Application of NUDT14 in improving sensitivity of Enzalocamide to treatment of prostatic cancer

Abstract

The invention relates to an application of NUDT14 in improving the sensitivity of Enzalocamide to prostatic cancer treatment. Through deep research, it is clear that NUDT14 can be used as a key factor for occurrence and development of prostate cancer and generation of enzalocamide treatment drug resistance, activities such as proliferation and metastasis of prostate cancer can be obviously inhibited by inhibiting NUDT14 or reducing the biological activity of NUDT14, the drug resistance of enzalocamide is reversed, the treatment sensitivity of enzalocamide is improved, and the survival rate of patients is prolonged; the prognosis is improved. According to the invention, related mechanisms of drug resistance generation and regulation in the process of treating prostatic cancer by enzalocamide are enriched, sufficient scientific basis and theoretical basis are provided for searching new prostatic cancer diagnosis, prognosis judgment and treatment molecular targets and developing new targeted drugs, accurate treatment can be better realized, and the application of enzalocamide in treatment of prostatic cancer is promoted. The method has important social value and scientific significance.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and relates to application of NUDT14 in improving sensitivity of enzalutamide to prostate cancer treatment. BACKGROUND

[0002] Prostate cancer has become the most common malignant tumor in men in Europe and the United States, and its mortality rate is only second to lung cancer. Although the morbidity and mortality of prostate cancer in China are relatively lower than those in western countries, due to factors such as aggravation of population aging, change of dietary habits and lifestyle, the morbidity of prostate cancer in China is showing a rapid upward trend year by year. Prostate cancer has high heterogeneity and usually grows slowly. Early localized prostate cancer usually adopts radical surgery treatment to effectively control disease progression, but due to lack of typical clinical symptoms in the early stage of tumor and restriction by examination means and other conditions, most patients in China are in the advanced stage when diagnosed, at which time endocrine therapy becomes the preferred treatment for patients in the advanced stage, but most patients still progress to the castration-resistant prostate cancer (CRPC) stage after treatment for a median time of about 2 years.

[0003] Enzalutamide is a new generation of androgen receptor antagonist (AR) developed in recent years, which was first approved for marketing by the US FDA in 2012 and was recommended by authoritative guidelines at home and abroad as a first-line treatment for metastatic / non-metastatic castration-resistant prostate cancer (nm / mCRPC). However, most patients inevitably develop enzalutamide resistance after a period of treatment. Once drug resistance occurs, patients often face the predicament of no medicine to cure. Therefore, it is particularly important to continue to explore the molecular mechanism of prostate cancer enzalutamide resistance, to screen new treatment targets for prostate cancer, and to provide new research ideas for delaying or even reversing drug resistance of tumor cells. SUMMARY

[0004] The present application aims at solving the problem of serious reduction of treatment effect caused by drug resistance in the prior art when enzalutamide is used to treat prostate cancer, and thus the molecular mechanism of enzalutamide resistance is studied in depth, and it is determined that NUDT14 is an important target related to enzalutamide resistance of prostate cancer, and by inhibiting NUDT14, the generation of enzalutamide resistance of prostate cancer can be effectively reduced, the treatment sensitivity of enzalutamide is improved, the survival rate of patients is prolonged, and the prognosis is improved. At the same time, by detecting the expression and / or mutation of NUDT14 in the body of patients, the prognosis of the patients receiving enzalutamide treatment can be reasonably evaluated, and a new strategy for the treatment and prognosis evaluation of prostate cancer is provided.

[0005] In order to solve the above technical problems, the present application is realized by the following technical scheme.

[0006] The present application provides the use of a NUDT14 inhibitor in the preparation of a drug for improving the treatment sensitivity of enzalutamide to prostate cancer in the first aspect of the present application.

[0007] Preferably, the NUDT14 inhibitor is selected from one or more of siRNA, shRNA and sgRNA designed based on the NUDT14 gene.

[0008] Preferably, the NUDT14 inhibitor is selected from siRNA and / or shRNA designed based on the NUDT14 gene.

[0009] Preferably, the siRNA is selected from one or more of siNUDT14#1 (the sequence is shown as SEQ ID NO: 1, 5'-GGAGCCTGGTGTTGGTGAATT-3'), siNUDT14#2 (the sequence is shown as SEQ ID NO: 2, 5'-GGCCGCTCACGCTGCATTATT-3'), and siNUDT14#3 (the sequence is shown as SEQ ID NO: 3, 5'-GGGACTTCATGAAGACGCATT-3').

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

[0011] The present application provides the use of a reagent for detecting the expression level of NUDT14 in the preparation of a product for evaluating the treatment sensitivity of enzalutamide to prostate cancer in the second aspect of the present application.

