KR102640230B1 - A method for diagnosing endometrial-related diseases and screening method for endometrial-related diseases using the mechanism of change in the mobility of endometrial stromal cells treated with C-peptide - Google Patents

Abstract

When C-peptide is treated with undifferentiated endometrial stromal cells that have not decidualized, MMP9, TIMP1, and TIMP3 increase, thereby increasing the mobility and invasiveness of the non-decidualized endometrial stromal cells. This increases significantly compared to the endometrial stromal cells that have undergone decidualization. The present invention can provide a method for diagnosing endometrial-related diseases and screening for therapeutic agents by confirming the increase in MMP9 and the decrease in TIMP1 and TIMP3, since endometrial-related diseases occur due to such increased undifferentiated cells.

Description

A method for diagnosing endometrial-related diseases and screening method for endometrial-related diseases using the mechanism of change in mobility of endometrial stromal cells treated with C-peptide the mechanism of change in the mobility of endometrial stromal cells treated with C-peptide}

This relates to a diagnostic method for endometrial-related diseases and a screening method for endometrial-related disease treatments using the mechanism of change in mobility of endometrial stromal cells treated with C-peptide.

The uterus is an organ composed of several layers with a thickness of 2 cm. The perimetrium consists of the outermost thin membrane, the myometrium is a muscle layer made of smooth muscle inside, and the inner layer is connected to the myometrium. It is made up of endometrium. Unlike the myometrium, the endometrium is a soft tissue layer and plays an important role in embryo implantation. Inside the endometrium is the empty space called the uterine cavity. The endometrium secretes growth factors and cytokines to provide an environment for embryo implantation and development, and is shaped according to a certain cycle by the regulation of the female hormones estrogen and progesterone. causes functional changes. Specifically, the endometrium is composed of a functional layer and a basal layer, and the functional layer repeatedly develops and regresses in accordance with the female reproductive cycle due to changes in female hormone secretion, and the egg ovulates in the ovary. During the period of about 10 days after conception, the endometrium thickens the most and becomes in a good condition for implantation of a fertilized egg. If the fertilized egg does not implant, the thickened and developed lining breaks down and becomes thinner, which is called menstruation.

Decidualization refers to the differentiation of endometrial stromal cells (ESC). During the decidualization process, morphological and functional changes occur.

ESCs that have undergone decidualization undergo transcriptional and morphological changes to prepare for future embryo implantation. The invasiveness and mobility of ESCs play an essential role in the remodeling of endometrial tissue during decidualization and implantation. ESCs that have undergone decidualization exhibit greater invasiveness than undifferentiated cells. Successful implantation is associated with migration of decidualized ESCs, subsequent secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) from ESCs, and abundance of extravillous trophoblast cells. there is. TIMP, a key regulator of MMP9 activity, plays an important role in maintaining homeostasis during endometrial migration and early implantation by altering the degree of trophoblast invasion.

Human proinsulin linking peptide (hereinafter referred to as C-peptide) is produced in the form of proinsulin in pancreatic β cells, and is separated from insulin through a proteolysis process to become C-peptide. Human C-peptide is composed of 31 amino acids and helps the insulin protein form an appropriate three-dimensional structure and helps insulin form interchain disulfide bonds.

C-peptide and insulin are secreted in equal amounts, and C-peptide has a longer half-life than insulin, so it is also used as an inert biomarker for insulin levels.

Although C-peptide has been reported to promote migration of other cell types, it is unclear whether C-peptide affects the mobility of ESCs. Migration of human endometrial stromal cells (HESC) is essential for successful decidualization and invasion of the embryonic trophoblast. HESCs that have undergone decidualization rapidly move toward the embryo in response to signals secreted from the embryo (trophoblast), thereby enhancing signal transmission between the mother and fetus (Gellersen et al. Hum Reprod 2010). Therefore, changes in motility and invasion between decidualized HESCs and non-deciduatized HESCs impede the interaction between decidualized HESCs and human embryos (trophoblasts), thereby affecting pregnancy. Reduces the probability of success.

