Application of PUS1 in improving sensitivity of Enzalocamide to treatment of prostatic cancer

Abstract

The invention relates to an application of PUS1 in improving the sensitivity of Enzalocamide to treatment of prostatic cancer. Through bioinformatics analysis and a series of in-vivo and in-vitro experiments, the key effect of the PUS1 expression level on sensitivity reduction in treatment of the prostatic cancer by the enzalocamide is found and clarified for the first time, the action mechanism of the PUS1 is deeply researched, and the PUS1 is clarified to promote the drug resistance of the prostatic cancer enzalocamide to depend on the Psi modification activity of the PUS1. The invention enriches related mechanisms of drug resistance generation and regulation in the process of treating prostatic cancer by the enzalocamide, provides sufficient scientific basis and theoretical basis for exploring new prostatic cancer diagnosis, prognosis judgment and treatment molecular targets and developing new targeted drugs, and has important social value and scientific significance.

Description

Technical Field

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

[0002] Prostate cancer is a common malignant tumor in men, with the highest incidence and the second highest mortality rate in Western countries. Although the incidence of prostate cancer in my country is relatively low, the incidence has shown a rapid growth trend in recent years due to changes in the environment and lifestyle, posing a threat to men's health. Early and middle stages of prostate cancer are mainly treated with surgery and radiotherapy, but most patients are in the advanced stage at the time of their first visit and mainly rely on endocrine therapy. Enzalutamide is a second-generation non-steroidal androgen receptor antagonist (ARSI) approved by the US FDA in 2012 for the treatment of castration-resistant prostate cancer (CRPC). However, the drug resistance of tumor cells has become a major obstacle to treatment. Enzalutamide resistance has been reported abroad, but most patients are older and cannot tolerate chemotherapy, facing a situation where no drug is available.

[0003] The mechanisms of enzalutamide resistance mainly include the following aspects: (1) AR gene mutation: patients with long-term ARSI treatment will have F876L mutation in the LBD region of the AR protein ligand binding domain, which causes enzalutamide to change from an antagonist to an agonist, making prostate cancer resistant; (2) AR splice variants (ARVs) formation: AR-V7 expression-positive prostate cancer is unresponsive to abiraterone and enzalutamide; (3) Neuroendocrineization: About 30-40% of mCRPC patients develop a neuroendocrine phenotype after endocrine therapy and become resistant to ARSI treatment; (4) Activation of other signaling pathways: increased expression of glucocorticoid receptors can bypass the AR signaling pathway to promote prostate cancer progression. The existing technology has revealed the important role of non-mutation epigenetic reprogramming in tumor occurrence and development, and incorporated it into new tumor phenotypic markers. This study first explained the important role of pseudouracil (Pseudouridine, Ψ) modification in epigenetic regulation of prostate cancer and conducted an in-depth discussion.

[0004] Ψ is the C5-glycoside isomer of uridine. It is the earliest and most abundant modified nucleoside in RNA and is known as the "fifth nucleoside" in RNA. Ψ was originally found in non-coding RNAs such as rRNA, tRNA, and snRNA. With the advancement of detection methods and technologies, Ψ modification sites have been found in almost all types of RNA, including mRNA. In eukaryotes, Ψ is modified mainly by two mechanisms: one is an RNA-independent mechanism, in which pseudouridine synthase directly recognizes and catalyzes the substrate; the other is an RNA-dependent mechanism, in which pseudouridine synthase relies on guide RNA and H / ACA ribonucleoprotein complex for catalysis. This modification mechanism mainly occurs in structural non-coding RNA. In the Ψ structure, the typical NC bond between ribose and base is replaced by a CC bond, generating additional hydrogen bond donors, making RNA more stable in spatial conformation. In addition, 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 Ψ in serum can be used as a tumor marker. DCK1 is the first Ψ-modifying enzyme discovered to have a pro-oncogenic effect. It promotes the growth of colorectal carcinoma (CRC) by binding and catalyzing ribosomal protein RPS3 mRNA, increasing its stability, and directly activating HIF-1α transcription to enhance angiogenesis and cell metastasis of CRC. In addition, the Ψ-modifying activity of PUS7 has also been shown to be closely related to the progression of various tumors. Shi et al. found through small RNA Ψ-seq detection that tRNA targeted by PUS7 is essential for the codon-specific translation control of key regulatory factors of brain glioma stem cells, and selective inhibition of PUS7 enzyme activity can significantly inhibit the growth of brain glioma. As the number of patients with advanced prostate cancer treated with enzalutamide is gradually increasing, the problem of drug resistance cannot be ignored. Therefore, it is of great significance to deeply explore the molecular mechanism of enzalutamide resistance in prostate cancer and provide new ideas and molecular targets for drug treatment. Summary of the invention

