Application of UFC1 as biomarker in preparation of reagent for diagnosing and / or treating benign prostatic hyperplasia

Abstract

The invention discloses an application of UFC1 as a biomarker in preparation of a reagent for diagnosing and / or treating benign prostatic hyperplasia. In the technical scheme provided by the invention, a cell experiment result shows that the multiplication capacity of WPMY-1 and BPH-1 cells can be remarkably inhibited by knocking down UFC1, and meanwhile, the apoptosis of the WPMY-1 and BPH-1 cells is powerfully promoted. The discovery reveals the core driving effect of UFC1 in BPH development, namely promoting tissue hyperplasia by maintaining unbalance of prostate cell proliferation and apoptosis. The mechanism indicates a precise intervention direction for targeted therapy: if the activity or expression of UFC1 can be specifically inhibited, abnormal proliferation of prostate cells can be blocked from the source, and meanwhile, a normal apoptosis procedure of the cells is recovered, so that bidirectional regulation is realized to reverse the proliferation process. Compared with an existing alpha receptor blocker only capable of relieving obstruction symptoms and a 5alpha-reductase inhibitor which needs to be used for a long time and possibly causes sexual dysfunction, a treatment strategy of targeting UFC1 directly strikes the cytobiology nature of hyperplasia, and the disease progress is expected to be more effectively controlled.

Description

Technical Field

[0001] This invention relates to the fields of medical diagnostics and treatment, specifically to the application of UFC1 as a biomarker in the preparation of reagents for the diagnosis and / or treatment of benign prostatic hyperplasia. Background Technology

[0002] Benign prostatic hyperplasia (BPH) is a common disease among middle-aged and elderly men, with its incidence increasing with age. This disease causes bladder outlet obstruction due to abnormal proliferation of the prostatic stroma and glands, leading to lower urinary tract symptoms (LUTS) such as urinary frequency, urgency, and difficulty urinating. This not only seriously affects patients' quality of life but may also induce complications such as urinary retention and renal insufficiency, posing a significant threat to the health of middle-aged and elderly men. Therefore, accurate detection of BPH is of great clinical significance.

[0003] Current clinical diagnostic methods for benign prostatic hyperplasia (BPH) have significant limitations and cannot meet the needs of precise diagnosis and treatment. Currently, they mainly rely on the International Prostate Symptom Score (IPSS), digital rectal examination, and imaging examinations. These methods not only struggle to differentiate between benign hyperplasia and malignant lesions in the early stages but also lack specific molecular markers. For example, while serum prostate-specific antigen (PSA) is a commonly used indicator, it significantly overlaps in BPH and prostate cancer. When PSA levels are in the 4-10 μg / L range, biopsy is often required for differentiation, which not only increases patient suffering but also raises medical costs.

[0004] In recent years, multi-omics research has provided new clues for the discovery of biomarkers for BPH and laid the foundation for the preparation of related diagnostic reagents. Studies have shown that the pathogenesis of BPH is closely related to gene expression imbalance and immune inflammatory response; for example, miR-143 / 145 can affect smooth muscle cell proliferation by inhibiting MAP4K4. However, most of these discovered biomarkers are still in the basic research stage and have not yet been translated into clinically usable diagnostic targets.

[0005] Currently, clinical treatments for benign prostatic hyperplasia (BPH) primarily include medication and surgery. Medication mainly uses alpha-blockers (such as tamsulosin) and 5α-reductase inhibitors (such as finasteride). While these can relieve symptoms, they have limitations such as poor long-term adherence, significant side effects (such as dizziness and sexual dysfunction), and the inability to reverse prostate enlargement. Surgical treatments, such as transurethral plasma resection of the prostate (TUPKP), have drawbacks including significant trauma and a high risk of postoperative complications (such as urinary incontinence and erectile dysfunction), particularly limiting their applicability to elderly patients or those with underlying medical conditions. Therefore, developing safer and more effective detection and treatment strategies is a crucial need in the field of BPH. Summary of the Invention

[0006] The main objective of this invention is to propose the application of UFC1 as a biomarker in the preparation of reagents for the diagnosis and / or treatment of benign prostatic hyperplasia, aiming to provide an effective and safe diagnostic and treatment strategy.

[0007] To achieve the above objectives, this invention proposes the application of reagents for detecting UFC1 as a biomarker in the preparation of reagents for the diagnosis and / or treatment of benign prostatic hyperplasia.

