Use of UFC1 as a biomarker in the preparation of reagents for the diagnosis and / or treatment of prostatic hyperplasia

Abstract

The application discloses application of UFC1 as a biomarker in preparation of reagents for diagnosing and / or treating prostatic hyperplasia. In the technical scheme, the cell experiment result shows that UFC1 can significantly inhibit the proliferation ability of WPMY-1 and BPH-1 cells, and can effectively promote the apoptosis of the cells. The finding reveals the core driving role of UFC1 in BPH progress, that is, maintaining the imbalance between the proliferation and apoptosis of prostate cells, and promoting the tissue hyperplasia. The mechanism indicates the precise intervention direction of the targeted treatment: if the activity or expression of UFC1 can be specifically inhibited, the abnormal proliferation of prostate cells can be blocked from the source, the normal apoptosis program of the cells can be restored, and the proliferation progress can be reversed by realizing the "two-way regulation". Compared with the existing alpha receptor blockers which can only relieve the obstruction symptoms, and the 5 alpha-reductase inhibitors which need long-term medication and can cause sexual dysfunction, the treatment strategy of targeting UFC1 directly attacks the biological nature of the hyperplastic cells, and is expected to more effectively control the disease progress.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical diagnosis and treatment technology, and particularly relates to application of UFC1 as a biomarker in preparation of a reagent for diagnosing and / or treating benign prostatic hyperplasia. BACKGROUND

[0002] Benign prostatic hyperplasia (BPH) is a common disease in middle-aged and elderly men, and its incidence increases with age. This disease causes bladder outlet obstruction due to abnormal hyperplasia of prostate stroma and glands, leading to lower urinary tract symptoms (LUTS) such as frequent urination, urinary urgency, and dysuria. It not only seriously affects the quality of life of patients, but also can induce complications such as urinary retention and renal insufficiency, posing a major threat to the health of middle-aged and elderly men. Therefore, accurate detection of BPH has important clinical significance.

[0003] Current clinical diagnostic methods for BPH have obvious limitations and cannot meet the needs of precise diagnosis and treatment. Currently, the main methods are the International Prostate Symptom Score (IPSS), rectal examination, and imaging examination. These methods not only have difficulty in distinguishing benign hyperplasia from malignant lesions in the early stage, but also lack specific molecular markers. For example, although serum prostate-specific antigen (PSA) is a commonly used indicator, there is significant overlap between BPH and prostate cancer. When PSA is in the range of 4-10 μg / L, biopsy is often needed for differentiation, which not only increases the pain of patients, but also increases the medical cost.

[0004] In recent years, multi-omics research has provided new clues for the discovery of BPH biomarkers and laid a foundation for the preparation of related detection 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 markers are still in the basic research stage and have not been converted into clinically useful diagnostic targets.

[0005] Currently, the main clinical treatment methods for BPH include drug therapy and surgical treatment. Drug therapy mainly uses alpha receptor blockers (such as tamsulosin) and 5 alpha-reductase inhibitors (such as finasteride). Although these drugs can relieve symptoms, they have limitations such as poor long-term medication compliance, significant side effects (such as dizziness and sexual dysfunction), and inability to reverse prostate volume enlargement. Surgical treatment such as transurethral plasmakinetic prostatectomy (TUPKP) has the disadvantages of large trauma, high risk of postoperative complications (such as urinary incontinence and erectile dysfunction), and limited applicability for elderly or patients with underlying diseases. Therefore, developing safer and more effective detection and treatment strategies is an important need in the field of BPH. SUMMARY

[0006] The main purpose of the present application is to provide a UFC1 as a biomarker for preparing a reagent for diagnosing and / or treating benign prostatic hyperplasia, aiming to provide a diagnostic and treatment strategy with effectiveness and safety.

[0007] To achieve the above-mentioned purpose, the present application proposes a reagent for detecting UFC1 as a biomarker for preparing a reagent for diagnosing and / or treating benign prostatic hyperplasia.

[0008] UFC1 (ubiquitin folding modifier binding enzyme 1) is a gene encoding ubiquitin folding modifier binding enzyme, and its expression product plays a key role in the ubiquitin-like modification system in cells, and its function is related to cell cycle regulation, endoplasmic reticulum stress response and immune inflammation regulation. Although UFC1 has been reported to be related to disease progression and prognosis in various disease studies, its role in BPH has not been fully explored.