In this process, the present inventor tested whether C-peptide regulates the migration of human ESCs in order to examine whether the high level of C-peptide that occurs when suffering from hyperinsulinemia can affect the mobility of HESCs. and investigated the regulatory mechanism of migration by C-peptide.

In the present invention, changes in mobility between ESCs that had decidualized and ESCs that had not decidualized upon treatment with C-peptide were confirmed. In addition, the regulatory mechanism of C-peptide-induced HESC mobility was further investigated by analyzing the expression of MMP2/9 and several TIMPs. In this process, the present invention was completed by confirming that C-peptide inhibits pregnancy, implantation, and maintenance of the endometrium by promoting the movement of HESCs that have not decidualized.

The present inventors examined proteins or genes whose expression levels change when C-peptide is treated in endometrial stromal cells that have not decidualized and in endometrial stromal cells that have decidualized, and as a result, the expression level or activity of a specific factor has changed. Changes were discovered, and the present invention was completed by focusing on the fact that such changes can cause diseases related to the endometrium.

Therefore, the purpose of the present invention is to provide information that can determine endometrial-related diseases by using genes or proteins of specific substances in endometrial stromal cells that have not decidualized as a kind of biomarker.

Another object of the present invention is to provide a method for treating endometrial stromal cells that have not decidualized with C-peptide and screening for a treatment for endometrial-related diseases using the gene or protein of the specific substance. .

In order to achieve the above purpose,

1) cultivating non-decidualized endometrial stromal cells from a patient suspected of having an endometrial-related disease and non-decidualized endometrial stromal cells from a healthy person, respectively;

2) Select from the group consisting of MMP9, GPR146, TIMP1, and TIMP3, which are substances that cause increased mobility of endometrial stromal cells in step 1) that have not been decidualized by C-peptide. Measuring the expression level or protein activity of one or more genes; and

3) Among the substances that cause increased mobility of endometrial stromal cells that have not undergone decidualization in step 2), the expression level or protein activity of the genes of MMP9 and GPR146 is increased compared to endometrial stromal cells of healthy people. , if the expression level or protein activity of the TIMP1 or TIMP3 gene is decreased compared to the endometrial stromal cells of a healthy person, determining that it is an endometrial-related disease;

Provides a method of providing information for determining endometrial stromal-related diseases, including.

Additionally, in order to achieve the above purpose,

1) Culturing endometrial stromal cells that have not undergone decidualization and then treating them with C-peptide;

2) After treating the test substance with the culture medium in which the endometrial stromal cells in step 1) were cultured, any one selected from the group consisting of MMP9, GPR146, TIMP1, and TIMP3, which are substances that cause increased mobility of endometrial stromal cells. Measuring the expression level of the above gene or the activity of the protein; and

3) Among the substances that cause increased mobility of endometrial stromal cells in step 2), the expression level or protein activity of the genes of MMP9 or GPR146 increases compared to the normal control group, or the expression level or protein of the genes of TIMP1 or TIMP3 Selecting a test substance whose activity decreases compared to the normal control group;

Provides a screening method for a treatment for endometrial stromal-related diseases, including.

By confirming the increase in MMP9 and GPR146, which are substances that cause increased mobility of endometrial stromal cells that have not decidualized, and the decrease in TIMP1 and TIMP3 in patients suspected of having an endometrial-related disease, we can determine whether they are currently suffering from an endometrial-related disease. It has the effect of quickly determining whether or not it is possible.

In addition, by selecting substances that increase MMP9 and GPR146, which are substances that cause increased mobility of endometrial stromal cells that have not deciduated, and reduce the expression level or protein activity of TIMP1 and TIMP3 genes, endometrial-related Disease treatments can be screened.