[0008] UFC1 (ubiquitin folding modification-binding enzyme 1) is a gene encoding a ubiquitin folding modification-binding enzyme. Its expression product plays a crucial role in the intracellular ubiquitination modification system, and its functions involve cell cycle regulation, endoplasmic reticulum stress response, and immune inflammation regulation. Although UFC1 has been reported to be associated with disease progression and prognosis in various disease studies, its role in BPH has not been fully explored.

[0009] Preferably, the interference sequence of the UFC1 includes: (a) a positive chain as shown in SEQ ID NO: 1 and a complementary antisense chain as shown in SEQ ID NO: 2; or (b) a positive chain as shown in SEQ ID NO: 3 and a complementary antisense chain as shown in SEQ ID NO: 4.

[0010] Preferably, the UFC1 is upregulated in patients with benign prostatic hyperplasia.

[0011] Preferably, the reagent is used to assess the severity of benign prostatic hyperplasia by detecting the expression level of UFC1, wherein the expression level of UFC1 increases with the severity of benign prostatic hyperplasia.

[0012] Preferably, the test sample for the reagent is a venous blood sample or a urine sample.

[0013] This invention also proposes the application of reagents for detecting UFC1 as a biomarker in the preparation of drug targets for the treatment of benign prostatic hyperplasia.

[0014] This invention also proposes the application of reagents for detecting UFC1 as a biomarker in the preparation of reagents for diagnosing different degrees of benign prostatic hyperplasia.

[0015] This invention also proposes the application of reagents for detecting UFC1 as a biomarker in the preparation of reagents for evaluating the efficacy of treatment for benign prostatic hyperplasia.

[0016] The present invention also proposes the use of a UFC1 inhibitor in the preparation of a drug for treating benign prostatic hyperplasia, wherein the UFC1 inhibitor comprises a small molecule that specifically inhibits the activity of UFC1.

[0017] The present invention also proposes a kit for diagnosing different degrees of benign prostatic hyperplasia, wherein the kit achieves diagnosis by detecting the mRNA or protein level of UFC1 in the sample.

[0018] This invention is the first to discover that UFC1 is specifically and highly expressed in the prostate tissue of patients with benign prostatic hyperplasia (BPH), and its expression level is significantly positively correlated with prostate volume and IPSS score. UFC1 promotes BPH progression by promoting prostate cell proliferation and inhibiting apoptosis. Based on this, UFC1 can serve as a novel biomarker for BPH treatment and prognostic assessment, providing a theoretical basis for developing targeted intervention strategies.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In the technical solution provided by this invention, from the perspective of the treatment mechanism, the cell experiment results clearly show that knocking down UFC1 can significantly inhibit the proliferation of WPMY-1 and BPH-1 cells, while strongly promoting their apoptosis. This discovery directly reveals the core driving role of UFC1 in the progression of BPH—by maintaining the imbalance between prostate cell proliferation and apoptosis, it promotes tissue hyperplasia. This mechanism points to a precise intervention direction for targeted therapy: if the activity or expression of UFC1 can be specifically inhibited, the abnormal proliferation of prostate cells can be blocked from the source, while restoring the normal apoptosis program of cells, achieving "bidirectional regulation" to reverse the hyperplasia process. Compared with existing α-receptor blockers that can only relieve obstructive symptoms and 5α-reductase inhibitors that require long-term use and may cause sexual dysfunction, the treatment strategy of targeting UFC1 directly addresses the cellular biological essence of hyperplasia and is expected to more effectively control disease progression.

[0020] (2) Animal experiments of this invention further verified the in vivo efficacy and safety of UFC1-targeted therapy. In a rat model of BPH induced by testosterone propionate, after injection of shUFC1 lentivirus, the volume of rat prostate tissue was significantly reduced, the thickness of connective tissue and epithelial cell layer was significantly decreased, and there was no significant effect on rat body weight. This result has important clinical implications: on the one hand, in vivo experiments confirmed that knocking down UFC1 can directly improve the histopathological characteristics of BPH, indicating that it has clear therapeutic activity in vivo; on the other hand, the characteristic of "not affecting body weight" suggests that UFC1-targeted therapy may have low systemic toxicity, which can avoid the tolerance risk of traditional surgery in elderly patients and overcome the interference of hormone-regulating drugs on systemic metabolism, providing a safer treatment option for elderly BPH patients or those with underlying diseases.