[0012] Preferably, the reagent for detecting the expression level of NUDT14 comprises primers for detecting the expression level of NUDT14 gene and / or reagents for detecting the content of NUDT14 protein.

[0013] As preferred, the primers for detecting the expression level of NUDT14 gene are selected from the following primer pairs:

[0014] The primer pair has an upstream sequence as shown in SEQ ID NO: 4 (5'-GGTCTGGAGTGGGACTGACT-3') and a downstream sequence as shown in SEQ ID NO: 5 (5'-CTGGCTGAGGAACCATGAGA-3').

[0015] As preferred, the reagent for detecting the content of NUDT14 protein is selected from anti-NUDT14 Antibody (NBP2-85412-0.1 mL; Novus Biologicals).

[0016] As preferred, the prostate cancer is castration-resistant prostate cancer.

[0017] The third aspect of the present application provides a pharmaceutical composition for preventing and / or treating prostate cancer, comprising a NUDT14 inhibitor and enzalutamide.

[0018] As preferred, the NUDT14 inhibitor is selected from one or more of siRNA, shRNA, sgRNA designed based on NUDT14 gene.

[0019] As preferred, the NUDT14 inhibitor is selected from siRNA and / or shRNA designed based on NUDT14 gene.

[0020] As preferred, the siRNA is selected from one or more of siNUDT14#1, siNUDT14#2, siNUDT14#3.

[0021] As preferred, the pharmaceutical composition can optionally comprise a pharmaceutically acceptable carrier.

[0022] As preferred, the pharmaceutically acceptable pharmaceutical adjuvant is selected from one or more of a filler, a disintegrant, a binder, a lubricant, a flavoring agent, a preservative, an antioxidant, a coloring agent.

[0023] As preferred, the prostate cancer is castration-resistant prostate cancer.

[0024] As preferred, the castration-resistant prostate cancer is neuroendocrine prostate cancer.

[0025] The fourth aspect of the present application provides use of a NUDT14 inhibitor in the preparation of a medicament for preventing and / or treating prostate cancer.

[0026] As preferred, the NUDT14 inhibitor is selected from one or more of siRNA, shRNA, sgRNA designed based on NUDT14 gene.

[0027] As preferred, the NUDT14 inhibitor is selected from siRNA and / or shRNA designed based on NUDT14 gene.

[0028] As preferred, the siRNA is selected from one or more of siNUDT14#1, siNUDT14#2, siNUDT14#3.

[0029] As preferred, the prostate cancer is castration-resistant prostate cancer.

[0030] The fifth aspect of the present application provides a kit for evaluating the sensitivity of enzalutamide in the treatment of prostate cancer, comprising reagents for detecting the expression level of NUDT14.

[0031] As preferred, the reagents for detecting the expression level of NUDT14 comprise reagents for detecting the expression level of NUDT14 gene and / or reagents for detecting the content of NUDT14 protein.

[0032] As preferred, the reagents for detecting the expression level of NUDT14 gene are selected from the following primer pairs:

[0033] The upstream sequence of the primer pair is shown in SEQ ID NO: 4, and the downstream sequence is shown in SEQ ID NO: 5.

[0034] As preferred, the reagents for detecting the content of NUDT14 protein are selected from anti-NUDT14 Antibody (NBP2-85412-0.1 mL; Novus Biologicals).

[0035] As preferred, the kit further comprises one or more of PCR enzyme, PCR buffer, dNTPs, and fluorescent substrate.

[0036] As preferred, the fluorescent substrate is selected from Syber Green or fluorescently labeled probes.

[0037] As preferred, the prostate cancer is castration-resistant prostate cancer.

[0038] It should be understood that, in the context of the present application, the NUDT14 includes NUDT14 nucleotide and NUDT14 protein encoded by NUDT14 nucleotide, etc. without special instructions. The NUDT14 inhibitor refers to a substance that can specifically down-regulate the expression level of NUDT14 and / or the transcription level of its mature mRNA and / or the expression level or activity of NUDT14 protein, such as down-regulating the expression level and / or activity of NUDT14 by using antisense oligonucleotide, siRNA, shRNA, sgRNA, antagomiRs, miRNA sponge, miRNA eraser, target masking and / or multi-target, etc. As long as it can achieve the reduction of the level and / or activity of NUDT14. The primer and / or primer pair refers to the PCR primer used for synthesizing the cDNA chain of NUDT14 gene in PCR, thereby for detecting the expression level of NUDT14 gene mRNA. In addition to the primers and / or primer pairs listed in the present application, those skilled in the art are fully capable of designing corresponding primers and / or primer pairs according to the gene sequence of NUDT14 by using conventional methods in the art including but not limited to molecular biology, and screening the designed primers and / or primer pairs by conventional experimental means, as long as it can achieve specific detection of the expression level of NUDT14; NUDT14 protein expression level can also be detected by using conventional reagents and methods in the art; the same is true for other genes / proteins, etc.