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

[0010] Preferably, the expression of UFC1 is up-regulated in patients with benign prostatic hyperplasia.

[0011] Preferably, the reagent is used for evaluating 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 detection sample of the reagent is a venous blood sample or a urine sample.

[0013] The present application also proposes a reagent for detecting UFC1 as a biomarker for preparing a drug target for treating benign prostatic hyperplasia.

[0014] The present application also proposes a reagent for detecting UFC1 as a biomarker for preparing a reagent for diagnosing different severities of benign prostatic hyperplasia.

[0015] The present application also proposes a reagent for detecting UFC1 as a biomarker for preparing a reagent for evaluating the therapeutic effect of benign prostatic hyperplasia.

[0016] The present application also proposes a UFC1 inhibitor for preparing a drug for treating benign prostatic hyperplasia, wherein the UFC1 inhibitor includes a small molecule that specifically inhibits the activity of UFC1.

[0017] The application further provides a kit for diagnosing different severities of benign prostatic hyperplasia, which realizes diagnosis by detecting the mRNA or protein level of UFC1 in a sample.

[0018] The application first discovers that UFC1 is specifically highly expressed in the prostate tissue of a BPH patient, and the expression level is significantly positively correlated with the prostate volume and IPSS score. UFC1 promotes the progression of BPH by promoting the proliferation of prostate cells and inhibiting cell apoptosis. Based on this, UFC1 can be used as a new biomarker for the treatment and prognosis evaluation of BPH, and provides a theoretical basis for the development of a targeted intervention strategy.

[0019] Compared with the prior art, the application has the beneficial effects that:

[0020] (1) From the treatment mechanism, the cell experiment results clearly show that knocking down UFC1 can significantly inhibit the proliferation ability of WPMY-1 and BPH-1 cells, and simultaneously strongly promote the apoptosis thereof. This discovery directly reveals the core driving role of UFC1 in the progression of BPH, that is, by maintaining the imbalance between the proliferation and apoptosis of prostate cells, the tissue hyperplasia is promoted. This mechanism indicates a precise intervention direction for targeted treatment: if the activity or expression of UFC1 can be specifically inhibited, the abnormal proliferation of prostate cells can be blocked from the source, and the normal apoptosis program of cells can be restored, so that the hyperplasia process is reversed through “bidirectional regulation”. Compared with the existing alpha receptor blockers which can only relieve the obstruction symptoms, and 5-alpha-reductase inhibitors which need long-term medication and may cause sexual dysfunction, the treatment strategy targeting UFC1 directly attacks the biological nature of hyperplasia cells, and is expected to more effectively control the progression of the disease.

[0021] (2) The animal experiment of the application further verifies the in-vivo effectiveness and safety of the UFC1 targeted treatment. In the testosterone propionate-induced BPH rat model, after injection of shUFC1 lentivirus, the prostate tissue volume of the rats is significantly reduced, the connective tissue and epithelial cell layer thickness are obviously reduced, and the body weight of the rats is not significantly affected. This result has important clinical implications: on the one hand, the in-vivo experiment proves that knocking down UFC1 can directly improve the histopathological features of BPH, which shows that it has definite therapeutic activity in vivo; on the other hand, the feature of “not affecting the body weight” suggests that the treatment targeting UFC1 may have lower systemic toxicity, which can not only avoid the tolerance risk of traditional surgery for elderly patients, but also overcome the interference of hormone-regulating drugs on the whole body metabolism, thereby providing a safer treatment option for BPH patients who are old or have underlying diseases.

[0022] (3) From the perspective of clinical transformation, the development path of UFC1 targeted therapy is clear and feasible. Based on the molecular function of UFC1 ubiquitin-like modifying enzyme, small molecule inhibitors can be designed to specifically block its enzyme activity, and RNA interference technology (such as siRNA, shRNA) can be used to precisely down-regulate its expression. Both strategies can directly inhibit the proliferation-apoptosis imbalance mediated by UFC1. In addition, the research results also provide a new idea for combination therapy: existing drugs (such as alpha receptor blockers) mainly aim at symptom relief, while UFC1 targeted therapy focuses on etiological control. The combination of the two may produce a synergistic effect of "quickly relieving symptoms + long-term inhibiting progression", thereby reducing the dose-dependent side effects of simple drug therapy and reducing the necessity of surgical intervention.

