Application of reagent for detecting expression level of LRRN3 in preparation of benign prostatic hyperplasia diagnosis product

Abstract

The invention discloses application of a reagent for detecting the expression level of LRRN3 in preparation of a benign prostatic hyperplasia diagnosis product. Cell function experiments and animal experiments prove that the overexpressed LRRN3 gene can inhibit proliferation of prostatic cells, retard the cell cycle and promote cell apoptosis, so that prostatic hyperplasia is effectively inhibited. The discovery provides a new target spot and a treatment strategy for the treatment of the prostatic hyperplasia, and provides a brand new thought and method for the treatment of the prostatic hyperplasia at the same time. Based on the research result, a gene therapy drug taking the LRRN3 gene as a target spot can be further developed, for example, a lentiviral vector carrying the LRRN3 gene or other gene delivery systems are constructed and used for treating the prostatic hyperplasia. In addition, the LRRN3 gene can be applied in combination with other treatment means, so that the treatment effect is improved.

Description

Technical Field

[0001] This invention relates to the fields of medical diagnostics and treatment, specifically to the application of reagents for detecting LRRN3 expression levels in the preparation of diagnostic products for benign prostatic hyperplasia. Background Technology

[0002] Benign prostatic hyperplasia (BPH) is a common disease among middle-aged and elderly men, typically beginning around age 40, with the incidence gradually increasing with age. The prevalence of BPH exceeds 50% in men over 50, and surpasses 80% in men over 70. Data from 2021 shows that there are over 110 million BPH patients worldwide, with 23.11 million in my country. With the accelerating aging of the population and increased life expectancy, the health burden and social medical pressure caused by this disease continue to intensify. Currently, the diagnosis of BPH mainly relies on a comprehensive assessment of clinical symptoms (such as urinary frequency, urgency, and difficulty urinating), the International Prostate Symptom Score (IPSS), digital rectal examination, prostate ultrasound, and serum prostate-specific antigen (PSA) testing. However, existing diagnostic methods still have limitations in early identification of disease progression risk and in differentiating symptomatic BPH from other prostate diseases (such as prostate cancer), making it difficult to accurately guide individualized treatment.

[0003] In terms of treatment, since androgens play a key role in the occurrence and progression of benign prostatic hyperplasia (BPH), 5α-reductase inhibitors targeting the androgen pathway have become the mainstream drugs in clinical treatment. Although they can relieve symptoms, they have limitations such as poor long-term medication adherence, side effects (e.g., dizziness, sexual dysfunction), and the inability to reverse prostate enlargement. Clinical practice shows that approximately 10% of patients still experience disease progression after receiving such drug treatment, and some patients have drug tolerance or adverse reactions (e.g., sexual dysfunction, breast tenderness). Existing treatment methods (including drugs and surgery) cannot completely stop the natural progression of BPH, especially for patients with moderate to severe symptoms or complications, where treatment outcomes remain unsatisfactory. Therefore, there is an urgent need to explore new therapeutic targets and develop more targeted and effective treatment plans to improve patient prognosis and reduce the disease burden. Summary of the Invention

[0004] The main objective of this invention is to propose the application of reagents for detecting LRRN3 expression levels in the preparation of diagnostic products for benign prostatic hyperplasia, aiming to provide a new therapeutic target and offer patients a more targeted and effective treatment plan.

[0005] To achieve the above objectives, this invention proposes the application of a reagent for detecting LRRN3 expression levels in the preparation of diagnostic products for benign prostatic hyperplasia.

[0006] The LRRN3 gene belongs to the family of leucine-rich repeat protein families. Previous studies have focused on the nervous system, but no studies have explored the role of this gene in prostate tissue.

[0007] Preferably, the nucleotide sequence of the LRRN3 is shown in SEQ ID NO: 1.

[0008] Preferably, the LRRN3 is downregulated in patients with benign prostatic hyperplasia.

[0009] Preferably, the product is used to detect the expression level of LRRN3 in blood leukocytes.

[0010] Preferably, the product includes gene chips, formulations, or reagent kits.

[0011] The present invention also proposes the application of the expression vector of the LRRN3 gene in the preparation of a pharmaceutical composition for the treatment of benign prostatic hyperplasia.

[0012] Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0013] The present invention also proposes the application of reagents expressing the LRRN3 gene in the preparation of drugs that inhibit the proliferation of benign prostatic hyperplasia cells.

[0014] This invention also proposes the application of reagents expressing the LRRN3 gene in the preparation of drugs that arrest the cell cycle of benign prostatic hyperplasia.

