Application of GPR1 as target spot in preparation of endometriosis related diagnosis and / or treatment preparation

Abstract

The invention discloses application of GPR1 as a target spot in preparation of endometriosis related diagnosis and / or treatment preparations, and belongs to the technical field of biological medicines. The invention provides application of a GPR1 target spot serving as a diagnostic marker in preparation of a reagent or a kit for diagnosing endometriosis. The endometriosis is selected from one of a uterus-sacral ligament type endometriosis, a peritoneal type endometriosis, a rectum-vagina type endometriosis, a deep infiltration type endometriosis and an ovarian type endometriosis. The invention discovers that the expression of GPR1 in endometriosis tissues and cells is obviously increased, which prompts that GPR1 can be used as a diagnostic marker of endometriosis. In-vitro and in-vivo models find that the knockout of GPR1 significantly inhibits the proliferation, migration and invasion ability of hEM15A cells and the growth of ectopic lesions in animals, indicating that GPR1 can be used as a new intervention target for clinical treatment of endometriosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of GPR1 as a diagnostic marker and therapeutic target for endometriosis. Background Art

[0002] Endometriosis (abbreviated as "endometriosis") is a common gynecological disease, which refers to the appearance of endometrium-like lesions outside the uterine cavity. Many studies have pointed out that endometriosis is a potential risk factor for a variety of diseases such as cancer, asthma, autoimmune diseases, and cardiovascular diseases.

[0003] The diagnosis of endometriosis is a difficult problem in the clinic at this stage. The diagnosis of endometriosis is usually based on clinical symptoms and signs, imaging examinations, laparoscopy, serum CA125 level examination, etc. Among them, the universal gold standard is the observation of the lesion morphology under laparoscopy, combined with the histopathological morphology level for diagnosis (endometrial glands and stroma can be seen in the lesions, accompanied by inflammatory reactions and fibrosis). However, in the clinical diagnosis of certain cases, histopathological evidence has not been found, and there is no clear molecular marker to assist in the detection of histopathological histology.

[0004] The treatment of endometriosis is mainly divided into surgical treatment and drug treatment. Surgical treatment has poor efficacy, and up to 75% of patients relapse within 5 years after surgery; the purpose of drug treatment is to block the development of lesions. Common drugs are mainly divided into five categories: non-steroidal anti-inflammatory drugs, oral contraceptives, high-efficiency progestins, androgen derivatives, and gonadotropin-releasing hormone agonists. Since drug treatment mainly controls the hypothalamus-pituitary-ovarian reproductive endocrine axis and has strong side effects, it is not suitable for young patients with fertility needs. Even more regrettable is that there is still no ideal molecular target for the targeted treatment of endometriosis. Therefore, the discovery and functional identification of important molecular targets are the key to breaking the deadlock, which will lay a molecular theoretical foundation for subsequent drug development.

[0005] G protein coupled receptors (GPCRs) are a class of seven transmembrane receptor superfamily, with more than 1,000 members in the human genome. They are involved in regulating almost all life activities such as perception, reproduction, development, immunity, and play an important role in the occurrence and development of major diseases such as tumors and metabolism. GPCRs have always been one of the important targets for drug development. According to statistics, the sales of drugs targeting GPCRs account for nearly one-third of the global market. However, there are still no non-hormonal targeted GPCRs drugs for the relief and treatment of endometriosis. In the field of endometriosis research, animal models have been used to find that antagonists or agonists targeting GPCRs such as Neuropeptide S Receptor 1 and PGE2 Receptors can significantly relieve pain and eliminate lesions, indicating that targeting GPCRs to treat endometriosis has good application prospects.

[0006] G protein coupled receptor-1 (GPR1) belongs to Class A of GPCRs. In 1994, Marchese et al. reported the cloning of GPR1 in a hypothalamic cDNA library. It was not until 2008 that the orphan receptor GPR1 was "deorphaned", and Barnea et al. used the Tango system to discover that Chemerin is the ligand of GPR1. In 2018, FAM19A1 was also identified as a ligand of GPR1. In 2019, a Cell article reported that Osteocrin-2-19, cholecystokinin (CCK-33) and gastrin-releasing peptide are potential ligands of GPR1, but these three endogenous peptides have not been confirmed at the biological function level.