[0023] (4) The present application proves by experiments 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 effect-the decrease of serum UFC1 level during treatment can be used as an objective basis for efficacy evaluation, but also suggests that it may become a "treatment response prediction marker", which helps to screen patients who are most likely to benefit from UFC1 targeted therapy and achieve individualized treatment. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, a brief introduction will be given below to the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0025] Figure 1 The figure provided by the present application shows the expression amount of UFC1 protein in the serum of prostate hyperplasia patients (N=277) and healthy people (N=121); * represents P <0.05, *** represents P <0.001.

[0026] Figure 2 The figure provided by the present application shows the expression level change of UFC1 in the serum of patients with different IPSS scores; * represents P <0.05, *** represents P <0.001.

[0027] Figure 3Figure provided by the present application shows the influence of UFC1 knockdown on prostate cell proliferation; 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 cell proliferation ability of WPMY-1 cells detected by flow cytometry after UFC1 knockdown; Figure D shows the cell proliferation ability of BPH-1 cells detected by flow cytometry after UFC1 knockdown; * represents P <0.05, *** represents P <0.001.

[0028] Figure 4 Figure provided by the present application shows the apoptosis of prostate cells after UFC1 knockdown; Figure A shows the apoptosis of WPMY-1 cells detected by flow cytometry before and after UCF1 knockdown; Figure B shows the apoptosis of BPH-1 cells detected by flow cytometry before and after UCF1 knockdown; * represents P <0.05, *** represents P <0.001.

[0029] Figure 5 Figure provided by the present application shows the influence of UFC1 knockdown on rat prostate tissue; Figure A shows the general observation pictures of rat prostate tissues of Sham+shNC group, Sham+shUFC1 group, T-BPH+shNC group and T-BPH+shUFC1 group; Figure B shows the histogram of prostate weight index (prostate weight / body weight) x 1000 and the body weight change curve of the four groups of rats; Figure C shows the representative pictures of hematoxylin-eosin (HE) staining of prostate tissues of Sham+shNC, Sham+shUFC1, T-BPH+shNC and T-BPH+shUFC1 four groups of rats and the epithelial thickness column chart; * represents P <0.05, *** represents P <0.001.

[0030] The implementation, functional features and advantages of the present application will be further described with reference to the accompanying drawings. DETAILED DESCRIPTION

[0031] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the manufacturers of the reagents or instruments are not specified, the conventional products that can be purchased in the market are adopted. In addition, the technical solutions in each embodiment can be combined with each other, but it should be considered that the combination of the technical solutions does not exist and is not within the protection scope of the present application when the combination of the technical solutions is contradictory or unachievable on the premise that the combination of the technical solutions can be realized by the ordinary skilled in the art. Based on the embodiments in the present application, all the other embodiments obtained by the ordinary skilled in the art without creative labor are within the protection scope of the present application.

[0032] The technical solutions of the present application will be further described in detail below in combination with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present application and are not used to limit the present application.

[0033] The reagents, instruments and cell sources used in the embodiments are as follows:

[0034] 1. Experimental reagents and instruments

[0035]

[0036] 2. Cell sources and culture

[0037] Human prostate cells BPH-1 cells and human normal prostate stroma immortalized cells WPMY-1 cells were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The cells were cultured 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) at 37°C under 5% CO2.

[0038] Example 1 Detection of serum UFC1 expression in healthy people and BPH patients

[0039] In this study, 398 serum samples were collected, including healthy people (N=121) and patients with benign prostatic hyperplasia (N=277), which were divided into G1 (healthy people group), G2 (conservative treatment group), G3 (drug treatment group), and G4 (surgery treatment group). First, the serum samples were subjected to protein extraction, and then the BCA method was used to detect the concentration of the extracted protein. After the detection was completed, Trypsin was added to the obtained protein for enzymatic treatment, and the treated sample was subjected to DIA mass spectrometry detection. Finally, based on the quantitative results obtained by mass spectrometry detection, the differential protein screening between different comparison groups was completed. This study was approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (Ethical Number 2022173).