[0039] NUDT14 is a uridine diphosphate glucose (UDPG) pyrophosphatase, which belongs to the Nudix hydrolase superfamily member. At present, the research on NUDT14 related to tumors is few, and is only limited to the discussion of the differential expression level of NUDT14 in different tumors, and there is no any report about the specific regulatory mechanism of NUDT14 in prostate cancer.

[0040] The application determines that the super-enhancer related gene Nudix Hydrolase 14 (NUDT14) is a key molecule for promoting prostate cancer enzalutamide resistance through prostate cancer transcriptomic data in public databases, combined with targeted cleavage and tagmentation technology (Cleavage Under Targets and Tagmentation, CUT&Tag), transcriptome sequencing, CRISPR-Cas9 gene editing technology and a series of in vivo and in vitro experiments. First, the application obtains the expression information of primary prostate cancer, mCRPC, CRPR-Adeno, CRPC-NE and other samples from public databases (GSE35988, GSE80609 and Beltran-2016), and through differential analysis, it can be seen that the expression level of NUDT14 in mCRPC is significantly higher than that in situ cancer, and its RNA expression level in neuroendocrine tumors is higher than that in adenocarcinoma. In the SU2C cohort, through KM survival curve analysis, it is found that the ARSI treatment sensitive time and overall survival of mCRPC patients with high expression of NUDT14 are shorter. In addition, by collecting prostate cancer clinical tissue samples, it is found by immunohistochemical analysis that NUDT14 is expressed lower in CRPC samples than in enzalutamide-resistant CRPC samples. In order to verify whether NUDT14 in drug-resistant cell lines is positively regulated by super-enhancer, the application constructs enzalutamide-resistant prostate cancer cell lines (C4-2B_ENZR and LNCaP_ENZR), and after treating C4-2B_ENZR and LNCaP_ENZR cell lines with BET inhibitor JQ1, it is found by qPCR detection that compared with the control group, JQ1 can cause the expression of NUDT14 in the two drug-resistant cell lines to be greatly reduced. By dividing the super-enhancer NUDT14-SE region into SE1-SE4, using CRISPR-Cas9 gene editing technology to target knock out SE2 sequence, and detecting NUDT14 expression by WB and qPCR, the results show that knocking out SE2 can significantly reduce the expression level of NUDT14 in drug-resistant cells. In order to reveal the role of NUDT14 in prostate cancer enzalutamide resistance, first, the expression of NUDT14 is knocked down in C4-2B_ENZR and LNCaP_ENZR cells, and its expression efficiency is obviously down-regulated by WB verification. Cell proliferation and plate cloning experiments show that knocking down NUDT14 expression can improve the sensitivity of drug-resistant cell lines to enzalutamide. The results of in vivo experiments show that compared with the control group, after overexpression of NUDT14, the growth of subcutaneous tumors treated with enzalutamide is significantly improved, and the treatment sensitivity is significantly reduced.

[0041] Overall, the present application clarifies that NUDT14 can be a key factor for the occurrence and development of prostate cancer and the generation of enzalutamide treatment resistance, and by inhibiting NUDT14 or reducing its biological activity, the proliferation and metastasis of prostate cancer can be significantly inhibited, the drug resistance of enzalutamide is reversed, the treatment sensitivity is improved, the survival rate of patients is prolonged, and the prognosis is improved. The present application enriches the related mechanism of drug resistance generation and regulation in the process of enzalutamide treating prostate cancer, provides sufficient scientific basis and theoretical basis for exploring new molecular targets for diagnosis, prognosis and treatment of prostate cancer, and developing new targeted drugs, and helps to better realize precision treatment, has important social value and scientific significance. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Figure 1 is a schematic diagram of the results of analyzing the expression level of NUDT14 in prostate cancer carcinoma in situ and mCRPC in the GSE35988 data set.

[0043] Figure 2 Figure 2 is a schematic diagram of the results of analyzing the expression level of NUDT14 in prostate cancer carcinoma in situ and mCRPC in the GSE80609 data set.

[0044] Figure 3 Figure 3 is a schematic diagram of the results of analyzing the expression difference of NUDT14 in CRPC-Adeno and CRPC-NE in the Beltran 2016 data set.

[0045] Figure 4 Figure 4 is a schematic diagram of the results of Kaplan-Meier survival analysis to study the relationship between NUDT14 expression and ARSI treatment sensitivity and treatment-related overall survival of mCRPC patients.