Figure 1a is for comparing the mobility of human endometrial stromal cells (HESC) that have been differentiated with 8-Br-cAMP and decidualized and undifferentiated endometrial stromal cells that have not been decidualized. This shows that a wound-healing scratch assay was performed.
Figure 1b shows the mobility of HESC differentiated with 8-Br-cAMP and decidualized HESC and undifferentiated HESC without decidualization treated with 50 nM C-peptide and analyzed by transwell.
Figure 1c shows the results of analyzing the mobility of HESCs using an invasion assay in collagen-coated transwells after treatment with 50 nM C-peptide in undifferentiated HESCs that had not undergone decidualization.
Figure 1d shows the results of wound-healing scratch assay 24 hours after treatment with 50 nM C-peptide on HESCs isolated from three subjects.
Figure 1e shows the results of transwell analysis of JEG-3 cells treated in the same manner as in Figure 1b.
Figure 1f shows the results of the same invasion analysis as Figure 1c for JEG-3 cells.
Figure 2a shows the results of HESCs cultured in serum-starvation medium for 18 hours, incubated with 50 nM C-peptide for 10 minutes, and then subjected to Western blotting for Akt, β-catenin, and ERK1/2.
Figure 2b is a graph showing the results of lysing HESCs that were differentiated with 8-Br-cAMP and decidualized and undifferentiated HESCs that were not decidualized and subjected to qRT-PCR for GPR146.
Figure 2c shows the results of HESCs pretreated with 1 μM Akti for 1 hour, cultured as in Figure 2a, and then subjected to Western blotting for β-catenin.
Figure 2d shows the results of HESC treated with 50 nM C-peptide as shown in Figure 2a, immunoprecipitated with IgG or anti-β-catenin, and then subjected to Western blotting for β-catenin and PP2A.
Figure 2e shows Western blot photographs and values of HESCs pretreated with 10 μM okadaic acid for 1 hour and then treated as in Figure 2a.
Figure 2f shows that HESCs were transduced using short hairpin RNA (shRNA) that inhibits the expression of PPP1C and selected for 5 days using 1.5 μM puromycin. Afterwards, the cells were serum-starved for 18 hours, then incubated with 50 nM C-peptide for 10 minutes, and the results of Western blotting for β-catenin and PP1 are shown.
Figure 3a shows the results of Western blotting for cytoplasmic and nuclear β-catenin after HESCs were serum-starved for 18 hours and incubated with 50 nM C-peptide for 10 minutes.
Figure 3b shows the results of Western blotting for β-catenin in the nucleus after HESCs were pretreated with 1 μM Akti and treated as in Figure 3a.
Figure 3b shows Western blotting for β-catenin in the nucleus after HESCs were pretreated with 10 nM OA and treated as in Figure 3a.
Figure 4a shows that HESCs were serum-starved for 18 hours, pretreated with 1 μM Akti or 10 nM OA for 1 hour, and treated with 50 nM C-peptdie for 1 hour, and then the mobility of HESCs was analyzed using the wound-recovery scratch method. It shows one result.
Figure 4b shows the results of analyzing the mobility of HESCs by transwell analysis after treating them as in Figure 4a.
Figure 4c shows the results of confirming the expression of PP1 by Western blot in cells transduced into HESCs using scramble and PPP1C shRNA, and the results of treating the cells transduced with PPP1C shRNA as shown in Figure 4a.
Figure 4d shows the results of lysing HESC transduced cells using scramble and β-catenin shRNA and Western blotting for β-catenin.
Figure 4e shows the results of analyzing the mobility of HESCs using the wound-recovery scratch assay method by treating HESC transduced cells using scramble and β-catenin shRNA as in Figure 4a.
Figure 4f shows the results of processing cells transduced into HESC using scramble and β-catenin shRNA as shown in Figure 4b.
Figure 4g shows the results of pre-treating HESCs with 10nM PNU-74654 for 1 hour, treating them as in Figure 4a, and analyzing the mobility of HESCs using a wound-recovery scratch analysis method.
Figure 4h shows the results of invasion by dividing HESCs on collagen-coated transwells, pretreating them with 10nM PNU-74654 for 1 hour, and then culturing them with 50nM C-peptide for 24 hours.
Figure 5a shows the results of HESCs being lysed by culturing with 50nM C-peptide for 24 hours, and mRNA expression of MMP2 and MMP9 confirmed by qRT-PCR.
Figure 5b shows the results of HESCs cultured with 50nM C-peptide for 24 hours, lysed, and subjected to Western blotting for MMP2 and MMP9.
Figure 5c shows the results of measuring MMP activity by treating HESCs as in Figure 4a and performing gelatin zymography.
Figure 5d shows the results of HESCs pretreated with 10 μM MMP2/9 inhibitor, treated with 50 nM C-peptide, and analyzed for mobility by wound-recovery scratch assay.
Figure 5e shows the results of qRT-PCR for TIMP1, TIMP2, and TIMP3 by dissolving HESCs treated as in Figure 5a.
Figure 5f shows qRT-PCR for MMP9, TIMP1, and TIMP3 after pretreatment with 1 uM Akti, 10 nM OA, or 10 nM PNU-74654 for 1 hour, followed by treatment with 50 nM C-peptide for 24 hours, and then dissolved. This shows the results of the implementation.
Figure 5g shows the predicted binding site of β-catenin in the regulatory region of the promoters of MMP9, TIMP1, and TIMP3 and the primers confirming it, and transduction of HESC using scramble and β-catenin shRNA. One cell was lysed, and the binding of β-catenin to the promoter regulatory region of MMP9, TIMP1, and TIMP3 was confirmed by chromatin immunoprecipitation. In HESCs, following scramble and β-catenin shRNA transduction. The results of confirming the expression level of β-catenin by Western blot are shown sequentially.
Figure 6 roughly shows the path from C-peptide to migration of HESCs that have not been decidualized.

