Application of HNRNPA3 in treatment and prognosis evaluation of prostate cancer

Abstract

The invention relates to application of HNRNPA3 in treatment and prognosis evaluation of prostate cancer. Through deep research, the HNRNPA3 is determined to be a key target spot for prognosis judgment and treatment of the neuroendocrine prostate cancer, can be used for treatment, auxiliary diagnosis and prognosis evaluation of the neuroendocrine prostate cancer, and meanwhile, improves the understanding of the pathogenesis of the neuroendocrine prostate cancer to a new height; the neuroendocrine prostate cancer pathogenesis content is greatly enriched, a sufficient scientific basis and theoretical basis are provided for searching new prostate cancer diagnosis, prognosis judgment and treatment molecular targets and developing new targeted drugs, and important social value and scientific significance are achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of HNRNPA3 in the treatment and prognosis evaluation of prostate cancer. Background Art

[0002] Prostate cancer (PCa) is the most common malignancy of the male genitourinary system. The clinical features of PCa vary significantly across regions. Most patients are diagnosed at an advanced stage, with high Gleason scores, and are primarily treated with systemic endocrine therapy.

[0003] PCa patients often irreversibly develop castration-resistant prostate cancer (CRPC) after receiving androgen deprivation therapy (ADT). Among them, neuroendocrine prostate cancer (NEPC), as the most malignant type of CRPC, occurs in 20-25% of metastatic CRPC patients and has extremely poor clinical characteristics of prognosis. NEPC can be divided into two subtypes: primary (accounting for <62% of newly diagnosed cases) and treatment-related (t-NEPC). The latter is often induced by long-term ADT or new endocrine drug treatment, and its median survival after diagnosis is less than 1 year. In terms of treatment principles, because the pathological characteristics of NEPC are similar to those of small cell lung cancer (SCLC), NEPC usually follows the treatment strategy of SCLC, but its clinical benefits are extremely limited. Therefore, it is of great clinical significance to deeply explore the molecular mechanism behind neuroendocrine differentiation (NED) of prostate cancer and find new targets for NEPC. Summary of the Invention

[0004] The present invention aims to address the existing challenges of NEPC, including its complex and poorly defined pathogenesis and prognosis. This study, led by researchers from the University of California Davis, has identified HNRNPA3 as a pro-oncogenic factor in the development and progression of NEPC. Inhibiting HNRNPA3 or reducing its bioactivity significantly inhibits NEPC proliferation and metastasis, prolonging patient survival and improving prognosis. This provides a novel strategy for the treatment, diagnosis, and prognostic assessment of NEPC.

[0005] In order to solve the above technical problems, the present invention is achieved through the following technical solutions.

[0006] In a first aspect, the present invention provides use of an HNRNPA3 inhibitor in the preparation of a medicament for preventing and / or treating prostate cancer.

[0007] Preferably, the HNRNPA3 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the HNRNPA3 gene.

[0008] Preferably, the HNRNPA3 inhibitor is selected from siRNA and / or shRNA designed based on the HNRNPA3 gene.

[0009] Preferably, the siRNA is selected from one or more of si#1 (sequence as shown in SEQ ID NO: 1, 5'-GGCGTGTAGTGGAACCAAA-3'), si#2 (sequence as shown in SEQ ID NO: 2, 5'-GGACAACAGCAATCAAATT-3'), and the shRNA is selected from sh#1 (sequence as shown in SEQ ID NO: 3, 5'-GGTGGAGGATATGATGGTTAC-3').

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

[0011] Preferably, the castration-resistant prostate cancer is neuroendocrine prostate cancer.

[0012] A second aspect of the present invention provides use of a reagent for detecting HNRNPA3 expression levels in the preparation of a product for auxiliary diagnosis and / or prognosis evaluation of prostate cancer.

[0013] Preferably, the reagent for detecting the expression level of HNRNPA3 includes primers for detecting the expression level of HNRNP A3 gene and / or a reagent for detecting the content of HNRNPA3 protein.