[0046] Figure 5 Figure 5 is a schematic diagram of the results of WB detection of NUDT14 expression in prostate cancer WT and drug-resistant cell lines (ENZR).

[0047] Figure 6 Figure 6 is a schematic diagram of the results of using IHC to verify the expression difference of NUDT14 in CRPC and enzalutamide treatment-resistant CRPC tissues.

[0048] Figure 7 Figure 7 is a schematic diagram of the results of qPCR detection of NUDT14 expression changes after JQ1 treatment of drug-resistant cells.

[0049] Figure 8 Figure 8 is a schematic diagram of the H3K27ac and H3K4me1 modification levels of NUDT14 in C4-2B_WT and C4-2B_ENZR cells.

[0050] Figure 9To detect NUDT14 expression after targeted knockout of SE2 by CRISPR-Cas9 technology using WB.

[0051] Figure 10 To detect NUDT14 expression after targeted knockout of SE2 by CRISPR-Cas9 technology using qPCR.

[0052] Figure 11 Schematic diagram of results for WB verification of NUDT14 knockdown efficiency in C4-2B_ENZR and LNCaP_ENZR cell lines.

[0053] Figure 12 Schematic diagram of results for WB verification of NUDT14 knockdown efficiency in LNCaP_ENZR cell lines.

[0054] Figure 13 Schematic diagram of results for cell proliferation experiment.

[0055] Figure 14 Schematic diagram of results for C4-2B_ENZR cell line clonogenicity experiment.

[0056] Figure 15 Schematic diagram of results for LNCaP_ENZR cell line clonogenicity experiment

[0057] Figure 16 Schematic diagram of results for WB verification of NUDT14 overexpression efficiency in C4-2B_ENZR and LNCaP_ENZR cell lines.

[0058] Figure 17 Schematic diagram of results for the effect of overexpressing NUDT14 on tumor growth in mice treated with enzalutamide.

[0059] Figure 18 Schematic diagram of quantitative analysis results for the effect of overexpressing NUDT14 on tumor growth rate and final weight in mice treated with enzalutamide. DETAILED DESCRIPTION

[0060] To make the objectives, technical solutions, and effects of the present application clearer and more explicit, the present application is further described in detail below with reference to the examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0061] The cell lines listed in the context of the present application, including LNCaP and C4-2B, were purchased from the American Type Culture Collection (Manassas, USA) and cultured according to the prior art, all cell lines were identified by short tandem repeat analysis at the China National Culture Collection (Wuhan) and verified for the presence of mycoplasma contamination using a PCR detection kit (Shanghai Biothrive Sci), and were frozen in liquid nitrogen and used for subsequent experiments. The reagents used in the present application were all obtained commercially.

[0062] The clinical samples used in the present application were signed by the patients with informed consent, the relevant procedures and methods were approved by the ethics committee, and met the requirements of medical ethics and the quality management specification for drug clinical trials, and the experimental process complied with the "Helsinki Declaration". The experimental methods used in the present application, such as bioinformatics analysis, molecular biology experiments, cell biology experiments, immunohistochemistry, animal experiments, etc. are conventional methods and techniques in the art. Among them, bioinformatics analysis is completed by R language software version 4.3, "limma" software package is used for differential analysis, and "survival" and "survminer" software packages are used for KM survival curve drawing. Representative results in repeated biological experiments are presented in the context of the drawings, and data are shown as mean ± SD and mean ± SEM according to the provisions in the drawings. 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. The differences in the mean values of two groups or more than two groups were compared using conventional medical statistical methods such as t-test, chi-square test, analysis of variance, etc. *p<0.05 is considered a significant difference.

[0063] Example 1

[0064] Firstly, the mCRPC sample expression profile data were obtained from public databases (GSE35988, GSE80609, Beltran-2016) and subjected to differential analysis. The results showed that the analysis of GSE35988 and GSE80609 databases found that the expression level of NUDT14 in metastatic castration-resistant prostate cancer patients was significantly higher than that in primary prostate cancer patients (*p<0.05, **p<0.01, ****p<0.0001), and the analysis of Beltran-2016 database found that the RNA expression level of NUDT14 in neuroendocrine (NE) tumors was higher than that in adenocarcinoma (Adeno) (see Figures 1-3). Kaplan-Meier survival analysis was performed in the SU2C cohort to investigate the relationship between NUDT14 expression and ARSI treatment sensitivity and treatment-related overall survival in mCRPC patients. It was found that mCRPC patients with high expression of NUDT14 had shorter ARSI treatment sensitivity time and overall survival (see Figure 4 ), that is, high expression of NUDT14 in vivo is more likely to develop resistance to ARSIs treatment.