[0007] At present, the knowledge about GPR1 receptor signaling is mainly provided by chemerin as an agonist. Chemerin is a widely distributed secreted protein with three main receptors: CMKLR1 (Chemerin Chemokine-Like Receptor 1), GPR1 and CCRL2 (CC Motif Chemokine Receptor Like 2). Studies have found that chemerin has a high affinity for GPR1 (KD: 0.21nM). After stimulation by chemerin, GPR1 does not bind to any type of G protein, but can produce fluctuations in calcium ion concentration, further recruit β-arrestin1 and β-arrestin2, and activate phosphorylated Erk1 / 2. Therefore, GPR1 operates in a G protein-independent, β-arrestin biased signaling manner under the stimulation of chemerin. The biological function of GPR1 is still not very clear. Summary of the invention

[0008] Endometriosis is a common gynecological disease that is difficult to diagnose and incurable, and there is currently no ideal diagnostic and therapeutic target. In view of the defects existing in the above-mentioned prior art, the purpose of the present invention is to design and provide the use of GPR1 as a target in the preparation of endometriosis-related diagnostic and / or therapeutic preparations. The present invention first discovered that the expression of GPR1 in endometriotic tissues and cells is significantly increased, suggesting that GPR1 can be used as a diagnostic marker for endometriosis. It was further found in in vitro and in vivo models that knocking out GPR1 significantly inhibited the proliferation, migration and invasion ability of hEM15A cells and the growth of ectopic lesions in animals, indicating that GPR1 can be used as a new intervention target for the clinical treatment of endometriosis.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides the use of a GPR1 target as a diagnostic marker in the preparation of a reagent or a kit for diagnosing endometriosis.

[0011] In the application, the endometriosis is selected from one of uterosacral ligament endometriosis, peritoneal endometriosis, rectovaginal endometriosis, deep infiltrating endometriosis, and ovarian endometriosis.

[0012] In a second aspect, the present invention provides the use of a reagent for detecting GPR1 molecules in the preparation of a kit for diagnosing diseases caused by endometriosis.

[0013] In the application, the disease is selected from the group consisting of chronic pelvic pain caused by endometriosis, implantation failure, recurrent miscarriage, and infertility.

[0014] In the application, the reagent for detecting the GPR1 molecule is selected from one of PCR primers, qPCR primers, FISH nucleic acid probes, antibodies, and binding polypeptides.

[0015] In a third aspect, the present invention provides the use of an inhibitor of a GPR1 molecule in the preparation of a drug for treating endometriosis.

[0016] In the application, the inhibitor is one of gRNA, dsRNA, siRNA, shRNA, microRNA or antisense RNA that targets the DNA or mRNA of GPR1 and inhibits gene transcription and protein expression.

[0017] In the application, the inhibitor is a biological or chemical agent that targets the protein sequence of the GPR1 molecule and produces an inhibitory or antagonistic effect on GPR1; the biological or chemical agent is selected from one of antibodies, polypeptides, and compounds.

[0018] In a fourth aspect, the present invention provides an endometriosis diagnostic kit comprising a GPR1 target as a diagnostic marker.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The experiment of the present invention shows that compared with the endometrial tissue in situ, the gene expression level of GPR1 in the peritoneal ectopic lesions, uterosacral ligament ectopic lesions, recto-vaginal lesions, deep infiltration lesions and ovarian ectopic lesions are significantly increased, and the qPCR and Western blot experiments confirm that the gene and protein expression levels of GPR1 in primary human ovarian endometriotic stromal cells are significantly increased. It shows that the detection of GPR1 can be used as a detection of various types of endometriotic tissues and assist in the identification of pathological tissues.

