellular PUS1 expression levels, and their knockdown efficiency was verified. The specific steps are as follows:

[0072] (1) Design siRNAs and shRNAs knockdown sequences on the Sigma website. Design and synthesize primers based on the siRNA and shRNA structures. Dilute the synthesized siRNAs and shRNAs primers with water to 100 μM. Take 10 μL of each of the forward and reverse primers, mix them evenly, and use a PCR instrument to gradually cool from 100℃ to room temperature and anneal to synthesize double strands. In this example, two siRNAs and shRNAs were used as examples, namely si-PUS1#1 (sequence as shown in SEQ ID NO: 2, 5'-GCTGATTGACGACATTCTATT-3'), si-PUS1#4 (sequence as shown in SEQ ID NO: 3, 5'-GGCCATTGTGAAGGGTTATTT-3'), shPUS1#2 (sequence as shown in SEQ ID NO: 4, 5'-CcggGAGCTTCATGATGCA TCAGATCTCGAGATCTGATGCATCATGAAGCTCTTTTTg-3'), and sh-PUS1#3 (sequence as shown in SEQ ID NO: 2). As shown in NO: 5, it is 5'-CcggTGTCGGGTCCTCACAATTCAAC TCGAGTTGAATTGTGAGGACCCGACATTTTTg-3').

[0073] (2) The pLKO.1 lentiviral vector was digested with EcoRI and AgeI restriction enzymes. After digestion, two single bands were obtained by gel running. The larger band was cut and recycled. Then, the annealed double strands were ligated into the vector after gel recycling.

[0074] (3) Logarithmic growth phase 293T cells were seeded in 10cm culture dishes at a seeding density of about 70%; 6000ng of target plasmid loaded with shPUS1#1 or shPUS1#1, 4500ng of PSPAX 2 and 1500ng of PMD2.G were transfected into 293T cells and cultured for 48h.

[0075] (4) After 48 hours, collect the culture medium and filter out the cell debris with a 0.45 μm filter to obtain the virus solution.

[0076] (5) One day before infection, logarithmic growth phase enzalutamide-resistant prostate cancer cells (LNCaP_ENZR, C4-2B_ENZR) were inoculated into 6cm culture dishes, the original culture medium was discarded, 3mL of virus solution was added, and 3μL of polybrene was added at the same time to promote virus infection. After 24 hours, the virus solution was discarded and fresh culture medium was replaced to continue culturing.

[0077] (6) Discard the culture medium 48 hours after infection and add a culture medium with an appropriate concentration of puromycin. At this time, the cells that have successfully integrated the foreign gene have puromycin resistance and can survive. After continuous screening for 7 to 10 days, a stable cell line can be obtained. The expression level of PUS1 can be detected by qPCR or WB to determine whether the knockdown has been successful.

[0078] Test results as follows Figure 6 As shown in the figure. The results show that the two siRNAs and shRNAs designed according to the present invention can all significantly inhibit PUS1 and reduce the expression level of PUS1 protein in prostate cancer cells.

[0079] The subsequent study investigated the effect of inhibiting PUS1 expression levels on prostate cancer cell proliferation and colony formation. The specific steps of the cell proliferation experiment are as follows:

[0080] (1) Enzalutamide-resistant prostate cancer cells (LNCaP_ENZR, C4-2B_ENZR) transfected with plasmids containing knockdown PUS1 gene expression sequences (si-PUS1#1, si-PUS1#4, sh-PUS1#2, or sh-PUS1#3) or blank control plasmids (shNC) in the logarithmic growth phase were digested with trypsin and counted. Appropriate cell densities (1.5 × 10⁻⁶) were selected based on the doubling time of each cell type. 5 / well), seeded into 96-well plates (3 replicates), with different concentrations of enzalutamide (10) added to each group. 0 10 0.5 10 1 10 1.5 10 2 (μM).

[0081] (2) The cells were cultured in a 37°C incubator and collected after 48 hours. 10 μL of CCK-8 was added to each well, and the culture plate was incubated in the incubator for 1-4 hours. The absorbance at 450 nm was measured to assess the cell proliferation status.

[0082] The specific steps of the clone formation experiment are as follows:

[0083] (1) Enzalutamide-resistant prostate cancer cells (LNCaP_ENZR, C4-2B_ENZR) transfected with plasmids containing knockdown PUS1 gene expression sequence (si-PUS1#1, si-PUS1#4, sh-PUS1#2 or sh-PUS1#3) or blank control plasmid (shNC) in the logarithmic growth phase were digested with 0.25% trypsin and pipetted into single cells. The cells were then suspended in RPMI-1640 medium containing 10% fetal bovine serum for later use.

