Application of prostate hyperplasia related markers in preparation of products for detecting prostate hyperplasia

Abstract

The application discloses an application of a prostate hyperplasia related marker in preparation of a product for detecting prostate hyperplasia, and the prostate hyperplasia related marker is at least one selected from MINP1, ACTBL and APOC4 proteins. In the technical scheme, MINP1, ACTBL and APOC4 are combined as a detection marker for prostate hyperplasia (BPH), so that the complementarity of the three in different stages of diseases can be fully exerted, and the comprehensive performance of the detection product is obviously improved. MINP1 and ACTBL participate in disease progression by regulating prostate cell proliferation and apoptosis, and APOC4 can present significant expression changes in blood cells of early BPH patients, so that BPH disease screening can be accurately performed. When the three are applied in cooperation, early screening of BPH can be realized, and the activity and severity of the lesion can be evaluated, so that a detection system covering the whole process of disease occurrence and development is formed, the limitation of a single marker in the diagnosis dimension is effectively made up, and the comprehensiveness and accuracy of BPH detection are greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical detection, and particularly relates to application of a prostate hyperplasia related marker in preparation of a product for detecting prostate hyperplasia. BACKGROUND

[0002] Benign prostatic hyperplasia (BPH) is a high-incidence urological disease in middle-aged and elderly men, and its incidence increases significantly with age. In China, the number of patients over 50 years old has exceeded 130 million. The disease is mainly manifested as lower urinary tract symptoms, which seriously affects the quality of life of patients and their families. With the expansion of the patient population, the social burden of the disease continues to increase. Early diagnosis is the key to improving the prognosis of BPH patients, but the current clinical understanding of the disease is still limited, and traditional diagnostic methods cannot achieve precise early intervention.

[0003] Current clinical diagnosis of BPH relies too much on traditional methods such as digital rectal examination and ultrasonography. These methods have inherent defects such as low sensitivity and poor specificity, making it difficult to capture disease signals in the early stages of the disease, resulting in a large number of patients missing the best treatment opportunity due to missed diagnosis or misdiagnosis. At the same time, existing diagnosis and treatment decisions are mainly based on objective prostate volume measurement and subjective symptom assessment, and existing evidence shows that there is not a simple linear relationship between prostate volume and lower urinary tract symptoms (LUTS). Diagnosis and treatment programs are easily influenced by individual experience of physicians, and lack of objective and unified standards. In particular, in the primary care setting, the lack of precise diagnostic capabilities is even more pronounced. These limitations highlight the urgent need for objective and accurate new detection targets to fill the gaps in the existing diagnostic system.

[0004] Therefore, finding specific markers for disease detection is of great practical significance for improving the efficiency of BPH diagnosis and treatment and reducing the burden of the disease. SUMMARY

[0005] The main purpose of the present application is to provide application of a prostate hyperplasia related marker in preparation of a product for detecting prostate hyperplasia, aiming at a new type of benign prostate diagnostic marker, which can perform early diagnosis and severity assessment of BPH through objective index detection, improve diagnostic accuracy, and provide strong support for early intervention and treatment of BPH.

[0006] To achieve the above purpose, the present application provides application of a reagent for detecting a prostate hyperplasia related marker in preparation of a product for detecting prostate hyperplasia, wherein the prostate hyperplasia related marker is at least one selected from MINP1, ACTBL and APOC4 proteins.

[0007] The expression of MINP1, ACTBL and APOC4 proteins is significantly different in patients with benign prostatic hyperplasia, and the difference is consistent with the progression of the disease, and the expression is consistent with the severity of the symptoms, but the mechanism of action in BPH is completely unknown.

[0008] Preferably, the prostate hyperplasia-related marker is MINP1, ACTBL and APOC4 protein.

[0009] Preferably, the MINP1 protein is encoded by the MINPP1 gene, and the overexpression interfering sequence of the MINPP1 gene is shown as SEQ ID NO: 1.