[0021] 2. The experiment of the present invention shows that the cell line hEM15A derived from the uterus of patients with ovarian ectopic endometriosis was used as the research object, and the GPR1 knockout cell line was constructed by the CRISPR Cas9 system. It was found that GPR1 knockout significantly inhibited the proliferation, migration and invasion of cells. The endometriosis animal model was constructed using Gpr1 knockout mice, and it was found that the endometriosis lesions of Gpr1 knockout significantly subsided. Inhibiting the expression of GPR1 can inhibit the biological function of endometrial stromal cells and the growth of lesions in vivo, indicating that GPR1 can be used as a therapeutic target for endometriosis, and its inhibitors can be used to prepare related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the analysis result of the dataset numbered GSE141549 in the GEO database;

[0023] Figure 2 The figure is a Western blot experiment result diagram, wherein A is the qPCR primer sequence diagram of GPR1; B is the expression result diagram of GPR1 gene; C is the expression result diagram of GPR1 protein content;

[0024] Figure 3 Figures showing gRNA sequences, qPCR experiments, Western blot results, and Transwell migration and invasion assay results;

[0025] Figure 4 The results of the lesion in model mice were constructed by allogeneic surgical induction. DETAILED DESCRIPTION

[0026] The following embodiments are only some embodiments of the present invention, rather than all embodiments. Therefore, the detailed description in the embodiments of the present invention provided below is not intended to limit the scope of the present invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the protection scope of the present invention.

[0027] In the following examples, the reagents, instruments, equipment, preparation methods or processes are all commonly used techniques in the art unless otherwise specified.

[0028] Embodiment 1:

[0029] The dataset numbered GSE141549 was searched in the public database website Gene Expression Omnibus (GEO, URL: https: / / www.ncbi.nlm.nih.gov / geo / ), and the file named GSE141549_batch Corrected normalizedArrays combined.xlsx was downloaded to extract the expression data of the GPR1 gene. Figure 1 As shown, compared with the uterine tissue in healthy women ( Figure 1 - Group A, n = 38), GPR1 gene expression level in peritoneal ectopic lesions ( Figure 1 - Group B, n = 76), ectopic lesions in the uterosacral ligament ( Figure 1 -C group, n = 26), rectal vaginal lesions ( Figure 1 -D group, n = 22), deep infiltration lesions ( Figure 1-E group, n = 40) and ectopic lesions in the ovary ( Figure 1 -F group, n=28) were significantly increased.

[0030] Embodiment 2:

[0031] 1. Tissue acquisition and primary cell extraction: Tissues were obtained from Shenzhen University General Hospital. All patients signed informed consent forms, and the procedures were approved by the Ethics Committee of Shenzhen University General Hospital and the Ethics Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.

[0032] After the tissue is obtained, it is manipulated in a sterile bench and minced with sterile ophthalmic scissors. Digestive solution (DNase I and Collagenase) is added and placed in a 37°C water bath for 1.5 h. The tissue is shaken for 10 s every 10 min until it is digested. After filtering through a 40 μm cell sieve, it is placed in a cell incubator for culture.

[0033] 2. Extraction of cell RNA: discard the culture medium of the cells, wash three times with PBS, aspirate 1ml of RNA lysis solution, blow the bottom of the dish, aspirate all the cell lysis solution into an RNase-free EP tube, and let it stand on the ice box for 5 minutes. Add 200μL of chloroform to the tube and let it stand in the ice box for 5 minutes. Centrifuge at 4℃, 12000rpm, 10min (take a new EP tube and place it on ice to precool). Aspirate the upper aqueous phase into a new EP tube, add 1000μL of isopropanol, and mix manually. Centrifuge at 4℃, 12000rpm, 10min. Discard the supernatant, add 1mL of 75% ethanol to the tube, centrifuge at 4℃, 12000rpm, 5min. Discard the supernatant and dry it to obtain RNA.

[0034] 3. cDNA reverse transcription: prepare the reverse transcription reaction solution according to the components given in the instruction manual, mix gently, incubate at 37°C for 15 min, 85°C for 5 s, and then place at 4°C to obtain cDNA.

[0035] 4.qPCR: Primers were synthesized by Shanghai Bioengineering. The reaction solution was prepared according to the components given in the instructions, and the Ct value was obtained using the 2-step PCR conditions. The relative expression content was calculated according to 2 -△△Ct calculate.

[0036] 5. Cell protein extraction: discard the culture medium of the cells, wash three times with PBS, add protein lysis buffer containing protease inhibitors, scrape the cells with a scraper and place on ice, shake once every 10 minutes, centrifuge at 12000 rpm at 4℃ after 30 minutes, take the supernatant and place it in a new EP tube for protein quantification.