[0065] Subsequently, Western Blot was used to detect the expression level of NUDT14 in wild-type and enzalutamide-resistant cell lines of two prostate cancer cells (LNCaP and C4-2B). The drug-resistant cell lines were constructed as follows: 25 μM or 10 μM enzalutamide was used to stimulate C4-2B or LNCaP cells for 6 months to induce enzalutamide-resistant cell lines (LNCaP_ENZR, C4-2B_ENZR). The specific steps of Western Blot are as follows:

[0066] (1) The prostate cancer cells were digested, collected, and added with lysis buffer, and lysed on ice for 1 h.

[0067] (2) Centrifuge at 15000xg for 15 min at 4°C, and take the supernatant and add loading buffer (1x) and 95°C water bath for 5 min.

[0068] (3) Take the sample prepared in step (2) for protein gel electrophoresis.

[0069] (4) After electrophoresis, take the protein gel for membrane transfer operation (PVDF membrane, 200mA constant current transfer for 2h).

[0070] (5) After membrane transfer, take out the PVDF membrane and place it in blocking solution, and block at room temperature for 2h on a vertical shaker at 10rpm.

[0071] (6) After blocking, wash the PVDF membrane and immerse it in the primary antibody (anti-NUDT14), and incubate overnight at 4°C on a vertical shaker at 10rpm.

[0072] (7) After incubation of the primary antibody, wash the PVDF membrane and then immerse it in the secondary antibody (HRP-linked anti-rabbit IgG), and incubate at room temperature for 2h on a vertical shaker at 10rpm.

[0073] (8) After incubation of the secondary antibody, wash the PVDF membrane and detect the target protein using a chemiluminescence instrument (α-tubulin as an internal reference).

[0074] The detection results are as follows: Figure 5As shown in the figure. The results showed that the expression level of NUDT14 was significantly higher in enzalutamide-resistant cell lines compared to wild-type cells.

[0075] Furthermore, immunohistochemical analysis was performed on clinical tissue samples from prostate cancer patients. The specific steps are as follows:

[0076] (1) The prostate cancer tissue was sliced ​​to a thickness of 4μm using a pathological tissue slicer, spread on a non-slip glass slide, and dried at 65℃ for 2 hours for later use.

[0077] (2) Dewaxing: Immerse the glass slide in xylene I for 5 min → xylene II for 5 min → xylene III for 5 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → 95% ethanol for 5 min → 85% ethanol for 5 min → 75% ethanol for 5 min → ddH2O I for 3 min → ddH2O II for 3 min.

[0078] (3) Blocking peroxidase: Soak in 3% hydrogen peroxide for 10 min, then wash with PBS for 5 min.

[0079] (4) High-pressure antigen retrieval: Prepare EDTA retrieval solution and add it to the pressure cooker. Place the glass slide in the cooker, heat at 800W for 20 minutes, and then let it cool naturally.

[0080] (5) Wash the slide twice with PBS for 3 minutes each time. Use a small piece of paper to absorb the moisture around the tissue. Use a biochemical pen to draw circles 0.5 cm away from the tissue boundary.

[0081] (6) Oily tissue surface: Immerse the glass slide in 0.1% PBST, lift it up and down and soak for 3 minutes.

[0082] (7) Primary antibody incubation: Dilute the antibody with antibody diluent according to the instructions, incubate at 4°C overnight with 50uL, wash with PBS for 3min, and then wash with PBST for 3min.

[0083] (8) Secondary antibody incubation: 1 drop of DAKO secondary antibody per tissue, covering the tissue surface, incubated at 37°C for 1 hour, washed with PBS for 3 minutes, and then washed with PBST for 3 minutes.

[0084] (9) Prepare DAB chromogenic solution: Prepare the chromogenic solution according to the ratio. After shaking off the liquid on the slide, add freshly prepared DAB chromogenic solution to the circle. Control the chromogenic time under the microscope. The positive result is brownish-yellow. Soak the slide in PBS to stop the chromogenic process.

[0085] (10) Counterstaining cell nuclei: After terminating DAB staining, the slides were counterstained with hematoxylin for about 3 minutes, rinsed with running water, and then mounted with 20 μL of mounting medium. The slides were then observed under a microscope.

[0086] The results showed that the expression level of NUDT14 in CRPC was significantly lower than that in enzalutamide-resistant CRPC tissues (see [link to study]). Figure 6 ).

[0087] The above results indicate that NUDT14 is highly expressed in prostate cancer tissues, especially in CRPC, where its expression is significantly elevated. Therefore, NUDT14 may influence the occurrence and development of prostate cancer and play a key role in the development of drug resistance, thus serving as a potential biomarker and therapeutic target for prostate cancer.