Hereinafter, the present invention will be described in detail.

Throughout the specification, “including” an element means that other elements may be further included rather than excluding other elements, unless specifically stated otherwise.

The present invention provides a method for cultivating 1) non-decidualized endometrial stromal cells from a patient suspected of having an endometrial-related disease and non-decidualized endometrial stromal cells from a healthy person, respectively. step;

2) Select from the group consisting of MMP9, GPR146, TIMP1, and TIMP3, which are substances that cause increased mobility of endometrial stromal cells in step 1) that have not been decidualized by C-peptide. Measuring the expression level or protein activity of one or more genes; and

3) Among the substances that cause increased mobility of endometrial stromal cells that have not undergone decidualization in step 2), the expression level or protein activity of the genes of MMP9 and GPR146 is increased compared to endometrial stromal cells of healthy people. , if the expression level or protein activity of the TIMP1 or TIMP3 gene is decreased compared to the endometrial stromal cells of a healthy person, determining that it is an endometrial-related disease;

Provides a method of providing information for determining endometrial stromal-related diseases, including.

Specifically, when treated with C-peptide, among endometrial stromal cells, undifferentiated cells that have not undergone decidualization move faster than cells that have decidualized (Figure 1a). This is because C-peptide improves the mobility and invasiveness of undifferentiated cells that have not decidualized (Figure 1b, Figure 1c). This increase in mobility is confirmed through HESCs from several people (Figure 1d).

In addition, treatment with C-peptide improves the migration of trophoblast-derived JEG-3 cells (Figure 1e) and improves invasiveness (Figure 1f).

In one embodiment of the present invention, in the patient suspected of having an endometrial-related disease in step 1), the mobility and invasiveness of non-decidualized endometrial stromal cells are increased compared to decidualized endometrial stromal cells. It may be a method of providing information for determining the characteristic endometrial stromal-related diseases.

Specifically, treatment with C-peptide enhances the phosphorylation of Akt (Ser 473) and β-catenin (Ser 552) and decreases the phosphorylation of β-catenin (Ser 33/37/41) in HESCs that have not decidualized. . On the other hand, in HESCs that have undergone decidualization, phosphorylation of Akt and β-catenin is not enhanced (Figure 2a).

In addition, the transcription level of GPR146, a G-protein coupled receptor presumed to be a C-peptide receptor, is higher in HESCs that have not undergone decidualization (Figure 2b). When treated with Akt inhibitor (Akti), phosphorylation of β-catenin (Ser 552) is reduced (Figure 2c).

In addition, treatment with C-peptide reduces the interaction between β-catenin and PP2A (Figure 2d), but treatment with OA, an inhibitor of PP, restores phosphorylation of β-catenin (Figure 2e). Likewise, C-peptide treatment did not reduce phosphorylation of β-catenin, but reduced β-catenin phosphorylation in scrambled shRNA-infected cells (Figure 2f).