[0015] This invention also proposes the application of reagents expressing the LRRN3 gene in the preparation of drugs that promote apoptosis of benign prostatic hyperplasia cells.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention is the first to discover that the expression level of the LRRN3 gene is downregulated in patients with benign prostatic hyperplasia (BPH). This type of reagent can be used as a novel biomarker detection tool to achieve early screening and diagnosis of the disease, improve the specificity and sensitivity of diagnosis, and help distinguish BPH from other prostate diseases (such as prostate cancer), avoiding misdiagnosis or missed diagnosis. At the same time, by dynamically monitoring the changes in the expression level of LRRN3, the degree of disease progression and prognosis can be assessed, providing precise molecular basis for the development of individualized treatment plans in clinical practice, making up for the shortcomings of existing diagnostic methods in early identification and disease assessment, and promoting the precise transformation of BPH diagnosis from symptom-oriented to molecular marker-oriented.

[0017] (2) This invention, through cell function experiments and animal experiments, confirms that overexpression of the LRRN3 gene can inhibit prostate cell proliferation, arrest the cell cycle, and promote apoptosis, thereby effectively inhibiting benign prostatic hyperplasia (BPH). This discovery provides a new target and treatment strategy for the treatment of BPH, and also offers a novel approach and method. Based on the results of this study, gene therapy drugs targeting the LRRN3 gene can be further developed, such as constructing lentiviral vectors or other gene delivery systems carrying the LRRN3 gene for the treatment of BPH. In addition, the LRRN3 gene may be used in combination with other treatment methods to improve the therapeutic effect. Future research can further optimize the gene delivery system, improve transfection efficiency, reduce immunogenicity, and lay the foundation for clinical translation. Attached Figure Description

[0018] 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.

[0019] Figure 1 Volcano plot of differential gene expression in leukocyte transcriptomics of BPH patients (N=277) and healthy individuals (N=121) provided by this invention; ***P < 0.001.

[0020] Figure 2 Box plots of LRRN3 gene expression levels in the blood of BPH patients and healthy individuals provided by this invention; ***P <0.001.

[0021] Figure 3 The following figures illustrate the effects of LRRN3 overexpression on the inhibition of BPH-1 and WPMY-1 cell proliferation and promotion of apoptosis, as provided by this invention: Figure A shows the mRNA level verification of BPH-1 (left) and WPMY-1 cells (right) after LRRN3 overexpression; Figure B shows the protein level verification of BPH-1 and WPMY-1 cells after LRRN3 overexpression; Figure C shows the cell proliferation curves of BPH-1 (left) and WPMY-1 cells (right) after LRRN3 overexpression detected by CCK-8 assay; Figure D shows the number of apoptotic cells detected by flow cytometry after LRRN3 overexpression; Figure E shows the cell cycle progression detected by flow cytometry after LRRN3 overexpression; *P<0.05, **P<0.01, ***P<0.001.

[0022] Figure 4The following figures illustrate the inhibition of prostate hyperplasia in rats after overexpression of LRRN3, as provided in this invention: Figure A shows the weight change curves of rats in the Sham+Vector, T-BPH+Vector, and T-BPH+ov-LRRN3 groups; Figure B shows the gross images of prostate tissue in rats in the Sham+Vector, T-BPH+Vector, and T-BPH+ov-LRRN3 groups; Figure C shows the histogram of prostate weight index (prostate weight / body weight (mg / g)) in rats in the Sham+Vector, T-BPH+Vector, and T-BPH+ov-LRRN3 groups; Figure D shows representative images of hematoxylin-eosin (HE) staining (top left), epithelial thickness (top right), representative images of Masson staining (bottom left), and statistical bar charts of smooth muscle and collagen fiber area percentages (bottom right) of prostate tissue in rats in the Sham+Vector, T-BPH+Vector, and T-BPH+ov-LRRN3 groups; *P<0.05, **P<0.01, ***P<0.001.

[0023] 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

[0024] 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, it should be considered that such a combination of technical solutions does not exist and is 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.

[0025] 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.

[0026] Reagents, instruments, and cell sources used in the examples: Table 1 Experimental Reagents and Instruments

[0027] 2. Cell source and culture Human prostatic hyperplasia cells (BPH-1) were purchased from Wuhan Pronosei Biotechnology Co., Ltd., and cultured in 1640 medium containing 10% fetal bovine serum. Human normal prostatic stromal cells (WPMY-1, catalog number: GNHu36) were obtained from the Cell Bank of the Chinese Academy of Sciences and cultured in DMEM medium (Gibco) containing 5% fetal bovine serum.

[0028] Example 1: Detection of LRRN3 expression in the transcriptome of blood leukocytes in healthy individuals and BPH patients This study collected blood samples from 398 individuals, including healthy individuals (N=121) and patients with benign prostatic hyperplasia (BPH) (N=277). Leukocytes were extracted from the collected blood samples, followed by leukocyte RNA extraction and quantification. Differentially expressed genes were screened and analyzed between different comparison groups based on the quantitative results. This study was approved by the Medical Ethics Committee of Zhongnan Hospital, Wuhan University (Ethics No. 2022173).

[0029] Transcriptomic sequencing of blood leukocytes from 398 healthy individuals and BPH patients was performed to analyze LRRN3 expression. The results showed that LRRN3 expression was significantly downregulated in the blood of BPH patients compared to healthy individuals. Figures 1-2 ).