[0088] Example 2

[0089] To verify whether NUDT14 in drug-resistant cell lines is positively regulated by a superenhancer, C4-2B_ENZR and LNCaP_ENZR cell lines were first treated with BET inhibitor JQ1 at varying concentrations for 48 hours, followed by detection via qPCR. The specific steps of the qPCR are as follows:

[0090] (1) Prostate cancer cells (LNCaP_ENZR and C4-2B_ENZR) in the logarithmic growth phase were digested and collected into 1.5 mL EP tubes. NucleoZol (740404.200, biofive) was added to each sample for cell lysis, followed by the addition of ddH2O at a ratio of 1 mL:200 µL (NucleoZol:ddH2O). The lysis buffer was centrifuged at 12,000 rpm for 15 minutes, and the supernatant was transferred to a fresh tube. An equal volume of isopropanol was then added, followed by another centrifugation at 12,000 rpm for 10 minutes. The supernatant was discarded, and the RNA was washed twice with 500 µL of 75% ethanol, centrifuged at 8000 rpm for 3 minutes each time. The RNA pellet was resuspended in 50–100 µL of ddH2O.

[0091] (2) RNA concentration was detected using NanoDrop 2000. Reverse transcription was performed using the TransScript Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit. qPCR was performed using the PerfectStart Green qPCR SuperMix kit. After the reaction, the NUDT14 mRNA expression level was calculated based on the CT value of each well, with the internal reference gene GAPDH as the internal reference. The primer pair sequences for detecting the NUDT14 expression level are as follows: the upstream sequence is shown in SEQ ID NO: 4 (5'-GGTCTGGAGTGGGACTGACT-3'), and the downstream sequence is shown in SEQ ID NO: 5 (5'-CTGGCTGAGGAACCATGAGA-3').

[0092] Test results as follows Figure 7 As shown in the figure. The results showed that, compared with the control group, JQ1 significantly reduced NUDT14 expression in both drug-resistant cell lines. Subsequently, the super-enhancer NUDT14-SE region was divided into SE1 to SE4, and the SE2 sequence was knocked out using CRISPR-Cas9 gene editing technology. NUDT14 expression was detected by Western blotting and qPCR, with the Western blotting and qPCR experiments performed according to the same procedures as above. The detection results are shown in the figure. Figures 8-10 As shown in the figure. The results indicate that knocking out SE2 significantly reduces the expression level of NUDT14 in drug-resistant cells.

[0093] Example 3

[0094] To further investigate the role of NUDT14 in enzalutamide resistance in prostate cancer, a series of in vitro and in vivo experiments were conducted.

[0095] First, C4-2B_ENZR and LNCaP_ENZR cells (constructed using the method described in Example 2) were transfected with NUDT14-targeting siRNAs (si-NUDT14#1, si-NUDT14#2, and si-NUDT14#3), respectively. The expression of NUDT14 in these cells was then detected using Western blotting. The sequence of si-NUDT14#1 is shown in SEQ ID NO: 1, which is 5'-GGAGCCTGGTGTTGGTGAATT-3'; the sequence of si-NUDT14#2 is shown in SEQ ID NO: 2, which is 5'-GGCCGCTCACGCTGCATTATT-3'; and the sequence of si-NUDT14#3 is shown in SEQ ID NO: 3, which is 5'-GGGACTTCATGAAGACGCATT-3'. The specific steps are as follows:

[0096] (1) Using siRNA-mate (Genepharma), small interfering RNAs (siRNAs) (Tsingke) targeting NUDT14 were transfected into C4-2B_ENZR and LNCaP_ENZR cells, respectively, and cells transfected with blank vector si-NC were set as controls. Cells were cultured under normal culture conditions. Cells in the logarithmic growth phase were digested, collected, and lysed with lysis buffer on ice for 1 h.

[0097] (2) Centrifuge at 15000×g at 4℃ for 15 min, take the supernatant and add loading buffer (to 1×), then incubate at 95℃ for 5 min.

[0098] (3) Take the sample prepared in step (2) and perform protein gel electrophoresis.

[0099] (4) After electrophoresis, the protein gel was transferred to a membrane (PVDF membrane, 200mA constant current transfer for 2h).

[0100] (5) After the transfer is completed, the PVDF membrane is removed and placed in the sealing solution and sealed at room temperature for 2 hours on a vertical shaker at 10 rpm.

[0101] (6) After sealing, clean the PVDF membrane, immerse it in the primary antibody (anti-NUDT14), and incubate it overnight on a vertical shaker at 10 rpm and 4°C.