In addition, when treated with C-peptide, β-catenin inside the nucleus increases while cytoplasmic β-catenin does not decrease (Figure 3a). However, when Akti and OA were treated together, the nuclear translocation of β-catenin decreased (Figure 3b, Figure 3c).

In addition, treatment with C-peptide increased cell mobility (Figure 4a), and treatment with Akti or OA decreased cell migration, as confirmed through wound-recovery scratch analysis and transwell analysis (Figure 4b).

Here, when PP1 is knocked down, cell migration is reduced (Figure 4c), and when β-catenin is knocked down (Figure 4d), cell migration is reduced (Figure 4e) and invasion is also reduced (Figure 4f). ). And when treated with PNU-74654, cell mobility decreased (Figure 4g) and invasion also decreased (Figure 4h).

The transcription amount of MMP9 increases due to C-peptide treatment (Figure 5a), and the protein amount also increases (Figure 5b). However, the expression of MMP2 is not significantly increased.

The amount of MMP9 dimers also increases (Figure 5c). When treated with an MMP9 inhibitor, the mobility increased by C-peptide was restored (Figure 5d).

Treatment with C-peptide decreased the transcript amounts of TIMP1 and TIMP3 (Figure 5e). However, TIMP2 is not significantly reduced. On the other hand, when treated with Akti (Akt inhibitor), OA, and PNU-74654, the expression of MMP9 was reduced to its original state, and the transcription levels of TIMP1 and TIMP3 were restored (Figure 5f).

β-catenin forms associations at the putative binding regions of MMP9, TIMP1, and TIMP3, and this association is reduced upon knockdown of β-catenin (Figure 5g).

In one aspect of the present invention, the step of measuring the expression level of the gene or the activity of the protein in step 2) includes immunoprecipitation, chromosome immunoprecipitation, Western blot, qRT-PCR, transwell assay, and invasion. Endometrial stromal-related diseases characterized by being performed by one or more methods selected from the group consisting of invasion assay, wound-healing scratch assay, and zymography. It may be a way to provide information for judgment.

The expression level of the gene in step 2) can be measured using qRT-PCR, competitive RT-PCR, real-time RT-PCR, and RNase protection assay (RPA). It may be a method of providing information for determining endometrial stromal-related diseases, characterized by using one or more of the following methods: , Northern blotting, and DNA chip.

Specifically, measuring the expression level of a gene is a process of confirming the presence and expression level of mRNA of the gene of a substance that causes increased mobility of endometrial stromal cells in a biological sample in order to provide information for determining endometrial-related diseases. The amount of mRNA is measured as, and according to one embodiment of the present invention, it can be measured using a primer or probe for mRNA, but is not limited to the above method.

The protein activity in step 2) can be measured using Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, and Zymo. It may be a method of providing information for the determination of endometrial stromal-related diseases, which is characterized by using one or more methods among photography, FACS, or protein chip.

Specifically, the measurement of protein activity confirms the presence and expression level of proteins expressed from the genes of substances that cause increased mobility of endometrial stromal cells in biological samples in order to provide information for determining endometrial-related diseases. As a process, according to one embodiment of the present invention, a protein can be identified using an antibody that specifically binds to the protein or a partial peptide having a binding domain, but the method is not limited to the above.

In another embodiment of the present invention, the endometrial stromal-related diseases include infertility, subfertility, endometriosis, adenomyosis, endometrial cancer, and endometrial stromal sarcoma. ), endometrial intraepithelial neoplasia, choriocarcinoma, endometritis, endometrial ectopia, endometrial glandular hyperplasia, endometrial atrophy, Provides information for determining endometrial stromal-related diseases characterized by one or more selected from the group consisting of endometrial stromatosis, hydatidiform mole, and villous adenoma. It could be a way.