[0040] The results showed that the expression of UFC1 in the serum of BPH patients (G234 group) was significantly higher than that in healthy people (G1 group) P < 0.05) Figure 1 . In addition, according to the IPSS score, BPH patients were divided into mild-LUTS (IPSS≤7), moderate-LUTS (8≤IPSS≤19), and severe-LUTS (20≤IPSS≤35), and it was found that the expression of UFC1 increased significantly with the severity of the symptoms Figure 2 . The above results show that the expression of UFC1 protein increases in patients with benign prostatic hyperplasia, and is positively correlated with clinical symptoms. UFC1 may become a potential therapeutic target for benign prostatic hyperplasia.

[0041] Example 2 Cell experiment

[0042] 1. Cell transfection

[0043] (1) One day before transfection, BPH-1 and WPMY-1 cells were plated at a cell density of 2x10 5 per well to ensure that the cells in the 6-well plate reached 70-90% confluence on the day of transfection. During this period, complete culture medium was used for culture; one day before transfection, the medium was replaced with antibiotic-free serum-containing medium (1 ml per well), and then returned to the 37°C incubator for 1 hour.

[0044] (2) Preparation of si-UCF1 mixture: 2 μL si-UCF1 + 100 μL Opti-MEM (single hole amount), incubated at room temperature for 5 minutes.

[0045] (3) Preparation of Lipofectamine 2000 mixture: 5 μL Lipofectamine 2000 + 100 μL Opti-MEM (single hole amount), incubated at room temperature for 5 minutes.

[0046] (4) Slowly mix the si-UCF1 mixture solution of step (2) with the Lipofectamine 2000 mixture solution of step (3), stand at room temperature for 20 minutes, and carefully add the mixed solution to the 6-well plate to be transfected.

[0047] (5) Put the 6-well plate back into the incubator at 37°C, incubate for 4-6 hours, and then replace it with preheated complete medium. After incubating the cells for 24-48 hours, perform subsequent experiments (RNA extraction, protein extraction, and other functional experiments, etc.).

[0048] The interference sequence of UFC1 is:

[0049]

[0050] 2、Cell proliferation

[0051] After trypsin digestion and centrifugation, the prostate cells transfected with si-UCF1 were resuspended. After cell counting, an appropriate amount of cell suspension was taken, 2000 cells per well, mixed thoroughly, and inoculated into a 96-well plate, and placed in an incubator. At 1 day, 2 days, 3 days, and 4 days, the old culture medium was removed, 100 μL of fresh culture medium and 10 μL of CCK8 detection reagent were added, and the absorbance at 450 nm was detected using an enzyme marker after 2 hours of incubation in the incubator. The absorbance at 450 nm was used to calculate cell proliferation.

[0052] 3、Cell apoptosis detection

[0053] (1) Collect 1-10 x 10 5 cells (including cells in the culture supernatant), centrifuge, discard the supernatant, wash the cells with pre-cooled PBS 1-2 times, then resuspend the cells with 500 μL of 1 x Binding Buffer.

[0054] (2) Staining treatment: add 5 μL of Annexin V-APC and 10 μL of PI to each tube.

[0055] (3) Incubation: mix gently after vortexing, and incubate at room temperature for 5 minutes in the dark.

[0056] (4) Analyze on the machine.

[0057] 4、Cell cycle detection

[0058] (1) After transfection, collect the cells and centrifuge, discard the supernatant to obtain the cell pellet.

[0059] (2) Add 1 mL of PBS buffer and mix by blowing, then centrifuge again and discard the supernatant.

[0060] (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.

[0061] (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.

[0062] Cellular experiment results:

[0063] (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).

[0064] (2) Flow cytometry results showed that knockdown of UCF1 significantly promoted apoptosis in WPMY-1 and BPH-1 cells. Figure 4 A, B).

[0065] Example 3: Effects of UFC1 knockdown on rat prostate tissue

[0066] 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).

[0067] 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).

[0068] 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).

[0069] 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.

[0070] 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 diagnosing benign prostatic hyperplasia.

2. The application of UFC1 interference sequences in the preparation of drugs for treating benign prostatic hyperplasia, characterized in that, The interference sequence of UFC1 is: (a) the positive chain shown in SEQ ID NO: 1 and the complementary antisense chain shown in SEQ ID NO: 2; or (b) the positive chain shown in SEQ ID NO: 3 and the complementary antisense chain 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 reagents for diagnosing different degrees of benign prostatic hyperplasia.

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

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