[0037] 6.Western blot: Perform SDS-PAGE electrophoresis with 30 micrograms of protein per well. After the electrophoresis, transfer the protein to a nitrocellulose membrane at 250 mA for 2 hours, and then block it with 5% skim milk powder. After 2 hours at room temperature, prepare GPR1 antibody (purchased from abcam, item number: ab249312) at a ratio of 1 / 1000, incubate the NC membrane, and place it in a 4°C refrigerator overnight. On the next day, elute the NC membrane with TBST solution, once for 10 minutes, three times prepare secondary antibodies at a ratio of 1 / 3000, incubate the NC membrane, and place it at room temperature for 1 hour. After eluting with TBST, cover the NC membrane with ECL to obtain the results. Figure 2 As shown, the qPCR primer sequence of GPR1 is Figure 2 A. The qPCR results using this primer as a probe showed that the expression level of the GPR1 gene in the ectopic endometrial stromal cells (Ovarian Endometriosis-Ecpotic, abbreviated as OvEM-Ec) of primary endometriosis patients (ovarian endometriosis) was significantly increased compared with the primary healthy eutopic endometrial stromal cells (Normal-Eupotic, abbreviated as Nor-Eu) and the eutopic endometrial stromal cells (Ovarian Endometriosis-Eupotic, abbreviated as OvEM-Eu) of primary endometriosis patients (ovarian endometriosis). Figure 2 B. In addition, compared with Nor-Eu and OVEM-Eu, the expression of GPR1 protein in OvEM-Ec was also significantly increased, such as Figure 2 C.

[0038] Embodiment 3:

[0039] 1. Knockout of GPR1 expression: A three-plasmid lentiviral system (pHBLV-U6-gRNA-EF1-CAS9-PURO vector, psPAX2 vector and pMD2G vector) was used to construct lentivirus. After concentrating the lentivirus, hEM15A cells were infected. After 24 hours, 2 μg / mL of puromycin was added for screening, and then single-cell clone screening was performed using the limiting dilution method. The results of qPCR and Western blot experiments showed that GPR1 was successfully knocked out in hEM15A cells. The sequence information of gRNA is as follows Figure 3 As shown in A. Figure 3 As shown in B and C, CRISPR Cas9 gene editing was performed with the gRNA to knock out GPR1 in hEM15A cells. The results of qPCR and Western blot experiments showed that the gene and protein expression of GPR1 were knocked out in two cell clones.

[0040] 2. Detection of cell proliferation ability: hEM15A cells were plated in a 96-well plate at 2000 cells / well (the serum concentration of the culture medium was 10%), and the assay was performed after 24, 48, 72 and 96 hours. The assay method was as follows: the culture medium was discarded, and CCK-8 reagent (purchased from Bio-Time) was mixed with serum-free culture medium at a volume ratio of 1 / 10 (total system per well was 100 μL), and the absorbance value was read at 450 nm using an ELISA reader to detect cell viability. The results were as follows: Figure 3 As shown in D, the results of the CCK8 experiment showed that the proliferation ability of the GPR1 knockout cell line was significantly inhibited.

[0041] 3. Cell migration ability: Transwell chamber (purchased from Corning) was used as a research model. hEM15A cells were placed in serum-free medium at 100,000 / well and added to the upper layer of the chamber. Medium containing 10% serum was added to the lower layer of the chamber as a chemoattractant. After 24 hours, the chamber was taken out, and the cells that migrated under the chamber membrane were fixed and stained with methanol and crystal violet, respectively. Then, the non-migrated cells on the upper side were wiped off with a cotton swab, and photos were taken and recorded. The migration rate was calculated. The results are shown in Figure 3 As shown in E, the migration of GPR1 knockout cell lines was significantly reduced.

[0042] 4. Cell invasion ability: Transwell chamber was used as the research model. 50 μL Metrial Gel (mixed with serum-free medium in a volume ratio of 1 / 10 per well, purchased from Corning) was pre-coated on the upper layer of the chamber and placed at 37°C for half an hour. hEM15A cells were placed in serum-free medium at 100,000 cells / well and added to the upper layer of the chamber. 10% serum medium was added to the lower chamber as a chemoattractant. After 48 hours, the chamber was removed, and the cells that invaded under the chamber membrane were fixed and stained with methanol and crystal violet, respectively. The non-invaded cells on the upper side of the membrane were wiped off with a cotton swab, and photos were taken and recorded. The invasion rate was calculated. The results are as follows: Figure 3 As shown in F, the invasion ability of GPR1 knockout cell lines was significantly reduced.