[0014] Preferably, the primers for detecting the expression level of the HNRNPA3 gene are selected from the following primer pairs:

[0015] The upstream sequence of the primer pair is shown in SEQ ID NO: 4 (5'-GTGCCCATCTAACA GTGAA-3'), and the downstream sequence is shown in SEQ ID NO: 5 (5'-GCCTGTCTTCC ATAACTTCT-3').

[0016] Preferably, the reagent for detecting the HNRNPA3 protein content is selected from anti-HNRN PA3 Antibody (Bethyl, A305-815A).

[0017] Preferably, the prostate cancer is castration-resistant prostate cancer.

[0018] Preferably, the castration-resistant prostate cancer is neuroendocrine prostate cancer.

[0019] A third aspect of the present invention provides a kit for auxiliary diagnosis and / or prognosis evaluation of prostate cancer, comprising a reagent for detecting the expression level of HNRNPA3.

[0020] Preferably, the reagent for detecting the expression level of HNRNPA3 includes a reagent for detecting the expression level of the HNRNPA3 gene and / or a reagent for detecting the content of the HNRNPA3 protein.

[0021] The reagent for detecting the expression level of the HNRNPA3 gene is selected from the following primer pairs:

[0022] 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.

[0023] Preferably, the reagent for detecting the HNRNPA3 protein content is selected from anti-HNRN PA3 Antibody (Bethyl, A305-815A).

[0024] Preferably, the kit further comprises one or more of PCR enzyme, PCR buffer, dNTPs, and fluorescent substrate.

[0025] Preferably, the fluorescent substrate is selected from Syber Green or a fluorescently labeled probe.

[0026] Preferably, the prostate cancer is castration-resistant prostate cancer.

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

[0028] A fourth aspect of the present invention provides a pharmaceutical composition for preventing and / or treating prostate cancer, comprising an HNRNPA3 inhibitor and a pharmaceutically acceptable carrier.

[0029] Preferably, the HNRNPA3 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the HNRNPA3 gene.

[0030] Preferably, the HNRNPA3 inhibitor is selected from siRNA and / or shRNA designed based on the HNRNPA3 gene.

[0031] Preferably, the siRNA is selected from one or more of si#1 (sequence as shown in SEQ ID NO: 1, 5'-GGCGTGTAGTGGAACCAAA-3'), si#2 (sequence as shown in SEQ ID NO: 2, 5'-GGACAACAGCAATCAAATT-3'), and the shRNA is selected from sh#1 (sequence as shown in SEQ ID NO: 3, 5'-GGTGGAGGATATGATGGTTAC-3').

[0032] Preferably, the pharmaceutically acceptable excipient is selected from one or more of a filler, a disintegrant, a binder, a lubricant, a flavoring agent, a preservative, an antioxidant, and a colorant.

[0033] Preferably, the prostate cancer is castration-resistant prostate cancer.

[0034] Preferably, the castration-resistant prostate cancer is neuroendocrine prostate cancer.

[0035] It should be understood that, unless otherwise specified, in the context of the present invention, HNRNPA3 includes components such as HNRNPA3 nucleotides and the HNRNPA3 protein encoded by the HNRNPA3 nucleotides. The HNRNPA3 inhibitor refers to a substance that can specifically downregulate the expression level of HNRNPA3 and / or the transcription level of its mature mRNA and / or the expression level or activity of the HNRNPA3 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 HNRNPA3 expression and / or activity, as long as the method can achieve a decrease in HNRNPA3 level and / or activity. The primers and / or primer pairs refer to PCR primers used to synthesize the cDNA chain of the HNRNPA3 gene in PCR, thereby detecting the expression level of the HNRNPA3 gene mRNA. 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 and / or primer pairs based on the HNRNPA3 gene sequence using conventional methods and means 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 they can achieve specific detection of HNRNPA3 expression levels; HNRNPA3 protein expression levels can also be detected using conventional reagents and methods in the art; the same applies to other genes / proteins.