[0084] (2) Dilute the cell suspension and seed it at a density of 1000 cells / well in a dish containing 10 mL of pre-warmed culture medium at 37°C. Gently rotate the dish to disperse the cells evenly. Add enzalutamide (10 μM in LNCaP_ENZR and 25 μM in C4-2B_ENZR). The control group was treated with an equal volume of DMSO. The cells were cultured in a cell culture incubator at 37°C, 5% CO2 and saturated humidity.

[0085] (3) When visible clones appear in the culture dish, stop the culture, discard the supernatant, carefully wash twice with PBS, add 1 mL of methanol containing 0.5% crystal violet to each well, and stain for 30 min; discard the methanol and wash off the residual methanol with water; cell clones can then be observed; under a microscope, a number of cells > 50 is considered a valid clone.

[0086] Test results as follows Figure 7-10 As shown in the figure, the results indicated that inhibiting intracellular PUS1 expression using siRNA or shRNA significantly reduced the resistance of prostate cancer cells to enzalutamide and enhanced the killing activity of enzalutamide against prostate cancer cells, with statistically significant differences. The cell colony formation assay showed that inhibiting PUS1 expression induced prostate cancer cell colony formation, and further inhibition of PUS1 expression on top of enzalutamide treatment significantly suppressed colony formation, with statistically significant differences. These results demonstrate that the intracellular PUS1 expression level is closely related to the cytotoxicity of enzalutamide; the lower the PUS1 expression level, the more significant the killing effect of 5-FU on prostate cancer cells.

[0087] Furthermore, the effect of PUS1 expression on tumor growth was investigated through in vivo experiments, with the specific steps as follows:

[0088] (1) Five-week-old male BALB / c-nu / nu mice were selected and divided into two groups. One group was subcutaneously injected with enzalutamide-resistant prostate cancer cells (C4-2B_ENZR) transfected with blank control plasmid (shNC), and the other group was subcutaneously injected with enzalutamide-resistant prostate cancer cells (C4-2B_ENZR) knocked down with PUS1 gene expression sequence plasmid (sh-PUS1#3).

[0089] (2) After tumor formation, the shNC group mice were randomly divided into two groups of 5 mice each, namely group 1 and group 3. Similarly, the sh-PUS1#3 group mice were randomly divided into two groups of 5 mice each, namely group 2 and group 4.

[0090] (3) Mice in groups 3 and 4 were treated with enzalutamide (10 mg / kg, administered by gavage for 4 consecutive days per week, followed by a 3-day break, for a total of 3 weeks); mice in groups 1 and 2 were treated with an equal volume of physiological saline containing 5% DMSO. Tumor volume was measured regularly during treatment. Three weeks after treatment, mice were sacrificed, tumors were dissected, and weighed to calculate the tumor inhibition rate.

[0091] Experimental results are as follows Figure 11-12 As shown in the figure. The results showed that inhibiting the expression of PUS1 in vivo alone could suppress the growth of prostate cancer tumors to some extent; while after treatment with enzalutamide, the tumor growth rate of mice with low PUS1 expression in vivo was significantly reduced and the tumor volume was significantly smaller, with statistically significant differences, further confirming that PUS1 is an important molecule mediating enzalutamide resistance in prostate cancer.

[0092] Example 3

[0093] Current research on the relationship between PUS1 and tumors is limited. Recent studies reveal that PUS1's Ψ-modification activity is related to the translation efficiency of various proto-oncogenes (IRS1, MYC, etc.), but the specific mechanisms remain unclear. Previous studies have found that PUS1 is associated with alternative splicing at the 3′ end of pre-mRNA. In vitro analysis has revealed significant Ψ-modification sites in the exons or flanking introns of genes such as PUM2, and significant splicing differences were observed in cells with PUS1 knockout. Furthermore, other studies have shown that PUS1 can modify the terminators of target mRNAs, causing a gene readthrough effect and altering the full-length amino acid sequence of the protein.

[0094] To determine whether PUS1 regulates enzalutamide resistance in prostate cancer through Ψ activity, mutations were performed at five sites (R116L, D118K, R171T, Y173E, and R267L), and overexpression plasmids were constructed (see [link to original text]). Figure 13Subsequently, the vector with mutations at five sites was transfected into C4-2B cells, and the intracellular PUS1 expression level was detected by Western blot according to the aforementioned method. The results showed that the mutations at the five sites were successful, and the intracellular PUS1 expression level was significantly reduced (see [link to relevant documentation]). Figure 14 ).

[0095] Subsequently, PUS1_wt, PUS1_mut, or a blank vector were transfected into C4-2B cells, and the effects of different concentrations of enzalutamide on cell proliferation were detected using the aforementioned method. The results are shown below. Figure 15 As shown in the figure. The results showed that under low to medium concentrations of enzalutamide treatment, the overexpression of PUS1 in cells transfected with PUS1_wt significantly reduced the cell's sensitivity to enzalutamide treatment and significantly increased cell proliferation activity. However, after transfection with PUS1_mut at 5 sites, the resistance of prostate cancer cells to enzalutamide was significantly reduced and cell proliferation activity was significantly inhibited.

