Use of a substance that inhibits FLNC gene expression in the preparation of a medicament for treating benign prostatic hyperplasia

Abstract

The present invention provides the application of the FLNC gene as a target gene in screening drugs for preventing, alleviating and / or treating benign prostatic hyperplasia. Through research, the present invention discovers that the reduction of FLNC level can reduce the prostate volume of rats and inhibit the hyperplasia of the epithelium and smooth muscle of the prostate tissue of rats. Therefore, the present invention for the first time verifies through human samples, cells and animal experiments that FLNC may be used as a clinical treatment target for BPH and has clinical diagnostic value, which is of great significance for the early diagnosis and treatment of BPH.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to the application of substances that inhibit the expression of the FLNC gene in the preparation of drugs for preventing, alleviating, and / or treating benign prostatic hyperplasia. Background Art

[0002] Benign Prostatic Hyperplasia (BPH) is one of the most common diseases in elderly men. The lower urinary tract symptoms (LUTS) caused by it seriously affect the quality of life of elderly men and impose a heavy economic burden. Currently, the drug treatment methods for BPH mainly include α-adrenergic antagonists, β-adrenergic agonists, 5α-reductase inhibitors, etc. Although there is a certain therapeutic effect in the early stage, with the progress of age and the aggravation of clinical symptoms, a large number of patients still need surgical treatment. Although transurethral resection of the prostate is an effective treatment method, it has relatively high risks and postoperative complications. Although a large number of studies actively explore the pathogenesis of BPH, the specific mechanism has not been clarified. Age and functional testes are still recognized as pathogenic factors. At the same time, as an age-progressive disease, there are also certain difficulties in the diagnosis of benign prostatic hyperplasia. Patients often seek medical treatment due to the appearance of LUTS symptoms. The filamin family includes three homologous genes: filamin A (FLNA), filamin B (FLNB), and filamin C (FLNC). The first two are widespread, while FLNC is specifically expressed in striated muscle. Currently, a large number of studies have shown that FLNC gene mutations are related to various familial cardiomyopathies, but their role in the occurrence and development of benign prostatic hyperplasia has not been reported, and it is not clear whether it can be used as a therapeutic target for benign prostatic hyperplasia.

[0003] Therefore, it is necessary to develop a target for screening drugs for preventing, alleviating, and / or treating benign prostatic hyperplasia and drugs. Summary of the Invention

[0004] The object of the present invention is to provide the application of substances that inhibit the expression of the FLNC gene in the preparation of drugs for preventing, alleviating, and / or treating benign prostatic hyperplasia. Through experiments, the present invention found that compared with healthy people, the expression level of FLNC in the serum of patients diagnosed with BPH was significantly increased (P < 0.01). The reduction of the FLNC level can reduce the prostate volume of rats and inhibit the hyperplasia of the epithelium and smooth muscle in the prostate tissue of rats. Therefore, FLNC can be used as a target for screening drugs for preventing, alleviating, and / or treating benign prostatic hyperplasia.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, there is provided the use of the FLNC gene as a target gene in screening drugs for preventing, alleviating and / or treating benign prostatic hyperplasia, and the screening method includes screening substances capable of inhibiting the expression of the FLNC gene.

[0007] In the second aspect of the present invention, there is provided the use of a substance that inhibits the expression of the FLNC gene in the preparation of a drug for preventing, alleviating and / or treating benign prostatic hyperplasia.

[0008] Further, the substance that inhibits the expression of the FLNC gene includes siRNA for knockdown.

[0009] Further, the sequences of the siRNA are as shown in SEQ ID NO.1-2 or SEQ ID NO.3-4.

[0010] Further, the drug prevents, alleviates and / or treats benign prostatic hyperplasia through at least one of the following effects: reducing the prostate volume and inhibiting the hyperplasia of the epithelium and smooth muscle of the prostate tissue.

[0011] In the third aspect of the present invention, there is provided a drug for preventing, alleviating and / or treating benign prostatic hyperplasia, characterized in that the drug contains a substance that inhibits the expression of the FLNC gene.