[0005] The purpose of the present invention is to address the problem that prostate cancer is prone to drug resistance during enzalutamide treatment in the prior art, thereby conducting in-depth research on its drug resistance mechanism, clarifying that PUS1 is an important target related to enzalutamide resistance in prostate cancer, and by inhibiting the expression of PUS1 or mutating PUS1 at specific sites, the generation of enzalutamide resistance in prostate cancer can be effectively reduced, thereby improving the therapeutic activity of enzalutamide. At the same time, by detecting the expression and / or mutation of PUS1 in the patient's body, a reasonable assessment can be made of the prognosis of the patient's treatment with enzalutamide, providing a new strategy for the treatment of prostate cancer.

[0006] In order to solve the above technical problems, the present invention is implemented through the following technical solutions.

[0007] The first aspect of the present invention provides the use of a PUS1 mutant in the preparation of a drug for improving the sensitivity of enzalutamide to the treatment of prostate cancer; 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 the present invention provides the use of a PUS1 inhibitor in the preparation of a drug for improving the sensitivity of enzalutamide to the treatment of prostate cancer.

[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'), 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'), 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 the expression level of PUS1 in the preparation of a product for evaluating the sensitivity of enzalutamide to the treatment of prostate cancer.

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

[0018] Preferably, the primers for detecting the expression level of the PUS1 gene are selected from the following primer pairs, the upstream sequence of the primer pair 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 the content of PUS1 protein is selected from anti-PUS1 antibody (ab203010, abcam).

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

[0021] The fourth aspect of the present invention provides a pharmaceutical composition for treating prostate cancer, comprising enzalutamide and an enzalutamide active agent; the enzalutamide active agent is selected from one or more of a PUS1 mutant and a PUS1 inhibitor; 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 a filler, a disintegrant, a binder, a lubricant, a flavoring agent, a preservative, an antioxidant, and a colorant.

[0030] It should be understood that, unless otherwise specified, in the context of the present invention, the PUS1 inhibitor refers to a substance that can specifically downregulate the expression level of PUS1 and / or the transcription level of its mature mRNA and / or the expression level or activity of PUS1 protein, for example, antisense oligonucleotides, siRNA, shRNA, sgRNA, antagomiRs, miRNA sponges, miRNAErasers, Target Masking and / or multi-target methods are used to downregulate the expression level and / or activity of PUS1, as long as the level and / or activity of PUS1 can be reduced. The primers and / or primer pairs refer to PCR primers used to synthesize the cDNA chain of the PUS1 gene in PCR, so as to detect the expression level of the mRNA of the PUS1 gene. In addition to the primers and / or primer pairs listed in the present invention, those skilled in the art are fully capable of designing corresponding primers, primer pairs, and antibodies according to the gene sequence of PUS1 using conventional methods in the field including but not limited to molecular biology, and screening the designed primers and / or primer pairs by conventional experimental means, or obtaining them commercially, as long as they can achieve specific detection of PUS1 expression levels; other conventional reagents and methods in the field can also be used to detect PUS1 protein expression levels.

[0031] Through a large number of studies, the present invention found that PUS1 expression was positively correlated with the Gleason score of prostate cancer; and it was expressed more highly in mCRPC. KM curve analysis showed that the ARSI treatment-related overall survival of patients with high expression of PUS1 was significantly shortened. In addition, detection in non-resistant and resistant cell lines of LNCaP and C4-2B found that PUS1 was highly expressed in resistant cell lines. Immunohistochemistry of clinical samples and PDX models found that compared with androgen-dependent prostate cancer, PUS1 was highly expressed in CRPC samples, and was more highly expressed in enzalutamide-resistant tissues. The above results indicate that PUS1 expression is associated with the malignancy and poor prognosis of prostate cancer. In vivo and in vitro experimental studies found that the expression of PUS1 was knocked down in C4-2B_ENZR and LNCaP_EZNR cell lines, and CCK-8 and plate cloning verification found that the resistance of both to enzalutamide was significantly reduced. The results of the subcutaneous transplant tumor model showed that in the enzalutamide-treated group, after knocking down the expression of PUS1, the growth of subcutaneous tumors was significantly slowed down, and the final tumor weight was smaller. The above results indicate that PUS1 is an important molecule that mediates enzalutamide resistance in prostate cancer.