[0010] The ACTBL protein is encoded by the ACTBL2 gene, and the interfering sequence of the ACTBL2 includes: (a) a sense strand as shown in SEQ ID NO: 2 and a complementary antisense strand as shown in SEQ ID NO: 3; or (b) a sense strand as shown in SEQ ID NO: 4 and a complementary antisense strand as shown in SEQ ID NO: 5.

[0011] Preferably, the product includes a gene chip, a preparation or a kit.

[0012] Preferably, the gene chip, the preparation or the kit includes specific primers for MINP1 and / or ACTBL; the specific primer pair for MINP1 includes an upstream primer as shown in SEQ ID NO: 6 and a downstream primer as shown in SEQ ID NO: 7; and the specific primer pair for ACTBL includes an upstream primer as shown in SEQ ID NO: 8 and a downstream primer as shown in SEQ ID NO: 9.

[0013] Preferably, the kit further includes reverse transcriptase, buffer, dNTPs, MgCl2, DEPC water, fluorescent probe, RNase inhibitor and Taq enzyme.

[0014] Preferably, the MINP1 and APOC4 are both down-regulated in patients with benign prostatic hyperplasia, and the ACTBL is up-regulated in patients with benign prostatic hyperplasia.

[0015] The application also provides a MINP1 agonist and / or an ACTBL inhibitor for use in the preparation of a medicament for treating benign prostatic hyperplasia.

[0016] Preferably, the MINP1 agonist and / or the ACTBL inhibitor can inhibit the proliferation of prostate cells or promote the apoptosis of prostate cells.

[0017] Preferably, the MINP1 agonist and / or ACTBL inhibitor is capable of reducing prostate volume, decreasing prostate index and / or reducing the degree of prostate tissue hyperplasia in an animal model.

[0018] The beneficial effects of the present application over the prior art are that:

[0019] (1) The technical solution provided by the present application combines MINP1, ACTBL and APOC4 as detection markers for benign prostatic hyperplasia (BPH), which can fully exert the complementarity of the three in different stages of the disease and significantly improve the comprehensive performance of the detection product. Among them, MINP1 and ACTBL participate in the progression of the disease by regulating prostate cell proliferation and apoptosis, and APOC4 can present significant expression changes in the blood cells of early BPH patients, accurately reflecting the severity and progression trend of the disease. When the three are used together, early screening of BPH can be achieved, and the activity and severity of the lesion can be evaluated, forming a detection system covering the entire process of disease occurrence and development, effectively making up for the limitations of single markers in the diagnosis dimension, and greatly improving the comprehensiveness and accuracy of BPH detection.

[0020] (2) The present application is based on the combined use of MINP1, ACTBL and APOC4, among which MINP1 and ACTBL have been verified by cell and animal experiments to be involved in the pathological process of BPH: MINP1 regulates cell proliferation and apoptosis, and ACTBL promotes disease development; APOC4 has been proved by proteomics sequencing technology to play an important role in early screening of the disease. Based on these characteristics, related detection products can not only diagnose the disease through the expression level of the markers, but also provide objective basis for disease severity grading and treatment target selection (such as intervention targeting MINP1 and ACTBL), helping to shift from "empirical diagnosis and treatment" to "precision management" in clinical practice. This multi-dimensional application value makes it irreplaceable in improving the efficiency of BPH diagnosis and treatment and improving patient prognosis, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. 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.

[0022] Figure 1Figure for expression of MINP1, ACTBL and APOC4 of the application; Figure A is a diagram of expression of MINP1, ACTBL and APOC4 protein in serum of BPH patients (N=193) and healthy population (N=86); Figure B is a diagram of expression of MINP1, ACTBL and APOC4 in subjects of BPH population requiring conservative treatment (mild-BPH, N=62), BPH patients requiring drug or surgical intervention (moderate-severe-BPH, N=131); *p<0.05, **p<0.01, ***P<0.001.