[0012] Further, the drug also includes pharmaceutically acceptable excipients.

[0013] Further, the excipient is selected from one of a filler, a disintegrant, a binder, a diluent, a lubricant, a sweetening agent or a coloring agent.

[0014] Further, the dosage form of the drug includes at least one of a granule, a tablet, a pill, a capsule, an injection and a dispersant.

[0015] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0016] The present invention provides the use of a substance that inhibits the expression of the FLNC gene in the preparation of a drug for preventing, alleviating, and / or treating benign prostatic hyperplasia. For the first time, through proteomic sequencing, it is found that compared with normal people, the expression level of FLNC protein is significantly increased in patients with prostatic hyperplasia, and at the same time, as the clinical symptoms worsen, its expression level gradually increases. At the same time, the change in FLNC level can affect the cell cycle process and apoptosis by influencing the expression levels of cycle and apoptosis proteins, thereby affecting the progression of BPH. On the other hand, by constructing a rat model of benign prostatic hyperplasia, it is verified in vivo that the reduction of FLNC level can reduce the prostate volume of rats and inhibit the hyperplasia of epithelial and smooth muscle in the rat prostate tissue. Therefore, for the first time, through human samples, cells, and animal experiments, the present invention verifies that FLNC may be used as a clinical treatment target for BPH and has clinical diagnostic value, which is of great significance for the early diagnosis and treatment of BPH. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0018] Figure 1 It shows the expression of FLNC protein in the sera of healthy and benign prostatic hyperplasia patients. A: Volcano plot of differential proteins detected by serum proteomics in prostatic hyperplasia patients (N = 30) and healthy individuals (N = 20). B: Expression levels of FLNC protein in the sera of prostatic hyperplasia patients and healthy individuals. C: Heat map of serum protein sequencing in benign prostatic hyperplasia patients and healthy individuals. **P < 0.01.

[0019] Figure 2 It shows that the expression level of FLNC protein is correlated with clinical symptoms. A: Immunohistochemical staining of FLNC protein in prostate tissues of mild, moderate, and severe prostatic hyperplasia patients. B: Quantitative results of immunohistochemical staining of FLNC protein. C - F: Statistical analysis of age, prostate volume, and quality of life scores in healthy and BPH patients at different stages. G - J: AUC curves of FLNC, PV, IPSS, and TPSA. **P < 0.01, ***P < 0.001.

[0020] Figure 3Knockdown of FLNC protein inhibits the proliferation and promotes the apoptosis of human primary prostate stromal cells. A: Verification of protein level after FLNC knockdown. B: Verification of mRNA level after FLNC knockdown. C: Detection of cell proliferation curve by CCK-8 assay after FLNC knockdown in cells. D: Detection of cell cycle progression by flow cytometry after FLNC knockdown in cells. E: Detection of apoptotic cell number by flow cytometry after FLNC knockdown in cells. F: Detection of the expression levels of proliferation, apoptosis and cyclin proteins by Western blotting after FLNC knockdown in cells. ** P<0.01, ***P<0.001.

[0021] Figure 4 Knockdown of FLNC can inhibit prostate hyperplasia in rats. A: Flow chart of BPH rat model and lentivirus treatment of FLNC and rat body weight curve. B: Microscopic view of rat prostate tissue samples. C: Prostate index of rats. D: H&E staining of rat prostate tissue and statistical analysis of epithelial components. *P<0.05, *** P<0.001. Specific embodiments

[0022] The present invention will be specifically described below in combination with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0023] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention belongs. In case of conflict, this specification shall prevail.

[0024] Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be obtained through market purchase or by existing methods.

[0025] The general idea of the present invention is as follows:

[0026] The filamin family includes three homologous genes: filamin A (FLNA), filamin B (FLNB) and filamin C (FLNC). The first two are ubiquitous, while FLNC is specifically expressed in striated muscle. At present, a large number of studies have shown that FLNC gene mutations are related to various familial cardiomyopathies, but its role in the occurrence and progression of benign prostatic hyperplasia has not been reported, and it is not clear whether it can be used as a therapeutic target for benign prostatic hyperplasia.