In one aspect of the present invention, the mobility can be confirmed as a change compared to a normal control group through one or more selected from the group consisting of a transwell assay, an invasion assay, and a wound-healing scratch assay. It may be a method of providing information for determining endometrial stromal-related diseases characterized by:

Specifically, the transwell assay and invasion assay can use a transwell chamber, and the transwell chamber refers to a chamber containing a microporous membrane at the bottom. The transwell chamber is also referred to as a transwell upper chamber or a transwell insert. The microporous membrane is a support that is permeable to substances such as buffers, cellular substances, or culture media, but not to cells. The pore size of the microporous membrane can be appropriately adopted by a person skilled in the art and is, for example, 0.4 μm to 0.8 μm. For example, microporous membranes are polycarbonate, polyester, and collagen-coated polytetrafluoroethylene. The transwell chamber is widely used in the art and may be a commercially available product.

The present invention includes the following steps: 1) culturing endometrial stromal cells that have not undergone decidualization and then treating them with C-peptide;

2) After treating the test substance with the culture medium in which the endometrial stromal cells in step 1) were cultured, any one selected from the group consisting of MMP9, GPR146, TIMP1, and TIMP3, which are substances that cause increased mobility of endometrial stromal cells. Measuring the expression level of the above gene or the activity of the protein; and

3) Among the substances that cause increased mobility of endometrial stromal cells in step 2), the expression level or protein activity of the genes of MMP9 or GPR146 increases compared to the normal control group, or the expression level or protein of the genes of TIMP1 or TIMP3 Selecting a test substance whose activity decreases compared to the normal control group;

Provides a screening method for a treatment for endometrial stromal-related diseases, including.

Specifically, the test substance may be a competitive inhibitor targeting GPR146, which is presumed to be a receptor for C-peptide, a non-competitive inhibitor, or a substance such as a miRNA containing a partial sequence of the GPR146 gene.

In one embodiment of the present invention, in step 2), the mobility and invasiveness of endometrial stromal cells that are not decidualized are increased compared to the endometrial stromal cells that are decidualized. It may be a screening method for treatments for endometrial stromal-related diseases.

In one aspect of the present invention, 4) the expression level or protein activity of one or more genes selected from the group consisting of MMP9, TIMP1, and TIMP3, which are substances that cause increased mobility of endometrial stromal cells changed in step 3). Confirming that there is no change when pretreated with Akti, OA (okadaic acid), or PNU-74654;

It may be a screening method for a treatment for diseases related to the endometrial stromal, characterized in that it additionally includes.

In another embodiment of the present invention, the expression level of the gene in step 2) is measured using qRT-PCR, competitive RT-PCR, real-time RT-PCR, and RNase. It may be a screening method for a treatment for diseases related to the endometrial stromal, characterized by using one or more of the following methods: RNase protection assay (RPA), Northern blotting, and DNA chip.

In one aspect of the present invention, the activity of the protein in step 2) is measured using Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, and immunoprecipitation assay. , It may be a screening method for a treatment for endometrial stromal-related diseases, characterized by using one or more of complement fixation analysis, zymography, FACS, or protein chip.

In one embodiment of the present invention, the endometrial stromal-related diseases include infertility, subfertility, endometriosis, adenomyosis, endometrial cancer, and endometrial stromal sarcoma. , endometrial intraepithelial neoplasia, choriocarcinoma, endometritis, endometrial ectopia, endometrial glandular hyperplasia, endometrial atrophy, uterus It may be a screening method for a treatment for endometrial stromal-related diseases, characterized by one or more selected from the group consisting of endometrial stromatosis, hydatidiform mole, and villous adenoma.

In another aspect of the present invention, the mobility and invasiveness are changed compared to a normal control group through one or more selected from the group consisting of a transwell assay, an invasion assay, and a wound-healing scratch assay. It may be a screening method for a treatment for diseases related to the endometrial stromal, which can be confirmed.

In the present invention, the endometrial stromal treatment is performed using endometrial stromal cells that have not undergone decidualization, or trophoblast cells or JEG-3 cells instead of the endometrial stromal cells. It may be a screening method for treatments for diseases.

Hereinafter, the present invention will be described in detail through examples and experimental examples.

However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

Meanwhile, the embodiments of the present invention may be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below. Additionally, the embodiments of the present invention are provided to more completely explain the present invention to those with average knowledge in the relevant technical field.