[0036] Heterogeneous nuclear ribonucleoproteins (HNRNPs) are a family of highly conserved RNA-binding proteins that are extensively involved in the entire process of RNA metabolism, including transcription, alternative splicing, transport, stability regulation, and translation. The ability of HNRNPs to bind and modify RNA has been implicated in the development and progression of various cancers. HNRNPA2B1 and HNRNPC are among the most studied HNRNPs involved in RNA modification. Both serve as methyl readers for m6A-modified RNA, mediating various post-transcriptional processes. In prostate cancer research, reports indicate that HNRNPs can splice EIF4G1 mRNA, promote the expression of EIF4G1 isoform a in CRPC cells, and mediate the c-Myc pathway to inhibit CXCL8 secretion in CRPC, thereby causing resistance to immunotherapy. However, research on RNA modification and the HNRNP family in prostate cancer NEDs remains underdeveloped.

[0037] HNRNPA3, a member of the HNRNPs family, may be involved in multiple stages of tumor development and progression, but reports on HNRNPA3 are limited. Preliminary reports suggest that high HNRNPA3 expression is associated with hepatocellular carcinoma progression and poor prognosis after radical cystectomy. Furthermore, the various mechanisms of HNRNPA3 remain unclear, and its role in prostate cancer has yet to be reported.

[0038] The present invention established a neuroendocrine prostate cancer cell line, analyzed the HNRNPA3 expression pattern using public databases, and detected its expression levels in cells and tissues, which were elevated compared to CRPC. The authors found that HNRNPA3 expression was significantly elevated in prostate cancer, particularly castration-resistant prostate cancer, and specifically in neuroendocrine prostate cancer cells. Furthermore, clinical data also showed that elevated HNRNPA3 expression was associated with poor progression-free survival in prostate cancer patients and a shortened response to ARSIs. These findings suggest that HNRNPA3 may act as an oncogene, playing a key role in the development of neuroendocrine prostate cancer. The present invention further validated these findings through a series of in vitro and in vivo functional experiments. In vitro experiments observed that inhibiting HNRNPA3 reduced the cloning and proliferation capacity of prostate cancer cells and decreased the expression of neuroendocrine markers. In vivo subcutaneous tumor formation experiments in mice revealed that inhibiting HNRNPA3 slowed tumor growth and reduced tumor volume in nude mice. The above results jointly confirm the view that inhibiting HNRNPA3 has anti-tumor activity in neuroendocrine prostate cancer, and clarify that HNRNPA3 is a key target for prognosis and treatment of neuroendocrine prostate cancer. It can be used for the treatment, auxiliary diagnosis and prognosis assessment of neuroendocrine prostate cancer. At the same time, it raises the understanding of the pathogenesis of neuroendocrine prostate cancer to a new level, greatly enriches the content of the pathogenesis of neuroendocrine prostate cancer, and provides sufficient scientific basis and theoretical foundation for exploring new molecular targets for diagnosis, prognosis and treatment of prostate cancer, and developing new targeted drugs. It has important social value and scientific significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the differential expression analysis results of HNRNPA3 in primary prostate cancer and CR PC using the GSE35988 dataset.

[0040] Figure 2 Schematic diagram of the differential expression analysis results of HNRNPA3 in CRPC and NEPC using the Beltran-2016 dataset.

[0041] Figure 3 Schematic diagram of the expression differences of HNRNPA3 in different GS scores and NEPC using the HuPSA-MoPSA dataset.

[0042] Figure 4 Schematic diagram of the KM survival curve analysis results of the correlation between HNRNPA3 expression and progression-free survival of prostate cancer patients.

[0043] Figure 5Schematic diagram of the KM survival curve analysis results of the correlation between HNRNPA3 expression and ARSI treatment response period in mCRPC patients.

[0044] Figure 6 Schematic diagram of the results of WB detection of HNRNPA3 expression in prostate cancer WT and NE cell lines.

[0045] Figure 7 Schematic diagram of the IHC verification results of the differential expression of HNRNPA3 in CRPC, NEPC, and NEPC_PDX tissues.

[0046] Figure 8 Schematic diagram of the knockdown efficiency and marker changes of NEPC cell lines after WB detection of HNRNPA3 knockdown (si#1, si#2).

[0047] Figure 9 Schematic diagram of the effect of knocking down HNRNPA3 using siRNA on NEPC cell proliferation.

[0048] Figure 10 Schematic diagram of the effect of knocking down HNRNPA3 using siRNA on NEPC clone formation.