[0027] Through human samples, cell and animal experiments, the present invention first discovers and verifies that FLNC may be used as a clinical treatment target for BPH and has clinical diagnostic value at the same time, which is of great significance for the early diagnosis and treatment of BPH.

[0028] The following will combine examples and experimental data to elaborate on the application of the FLNC gene of the present application in the preparation of drugs for preventing and / or treating benign prostatic hyperplasia.

[0029] Example 1: FLNC can be used as a diagnostic marker for BPH

[0030] 1. Method

[0031] 1.1 Experimental reagents and instruments:

[0032] 1.1.1 Main reagents and consumables

[0033] Table 1

[0034]

[0035] 1.1.2 Main instruments

[0036] Table 2

[0037]

[0038] 1.2 Detection of serum FLNC expression in healthy people and BPH patients

[0039] A total of 50 serum samples from healthy people and BPH patients were collected clinically. The serum samples were subjected to protein extraction, followed by protein enrichment, quality control, peptide separation and High pH RP separation. Finally, DDA (data dependent acquisition, DDA) library construction and DIA (data independent acquisition, DIA) quantitative detection were carried out, and differential protein screening and analysis were performed between different comparison groups based on the quantitative results. This study was approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (Ethical number 2022173).

[0040] 1.3 Detection of FLNC expression in prostate tissues of BPH patients

[0041] Thirteen BPH patients were selected and divided into mild-LUTS (IPSS≤7), moderate-LUTS (8≤IPSS≤19), and severe-LUTS (20≤IPSS≤35) according to the IPSS score. IHC staining of prostate tissues of BPH patients in different groups was performed with FLNC antibody. Specific method:

[0042] 1. Place the paraffin sections in an oven at 60 °C for 1 h to dry the sections.

[0043] 2. Then dewax with 100% xylene three times, 5 min each time, until completely dewaxed.

[0044] 3. Place them successively in gradient alcohols of 100%×2, 95%, and 70% for 5 min each for hydration, and finally wash twice with double-distilled water, 5 min each time.

[0045] 4. Take a certain amount of citrate antigen retrieval working solution in a retrieval box. The amount of the retrieval solution must be sufficient to completely immerse the whole section. Place the retrieval box in a pressure cooker with an appropriate amount of tap water added. Preheat it over high heat until boiling. Place the dewaxed and hydrated tissue sections on a high-temperature-resistant staining rack, and then slowly place the staining rack into the retrieval box. Cover the pot lid and continue heating until it starts to steam. Start timing. After 5 min, turn off the power of the pressure cooker, remove the valve and open the lid, and let it cool naturally at room temperature for 20 - 30 minutes. Then wash twice with double-distilled water, 5 min each time.

[0046] 5. Add 100 μL of 3% hydrogen peroxide solution to each section, place it in a wet box, and incubate in an oven at 37 °C for 20 min. Wash with PBS three times, 5 min each time.

[0047] 6. Add 100 μL of 10% goat serum to each section for blocking, place it in a wet box, and incubate in an oven at 37 °C for 30 min.

[0048] 7. Discard the blocking solution, and add 50 μL of FLNC primary antibody dilution (diluted 1:200) to each section, and incubate overnight at 4 °C.

[0049] 8. Discard the primary antibody, and wash with PBS three times, 5 min each time.

[0050] 9. Add 100 μL of the corresponding secondary antibody dilution (1:1000) to each section, and incubate at 37 °C for 30 min.

[0051] 10. Discard the secondary antibody, and wash with PBS three times, 5 min each time.

[0052] 11. Add 100 μL of freshly prepared DAB chromogenic solution to each section. Immediately rinse with running water when the section shows brownish-yellow to stop the reaction.

[0053] 12. Subsequently, counterstain with hematoxylin for 1 min, differentiate with 1% hydrochloric acid alcohol for 2 - 3 s, and then immediately rinse with tap water to turn blue.

[0054] 13. Place them successively in gradient alcohols of 70%, 95%, and 100% for 3 min for dehydration, put them in xylene solution twice, 5 min each time for clearing, and after air-drying, add neutral balsam to cover the sections.