[0021] (3) From a clinical translational perspective, the development path of UFC1-targeted therapy is clear and feasible. Based on the molecular function of UFC1 as a ubiquitin-like modifying enzyme, small molecule inhibitors can be designed to specifically block its enzyme activity, or its expression can be precisely downregulated through RNA interference technology (such as siRNA and shRNA). Both strategies can directly inhibit UFC1-mediated proliferation-apoptosis imbalance. In addition, the results of this study also provide new ideas for combination therapy: existing drugs (such as α-receptor blockers) mainly target symptom relief, while UFC1-targeted therapy focuses on etiological control. The combination of the two may produce a synergistic effect of "rapid symptom relief + long-term inhibition of progression", thereby reducing the dose-dependent side effects of drug therapy alone and reducing the necessity of surgical intervention.

[0022] (4) This invention experimentally confirms that UFC1 is highly expressed in the serum of BPH patients and is positively correlated with the severity of symptoms. This feature not only provides a convenient indicator for dynamic monitoring of treatment effects—the decrease in serum UFC1 levels during treatment can serve as an objective basis for efficacy assessment—but also suggests that it may become a "treatment response predictive biomarker," which can help screen patients most likely to benefit from UFC1-targeted therapy and achieve individualized treatment. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a graph showing the expression levels of UFC1 protein in the serum of patients with benign prostatic hyperplasia (N=277) and healthy individuals (N=121) provided by this invention; * indicates P < 0.05, *** indicates P < 0.001.

[0025] Figure 2 This invention provides a graph showing the changes in serum UFC1 expression levels in patients with different IPSS scores; * indicates... P <0.05, *** indicates P < 0.001.

[0026] Figure 3The following figures illustrate the effect of UFC1 knockdown on prostate cell proliferation, as provided by this invention: Figure A shows the mRNA level verification (left) and protein level verification (right) of WPMY-1 cells after UFC1 knockdown; Figure B shows the mRNA level verification (left) and protein level verification (right) of BPH-1 cells after UFC1 knockdown; Figure C shows the proliferation capacity of WPMY-1 cells after UFC1 knockdown detected by flow cytometry; Figure D shows the proliferation capacity of BPH-1 cells after UFC1 knockdown detected by flow cytometry; * represents... P < 0.05, *** indicates P < 0.001.

[0027] Figure 4 Figure A shows the apoptosis of prostate cells after UFC1 knockdown, as provided by this invention; Figure B shows the apoptosis of WPMY-1 cells before and after UFC1 knockdown, detected by flow cytometry; * represents... P < 0.05, *** indicates P < 0.001.

[0028] Figure 5 The following figures illustrate the effect of UFC1 knockdown on rat prostate tissue provided by this invention: Figure A shows gross images of rat prostate tissue from the Sham+shNC, Sham+shUFC1, T-BPH+shNC, and T-BPH+shUFC1 groups; Figure B shows histograms of prostate weight index (prostate weight / body weight) × 1000 and curves of body weight change in the four groups; Figure C shows representative images of hematoxylin-eosin (HE) staining and bar charts of epithelial thickness of prostate tissue from the Sham+shNC, Sham+shUFC1, T-BPH+shNC, and T-BPH+shUFC1 groups; * represents... P < 0.05, *** indicates P < 0.001.

[0029] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0032] Reagents, instruments, and cell sources used in the examples: 1. Experimental reagents and instruments

[0033] 2. Cell source and culture Human prostate cells BPH-1 and human normal prostate stromal immortalized cells WPMY-1 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured at 37°C and 5% CO2 in RPMI-1640 (Gibco, LifeTechnology, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco) or DMEM (Gibco, LifeTechnology, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (Gibco).

[0034] Example 1: Detection of serum UFC1 expression in healthy individuals and BPH patients This study collected serum samples from 398 individuals, including healthy individuals (N=121) and patients with benign prostatic hyperplasia (BPH) (N=277), who were divided into four groups: G1 (healthy population), G2 (conservative treatment group), G3 (drug treatment group), and G4 (surgical treatment group). First, proteins were extracted from these serum samples, and the concentration of the extracted proteins was then determined using the BCA method. After detection, trypsin was added to the obtained proteins for enzymatic digestion, and the treated samples were then analyzed by DIA mass spectrometry. Finally, based on the quantitative results obtained from mass spectrometry, differentially expressed proteins were screened among the different comparison groups. This study was approved by the Medical Ethics Committee of Zhongnan Hospital, Wuhan University (ethics number 2022173).