<Example 1> Preparation of antibodies and other reagents

C-peptide was produced and used by Peptron (Daejeon, Korea).

Other reagents were prepared as follows.

Cell Tracking Dye Kit-Green-CytoPainter (catalog number ab138891) from Abcam (Cambridge, UK); Vectashield, anti-fade mounting medium containing 4′, 6-diamidino-2-phenylindole (DAPI) (catalog number H1200) from VECTOR (Malven, PA, USA); Okadasan (OA) (catalog number ALX-350-003-C100) from Cell Signaling Technology (Danvers, MA, USA); 8-bromo adenosine 35′-cyclic adenosine monophosphate (8-Br-cAMP) (catalog no. B5386) and Akti (catalog no. 124018) from Sigma-Aldrich (St. Louis, MO, USA); PNU-74654 (catalog number 74654) from Selleckchem (Houston, TX, USA); and MMP2/MMP9 inhibitor from Abcam (catalog number ab1415190). Akt (catalog number 9272S), pS473-Akt (catalog number 4051L), β-catenin (catalog number 9582S), pS522-β-catenin (catalog number 9566S), pS33/37/41-β-catenin (catalog number 9561S) , pT202/Y204-ERK1/2 (catalog number 9101S), ERK1/2 (catalog number 9102S), MMP2 (catalog number 87809S), MMP9 (catalog number 13667S), and PP2A (catalog number 2259S) were purchased from Cell Signaling Technology. . Antibodies against tubulin (catalog number ab11304) and PP1A (catalog number MAB3000) were purchased from Abcam and R&D Systems (Minneapolis, MN, USA), respectively. Anti-mouse secondary antibody (Cat. No.115-035-003) and anti-rabbit antibody (Cat. No.211-002-171) were obtained from Jackson Immuno Research Laboratories Inc. (West Grove, PA, USA). .

<Example 2> Culture and isolation process of endometrial stromal cells (HESC)

Human endometrial stromal cells (HESC) were isolated from the endometrium of 25 premenopausal women aged 45-50 years through hysterectomy.

Sixteen endometrial stromal cells (HESC) were isolated and incubated at 37° in Dulbecco's modified Eagle's medium (DMEM, Welgene) containing 1 g/L glucose (Welgene, Gyeongsangbuk-do, Korea) and 10% fetal bovine serum (FBS). Grown at C and 5% CO2.

Then, it was separated from the plate using 1% penicillin/streptomycin (P/S, 10,000 U/mL, Welgene) and 0.05% trypsin Ethylenediaminetetraacetic acid (EDTA) (Welgene). In vitro cells were expanded until 100% confluence and decidualized by exposure to DMEM containing 10% FBS, 1% P/S, and 0.5mM 8-Br-cAMP, with medium changed every other day. proceeded.

<Example 3> Cell lysis process, immunoprecipitation process, and western blot process

The dropped cells were washed with phosphate-buffered saline (PBS), lysed using lysis buffer (Cat. No. 9303, Cell Signaling Technology), and then microcentrifuged at 13,000 x g for 10 minutes at 4°C. The supernatant was collected and boiled in sodium dodecyl sulfate (SDS) sample buffer for 3 min. Proteins were separated using SDS-polyacrylamide gel electrophoresis. For immunoprecipitation of β-catenin, RIPA buffer I (50mM Tris-HCl, pH 7.4, 150mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40, protease inhibitor cocktail (Cat. No.P8340, Sigma)) were then microcentrifuged at 13,000 x g for 10 minutes at 4°C.

Afterwards, the supernatant of the centrifuged solution was collected, immunoprecipitated with β-catenin in RIPA buffer overnight at 4°C, and then incubated with protein G-agarose for 1 hour at 4°C.

The resulting beads were washed three times using RIPA buffer I and analyzed using SDS-polyacrylamide gel electrophoresis. The resolved gel was transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Each membrane was blocked for 30 min in phosphate-buffered saline Tween (PBST) with 5% (w/v) non-fat skim milk at room temperature and then incubated with primary antibody in PBST with 5% (w/v) non-fat skim milk. Together, they were incubated at -4°C overnight. Afterwards, horseradish peroxidase-conjugated secondary antibodies were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). Western blot band intensities were quantified by densitometry of X-ray film images using ImageJ.