[0023] Figure 2 Figure for proliferation of BPH-1 and WPMY-1 cells after overexpression of MINPP1 gene (MINP1 protein) of the application; Figure A is a diagram of detection of overexpression effect at mRNA and protein levels by qRT-PCR and WB experiment; Figure B is a diagram of cell proliferation curve detected by CCK-8 analysis after overexpression of MINPP1 in cells; **P<0.05, ***P<0.001.

[0024] Figure 3 Figure for functional influence of interference of ACTBL2 gene (ACTBL protein) on prostate cells of the application; Figure A is a diagram of detection of interference efficiency of ACTBL2 by PCR and WB; Figure B is a diagram of determination of proliferation ability of WPMY-1 and BPH-1 cells before and after interference of ACTBL2 by CCK8 method; Figure C is a diagram of detection of contraction ability of WPMY-1 cells before and after interference of ACTBL2 by gel contraction experiment; Figure D is a diagram of detection of apoptosis of prostate cells before and after interference of ACTBL2 by flow cytometry; Figure E is a diagram of detection of cycle of prostate cells before and after interference of ACTBL2 by flow cytometry; *P<0.05, **P<0.01, ***P<0.001.

[0025] Figure 4 Figure for inhibition of rat prostate hyperplasia after overexpression of MINPP1 of the application; Figure A is a diagram of observation under microscope of rat prostate; Figure B is a diagram of body weight curve of rats and prostate index of rats; Figure C is a diagram of H&E staining of rat prostate tissue and statistical analysis of epithelial components; Figure D is a diagram of masson staining of rat prostate tissue and statistical analysis of collagen components; *P<0.05.

[0026] Figure 5Figure A is a picture of the general observation of the prostate tissue of the rats in the Control+shNC, Control+shACTBL2, E / T-BPH+shNC and E / T-BPH+shACTBL2 groups; Figure B is a histogram of the body weight and prostate weight index (prostate weight (mg) / body weight (g)) of the rats in the four groups; Figures C and D are the HE (C) and masson (D) staining results and statistical analysis columnar chart of the rats in the four groups, respectively, ns is not significant, *p<0.05, **p<0.01.

[0027] Figure 6 Figure is a ROC curve diagram for verifying the ability of ACTBL and APOC4 to distinguish between BPH patients and healthy people in the verification set of the present application.

[0028] Figure 7 Figure is a ROC curve diagram for verifying the ability of ACTBL to distinguish between BPH patients of different severity in the verification set of the present application.

[0029] Figure 8 Figure is a ROC curve diagram for verifying the ability of MINP1 to distinguish between BPH patients of different severity in the verification set of the present application.

[0030] Figure 9 Figure is a ROC curve diagram for verifying the ability of ACTBL and MINP1 to distinguish between BPH patients of different severity in the verification set of the present application.

[0031] The purposes, functional features and advantages of the present application will be further described with reference to the accompanying drawings. DETAILED DESCRIPTION

[0032] 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 described clearly and completely below. The specific conditions not noted in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not noted by the manufacturers are all conventional products that can be obtained by market purchase. In addition, the technical solutions in each embodiment can be combined with each other, but it must be based on the premise that the technical solutions can be realized by the ordinary skilled in the art, and when the combination of the technical solutions appears contradictory or unachievable, it should be considered that the combination of the technical solutions does not exist and is not within the protection scope required by the present application. Based on the embodiments in the present application, all other embodiments obtained by the ordinary skilled in the art without making creative efforts are within the protection scope of the present application.

[0033] The technical solutions of the present application are 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 do not limit the present application.