[0032] Since there are few studies related to PUS1 and tumors, the latest studies have revealed that the Ψ modification activity of PUS1 is related to the translation efficiency of various proto-oncogenes (IRS1, MYC, etc.), but the specific mechanism is unknown. Previous studies have found that PUS1 is related to the variable splicing of the 3′ end of pre-mRNA. In vitro verification has found that there are obvious Ψ modification sites in the exons or flanking introns of genes such as PUM2, and obvious splicing differences appear in cells knocked out of PUS1. In addition, studies have shown that PUS1 can modify the terminator of the target mRNA, causing a gene read-through effect and changing the full-length amino acid sequence of the protein. In order to clarify whether PUS1 regulates enzalutamide resistance in prostate cancer through Ψ activity, mutations were made at these five sites (R116L, D118K, R171T, Y173E, R267L), and an overexpression plasmid was constructed. CCK-8 and plate cloning assays found that overexpression of mutant PUS1_mut cells helped reduce the resistance of prostate cancer cells to enzalutamide, indicating that PUS1 promoted enzalutamide resistance in prostate cancer by relying on its Ψ modification activity.

[0033] In general, the present invention, through bioinformatics analysis and a series of in vitro and in vivo experiments, has for the first time discovered and clarified the key role of PUS1 expression level in reducing the sensitivity of enzalutamide in the treatment of prostate cancer, and has deeply studied its mechanism of action, that is, PUS1 promotes enzalutamide resistance in prostate cancer and depends on its Ψ modification activity. The present invention enriches the relevant mechanisms of drug resistance generation and regulation in the process of enzalutamide treatment of prostate cancer, provides sufficient scientific basis and theoretical foundation for exploring new molecular targets for prostate cancer diagnosis, prognosis determination and treatment, and developing new targeted drugs, and has important social value and scientific significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the relationship between PUS1 expression and Gleason score in the TCGA-PRAD dataset.

[0035] Figure 2 Schematic diagram of the differential expression results of PUS1 in normal prostate tissue, prostate cancer in situ and mCRPC in the GSE35988 dataset.

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

[0037] Figure 4 Schematic diagram of WB verification of the protein expression changes of PUS1 in resistant and non-resistant prostate cancer cells.

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

[0039] Figure 6 Schematic diagram of WB verification of PUS1 knockdown efficiency in C4-2B_ENZR and LNCaP_ENZR cells.

[0040] Figure 7 Schematic diagram of the results of CCK8 detection of changes in the sensitivity of C4-2B_ENZR cells to enzalutamide after knockdown of PUS1.

[0041] Figure 8 Schematic diagram of the results of CCK8 detection of changes in the sensitivity of LNCaP_ENZR cells to enzalutamide after knockdown of PUS1.

[0042] Fig. 9 Schematic diagram of the results of the plate cloning experiment to detect the changes in the cloning ability of C4-2B_ENZR cells under enzalutamide stimulation after knocking down PUS1.

[0043] Fig.10 Schematic diagram of the results of the plate cloning experiment to detect the changes in the cloning ability of LNCaP_ENZR cells under enzalutamide stimulation after knocking down PUS1.

[0044] Fig.11 Schematic diagram of the effect of PUS1 expression level on tumor growth in mice treated with enzalutamide.

[0045] Fig.12 Schematic diagram of the quantitative analysis results of the effect of PUS1 expression level on tumor growth in mice treated with enzalutamide.

[0046] Fig.13 Schematic diagram of the results of PUS1 enzyme modification active site mutation.

[0047] Fig.14 Schematic diagram of WB verification of the expression efficiency of wild-type PUS1_wt and mutant PUS1_mut plasmids.

[0048] Fig.15 Schematic diagram of the results of CCK8 detection of changes in the sensitivity of prostate cancer cells to enzalutamide after overexpression of PUS1_wt and PUS1_mut.