[0030] Example 2 Cell Experiment 1. Cell transfection BPH-1 and WPMY-1 cells were mixed at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and cultured for 24 hours. The next day, a mixture of ov-LRRN3 lentivirus and negative control lentivirus was added to the 6-well plates, and the cells were cultured for at least 48 hours. The overexpression of LRRN3 at the mRNA and protein levels was detected by qRT-PCR and Western blotting. The nucleotide sequence of LRRN3 is shown in SEQ ID NO: 1, and the ov-LRRN3 lentivirus information is shown in Table 2.

[0031] Table 2. Information on LRRN3 overexpression lentiviruses

[0032] 2. Cell proliferation Overexpressed BPH-1 and WPMY-1 cells were cultured at 2 × 10⁶ cells per well. 3 Cells were seeded at a density of 1000 cells / well in 96-well plates and cultured for 0, 1, 2, 3, 4 and 5 days. At different time points, 10 µL of CCK-8 solution was added to each well and the cells were incubated in the dark for 2 hours. The absorbance at 450 nm was measured.

[0033] 3. Cell cycle detection After overexpressing BPH-1 and WPMY-1 cells, the cells were washed twice with PBS, digested with trypsin, and centrifuged. The cell pellet was resuspended in 1 mL of DNA staining solution and 10 µL of permeabilization buffer, and incubated in a 37˚C cell culture incubator in the dark for 30 minutes. Immediately afterwards, the number of cells in G0 / G1, S, and G2 phases was detected by flow cytometry.

[0034] 4. Apoptosis detection After overexpressing BPH-1 and WPMY-1 cells, the cells were washed twice with PBS, digested with trypsin, and centrifuged. The cell pellet obtained after centrifugation was resuspended in 1 mL buffer, and 5 µL Annexin V-FITC and 10 µL propidium iodide staining solution were added and incubated in the dark for 5 minutes. The apoptosis level was immediately detected by flow cytometry.

[0035] Cellular experiment results: LRRN3 overexpression cell models were constructed in BPH-1 and WPMY-1 cells using lentiviruses. The overexpression effect was detected at the mRNA and protein levels using qRT-PCR and Western blotting experiments. Figure 3 A, B).

[0036] LRRN3 overexpression significantly inhibited the proliferation of BPH-1 and WPMY-1 cells. Figure 3 C).

[0037] At the same time, the cell cycle process is significantly blocked ( Figure 3 E). On the other hand, LRRN3 overexpression significantly increased the number of apoptotic cells with statistical significance. Figure 3 D).

[0038] Example 3: Effects of LRRN3 overexpression on rat prostate tissue This study established a rat model of benign prostatic hyperplasia (BPH) using the classic castration plus testosterone propionate method, and verified the model's effectiveness and the effect of LRRN3 gene overexpression on prostate tissue through experimental indicators. The experimental design included a sham-vector group, a lentivirus control group (T-BPH-vector), and an ov-LRRN3 lentivirus injection group (T-BPH-ov-LRRN3). First, rats underwent castration surgery. Postoperatively, prostate hyperplasia was induced by subcutaneous injection of testosterone propionate. The body weight changes of rats in each group were recorded. Figure 4 A) provides basic physiological data for subsequent indicator analysis.

[0039] The results showed that, compared with the lentivirus control group, the prostate volume of rats in the ov-LRRN3 lentivirus injection group was significantly reduced ( Figure 4B); at the same time, the prostate index (prostate weight / body weight (mg / g)) showed a significant statistical difference ( Figure 4 C); H&E staining showed that the prostate epithelial thickness of rats in the lentivirus control group was significantly increased, proving the successful establishment of the BPH model. Meanwhile, the prostate epithelial thickness of rats in the ov-LRRN3 lentivirus injection group was lower than that of rats in the lentivirus control group, indicating that overexpression of LRRN3 can inhibit the progression of prostate tissue hyperplasia. Figure 4 D).

[0040] 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 LRRN3 expression levels in the preparation of diagnostic products for benign prostatic hyperplasia.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the LRRN3 is shown in SEQ ID NO:

1.

3. The application according to claim 1, characterized in that, The expression of LRRN3 is downregulated in patients with benign prostatic hyperplasia.

4. The application according to claim 1, characterized in that, The product is used to detect the expression level of LRRN3 in blood leukocytes.

5. The application according to claim 1, characterized in that, The products include gene chips, formulations, or reagent kits.

6. Application of LRRN3 gene expression vector in the preparation of drug compositions for the treatment of benign prostatic hyperplasia.

7. The application according to claim 6, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

8. Application of reagents that overexpress the LRRN3 gene in the preparation of drugs that inhibit the proliferation of benign prostatic hyperplasia cells.

9. Application of reagents that overexpress the LRRN3 gene in the preparation of drugs that arrest the cell cycle of benign prostatic hyperplasia.

10. Application of reagents that overexpress the LRRN3 gene in the preparation of drugs that promote apoptosis of benign prostatic hyperplasia cells.

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