[0034] Reagents / consumables, instruments and cell sources used in the embodiments:

[0035] Table 1 Major reagents and consumables

[0036]

[0037] Table 2 Major instruments

[0038]

[0039] Cell sources and culture

[0040] Human prostate hyperplasia cells (BPH-1) were purchased from Wuhan Punsai Life Science and Technology Co., Ltd. and were cultured in 1640 culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin;

[0041] Human normal prostate stroma immortalized cells WPMY-1 and RWPE-1 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Cell WPMY-1 was cultured in DMEM culture medium (Gibco) containing 5% fetal bovine serum, and RWPE-1 cells were cultured in Prostate Epithelial Cells Medium culture medium containing 1% penicillin-streptomycin.

[0042] Interference sequence

[0043] ov-MINPP1 overexpression interference sequence (SEQ ID NO: 1) is:

[0044]

[0045] Table 3 Interference sequences of ACTBL2 are as follows:

[0046]

[0047] Table 4 Primers for qRT-PCR

[0048]

[0049] Example 1 Detection of blood protein group MINP1, ACTBL and APOC4 expression in healthy people and BPH patients

[0050] To explore the key biomarkers of benign prostatic hyperplasia (BPH), 279 training set subjects and 148 validation set subjects were systematically collected. Among the 279 training set subjects, there were 86 healthy controls, 62 BPH patients requiring conservative treatment (mild-BPH), and 131 BPH patients requiring drug or surgical intervention (moderate-severe-BPH). Among the 148 validation set subjects, there were 46 healthy controls, 33 BPH patients requiring conservative treatment (mild-BPH), and 69 BPH patients requiring drug or surgical intervention (moderate-severe-BPH). This study was approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (Ethics Number 2022173). Proteinomic sequencing technology was used for deep analysis of the samples, and the results showed that in the training set, the proteins MINP1 and APOC4 were significantly reduced in the serum of BPH patients, and the expression decreased with the progression of BPH, and the ACTBL protein was significantly increased in the serum of BPH patients, and showed an upward trend with the severity of the disease (Figures 1A-1B). Figure 1 The above results show that MINP1, ACTBL and APOC4 can be used as potential biomarkers for evaluating the occurrence, development and progression of BPH, and provide a new research direction for the clinical diagnosis and disease monitoring of BPH.

[0051] Example 2 Cell experiment

[0052] 1. Cell transfection

[0053] (1) BPH-1 and WPMY-1 cells were seeded in 6-well plates at a density of 70-80% confluence, and cultured for 24 hours. Fresh medium was replaced, and MINPP1 overexpression lentivirus was mixed with 40 µL transfection reagent per well at a MOI of 10 to prepare transfection complexes. The complexes were added to the 6-well plates, and the plates were gently shaken to ensure uniform distribution of the complexes. The cells were incubated in a 37°C incubator for 6-8 hours, and then replaced with complete medium. After 48 hours of transfection, puromycin was added for cell selection. When the control group (untransfected cells) died completely, the surviving cells were collected, which were MINPP1 overexpression cell lines for subsequent experiments.

[0054] (2) BPH-1 and WPMY-1 cells were seeded in 6-well plates at a density of 2x10 5 per well and cultured for 24 hours. The next day, si-ACTBL2 and control si-NC were mixed with 40 µL transfection reagent and added to the 6-well plates. The cells were cultured for at least 72 hours. qRT-PCR and WB were used to detect the knockdown effect of ACTBL2 at the mRNA and protein levels.

[0055] 2. Cell proliferation

[0056] The above stable ov-MINPP1 overexpression or si-ACTBL2 transfected prostate cells were digested with trypsin, centrifuged, and resuspended. After cell counting, an appropriate amount of cell suspension was taken at 2000 cells per well, mixed thoroughly, and inoculated into a 96-well plate. The old culture medium was removed at 8 hours, 1 day, 2 days, 3 days, 4 days, and 5 days, and 100 µL of fresh culture medium and 10 µL of CCK8 detection reagent were added. After 2 hours of incubation in the incubator, the absorbance at 450 nm was detected using a microplate reader for cell proliferation calculation.