[0049] Fig.16 Schematic diagram of the results of the plate cloning experiment to detect the changes in the cloning ability of prostate cancer cells after overexpressing PUS1_wt and PUS1_mut under enzalutamide treatment. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

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

[0052] The clinical samples used in the present invention were all signed with informed consent by the patients; the pathology and tissue morphology of all tissue samples were confirmed by the pathology department. The procedures and methods of the present invention were approved by the Institutional Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University, in line with the requirements of medical ethics and the quality management standards for drug clinical trials, and the experimental processes all complied with the Declaration of Helsinki. The experimental methods used in the present invention, such as bioinformatics analysis, molecular biology experiments, cell biology experiments, immunohistochemistry, etc., are conventional methods and techniques in the field. Representative results selected from the biological experiment repetitions are presented in the context figures, and the data are displayed as mean±SD and mean±SEM as specified in the figure. All in vitro experiments were repeated at least three times, and animal experiments were repeated twice. The data were analyzed using GraphPad Prism 8.0 software. Conventional medical statistical methods such as t-test, chi-square test, and analysis of variance were used to compare the mean differences between two or more groups. *p<0.05 is considered a significant difference.

[0053] Example 1

[0054] First, we analyzed the TCGA-PRAD dataset and found that PUS1 expression was positively correlated with the Gleason score of prostate cancer (see Figure 1). Subsequently, the expression of PUS1 in normal prostate tissue (normal), prostate cancer in situ (primary) and metastatic castration-resistant prostate cancer (mCRPC) was analyzed in the GSE35988 dataset, and it was found that the expression of PUS1 in tumor tissue of mCRPC patients was significantly increased (see Figure 2 ). Kaplan-Meier curve analysis found that PUS1 expression was significantly correlated with ARSI treatment-related overall survival in mCRPC patients, that is, the ARSI treatment-related overall survival of patients with high PUS1 expression was significantly shortened (see Figure 3 ).

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

[0056] (1) Prostate cancer cells in the logarithmic growth phase were collected after digestion into 1.5 mL EP tubes, washed twice with PBS, added with cell lysis buffer containing 1× protease inhibitor and phosphatase inhibitor, and placed on ice for 30 min; after cell lysis, collected into 1.5 mL EP tubes, and centrifuged at 12000 rpm and 4°C for 10 min.

[0057] (2) After protein quantification, add 5× protein loading buffer according to the sample volume and mix well, then incubate at 95°C for 5 min. Perform protein electrophoresis on the obtained protein sample, set the starting voltage to 80 V, and adjust the voltage to 120 V when the protein marker bands are clearly separated.

[0058] (3) After the protein electrophoresis is completed, the membrane can be transferred. First, place the protein gel in the transfer solution for balance, then add an appropriate amount of transfer solution into the container, and place the sponge, filter paper, PVD F membrane (activated with methanol), gel, filter paper, and sponge in order. Expel the gas and place the transfer tank in ice water. The transfer condition is 300mA constant current for 1h.

[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 required bands, add the corresponding primary antibody, and place it on a shaker at 4°C overnight. Recover the primary antibody and wash the membrane three times with 1×TBST buffer, each for 10 minutes. Incubate the secondary antibody at room temperature for 1 hour, and then wash the membrane three times with TBST buffer, each for 10 minutes.

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

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

[0062] (1) The sample tissue is embedded in paraffin for sealing. The paraffin specimens are stored at room temperature, and the cut white slices are stored in a refrigerator at 4°C. During the experimental test, the paraffin slices are taken out and placed in a 60°C oven for about 2 hours to make the slices fit the glass slides.

[0063] (2) Dewax the sections in two portions of xylene for 10 min each. Hydrate the sections in gradient alcohol (100%, 100%, 95%, 85%, 70%) for 5-7 min each, and then wash with PBS three times for 3 min each.

[0064] (3) Immerse the sections in citrate antigen retrieval solution and heat them in a microwave oven at medium-high temperature for 20 min for antigen retrieval. After heating, let the sections cool naturally to room temperature in the antigen retrieval solution and wash them three times with PBS, each time for 3 min.

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

[0066] (5) Shake off the blocking solution, add the primary antibody to cover the tissue without washing, and place it in a wet box at 4°C for blocking overnight. On the second day, place it at room temperature for 1 hour, then wash it with PBS for 5 minutes, and repeat 3 times. Add the reaction sensitization solution and incubate it at room temperature for 20 minutes, wash it three times with PBS; add the biotin-labeled secondary antibody, incubate it at room temperature for 30 minutes, wash it with PBS for 5 minutes, and repeat 3 times.