[0035] The results showed that, compared with healthy individuals (G1 group), serum UFC1 expression was significantly increased in BPH patients (G234 group). P < 0.05) Figure 1 Furthermore, based on the IPSS score, BPH patients were divided into mild-LUTS (IPSS≤7), moderate-LUTS (8≤IPSS≤19), and severe-LUTS (20≤IPSS≤35). It was found that UFC1 expression significantly increased with increasing symptom severity. Figure 2 The above results indicate that UFC1 protein expression is increased in patients with benign prostatic hyperplasia (BPH) and is positively correlated with clinical symptoms, suggesting that UFC1 may be a potential therapeutic target for BPH.

[0036] Example 2 Cell Experiment 1. Cell transfection (1) One day before transfection, BPH-1 and WPMY-1 cells were seeded into plates, with a cell density of 2 × 10⁶ cells per well. 5 Ensure that the cells in the 6-well plate reach a confluence density of 70%-90% on the day of transfection, and use complete culture medium during this period; before transfection, change to antibiotic-free, serum-containing culture medium (1 ml per well), and then incubate at 37°C for 1 hour.

[0037] (2) Preparation of Si-UCF1 mixture: 2 μL Si-UCF1 + 100 μL Opti-MEM (amount per well), incubate at room temperature for 5 minutes.

[0038] (3) Prepare Lipofectamine 2000 mixture: 5 μL Lipofectamine 2000 + 100 μL Opti-MEM (single well amount), incubate at room temperature for 5 minutes.

[0039] (4) Slowly mix the si-UCF1 mixture from step (2) with the Lipofectamine 2000 mixture from step (3), let it stand at room temperature for 20 minutes, and carefully drop the mixed liquid into the 6-well plate to be transfected.

[0040] (5) Place the 6-well plate back into the incubator at 37°C and incubate for 4-6 hours. Then replace it with preheated complete culture medium. After the cells have been incubated for 24-48 hours, proceed with subsequent experiments (RNA, protein extraction and other functional experiments, etc.).

[0041] The UFC1 interference sequence is as follows:

[0042] 2. Cell proliferation The prostate cells transfected with si-UCF1 were digested with trypsin, centrifuged, and resuspended. After cell counting, an appropriate amount of cell suspension was taken at 2000 cells per well, thoroughly mixed, and seeded into 96-well plates and placed in an incubator. On days 1, 2, 3, and 4, the old culture medium was removed, and 100 µL of fresh culture medium and 10 µL of CCK8 assay reagent were added. After incubation for 2 hours, the absorbance at 450 nm was measured using a microplate reader to calculate cell proliferation.

[0043] 3. Apoptosis detection (1) Collect 1-10×10 5 Centrifuge each cell (including those in the culture supernatant), discard the supernatant, wash the cells 1-2 times with pre-cooled PBS, and then resuspend the cells in 500 μL of 1×Binding Buffer.

[0044] (2) Staining treatment: Add 5 μL Annexin V-APC and 10 μL PI to each tube.

[0045] (3) Incubation: After gently vortexing and mixing, incubate at room temperature in the dark for 5 minutes.

[0046] (4) Perform analysis on the computer.

[0047] 4. Cell cycle detection (1) After transfection, cells were collected and centrifuged, and the supernatant was discarded to obtain cell pellet.

[0048] (2) Add 1 mL of PBS buffer, mix well by pipetting, and centrifuge again to discard the supernatant.

[0049] (3) Use a cell cycle assay kit (Lianke Biotechnology). Add 1 ml of DNA Staining solution and 10 μl of Permeabilization solution to the cell pellet, mix gently, and incubate at room temperature in the dark for 20 minutes.

[0050] (4) Flow cytometry detection: Select the lowest loading speed for the flow cytometer, excitation wavelength 488nm, and collect an appropriate amount of cells for analysis.