[0049] Figure 11 Schematic diagram of the quantitative analysis results of NEPC clone formation after knocking down HNRNPA3 using siRNA.

[0050] Figure 12 Schematic diagram of the knockdown efficiency and marker changes of NEPC cell lines after WB detection of HNRNPA3 (sh#1) knockdown.

[0051] Figure 13 Schematic diagram of the effect of HNRNPA3 inhibition on prostate cancer tumor volume and growth curve in vivo.

[0052] Figure 14 Schematic diagram of the effect of HNRNPA3 inhibition on prostate cancer tumor growth curve and tumor mass in vivo. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0054] Unless otherwise specified, the cell lines listed in the present invention, including LNCaP and C4-2B, were purchased from the American Type Culture Collection (Manassas, USA) and cultured according to existing technologies. All cell lines were identified by short tandem repeat analysis at the China Type Culture Collection (Wuhan) and verified for mycoplasma contamination using a PCR detection kit (Shanghai Biothrive Sci). They were then frozen in liquid nitrogen and used for subsequent experiments. The reagents used in the present invention were all commercially available.

[0055] All clinical samples used in this invention were collected with informed consent from patients. The relevant procedures and methods were approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University and adhered to medical ethics requirements and Good Clinical Practice guidelines for pharmaceutical 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 conventional methods and techniques in the art. Bioinformatics analysis was performed using R version 4.3. Analysis of variance was performed using the "limma" package, and KM survival curves were constructed using the "survival" and "survminer" packages. Representative results from replicates of biological experiments are presented in the accompanying figures. Data are presented as mean ± SD and mean ± SEM as indicated 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. Differences in mean values between two or more groups were compared using standard medical statistical methods, such as t-tests, chi-square tests, and analysis of variance. *P < 0.05 was considered significant.

[0056] Example 1

[0057] First, we obtained the expression information of primary prostate cancer, CRPC, and NEPC samples from public databases (GSE35988, Beltran-2016, and HuPSA-MoPSA) to perform differential expression analysis of HNRNPA3. Figure 1-3As shown. The results showed that the GSE35988 database analysis found that the expression level of HNRNPA3 in patients with castration-resistant prostate cancer was significantly higher than that in patients with primary prostate cancer (****p < 0.0001). The Beltran-2016 database analysis found that compared with castration-resistant prostate cancer, the expression level of HNRNPA3 in patients with neuroendocrine prostate cancer was more significant (*p < 0.05). The HuPSA-MoPSA database analysis found that compared with primary prostate cancer patients with different GS scores, the expression level of HNRNPA3 in patients with neuroendocrine prostate cancer was significantly higher (**p < 0.01, ***p < 0.001, ****p < 0.0001). These results show that the expression level of HNRNPA3 in CRPC is significantly higher than that in primary prostate cancer, especially in neuroendocrine prostate cancer.

[0058] Subsequently, the effect of HNRNPA3 on progression-free survival (PFS) of prostate cancer patients was analyzed by KM survival curve analysis in TCGA-PRAD. The results showed that the progression-free survival of prostate cancer patients with high expression of HNRNPA3 was significantly shorter than that of prostate cancer patients with low expression of HNRNPA3 (p = 0.0037) (see Figure 4 ). Clinically, a series of androgen receptor signaling inhibitors (AR signaling inhibitors, ARSIs), including androgen synthesis inhibitors (Abiraterone, etc.) and AR antagonists (Enz alutamide, Darolutamide, Apalutamide, etc.), have been used to treat CRPC and can significantly prolong the survival of patients. However, even so, due to AR splicing variants or bypass activation, CRPC will eventually become less sensitive to ARSIs and gradually develop drug resistance. In this regard, an analysis in SU2C found that mCRPC patients with high expression of HNRNPA3 had a shorter median time to respond to ARSI drug treatment (see Figure 5 ), that is, high expression of HNRNPA3 in vivo is more likely to cause resistance to the treatment of ARSIs.