[0055] 14. The scanning microscope was used to detect the staining intensity of FLNC.

[0056] 1.4 Receiver Operating Characteristic (ROC) curve plotting for serum FLNC expression levels, prostate volume (PV), and international prostate symptom score (IPSS) in healthy individuals and BPH patients: ROC curves were plotted based on the serum FLNC expression levels, PV, and IPSS of healthy individuals and BPH patients, respectively. The cut-off value was determined using Graphpad Prism 8.0 software. The sensitivity calculation formula is: TP / (TP+FN)×100%, where TP is the true positive in BPH patients and FN is the false negative in BPH patients; the specificity calculation formula is: TN / (TN+FP)×100%, where TN is the true negative in healthy controls and FP is the false positive in healthy controls.

[0057] 1.5 Cell source

[0058] Human primary prostate stromal cells were extracted from the prostate tissues of patients with benign prostatic hyperplasia. After weighing and washing the tissues, they were cut into small pieces, and then added to the culture medium and collagenase and digested thoroughly on a thermostatic shaker. The supernatant was discarded and the cells were washed three times with the culture medium. Then the cell pellet was placed in a culture flask and cultured with an appropriate amount of culture medium.

[0059] 1.6 Cell transfection

[0060] Two different sequences of small interfering RNAs and the negative control sequence RNA were diluted in Opti-MEM medium and mixed with Lipofectamine 3000. The mixture was added to a six-well plate, and the cells were inoculated and cultured for at least 48 hours. The interference effects of FLNC at the mRNA and protein levels were detected by qRT-PCR and WB.

[0061] 1.7 CCK8 assay:

[0062] The transfected prostate stromal cells were seeded at a density of 2×10 3 into 96-well plates and cultured for 0, 1, 2, and 3 days. At different time points, 10 µL of CCK8 solution was added to each well, and the cells were incubated in the dark for 2 hours. The absorbance was measured at 450 nm.

[0063] 1.8 Cell cycle detection:

[0064] The interfered prostate stromal cells were washed twice with PBS and then digested and centrifuged with trypsin. The cell pellet after centrifugation was resuspended with 1 mL of DNA staining solution and 10 μL of permeabilization solution, incubated in the dark at 37˚C in a cell culture incubator for 30 minutes, and immediately the number of cells in G0 / G1, S phase and G2 phase was detected by flow cytometry.

[0065] 1.9 Detection of apoptosis:

[0066] The interfered prostate stromal cells were washed twice with PBS and then digested and centrifuged with trypsin. The cell pellet was resuspended with 1 mL of buffer and 5 μL of Annexin V-FITC and 10 μL of propidium iodide staining solution were added, incubated in the dark for 5 minutes, and immediately the apoptosis level of the cells was detected by flow cytometry.

[0067] 2. Results

[0068] 2.1 Increased expression level of FLNC protein in the serum of BPH patients

[0069] We analyzed the expression of FLNC protein in the sera of 50 normal and benign prostatic hyperplasia patients by proteomic sequencing. The results showed that the expression level of FLNC in the sera of patients diagnosed with BPH was significantly increased compared with that of healthy people (P<0.01) ( Figure 1 A-C).

[0070] 2.2 Correlation between the expression level of FLNC protein and clinical symptoms

[0071] We divided the patients into mild-LUTS (IPSS≤7), moderate-LUTS (8≤IPSS≤19), and severe-LUTS (20≤IPSS≤35) according to the IPSS score, and found that the expression level of FLNC in the prostate tissue increased with the increase of the severity of the included patients ( Figure 2 A, B). At the same time, we performed statistical analysis on the age, prostate volume, quality of life score and IPSS score of the included patients according to the different grades of benign prostatic hyperplasia, and found that with the aggravation of the clinical stage, the above clinical indicators showed an upward trend ( Figure 2 C-F).

[0072] 2.3 Receiver operating characteristic (ROC) curves were plotted based on the expression level of FLNC protein, the prostate volume (PV) of the patients, and the International Prostate Symptom Score (IPSS) in the serum proteomic sequencing of the above 50 normal and BPH patients to evaluate the sensitivity and specificity of FLNC as a diagnostic marker for differentiating healthy and BPH patients. The results are shown in Table 3 and Figure 2 G.