[0051] Cellular experiment results: (1) In the above experiments, we used si-UFC1 to construct UFC1 knockdown cell models in WPMY-1 and BPH-1 cells, and used qRT-PCR and WB experiments to detect the knockdown effect at the mRNA and protein levels. Figure 3 A, B). CCK8 assay results showed that knockdown of UCF1 significantly reduced the proliferation ability of WPMY-1 and BPH-1 cells. Figure 3 C, D).

[0052] (2) Flow cytometry results showed that knockdown of UCF1 significantly promoted apoptosis in WPMY-1 and BPH-1 cells. Figure 4 A, B).

[0053] Example 3: Effects of UFC1 knockdown on rat prostate tissue This study investigated the morphological and histopathological changes of rat prostate tissue by injecting shUFC1 lentivirus into the rats. Four groups of rats were included in the experiment: sham-operated group (Sham+shNC), sham-operated group treated with shUFC1 (Sham+shUFC1), testosterone propionate-induced benign prostatic hyperplasia (BPH) group (T-BPH+shNC), and testosterone propionate-treated BPH group treated with shUFC1 (T-BPH+shUFC1).

[0054] The results showed that, from the gross examination of rat prostate tissue ( Figure 5 A) As can be seen, compared with the sham-operated group (Sham+shNC), the prostate tissue appearance of the Sham+shUFC1 group was significantly smaller; compared with the testosterone propionate-induced BPH control group (T-BPH+shNC), the prostate tissue appearance of the BPH group treated with testosterone propionate combined with shUFC1 (T-BPH+shUFC1) was significantly smaller. Statistical analysis of rat prostate weight and body weight revealed that shUFC1 treatment had no significant effect on rat body weight. Figure 5 B).

[0055] Regarding the prostate index, the prostate index of the Sham+shUFC1 group was significantly lower than that of the Sham+shNC group. P <0.05); while compared with the T-BPH+shNC group, there was no significant difference in prostate index in the T-BPH+shUFC1 group ( P>0.05). Histopathological observation showed that, compared with the control group, the thickness of the prostate connective tissue and epithelial cell layer in rats treated with shUFC1 was reduced ( Figure 5 C).

[0056] In summary, this invention, through cell and animal experiments, demonstrates that targeted inhibition of UFC1 is a novel treatment strategy for BPH that combines efficacy and safety. Its core value lies in overcoming the limitations of existing treatments that only address the symptoms, not the underlying cause, by blocking disease progression at the root of cell proliferation-apoptosis regulation. Furthermore, it possesses advantages in clinical translation, including low toxicity, monitorability, and ease of combination therapy. Future research could further develop UFC1-specific inhibitors and conduct preclinical pharmacodynamic studies, propelling this target from experimental evidence to clinical application and providing better treatment options for BPH patients.

[0057] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. Application of reagents for detecting UFC1 as a biomarker in the preparation of reagents for the diagnosis and / or treatment of benign prostatic hyperplasia.

2. The application according to claim 1, characterized in that, The interference sequence of the UFC1 includes: (a) a positive chain as shown in SEQ ID NO: 1 and a complementary antisense chain as shown in SEQ ID NO: 2; or (b) a positive chain as shown in SEQ ID NO: 3 and a complementary antisense chain as shown in SEQ ID NO:

4.

3. The application according to claim 1, characterized in that, The UFC1 is upregulated in patients with benign prostatic hyperplasia.

4. The application according to claim 1, characterized in that, The reagent is used to assess the severity of benign prostatic hyperplasia by detecting the expression level of UFC1, wherein the expression level of UFC1 increases with the severity of benign prostatic hyperplasia.

5. The application according to claim 1, characterized in that, The reagent is used to test venous blood or urine samples.

6. Application of reagents for detecting UFC1 as a biomarker in the preparation of drug targets for the treatment of benign prostatic hyperplasia.

7. Application of reagents for detecting UFC1 as a biomarker in the preparation of reagents for diagnosing different degrees of benign prostatic hyperplasia.

8. Application of reagents for detecting UFC1 as a biomarker in the preparation of reagents for evaluating the efficacy of benign prostatic hyperplasia treatment.

9. The use of a UFC1 inhibitor in the preparation of a drug for treating benign prostatic hyperplasia, characterized in that, The UFC1 inhibitors include small molecules that specifically inhibit UFC1 activity.

10. A kit for diagnosing different degrees of benign prostatic hyperplasia, characterized in that, The kit enables diagnosis by detecting the mRNA or protein level of UFC1 in a sample.

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