Use of reagent for detecting expression level of LRRN3 in preparation of diagnostic product for benign prostatic hyperplasia

Abstract

The application discloses application of a reagent for detecting LRRN3 expression level in preparation of a benign prostatic hyperplasia diagnosis product. It is proved through cell function experiments and animal experiments that overexpression of the LRRN3 gene can inhibit the proliferation of prostate cells, block the cell cycle and promote cell apoptosis, thereby effectively inhibiting the prostatic hyperplasia. The discovery provides a new target and treatment strategy for the treatment of the prostatic hyperplasia, and provides a brand-new thought and method for the treatment of the prostatic hyperplasia. Based on the research results, a gene therapy drug taking the LRRN3 gene as a target can be further developed, for example, a lentivirus vector carrying the LRRN3 gene or other gene delivery systems are constructed and used for the treatment of the prostatic hyperplasia. In addition, the LRRN3 gene can be combined with other treatment methods to improve the treatment effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical diagnosis and treatment technology, and particularly relates to application of a reagent for detecting LRRN3 expression level in preparation of a benign prostatic hyperplasia diagnosis product. BACKGROUND

[0002] Benign prostatic hyperplasia (BPH) is a common disease in middle-aged and elderly men, which usually starts at the age of 40 and the incidence gradually increases with age. The prevalence of BPH in men over 50 years old is more than 50%, and the prevalence of BPH in men over 70 years old is more than 80%. At present, the diagnosis of BPH mainly depends on the comprehensive judgment of clinical symptoms (such as frequent urination, urinary urgency, dysuria, etc.), international prostate symptom score (IPSS), rectal examination, prostate ultrasonography and serum prostate specific antigen (PSA) detection, etc. However, the existing diagnostic methods still have certain limitations in early identification of disease progression risk and differentiation of symptomatic BPH from other prostate diseases (such as prostate cancer), which is difficult to accurately guide individualized treatment.

[0003] In terms of treatment, since androgens play a key role in the occurrence and progression of BPH, 5α-reductase inhibitors targeting the androgen pathway have become the mainstream drugs for clinical treatment. Although these drugs can alleviate symptoms, they have limitations such as poor long-term medication compliance, side effects (such as dizziness, sexual dysfunction) and inability to reverse prostate volume enlargement. Clinical practice shows that about 10% of patients still have disease progression after receiving such drug treatment, and some patients have drug resistance or adverse reactions (such as sexual dysfunction, breast pain, etc.). The existing treatment methods (including drugs and surgery) cannot completely prevent the natural progression of BPH, especially for patients with moderate to severe symptoms or complications, the treatment effect is still not ideal, so it is urgent to explore new treatment targets and develop more targeted and effective treatment programs to improve patient prognosis and reduce disease burden. SUMMARY

[0004] The main purpose of the present application is to provide a new treatment target for patients by providing a reagent for detecting LRRN3 expression level in the preparation of a benign prostatic hyperplasia diagnosis product.

[0005] To achieve the above-mentioned purpose, the present application provides a reagent for detecting LRRN3 expression level in the preparation of a benign prostatic hyperplasia diagnosis product.

[0006] LRRN3 gene belongs to the leucine-rich repeat protein family, and 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 LRRN3 is shown as SEQ ID NO: 1.

[0008] Preferably, the LRRN3 is down-regulated in benign prostatic hyperplasia patients.

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

[0010] Preferably, the product comprises a gene chip, a preparation or a kit.

[0011] The present application also provides an expression vector of LRRN3 gene for use in the preparation of a pharmaceutical composition for treating benign prostatic hyperplasia.

[0012] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0013] The present application also provides a reagent for over-expressing LRRN3 gene for use in the preparation of a medicament for inhibiting the proliferation of benign prostatic hyperplasia cells.

[0014] The present application also provides a reagent for over-expressing LRRN3 gene for use in the preparation of a medicament for blocking the cell cycle of benign prostatic hyperplasia cells.

[0015] The present application also provides a reagent for over-expressing LRRN3 gene for use in the preparation of a medicament for promoting the apoptosis of benign prostatic hyperplasia cells.