[0059] Example 2

[0060] Western blotting was used to examine HNRNPA3 expression in wild-type prostate cancer cells (LNCaP and C4-2B) and NEPC cell lines. NEPC cell lines were constructed using the following method: C4-2B or LNCaP cells were stimulated with 25 μM or 10 μM enzalutamide for 6 months. Cell morphology was observed and neuroendocrine markers were measured to induce neuroendocrine differentiation. The specific steps for Western blotting are as follows:

[0061] (1) Prostate cancer cells were digested and collected, and lysed on ice for 1 h after adding lysis buffer.

[0062] (2) Centrifuge at 15,000 × g for 15 min at 4°C. Take the supernatant and add loading buffer (to 1×), and then water bath at 95°C for 5 min.

[0063] (3) Perform protein gel electrophoresis on the sample prepared in step (2).

[0064] (4) After electrophoresis, the protein gel was transferred to a membrane (PVDF membrane, 200 mA constant current transfer for 2 h).

[0065] (5) After the transfer is completed, remove the PVDF membrane and place it in the blocking solution. Block it at room temperature for 2 hours on a vertical shaker at 10 rpm.

[0066] (6) After blocking, the PVDF membrane was washed, immersed in the primary antibody (anti-HNRNPA3), and incubated overnight on a vertical shaker at 10 rpm and 4°C.

[0067] (7) After the primary antibody incubation is completed, the PVDF membrane is washed and then immersed in the secondary antibody (HRP-linked anti-rabbit IgG) and incubated on a vertical shaker at 10 rpm at room temperature for 2 h.

[0068] (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 reference).

[0069] Test results such as Figure 6 The results showed that the expression of HNRNPA3 in NEPC cell lines was higher than that in wild-type cells.

[0070] Furthermore, multiple clinical tissue samples of prostate cancer (CRPC and NEPC) were collected, and a PDX model was constructed. The specific steps are as follows: a vessel filled with physiological saline was used to collect the in vitro prostate cancer gross tissue, part of the tissue was embedded for HE staining and IHC identification, and the other part was sterilized with completely sterilized tissue scissors and straight forceps to remove the necrotic tissue. The tissue was then cut to a diameter of about 5 mm and implanted on the back of NCG mice. After the tumor grew, it was defined as the F0 generation; the tumor tissue was separated, part of it was embedded and identified by IHC, and the rest was cut to a diameter of 5 mm. Part of it was frozen at -80°C or in liquid nitrogen, and part was continued to be inoculated on the back of NCG mice, which was the F1 generation. This process was repeated until the F4 generation, and the tumor grew to 200 mm. 3 When the tumor size is approximately 100 μg / kg, the tumor-bearing mice are castrated and given enzalutamide (10 mg / kg) by gavage. The mice's physiological status and tumor growth trends are regularly observed. Generally, tumors shrink or even disappear under enzalutamide treatment. However, after developing drug resistance, tumors resume growth and continue to grow despite enzalutamide. These tumors are considered enzalutamide-resistant PDX. After isolation, some of the resistant PDX are embedded in paraffin and characterized by IHC, while others are stored for seed preservation or further experimental verification.

[0071] HE staining and immunohistochemical analysis revealed that HNRNPA3 expression in NEPC samples was significantly higher than that in CRPC samples (see Figure 7 ), and the levels of neuroendocrine markers (CHGA, SYP, NSE) were significantly increased. The immunohistochemical analysis steps are as follows:

[0072] (1) Slice the prostate cancer tissue into 4 μm thick slices using a pathological tissue slicer, spread them on a non-slip glass slide, and bake them at 65°C for 2 hours until dry.

[0073] (2) Dewaxing: Soak the slides 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.

[0074] (3) Blocking peroxidase: Soak in 3% hydrogen peroxide for 10 min and wash with PBS for 5 min.

[0075] (4) High-pressure antigen repair: Prepare EDTA repair solution and add it to the pressure cooker. Place the slide in the pot and heat at 800W for 20 minutes, then let it cool naturally.

[0076] (5) Wash the slides twice with PBS, 3 minutes each time, use a small piece of paper to absorb the moisture around the tissue, and use a tissue pen to draw a circle 0.5 cm away from the tissue boundary.

[0077] (6) Oiling the tissue surface: Soak the slide in 0.1% PBST, lift it up and down, and soak it for 3 minutes.