[0067] (6) Add DAB for color development, and place in water to terminate the reaction after color development is completed. Add hematoxylin for 25-30 seconds for re-staining, and place in water to terminate the coloring; then rinse in running water for 30-45 minutes until the color of the slice is stable for anti-blue treatment. Gradient alcohol dehydration: Soak in 70%, 85%, 95%, 100%, 100% alcohol, and xylene in sequence for dehydration, each time for 5 minutes, and finally seal the slide.

[0068] Two independent researchers analyzed the clinical pathological immunohistochemical sections of the patients without knowing the patient information. Figure 5As shown. The results showed that compared with ADPC, the expression of PUS1 in CRPC samples was significantly increased, and the expression was higher in enzalutamide-resistant tissues. The above results indicate that PUS1 expression is associated with the malignancy and poor prognosis of prostate cancer.

[0069] Example 2

[0070] The above examples clearly show the adverse effects of high expression of PUS1 and enzalutamide on the drug resistance of prostate cancer. In order to further study the effect of PUS1 on the therapeutic activity of enzalutamide on prostate cancer, a series of in vivo and in vitro experiments were conducted to further explore.

[0071] First, siRNA and shRNA were designed to reduce the expression level of PUS1 in cells 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 primers according to the siRNA and shRNA structures and synthesize primers, dilute the synthesized siRNAs and shRNAs primers with water to 100 μM, take 10 μL of the upstream and downstream primers respectively, mix them evenly, and then use a PCR instrument to gradually cool from 100°C to room temperature to anneal and synthesize double strands; this embodiment uses two siRNAs and shRNAs 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 As shown in NO: 5, it is 5'-CcggTGTCGGGTCCTCACAATTCAAC TCGAGTTGAATTGTGAGGACCCGACATTTTTg-3').

[0073] (2) The pLKO.1 lentiviral vector was double-digested with EcoRI and AgeI endonucleases, and two single bands were obtained by gel running after digestion. The larger band was cut out for gel recovery; then the annealed double-stranded DNA was ligated to the vector after gel recovery.

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

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

[0076] (5) One day before infection, inoculate the enzalutamide-resistant prostate cancer cells (LNCaP_ENZR, C4-2B_ENZR) in the logarithmic growth phase into a 6-cm culture dish, discard the original culture medium, add 3 mL of virus solution, and add 3 μL of polybrene to promote virus infection. After 24 hours, discard the virus solution and replace with fresh culture medium for continued culture.

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

[0078] Test results such as Figure 6 The results show that the two siRNAs and shRNAs designed according to the present invention can produce significant inhibitory activity on PUS1 and reduce the expression level of PUS1 protein in prostate cancer cells.

[0079] Subsequently, the effects of inhibiting PUS1 expression levels on the proliferation and clone formation of prostate cancer cells were studied. 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 with knockdown PUS1 gene expression sequences (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 trypsin and counted. The appropriate cell density (1.5×10 5 / well) and inoculated into 96-well plates (3 replicates). Different concentrations of enzalutamide (10 0 , 10 0.5 , 10 1 , 10 1.5 , 10 2 , μM).

[0081] (2) Culture the cells in a 37°C incubator and collect the cells after 48 hours. Add 10 μL of CCK-8 to each well and incubate the culture plate in the incubator for 1-4 hours. Measure the absorbance at 450 nm to evaluate the cell proliferation.

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

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

[0084] (2) The cell suspension was diluted and inoculated at a density of 1000 / well in a dish containing 10 mL of 37°C pre-warmed culture medium. The cells were gently rotated to disperse the cells evenly. Enzalutamide (10 μM in LNCaP_ENZR and 25 μM in C4-2B_ENZR) was added. The control group was treated with an equal volume of DMSO and placed at 37°C with 5% CO. 2 The cells were cultured in a cell culture incubator with saturated humidity.

[0085] (3) When clones visible to the naked eye appear in the culture dish, terminate the culture, discard the supernatant, carefully rinse 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 the remaining methanol with water; cell clones can be observed; observe under a microscope, and a valid clone is counted when the number of cells is greater than 50.