[0057] 3. Cell cycle detection

[0058] The above si-ACTBL2 transfected BPH-1 and WPMY-1 cells were washed twice with PBS, digested with trypsin, and then centrifuged. 1 mL of DNA staining solution and 10 µL of permeabilization solution were added to the cell pellet, and the cells were resuspended. The cell suspension was placed in a 37°C cell incubator and incubated in the dark for 30 minutes. After incubation, the number of G0 / G1, S, and G2 cells was immediately detected by flow cytometry.

[0059] 4. Apoptosis detection

[0060] The prostate cells stably overexpressing ov-MINPP1 or transfected with si-ACTBL2 were washed twice with PBS, and then trypsinized and centrifuged. The cell pellet was resuspended in 1 mL buffer, and stained with 5 μL Annexin V-APC and 10 μL propidium iodide in the dark for 5 min. The level of apoptosis was immediately detected by flow cytometry.

[0061] The results showed that MINPP1 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 2 A). The proliferation level of WPMY-1 cells was significantly inhibited after MINPP1 overexpression Figure 2 B).

[0062] To verify the key role of ACTBL2 in BPH, we constructed ACTBL2 knockdown cell models in WPMY-1 and BPH-1, and the knockdown effect was verified by qRT-PCR and WB experiments. Figure 3 A). The proliferation of prostate cells WPMY-1 and BPH-1 was slowed down after ACTBL2 knockdown Figure 3 B), had no significant effect on the cell contraction ability of WPMY-1 Figure 3 C), the apoptosis rate of BPH-1 cells increased Figure 3 D), and the cell cycle appeared S phase arrest Figure 3 E). The above in vitro experiments showed that the knockdown of ACTBL2 could inhibit the growth of prostate hyperplasia cells.

[0063] Example 3 Animal experiment

[0064] (1) The present study used the classical castration + testosterone propionate method to construct a rat prostate hyperplasia model, and verified the effectiveness of the model and the effect of MINPP1 gene overexpression on prostate tissue through experimental indicators. The experimental design included sham operation group (sham-vector), sham operation overexpression group (sham+ovMINPP1), lentivirus control group (T-BPH+vector), and ov-MINPP1 lentivirus injection group (T-BPH+ovMINPP1). First, the rats were subjected to castration surgery, and then testosterone propionate was injected subcutaneously to induce prostate hyperplasia. The body weight changes of the rats in each group were recorded to provide basic physiological data for subsequent indicator analysis. The results showed that there was no significant difference in prostate tissue morphology between the sham+ovMINPP1 group and the sham+vector group, indicating that overexpression of MINPP1 alone had little effect on tissue morphology without BPH disease stimulation. Compared with the T-BPH+vector group, the T-BPH+ovMINPP1 group had a more normal morphology (smaller volume and more normal shape), suggesting that overexpression of MINPP1 could alleviate the tissue abnormalities caused by BPH disease. At the same time, there was a significant statistical difference in prostate index (prostate weight / body weight) between the two groups (p<0.05) Figure 4 A-B); H&E staining showed that the prostate epithelium of the lentivirus control group increased significantly, indicating that the BPH model was successfully established. At the same time, the prostate epithelium of the ov-MINPP1 lentivirus injection group was slightly lower than that of the lentivirus control group, but the difference was not statistically significant (p>0.05) Figure 4 C), and the collagen fiber and smooth muscle content of the prostate tissue of the ov-MINPP1 lentivirus injection group did not change significantly compared with the lentivirus control group (p>0.05) Figure 4 D).