[0102] (7) After the primary antibody incubation is complete, wash the PVDF membrane and then immerse it in the secondary antibody (HRP-linked anti-rabbit IgG) and incubate it at room temperature for 2 hours at 10 rpm on a vertical shaker.

[0103] (8) After the secondary antibody incubation is completed, the PVDF membrane is washed and the target protein is detected using a chemiluminescence analyzer (α-Tubulin is used as an internal control).

[0104] Test results as follows Figures 11-12 As shown in the figure. The results showed that, compared with the blank vector si-NC group, the expression of NUDT14 in prostate cancer cells transfected with si-NUDT14#1, si-NUDT14#2 or si-NUDT14#3 was significantly inhibited (**p<0.01, ***p<0.001).

[0105] Subsequently, cell proliferation and colony formation experiments were conducted. The specific steps of the cell proliferation experiment are as follows:

[0106] (1) siRNAs targeting NUDT14 (si-NUDT14#1, si-NUDT14#2) were transfected into C4-2B_ENZR and LNCaP_ENZR cells (constructed using the aforementioned method), and cells transfected with the blank vector si-NC were set up as controls; different concentrations (e.g., 10) were used in each group. 0 10 0.5 10 1 10 1.5 10 2 The sample was treated with enzalutamide (μM).

[0107] (2) When the cells grow to the logarithmic phase, trypsin digest and count them. Select the appropriate cell density according to the doubling time of various cells and seed them into 96-well plates (3 replicates).

[0108] (3) The cells were cultured in a 37°C incubator and collected after 48 hours of culture. 10 μL of CCK-8 was added to each well, and the culture plate was incubated in the incubator for 2 hours. The absorbance at 450 nm was measured to assess the cell proliferation status.

[0109] Experimental results are as follows Figure 13 As shown in the figure. The results showed that, compared with the control group (si-NC), inhibiting the expression of NUDT14 using the NUDT14 inhibitor could effectively inhibit the proliferation of prostate cancer cells, improve the inhibitory activity of enzalutamide on drug-resistant cell lines, and reverse their drug resistance (*p<0.05, **p<0.01).

[0110] The specific experimental steps for cell clone formation are as follows:

[0111] (1) siRNAs targeting NUDT14 (si-NUDT14#1, si-NUDT14#2) were transfected into C4-2B_ENZR and LNCaP_ENZR cells (constructed using the aforementioned method), and cells transfected with the blank vector si-NC were set as controls.

[0112] (2) When the cells grow to the logarithmic phase, digest them with trypsin and count them. Select an appropriate cell density (about 1000 cells / well) according to the doubling time of various cells. Seed them into a 6-well plate containing 2 mL of 37℃ pre-warmed culture medium and gently rotate it to disperse the cells evenly. Incubate the cells in a cell culture incubator at 37℃ with 5% CO2 and saturated humidity.

[0113] (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 0.1% crystal violet to each well, stain for 30 min; discard the methanol, wash the residual methanol with water; cell clones can then be observed; under a microscope, a number of cells > 50 is considered a valid clone.

[0114] Test results as follows Figures 14-15 As shown in the figure. The results showed that, compared with the blank vector si-NC group, silencing the NUDT14 gene with siRNA significantly reduced the clonogenic ability of prostate cancer cells, significantly inhibited the clonogenic formation of prostate cancer cells, and improved the sensitivity of drug-resistant cell lines to enzalutamide. The difference was statistically significant.

[0115] Furthermore, NUDT14 was constructed using a method similar to that described for si-NUDT14, and its overexpression efficiency was verified in C4-2B_WT cells (constructed using the method in Example 2) using the Western Blot method described above. The results are as follows: Figure 16 As shown in the figure. The results indicate that the constructed NUDT14 can effectively increase the level of NUDT14 in prostate cancer cells.

[0116] After confirming the overexpression efficiency of the constructed NUDT14, in vivo research experiments were conducted using nude mice. The specific steps are as follows:

[0117] (1) The day before the experiment, the pre-packaged Matrigel matrix gel was placed in a 4°C refrigerator overnight from -20°C to melt from a solid state to a liquid state.

[0118] (2) Four-week-old male NCG mice (Jicui Yaokang) were randomly divided into four groups, referred to as group 1 to group 4. Group 3 and 4 mice were subcutaneously injected with C4-2B_WT cells transfected with NUDT14 (constructed using the aforementioned method), while group 1 and 2 mice were subcutaneously injected with C4-2B_WT cells transfected with vector (constructed using the aforementioned method) as negative controls.

[0119] (3) Observe the growth and mental state of mice daily. After tumor formation, treat with enzalutamide (10 mg / kg, administered by gavage for 5 consecutive days per week, followed by a 2-day break, for a total of 2 weeks). Measure the tumor size of each group of mice every 4 days after injection 12 days later and calculate the tumor volume using the following formula: Volume (mm) 3 = Length (mm) × Width 2 (mm) 2 ) / 2.