[0086] Test results such as Figure 7-10 As shown. The results showed that after using siRNA or shRNA to inhibit the expression of PUS1 in cells, the resistance of prostate cancer cells to enzalutamide was significantly reduced, and the killing activity of enzalutamide on prostate cancer cells was improved, and the difference was statistically significant; the results of the cell clone formation experiment showed that the clone formation of prostate cancer cells could be achieved by inhibiting the expression of PUS1, and when the expression of PUS1 was further inhibited on the basis of enzalutamide treatment, the clone formation of prostate cancer cells could be very significantly inhibited, and the difference was statistically significant. The above results show that the expression level of PUS1 in cells is closely related to the cytotoxicity of enzalutamide. The lower the expression level of PUS1, the more significant the killing effect of 5-FU on prostate cancer cells.

[0087] Furthermore, the effect of PUS1 expression on tumor growth was studied through in vivo experiments. The specific steps are as follows:

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

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

[0090] (3) Mice in group 3 and group 4 were treated with enzalutamide (10 mg / kg, gavage for 4 consecutive days per week, 3 days off, for a total of 3 weeks); mice in group 1 and group 2 were treated with an equal volume of saline containing 5% DMSO. Tumor volume was measured regularly during treatment. Mice were killed 3 weeks after treatment, and the tumors were removed and weighed to calculate the tumor inhibition rate.

[0091] The experimental results are as follows Figure 11-12 The results showed that inhibiting the expression of PUS1 in vivo alone could inhibit the growth of prostate cancer tumors to a certain extent; and after treatment with enzalutamide, the tumor volume growth rate of mice with low PUS1 expression in vivo was significantly reduced, and the tumor volume was significantly reduced. The difference was statistically significant, further confirming that PUS1 is an important molecule that mediates enzalutamide resistance in prostate cancer.

[0092] Example 3

[0093] There are few studies related to PUS1 and tumors in the prior art. The latest study revealed that the Ψ modification activity of PUS1 is related to the translation efficiency of various proto-oncogenes (IRS1, MYC, etc.), but the specific mechanism is not clear. Previous studies have found that PUS1 is related to the variable splicing of the 3′ end of pre-mRNA. In vitro analysis found that there are obvious Ψ modification sites in the exons or flanking introns of genes such as PUM2, and obvious splicing differences appeared in cells knocked out of PUS1. In addition, studies have shown that PUS1 can cause gene read-through effects and change the full-length amino acid sequence of proteins by modifying the terminator of target mRNA.

[0094] To clarify whether PUS1 regulates enzalutamide resistance in prostate cancer through Ψ activity, five sites were mutated (R116L, D118K, R171T, Y173E, R267L) and overexpression plasmids were constructed (see Fig.13). Subsequently, the vectors with mutations at the five sites were transfected into C4-2B cells, and the intracellular PUS1 expression level was detected by Western blot according to the above method. The results showed that the mutation of the site was successful and the intracellular PUS1 expression level was significantly reduced (see Fig.14 ).

[0095] Subsequently, PUS1_wt, PUS1_mut or 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 test results are shown in Fig.15 The results showed that under low and medium concentrations of enzalutamide treatment, the overexpression of PUS1 in cells transfected with PUS1_wt significantly reduced the sensitivity of cells to enzalutamide treatment, and significantly increased the cell proliferation activity; after transfection with PUS1_mut with 5 mutation sites, the drug resistance of prostate cancer cells to enzalutamide was significantly reduced, and the cell proliferation activity was significantly inhibited.

[0096] Furthermore, PUS1_wt, PUS1_mut or blank vector were transfected into C4-2B cells, and the effect of enzalutamide treatment at a specific concentration (25 μM) on cell clone formation was detected using the aforementioned method. The test results are as follows: Fig.16 As shown. The results showed that in cells transfected with PUS1_wt, due to the overexpression of PUS1, the therapeutic sensitivity of cells to enzalutamide was significantly reduced, and the cell clone formation ability was significantly improved; after transfection with PUS1_mut with 5 mutation sites, the resistance of prostate cancer cells to enzalutamide was significantly reduced, and the clone formation ability of cells was significantly inhibited. The above results show that R116, D118, R171, Y173, and R267 are the key sites for PUS1 to exert Ψ modification enzyme activity. PUS1 promotes enzalutamide resistance in prostate cancer by relying on its Ψ modification activity. By mutating the above sites, the Ψ modification activity of PUS1 can be effectively inhibited, thereby promoting the therapeutic sensitivity of prostate cancer to enzalutamide.