[0065] (2) To explore the effect of shACTBL2 on rat prostate tissue, the present study used an animal experiment to construct a rat BPH animal model using estrogen and testosterone induction. shACTBL2 lentivirus was injected into the rat prostate tissue to observe its morphological and histopathological changes and to explore its effect on rat prostate tissue. The experiment set up four groups of rats: sham operation interference control group (Control+shNC), sham operation interference group (Control+shACTBL2), estrogen and testosterone induction-induced BPH interference control group (E / T-BPH+shNC), and estrogen and testosterone induction-induced BPH interference group (E / T-BPH+shACTBL2). The experimental results showed that after ACTBL2 knockdown, the mouse prostate hyperplasia was alleviated, and the prostate volume and prostate index were significantly reduced (p<0.05) Figure 5 A, B) There was no significant difference in body weight between the rats in each group (p>0.05) Figure 5C). In addition, HE staining results of rat prostate tissue showed that the prostate interstitial hyperplasia was reduced after ACTBL2 knockdown, and the epithelial thickness had no significant change Figure 5 D). Masson staining results also showed that ACTBL2 knockdown could significantly reduce the level of prostate collagen fibers without changing the level of smooth muscle Figure 5 E).

[0066] Example 4. Ability of ACTBL and APOC4 respectively and in combination to distinguish healthy population and BPH patients

[0067] The sensitivity and specificity of ACTBL and APOC4 respectively and in combination to distinguish healthy population and BPH patients were evaluated by selecting the training set in Example 1 and in the validation set. The results are shown in Table 5 and Figure 6 .

[0068] Table 5. Statistical analysis of receiver operating characteristic (ROC) curve of BPH and healthy population

[0069]

[0070] Note: AUC (Area under the curve); CI (Confidence interval); PPV (Positive predictive value); NPV (Negative predictive value).

[0071] Example 5. Ability of ACTBL and MINP1 respectively and in combination to distinguish BPH patients with different severity

[0072] The sensitivity and specificity of ACTBL and MINP1 respectively and in combination to distinguish whether the population needs drug or surgical treatment were evaluated by selecting the training set in Example 1 and in the validation set. The results are shown in Table 6 and Figures 7~9 .

[0073] Table 6. Statistical analysis of receiver operating characteristic (ROC) curve of BPH conservative treatment and drug and surgical treatment population

[0074]

[0075] Note: AUC (Area under the curve); CI (Confidence interval); PPV (Positive predictive value); NPV (Negative predictive value).

[0076] The preferred embodiments of the present application are described above with reference to the drawings, and the patent range of the present application is not limited thereby. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the patent protection range of the present application.

Claims

1. The application of reagents for detecting biomarkers related to benign prostatic hyperplasia (BPH) in the preparation of products for detecting BPH, characterized in that, The biomarkers associated with benign prostatic hyperplasia are MINP1, ACTBL, and APOC4 proteins.

2. The application according to claim 1, characterized in that, The MINPP1 protein is encoded by the MINPP1 gene, the coding sequence of which is shown in SEQ ID NO: 1; The ACTBL protein is encoded by the ACTBL2 gene, and the interfering sequence of the ACTBL2 includes: (a) the positive strand as shown in SEQ ID NO: 2 and the complementary antisense strand as shown in SEQ ID NO: 3; or (b) the positive strand as shown in SEQ ID NO: 4 and the complementary antisense strand as shown in SEQ ID NO:

5.

3. The application according to claim 1, characterized in that, The products include formulations or kits.

4. The application according to claim 3, characterized in that, The formulation or kit includes specific primers for MINP1 and / or ACTBL; the specific primer pair for MINP1 includes an upstream primer as shown in SEQ ID NO: 6 and a downstream primer as shown in SEQ ID NO: 7; the specific primer pair for ACTBL includes an upstream primer as shown in SEQ ID NO: 8 and a downstream primer as shown in SEQ ID NO:

9.

5. The application according to claim 4, characterized in that, The kit also includes reverse transcriptase, buffer, dNTPs, MgCl2, DEPC water, fluorescent probe, RNase inhibitor and Taq enzyme.

6. The application according to claim 1, characterized in that, Both MINP1 and APOC4 were downregulated in patients with benign prostatic hyperplasia (BPH), while ACTBL was upregulated in patients with BPH.

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