[0078] (7) Primary antibody incubation: dilute the antibody with antibody diluent according to the instructions, incubate 50 μL at 4°C overnight, wash with PBS for 3 minutes, and then wash with PBST for 3 minutes.

[0079] (8) Secondary antibody incubation: 1 drop of DAKO secondary antibody was used to cover the tissue surface. After incubation at 37°C for 1 hour, the tissue was washed with PBS for 3 minutes and then with PBST for 3 minutes.

[0080] (9) Prepare DAB colorimetric solution: Prepare the colorimetric solution in proportion, shake off the liquid on the slide, and then drop freshly prepared DAB colorimetric solution into the circle. Control the colorimetric time under a microscope. The positive color is brown-yellow. Soak the slide in PBS to stop the colorimetric development.

[0081] (10) Re-staining of cell nuclei: After DAB staining, the slides were re-stained with hematoxylin for about 3 minutes, rinsed with running water, and sealed with 20 μl of mounting medium before observation under a microscope.

[0082] The above results show that HNRNPA3 is highly expressed in prostate cancer tissues, especially in CRP C, and more significantly in NEPC. HNRNPA3 may affect the occurrence and development of prostate cancer and can serve as a potential biomarker and therapeutic target for prostate cancer.

[0083] Example 3

[0084] The aforementioned examples have demonstrated the impact of HNRNPA3 expression levels on prostate cancer in clinical samples. To further investigate the role of HNRNPA3 in prostate cancer NED, a series of in vitro and in vivo experiments were conducted.

[0085] First, C4-2_NE and LNCaP_NE cells (constructed using the method in Example 2) were transfected with siRNA targeting HNRNPA3 (si#1 and si#2), respectively, and the expression of HNRNPA3 was detected by Western Blot. The sequence of si#1 is shown in SEQ ID NO: 1, which is 5'-GGCGTGTAGTGGAACCAAA-3'; the sequence of si#2 is shown in SEQ ID NO: 2, which is 5'-GGACAACAGCAATCAAATT-3'. The specific steps are as follows:

[0086] (1) siRNA targeting HNRNPA3 (si#1, si#2) was transfected into C4-2_NE and LNCaP_NE cells, respectively. Cells transfected with blank vector si-NC were set as controls and cultured under conventional culture conditions. Cells in the logarithmic growth phase were digested and collected, lysed in lysis buffer, and lysed on ice for 1 h.

[0087] (2) Centrifuge at 15,000 × g for 15 min at 4°C. Take the supernatant and add loading buffer (to 1×), and then water bath at 95°C for 5 min.

[0088] (3) Perform protein gel electrophoresis on the sample prepared in step (2).

[0089] (4) After electrophoresis, the protein gel was transferred to a membrane (PVDF membrane, 200 mA constant current transfer for 2 h).

[0090] (5) After the transfer is completed, remove the PVDF membrane and place it in the blocking solution. Block it at room temperature for 2 hours on a vertical shaker at 10 rpm.

[0091] (6) After blocking, the PVDF membrane was washed, immersed in the primary antibody (anti-HNRNPA3), and incubated overnight on a vertical shaker at 10 rpm and 4°C.

[0092] (7) After the primary antibody incubation is completed, the PVDF membrane is washed and then immersed in the secondary antibody (HRP-linked anti-rabbit IgG) and incubated on a vertical shaker at 10 rpm at room temperature for 2 h.

[0093] (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 reference).

[0094] Test results such as Figure 8 The results showed that compared with the blank vector si-NC group, the expression of HNRNPA3 in prostate cancer cells transfected with si#1 or si#2 was significantly inhibited, and the expression of neuroendocrine markers (CHGA, SYP, NSE) was downregulated, and the difference was statistically significant.

[0095] Subsequently, cell proliferation assay and clone formation assay were performed. The specific steps of the cell proliferation assay are as follows:

[0096] (1) siRNA targeting HNRNPA3 (si#1, si#2) was transfected into C4-2_NE and LNCaP_NE cells (constructed using the method in Example 2), respectively, and cells transfected with blank vector si-NC were set as controls.