[0097] From the above, it can be clearly seen that PUS1 expression is positively correlated with the Gleason score of prostate cancer; and it is more expressed in mCRPC. KM curve analysis showed that the ARSI treatment-related overall survival of patients with high PUS1 expression was significantly shortened. In addition, detection in non-resistant and resistant cell lines of LNCaP and C4-2B found that PUS1 was highly expressed in resistant cell lines. Immunohistochemistry of clinical samples and PDX models found that compared with androgen-dependent prostate cancer, PUS1 was highly expressed in CRPC samples, and was more highly expressed in enzalutamide-resistant tissues. The above results indicate that PUS1 expression is associated with the malignancy and poor prognosis of prostate cancer. In vivo and in vitro experimental studies found that knocking down PUS1 expression in C4-2B_ENZR and LNCaP_EZNR cell lines, CCK-8 and plate cloning verification found that the resistance of both to enzalutamide was significantly reduced. The results of the subcutaneous transplant tumor model showed that in the enzalutamide-treated group, after knocking down the expression of PUS1, the growth of subcutaneous tumors was significantly slowed down, and the final tumor weight was smaller. The above results indicate that PUS1 is an important molecule that mediates enzalutamide resistance in prostate cancer.

[0098] Since there are few studies related to PUS1 and tumors, the latest studies have revealed that the Ψ modification activity of PUS1 is related to the translation efficiency of various proto-oncogenes (IRS1, MYC, etc.), but the specific mechanism is unknown. Previous studies have found that PUS1 is related to the variable splicing of the 3′ end of pre-mRNA. In vitro verification has found that there are obvious Ψ modification sites in the exons or flanking introns of genes such as PUM2, and obvious splicing differences appear in cells knocked out of PUS1. In addition, studies have shown that PUS1 can modify the terminator of the target mRNA, causing a gene read-through effect and changing the full-length amino acid sequence of the protein. In order to clarify whether PUS1 regulates enzalutamide resistance in prostate cancer through Ψ activity, mutations were made at these five sites (R116L, D118K, R171T, Y173E, R267L), and an overexpression plasmid was constructed. CCK-8 and plate cloning assays found that overexpression of mutant PUS1_mut cells helped reduce the resistance of prostate cancer cells to enzalutamide, indicating that PUS1 promoted enzalutamide resistance in prostate cancer by relying on its Ψ modification activity.

[0099] The present invention enriches the relevant mechanisms of drug resistance generation and regulation during the treatment of prostate cancer with enzalutamide, provides sufficient scientific basis and theoretical foundation for exploring new molecular targets for prostate cancer diagnosis, prognosis determination and treatment, and developing new targeted drugs, helps to better achieve 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 specific implementation method part specifically introduces the analytical method involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the method and ideas of the present invention, rather than limiting the relevant content. Without departing from the principle of the present invention, those skilled in the art may also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. Use of a PUS1 mutant in the preparation of a drug for improving the sensitivity of enzalutamide to the treatment of prostate cancer, characterized in that: The mutation site of the PUS1 mutant is selected from one or more of R116, D118, R171, Y173, and R267.

2. The use according to claim 1, characterized in that: The mutation sites of the PUS1 mutant are R116, D118, R171, Y173, and R267.

3. The use according to claim 1, characterized in that: The prostate cancer is selected from castration-resistant prostate cancer.

4. Application of PUS1 inhibitors in the preparation of drugs for improving the sensitivity of enzalutamide in the treatment of prostate cancer.

5. The use according to claim 4, characterized in that: The PUS1 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the PUS1 gene.

6. The use according to claim 4, characterized in that: The prostate cancer is selected from castration-resistant prostate cancer.

7. Use of a reagent for detecting the expression level of PUS1 in the preparation of a product for evaluating the sensitivity of enzalutamide to the treatment of prostate cancer.

8. The use according to claim 7, characterized in that: The reagent for detecting the expression level of PUS1 includes primers for detecting the expression level of PUS1 gene and / or reagents for detecting the content of PUS1 protein.

9. The use according to claim 7, characterized in that: The prostate cancer is selected from castration-resistant prostate cancer.

10. A pharmaceutical composition for treating prostate cancer, characterized in that: It includes enzalutamide and an enzalutamide active agent; the enzalutamide active agent is selected from one or more of a PUS1 mutant and a PUS1 inhibitor; the mutation site of the PUS1 mutant is selected from one or more of R116, D118, R171, Y173, and R267.

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