[0043] Embodiment 4:

[0044] The peritoneal endometriosis mouse model (PeEM model for short) is a widely recognized model in the field of endometriosis research and is widely used to analyze the functional effects of specific genes and compounds on ectopic lesions. PeEM models are divided into surgical induction and injection induction according to the modeling method, and are divided into autologous and allogeneic according to the source of endometrium.

[0045] The present invention adopts the allogeneic surgery induction method. The model has a high success rate and strong repeatability. Compared with the sham group, the PeEM mice showed reduced fertility and increased pain response, which better reflected the clinical symptoms. The construction process is as follows:

[0046] 1. Place 7-week-old C57BL / 6J female mice in the animal room and conduct the experiment one week later;

[0047] 2. Both donor and recipient mice in estrus were selected as the model construction objects;

[0048] 3. Kill the donor mouse by cervical dislocation, remove the uterus, and remove the ovaries, fallopian tubes, blood vessels and adipose tissue;

[0049] 4. Divide the bilateral uterus into two halves and cut them open longitudinally with surgical scissors to expose the endometrial layer;

[0050] 5. Use a scalpel to divide the two uterine sections into four equal pieces and place them in normal saline;

[0051] 6. Anesthetize the recipient mice with 2.5% Avertin (intraperitoneal injection, 300 μl);

[0052] 7. Open the abdominal cavity along the midline;

[0053] 8. Use 6-0 absorbable surgical sutures to fix the four pieces of uterus to both sides of the abdominal wall (two pieces / one side, with the endometrial surface close to the abdominal wall);

[0054] 9. Suture the muscles and skin, disinfect, observe every other day, terminate after four weeks, collect samples, weigh and count the lesions. Figure 4 As shown, wild type (WT) and Gpr1 knockout (Gpr1 - / - ) mouse uterus (divided into four equal parts) was surgically transplanted into the abdominal wall of WT mice to construct WT→WT and Gpr1 - / - →WT mice, the mice were killed one month after construction, and the abdominal wall lesions of each mouse were collected, such as Figure 4 As shown in A. The lesions on the abdominal wall were peeled off and weighed. The results showed that compared with the WT→WT group, Gpr1 - / - →The weight of the lesions in the WT group was significantly reduced (e.g. Figure 4 B) In addition, the statistical results showed that about 8.8% of the lesions in the WT→WT group regressed, while Gpr1 - / - → In the WT group, approximately 72.5% of lesions disappeared (e.g. Figure 4 C).

[0055] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Use of GPR1 as a target in the preparation of diagnostic and / or therapeutic preparations for endometriosis-related diseases.

2. Application of GPR1 target as a diagnostic marker in the preparation of reagents or kits for diagnosing endometriosis.

3. The use according to claim 2, characterized in that The endometriosis is selected from one of uterosacral ligament endometriosis, peritoneal endometriosis, rectovaginal endometriosis, deep infiltrating endometriosis and ovarian endometriosis.

4. Use of a reagent for detecting GPR1 molecules in the preparation of a kit for diagnosing diseases caused by endometriosis.

5. The use according to claim 4, characterized in that The disease is selected from the group consisting of chronic pelvic pain caused by endometriosis, implantation failure, recurrent miscarriage, and infertility.

6. The use according to claim 4, characterized in that The reagent for detecting the GPR1 molecule is selected from one of PCR primers, qPCR primers, FISH nucleic acid probes, antibodies, and binding polypeptides.

7. Use of inhibitors of GPR1 molecules in the preparation of drugs for the treatment of endometriosis.

8. The use according to claim 7, characterized in that The inhibitor is one of gRNA, dsRNA, siRNA, shRNA, microRNA or antisense RNA that targets the DNA or mRNA of GPR1 and inhibits gene transcription and protein expression.

9. The use according to claim 7, characterized in that The inhibitor is a biological or chemical agent that targets the protein sequence of the GPR1 molecule and produces an inhibitory or antagonistic effect on GPR1; the biological or chemical agent is selected from one of antibodies, polypeptides, and compounds.

10. A diagnostic kit for endometriosis, characterized in that: The GPR1 target was included as a diagnostic marker.

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