[0096] Furthermore, PUS1_wt, PUS1_mut, or a blank vector were transfected into C4-2B cells, respectively, and the effect of a specific concentration (25 μM) of enzalutamide on cell colony formation was detected using the aforementioned method. The results are as follows: Figure 16 As shown in the figure, the results indicated that overexpression of PUS1 in cells transfected with PUS1_wt significantly reduced the cell's sensitivity to enzalutamide and significantly increased its clonogenic ability. However, transfection with PUS1_mut at five mutated sites significantly reduced the enzalutamide resistance of prostate cancer cells and significantly inhibited cell clonogenic ability. These results suggest that R116, D118, R171, Y173, and R267 are key sites for PUS1 to exert its Ψ-modifying enzyme activity. PUS1's promotion of enzalutamide resistance in prostate cancer depends on its Ψ-modifying activity. Mutating these sites can effectively inhibit the Ψ-modifying activity of PUS1, thereby promoting the sensitivity of prostate cancer cells to enzalutamide.

[0097] As clearly demonstrated above, PUS1 expression is positively correlated with Gleason scores in prostate cancer, and its expression is even higher in mCRPC. KM curve analysis showed that patients with high PUS1 expression had significantly shorter ARSI-related overall survival. Furthermore, in both non-resistant and drug-resistant LNCaP and C4-2B cell lines, PUS1 expression was found to be higher in drug-resistant cell lines. Immunohistochemical analysis of clinical samples and PDX models revealed that PUS1 was highly expressed in CRPC samples compared to androgen-dependent prostate cancer, and its expression was even higher in enzalutamide-resistant tissues. These results indicate that PUS1 expression is associated with the malignancy and poor prognosis of prostate cancer. In vivo and in vitro studies showed that knockdown of PUS1 expression in C4-2B_ENZR and LNCaP_EZNR cell lines, and CCK-8 and plate clone validation, significantly reduced resistance to enzalutamide in both cell lines. Results from a subcutaneous xenograft model showed that in the enzalutamide treatment group, knockdown of PUS1 expression significantly slowed subcutaneous tumor growth and resulted in smaller final tumor weight. These results indicate that PUS1 is an important molecule mediating enzalutamide resistance in prostate cancer.

[0098] Due to limited research on the relationship between PUS1 and tumors, recent studies have revealed that PUS1's Ψ-modification activity is related to the translation efficiency of various proto-oncogenes (IRS1, MYC, etc.), but the specific mechanisms remain unclear. Previous studies have found that PUS1 is associated with alternative splicing at the 3′ end of pre-mRNA. In vitro validation revealed significant Ψ-modification sites in the exons or flanking introns of genes such as PUM2, and significant splicing differences were observed in cells with PUS1 knockout. In addition, studies have shown that PUS1 can modify the terminator of target mRNA, causing a gene readthrough effect and altering the full-length amino acid sequence of the protein. To clarify whether PUS1 regulates enzalutamide resistance in prostate cancer through Ψ-activity, overexpression plasmids were constructed by mutagenizing five sites (R116L, D118K, R171T, Y173E, R267L). CCK-8 and plate clone assays revealed that overexpression of mutant PUS1_mut cells helped reduce enzalutamide resistance in prostate cancer cells, indicating that PUS1's promotion of enzalutamide resistance in prostate cancer depends on its Ψ-modification activity.

[0099] This invention enriches the understanding of the mechanisms of drug resistance development and regulation during enzalutamide treatment of prostate cancer. It provides sufficient scientific evidence and theoretical basis for exploring new molecular targets for prostate cancer diagnosis, prognosis and treatment, and developing new targeted drugs. It helps to achieve better precision treatment and provides a new drug treatment target for humans to conquer prostate cancer. It has important social value and scientific significance.

[0100] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. The use of PUS1 inhibitors in the preparation of drugs for treating enzalutamide-resistant prostate cancer, characterized in that, The PUS1 inhibitor is selected from one or more of siRNA and shRNA designed based on the PUS1 gene; the sequence of the siRNA designed based on the PUS1 gene is selected from one or more of SEQ ID NO: 2 and SEQ ID NO: 3; the sequence of the shRNA designed based on the PUS1 gene is selected from one or more of SEQ ID NO: 4 and SEQ ID NO:

5.

2. A pharmaceutical composition for treating prostate cancer, characterized in that, The invention includes enzalutamide and an enzalutamide active agent; the enzalutamide active agent is selected from PUS1 inhibitors; the PUS1 inhibitor is selected from one or more of siRNA and shRNA designed based on the PUS1 gene; the sequence of the siRNA designed based on the PUS1 gene is selected from one or more of SEQ ID NO: 2 and SEQ ID NO: 3; the sequence of the shRNA designed based on the PUS1 gene is selected from one or more of SEQ ID NO: 4 and SEQ ID NO: 5.

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