[0120] (4) On day 32, the mice were sacrificed. After being euthanized by overdose, the tumors of each group of mice were removed, photographed, weighed and the tumor volume was measured.

[0121] Test results as follows Figures 17-18 As shown in the figure. The results showed that, compared with group 2, mice in group 4 exhibited significantly faster tumor growth and larger tumor volume after NUDT14 overexpression, and this difference was statistically significant (*p<0.05, **p<0.01). Therefore, overexpression of NUDT14 significantly accelerates the growth of subcutaneous tumors treated with enzalutamide and reduces the sensitivity of tumors to enzalutamide treatment.

[0122] As can be clearly seen from the above, this invention first obtained expression information of primary prostate cancer, mCRPC, CRPR-Adeno, and CRPC-NE samples from public databases (GSE35988, GSE80609, and Beltran-2016). Differential analysis showed that the expression level of NUDT14 in mCRPC was significantly higher than that in carcinoma in situ, and its RNA expression level in neuroendocrine tumors was higher than that in adenocarcinoma. In the SU2C cohort, KM survival curve analysis revealed that mCRPC patients with high NUDT14 expression had shorter ARSI treatment sensitivity time and overall survival. Furthermore, by collecting clinical tissue samples from prostate cancer, immunohistochemical analysis revealed that NUDT14 expression in CRPC samples was lower than in enzalutamide-resistant CRPC samples. To verify whether NUDT14 is positively regulated by a superenhancer in drug-resistant cell lines, this invention constructed enzalutamide-resistant prostate cancer cell lines (C4-2B_ENZR and LNCaP_ENZR). After treating C4-2B_ENZR and LNCaP_ENZR cell lines with the BET inhibitor JQ1, qPCR analysis revealed that JQ1 significantly reduced NUDT14 expression in both drug-resistant cell lines compared to the control group. By dividing the superenhancer NUDT14-SE region into SE1 to SE4, CRISPR-Cas9 gene editing technology was used to target and knock out the SE2 sequence. Western blotting and qPCR analysis of NUDT14 expression showed that SE2 knockout significantly reduced NUDT14 expression levels in drug-resistant cells. To elucidate the role of NUDT14 in enzalutamide resistance in prostate cancer, NUDT14 expression was first knocked down in C4-2B_ENZR and LNCaP_ENZR cells, and Western blotting confirmed a significant downregulation of its expression efficiency. Cell proliferation and plate colony assays revealed that knocking down NUDT14 expression increased the sensitivity of drug-resistant cell lines to enzalutamide. In vivo experiments showed that, compared to the control group, overexpression of NUDT14 significantly accelerated the growth of enzalutamide-treated subcutaneous tumors and markedly reduced their therapeutic sensitivity.

[0123] In summary, this invention clarifies that NUDT14 can serve as a key factor in the development and progression of prostate cancer and the emergence of enzalutamide resistance. Inhibiting NUDT14 or reducing its biological activity can significantly suppress prostate cancer proliferation and metastasis, reverse enzalutamide resistance, improve treatment sensitivity, prolong patient survival, and improve prognosis. This invention enriches the 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 developing new targeted drugs. It contributes to achieving better precision medicine and has significant social and scientific value.

[0124] 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. Application of NUDT14 inhibitors in the preparation of drugs that enhance the sensitivity of enzalutamide to prostate cancer treatment.

2. The application according to claim 1, characterized in that, The NUDT14 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the NUDT14 gene.

3. The application according to claim 1, characterized in that, The prostate cancer mentioned is castration-resistant prostate cancer.

4. Application of reagents for detecting NUDT14 expression levels in the preparation of products for evaluating the sensitivity of enzalutamide to prostate cancer treatment.

5. The application according to claim 4, characterized in that, The reagents for detecting NUDT14 expression levels include primers for detecting NUDT14 gene expression levels and / or reagents for detecting NUDT14 protein content.

6. The application according to claim 4, characterized in that, The prostate cancer mentioned is castration-resistant prostate cancer.

7. A pharmaceutical composition for the prevention and / or treatment of prostate cancer, characterized in that, This includes NUDT14 inhibitors and enzalutamide.

8. The pharmaceutical composition according to claim 7, characterized in that, The NUDT14 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the NUDT14 gene.

9. The pharmaceutical composition according to claim 7, characterized in that, The prostate cancer mentioned is castration-resistant prostate cancer.

10. Use of NUDT14 inhibitors in the preparation of drugs for the prevention and / or treatment of prostate cancer.

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