[0016] Compared with the prior art, the present application has the following advantages:

[0017] (1) The present application first discovers that the expression amount of LRRN3 gene is down-regulated in patients with benign prostatic hyperplasia. The reagent can be used as a new type of biomarker detection tool to realize early screening and diagnosis of the disease, improve the specificity and sensitivity of diagnosis, and help to distinguish benign prostatic hyperplasia from other prostate diseases (such as prostate cancer), thereby avoiding misdiagnosis or missed diagnosis. At the same time, by dynamically monitoring the expression level of LRRN3, the degree of disease progression and prognosis can be evaluated, and precise molecular basis can be provided for formulating individualized treatment plan in clinic, thereby making up for the shortcomings of existing diagnostic methods in early identification and disease assessment, and promoting the precise transformation of benign prostatic hyperplasia diagnosis from symptom-oriented to molecular marker-oriented.

[0018] (2) The present application is confirmed by cell function experiment and animal experiment, overexpression of LRRN3 gene can inhibit the proliferation of prostate cells, block the cell cycle, promote the apoptosis, thereby effectively inhibiting the prostate hyperplasia. This finding provides a new target and treatment strategy for the treatment of prostate hyperplasia, and provides a new idea and method for the treatment of prostate hyperplasia. Based on the research results, the gene therapy drugs taking LRRN3 gene as a target can be further developed, for example, the lentiviral vector carrying LRRN3 gene or other gene delivery system is constructed, which is used for the treatment of prostate hyperplasia. In addition, LRRN3 gene can be combined with other treatment methods to improve the treatment effect. Future research can further optimize the gene delivery system, improve the transfection efficiency, reduce the immunogenicity, and lay a foundation for clinical transformation. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description only some embodiments of the present application, and for those skilled in the art, other related drawings can be obtained without creative labor.

[0020] Figure 1 The volcano plot analysis of the differential genes of the leukocyte transcriptome of the BPH patients (N=277) and the healthy population (N=121) provided by the present application; ***P<0.001.

[0021] Figure 2 The box plot of the expression amount of LRRN3 gene in the blood of the BPH patients and the healthy population provided by the present application; ***P<0.001.

[0022] Figure 3 The graph of inhibiting BPH-1 and WPMY-1 cell proliferation and promoting apoptosis after overexpression of LRRN3 provided by the present application; Figure A is the verification graph of BPH-1 (left) and WPMY-1 cell (right) mRNA level after overexpression of LRRN3; Figure B is the verification graph of BPH-1 and WPMY-1 cell protein level after overexpression of LRRN3; Figure C is the cell proliferation curve graph of BPH-1 (left) and WPMY-1 cell (right) after overexpression of LRRN3 detected by CCK-8 analysis; Figure D is the cell apoptosis number graph detected by flow cytometry after overexpression of LRRN3; Figure E is the cell cycle progress graph detected by flow cytometry after overexpression of LRRN3; *P<0.05, **P<0.01, ***P<0.001.

[0023] Figure 4Figure A is a graph of the body weight change of rats in the Sham+Vector group, the T-BPH+Vector group and the T-BPH+ov-LRRN3 group; Figure B is a picture of the general observation of the prostate tissue of rats in the Sham+Vector group, the T-BPH+Vector group and the T-BPH+ov-LRRN3 group; Figure C is a histogram of the prostate weight index (prostate weight / body weight (mg / g)) of rats in the Sham+Vector group, the T-BPH+Vector group and the T-BPH+ov-LRRN3 group; Figure D is a representative picture of hematoxylin-eosin (HE) staining of the prostate tissue of rats in the Sham+Vector group, the T-BPH+Vector group and the T-BPH+ov-LRRN3 group (top left), a column chart of epithelial thickness (top right), a representative picture of Masson staining (bottom left) and a column chart of the percentage of smooth muscle and collagen fiber area (bottom right) of rats in the Sham+Vector group, the T-BPH+Vector group and the T-BPH+ov-LRRN3 group; *P<0.05, **P<0.01, ***P<0.001.

[0024] The implementation, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0025] In order to make the objectives, 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 by the ordinary skilled in the art. Based on the embodiments in the present application, all other embodiments obtained by the ordinary skilled in the art without creative labor are within the protection scope of the present application.