[0073] Table 3. Statistical analysis of the receiver operating characteristic (ROC) curve and markers in BPH subjects

[0074]

[0075] From Figure 2 G and the results in Table 3, it can be seen that the AUC of FLNC is 0.719, the sensitivity is 87.0%, and the specificity is 89.5%.

[0076] Example 2. FLNC can be used as a clinical treatment target for BPH

[0077] 1. After knocking down FLNC, the proliferation of human primary prostate stromal cells can be inhibited, and apoptosis can be promoted at the same time

[0078] We constructed a FLNC knockdown cell model in primary prostate stromal cells using small interfering RNA (the sequences are shown in Table 4).

[0079] Table 4

[0080]

[0081] The steps for establishing a benign prostatic hyperplasia animal model and FLNC intervention treatment are as follows

[0082] In the sham operation group, after castration for 1 week, normal saline was continuously injected for 14 days. In the lentivirus control group and the FLNC lentivirus treatment group, after castration for 1 week, testosterone propionate was continuously injected for 28 days, and at 14 days after the injection of testosterone propionate, lentivirus blank control and FLNC lentivirus were respectively injected into the ventral lobe of the prostate. After the animal modeling was completed, the three groups of rats were sacrificed simultaneously by the method of overdose anesthesia injection, and the prostate tissues were extracted, photographed, weighed, and then stored in formaldehyde fixative for subsequent detection. The rats need to be weighed daily and their activity status observed.

[0083] Then, qRT-PCR (the primers used for detection are shown in Table 5) and WB experiments were used to detect the interference effect at the mRNA and protein levels ( Figure 3 A, B).

[0084] Table 5. Primers used for qRT-PCR

[0085]

[0086] After knocking down FLNC, the proliferation level of primary prostate stromal cells was significantly inhibited ( Figure 3 C). At the same time, the cell cycle process was significantly blocked ( Figure 3 D). On the other hand, after knocking down FLNC, the number of apoptotic cells increased significantly and showed statistical differences ( Figure 3 E).

[0087] Secondly, we used Western blotting to detect the cell proliferation, apoptosis, and cycle-related proteins Bax, Bcl-2, CDK4, and CyclinD1, and found that after FLNC knockdown, the expression levels of CyclinD1, CDK4, and Bax increased, while the expression level of Bcl-2 decreased ( Figure 3 F).

[0088] 2. FLNC knockdown can inhibit rat prostate hyperplasia

[0089] We used the classic castration + testosterone propionate method to establish a rat prostate hyperplasia model and recorded the body weight curve at the same time ( Figure 4 A). We found that compared with the rats in the sham operation group, the prostate volume of the rats in the lentivirus blank control group increased significantly, while the prostate volume of the rats in the FLNC lentivirus injection group decreased significantly ( Figure 4 B), and there were significant statistical differences in the prostate index (prostate weight / body weight) ( Figure 4 C). H&E staining showed that the prostate epithelial thickness of the rats in the lentivirus blank control group increased significantly, proving that the BPH model was successfully established. At the same time, the prostate epithelial thickness of the rats in the FLNC lentivirus injection group decreased compared with that of the rats in the sham operation group, indicating that reducing the FLNC level can delay the progression of prostate tissue hyperplasia ( Figure 4 D).

[0090] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device.

[0091] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concepts. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0092] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Use of a substance that inhibits the expression of the FLNC gene in the preparation of a drug for preventing, alleviating, and / or treating benign prostatic hyperplasia, wherein the substance that inhibits the expression of the FLNC gene includes siRNA for knockdown, and the sequences of the siRNA are shown as SEQ ID NO.1-4.

2. The application according to claim 1, wherein The drug prevents, alleviates, and / or treats benign prostatic hyperplasia through at least one of the following effects: reducing the prostate volume and inhibiting the hyperplasia of the epithelium and smooth muscle of the prostate tissue.

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