[0097] (2) When the cells grow to the logarithmic phase, trypsinize and count them. According to the doubling time of various cells, the appropriate cell density is selected and the cells are inoculated into 96-well plates (3 replicates).

[0098] (3) Culture the cells in a 37°C incubator and collect the cells at 24 h, 48 h, 72 h, 96 h, and 120 h. Add 10 μL of CCK-8 to each well and incubate the culture plate in the incubator for 1-4 h. Measure the absorbance at 450 nm to evaluate the cell proliferation.

[0099] The experimental results are as follows Figure 9 The results showed that compared with the control group (si-NC), the proliferation of prostate cancer cells could be effectively inhibited after the expression of HNRNPA3 was inhibited by HNRNPA3 inhibitor (*p < 0.05, **p < 0.01).

[0100] The specific experimental steps for cell clone formation are as follows:

[0101] (1) siRNA targeting HNRNPA3 (si#1, si#2) was transfected into C4-2_NE and LNCaP_NE cells (constructed using the method in Example 2), respectively, and cells transfected with blank vector si-NC were set as controls.

[0102] (2) When the cells grow to the logarithmic phase, trypsinize and count them. Select the appropriate cell density according to the doubling time of each cell type, inoculate them into a dish containing 10 mL of 37°C pre-warmed culture medium, and gently rotate to evenly disperse the cells. Place them in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for culture.

[0103] (3) When visible clones 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 minutes; discard the methanol, and wash away the residual 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.

[0104] Test results such as Figure 10-11 The results showed that compared with the blank vector si-NC group, after silencing the HNRNPA3 gene using siRNA, the cloning ability of prostate cancer cells was significantly weakened, and the cloning of prostate cancer cells was significantly inhibited, with a statistically significant difference (**p < 0.01).

[0105] Furthermore, sh-HNRNP A3 (sh#1, sequence as shown in SEQ ID NO: 3, 5'-GGTGGAGGATATGATGGT TAC-3') was constructed using a method similar to the above-mentioned si-HNRNPA3, and the above-mentioned Western Blot method was used to verify the inhibitory efficiency of sh#1 in C4-2B_NE cells (constructed using the method in Example 2). The results are as follows: Figure 12 The results showed that the constructed sh#1 could effectively reduce the levels of HNRNPA 3 and related neuroendocrine markers (CHGA) in prostate cancer cells.

[0106] After confirming the inhibitory efficiency of the constructed sh#1, in vivo research experiments were conducted using nude mice. The specific steps are as follows:

[0107] (1) One day before the experiment, place the already packaged Matrigel from -20℃ into a 4℃ refrigerator overnight to melt it from solid to liquid.

[0108] (2) Take 8 4-week-old NOD / ShiLtJGpt-Prkdc em26Cd52 Il2rg em26Cd22 / Gpt mice (Jicui Yaokang Company) were randomly divided into two groups, designated as Group 1 and Group 2. The mice in Group 1 were subcutaneously injected with C4-2B_NE cells transfected with sh-HNRNPA3 (constructed using the method in Example 2), and the mice in Group 2 were subcutaneously injected with C4-2B_NE cells transfected with sh-NC (constructed using the method in Example 2) as a negative control.

[0109] (3) The growth and mental state of the mice were observed daily. The tumor size of each group of mice was measured every 4 days after 12 days of injection, and the tumor volume was calculated using the following formula: Volume (mm 3 )=Length(mm)×Width 2 (mm 2 ) / 2.

[0110] (4) On the 32nd day, the mice were killed by over-anesthesia, and the tumors of each group of mice were removed, photographed, and measured in volume.

[0111] Test results such as Figure 13-14 The results showed that compared to Group 2, mice in Group 1 had significantly slower tumor growth and smaller tumor volume after HNRNPA3 expression was inhibited by sh-HNRNPA3, with statistically significant differences (*p < 0.05, **p < 0.01). This indicates that inhibiting HNRNPA3 can significantly inhibit the growth of prostate cancer cells in vivo.