[0026] The technical solutions of the present application will be further described in detail below in combination with specific embodiments and the accompanying drawings. It should be understood that the following embodiments are only used to explain the present application and do not limit the present application.

[0027] The reagents, instruments and cell sources used in the embodiments are as follows:

[0028] Table 1 Experimental reagents and instruments

[0029]

[0030] 2、Cell source and culture

[0031] Human prostate hyperplasia cells (BPH-1) were purchased from Wuhan Pons Life Science Co., Ltd., and were cultured in 1640 medium containing 10% fetal bovine serum. Human prostate normal interstitial cells (WPMY-1, catalog number: GNHu36) were from the Chinese Academy of Sciences Cell Bank, and were cultured in DMEM medium (Gibco) containing 5% fetal bovine serum.

[0032] Example 1 Detection of LRRN3 expression in blood leukocytes of healthy people and BPH patients

[0033] A total of 398 blood samples were collected in this study, covering healthy people (N = 121) and prostate hyperplasia patients (N = 277). The collected blood samples were subjected to leukocyte extraction, followed by leukocyte RNA extraction, quantitative detection, and differential gene screening and analysis between different comparison groups based on the quantitative results. This study was approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (Ethics Number 2022173).

[0034] The expression of LRRN3 was analyzed by transcriptome sequencing of the blood leukocytes of 398 normal people and BPH patients. The results showed that the expression of LRRN3 in the blood of BPH patients was significantly down-regulated compared with healthy people (P < 0.05). Figures 1-2 )。

[0035] Example 2 Cell experiment

[0036] 1. Cell transfection

[0037] BPH-1 and WPMY-1 cells were seeded in a 6-well plate at a density of 2 x 10 5 cells per well, cultured for 24 h, and the next day, a mixture of ov-LRRN3 lentivirus and negative control lentivirus was added to the six-well plate, and the cells were cultured for at least 48 hours. The overexpression effect of LRRN3 at the mRNA and protein levels was detected by qRT-PCR and WB. The nucleotide sequence of LRRN3 is shown in SEQ ID NO: 1, and the ov-LRRN3 lentivirus information is shown in Table 2.

[0038] Table 2 LRRN3 overexpression lentivirus information

[0039]

[0040] 2. Cell proliferation

[0041] The overexpressed BPH-1 and WPMY-1 cells were seeded in a 6-well plate at a density of 2 x 10 3The density of individual cells was seeded into a 96-well plate for culture for 0, 1, 2, 3, 4, 5 days, at different time points, 10 μL CCK-8 solution was added to each well, and the cells were incubated in the dark for 2 hours, and the absorbance at 450 nm was measured.

[0042] 3. Cell cycle detection

[0043] After overexpression of BPH-1 and WPMY-1 cells, the cells were washed twice with PBS, then digested and centrifuged with trypsin. The cell pellet obtained after centrifugation was resuspended with 1 mL DNA staining solution and 10 μL permeabilization solution, and immediately detected for the number of G0 / G1, S and G2 cells by flow cytometry after incubation in a 37°C cell incubator in the dark for 30 minutes.

[0044] 4. Apoptosis detection

[0045] After overexpression of BPH-1 and WPMY-1 cells, the cells were washed twice with PBS, then digested and centrifuged with trypsin. The cell pellet obtained after centrifugation was resuspended with 1 mL buffer and 5 μL Annexin V-FITC and 10 μL propidium iodide staining solution, and immediately detected for the apoptosis level by flow cytometry after incubation in the dark for 5 minutes.

[0046] Cell experiment results:

[0047] LRRN3 overexpression cell models were constructed in BPH-1 and WPMY-1 cells using lentivirus, and the overexpression effect was detected at the mRNA and protein levels by qRT-PCR and WB experiments Figure 3 A, B).

[0048] After overexpression of LRRN3, the proliferation level of BPH-1 and WPMY-1 cells was significantly inhibited Figure 3 C).

[0049] At the same time, the cell cycle process was significantly blocked Figure 3 E). On the other hand, after overexpression of LRRN3, the number of apoptotic cells increased significantly and had a statistical difference Figure 3 D).

[0050] Example 3 Effect of LRRN3 overexpression on rat prostate tissue

[0051] 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.

[0052] 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 4 B); 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).

[0053] 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.

Images