[0112] The present invention first obtained the expression information of primary prostate cancer, CRPC, and NEPC samples from public databases (GSE35988, Beltran-2016, HuPSA-MoPSA). Through differential analysis, it can be seen that HNRNPA3 is most highly expressed in NEPC, and its expression in CRPC is also higher than that in primary prostate cancer. In TCGA-PRAD, KM survival curve analysis found that prostate cancer patients with high expression of HNRNPA3 had a shorter progression-free survival, while in the SU2C cohort (CRPC patients), mCRPC patients with high expression of HNRNPA3 had a shorter median time to ARSI drug treatment response. In NEPC cell lines, the expression of HNRNPA3 was higher than that of wild-type strains. In addition, by collecting clinical tissue samples of prostate cancer and constructing PDX models, HE staining and immunohistochemical analysis found that HNRNPA3 was expressed in NEPC samples at a higher level than in CRPC samples. To reveal the role of HNRNPA3 in prostate cancer NED, the present invention constructed HNRNPA3 knockdown cell lines in C4-2B_NE and LNCaP_NE cell lines. Its expression efficiency was significantly downregulated by WB verification, and its neuroendocrine marker expression was downregulated. By CCK-8 and plate cloning verification, it was found that the proliferation capacity of the two knockdown cell lines was weakened. Then, sh-HNRNPA3 was constructed and its efficiency was verified, and a subcutaneous transplant tumor model was constructed using the sh-HNRNPA3 cell line. The results showed that after knocking down HNRNPA3, the subcutaneous tumor growth of the NEPC cell line was significantly slower than that of the control group, and the final tumor weight was smaller.

[0113] The above findings clearly demonstrate that HNRNPA3 may act as a tumorigenic factor in the development and progression of prostate cancer. Inhibiting HNRNPA3 or reducing its biological activity can significantly inhibit prostate cancer proliferation and metastasis, prolonging patient survival and improving prognosis. Furthermore, the present invention demonstrates for the first time that elevated HNRNPA3 expression may be associated with the development of resistance to ARSI drug therapy. This research clarifies that HNRNPA3 is a key target for prognosis and treatment of prostate cancer, and can be used for treatment, auxiliary diagnosis, and prognostic assessment of prostate cancer. This research also elevates our understanding of the pathogenesis of prostate cancer to a new level, significantly enriching the understanding of prostate cancer pathogenesis. It provides a sufficient scientific basis and theoretical foundation for exploring new molecular targets for prostate cancer diagnosis, prognosis, and treatment, and for developing new targeted drugs, thus possessing significant social value and scientific significance.

[0114] The above detailed description of the analytical methods involved in the present invention provides a detailed introduction. It should be noted that the above description is intended solely to help those skilled in the art better understand the methods and concepts of the present invention, and is not intended to limit the relevant content. Without departing from the principles of the present invention, those skilled in the art may make appropriate adjustments or modifications to the present invention, and such adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. Use of an HNRNPA3 inhibitor in the preparation of a medicament for preventing and / or treating prostate cancer.

2. The use according to claim 1, characterized in that The HNRNPA3 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the HNRNPA3 gene.

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

4. The use according to claim 3, characterized in that The castration-resistant prostate cancer is neuroendocrine prostate cancer.

5. Use of a reagent for detecting HNRNPA3 expression levels in the preparation of a product for auxiliary diagnosis and / or prognosis evaluation of prostate cancer.

6. The use according to claim 5, characterized in that The reagents for detecting the expression level of HNRNPA 3 include primers for detecting the expression level of HNRNPA 3 gene and / or reagents for detecting the content of HNRNPA 3 protein.

7. The use according to claim 5, characterized in that The reagents for measuring HNRNPA3 gene expression levels are selected from the following primer pairs: The upstream sequence of the primer pair is shown in SEQ ID NO: 4, and the downstream sequence of the primer pair is shown in SEQ ID NO:

5.

8. A kit for auxiliary diagnosis and / or prognosis evaluation of prostate cancer, characterized in that: Includes reagents for detecting HNRNPA3 expression levels.

9. A pharmaceutical composition for preventing and / or treating prostate cancer, characterized in that: The invention comprises an HNRNPA3 inhibitor and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, characterized in that The HNR NPA3 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the HNRNPA3 gene.

Images