<Example 4> qRT-PCR performance process

Total RNA was extracted from isolated HESCs using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and performed according to the manufacturer's instructions (Enzynomics, Daejeon).

Next, qRT-PCR was performed using TOPreal ^TM qPCR 2X PreMIX (SYBR Green with high ROX, Enzynomics), CFX384 C1000 thermal cycler (Bio Rad, Hercules, CA, USA), and the following primers. MMP2 (primerbank ID 189217851c1), MMP9 (primerbank ID 74272286c3), TIMP1 (primerbank ID 73858576c1), TIMP2 (primerbank ID 73858577c2), TIMP3 (primerbank ID 75905820c1), GPR146 (primerbank ID 41349503c3) and glyceraldehyde 3- phosphate dehydrogenase GAPDH.

Gene expression levels were normalized to that of human GAPDH.

<Example 5> Process of conducting wound-healing scratch assay in vitro

Isolated HESCs were cultured in DMEM containing 1.0 g/L glucose, 10% FBS, and 1% P/S at 37°C and 5% CO ₂ for one week. After replacing the medium with DMEM medium without serum, HESCs were cultured at 37°C and 5% CO ₂ for 18 hours to minimize cell proliferation, and a scratch was made with a sterile plastic pipette tip (200 μL). Migration analysis of HESCs and JEG-3 was performed after treatment with 50 nM C-peptide. After 24 hours, cells were stained using CytoPainter Cell Tracking Staining Kit and observed using a fluorescence microscope with a 4X objective (Olympus CKX3-Houn Microscope, Olympus, Tokyo, Japan).

ImageJ was used to calculate the number of migrated cells and the distance traveled.

<Example 6> Transwell and invasion assay

Isolated HESCs were cultured with 0.05% trypsin-EDTA (Welgene) and suspended in serum-free medium (DMEM containing 1.0 g/L glucose and 1% P/S). Transwell migration assays were performed in 24-well cell culture inserts (pore size 8 μm).

The process involved adding 300 μL cell suspension (5 -peptide) was added to the lower chamber. After culturing in an environment of 37°C and 5% CO ₂ for 24 hours, cells on the upper surface of the chamber were removed by wiping with a cotton swab. The migrated cells were fixed in 10% formaldehyde for 10 minutes and stained with DAPI. Images were captured using a fluorescence microscope (Olympus CKX3-Houn, 4X), and migrated cells were counted in five randomly selected fields using ImageJ.

Cell invasion was assessed using the QCM Collagen Cell Invasion Assay Kit (Cat no. ECM551, Millipore, Bedford, MA, USA).

300 μL cell suspension and 500 μL C-peptide-containing growth medium were added to the collagen-coated insert and lower chamber, respectively. After 24 hours of incubation in an environment of 37°C and 5% CO ₂ , non-invading cells were removed using a cotton swab, and the invaded cells were fixed in 10% formaldehyde for 10 minutes, stained, and treated with extract buffer. Manufacturer's protocol. The number of invading cells was quantified by measuring optical density at 560 nm.

<Example 7> Nuclear fractionation process

Isolated HESCs were grown to 100% confluence in DMEM containing 1g/L glucose, 10% FBS, and 1% P/S, then cultured in serum-free medium for 48 hours, and Akti After pretreatment with (1 μM) and okadaic acid (OA) (30 nM) for 1 hour, 50 nM C-peptide was treated for 10 minutes. Cells were then washed three times using cold PBS and incubated in 300 μL cytosolic protein extraction buffer (5 mM KCl, 5 mM HEPES, 0.05 mM ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA), 0.05 mM EDTA. and 0.075% NP-40). After lysis, cells were incubated on ice for 30 min with shaking every 10 min and then centrifuged at 1500 × g for 10 min at 4°C.

After centrifugation, the cytoplasmic supernatant was transferred to a new tube. Meanwhile, the remaining nuclear pellet was washed three times using cytosolic protein extraction buffer and incubated in 100 μL nuclear protein extraction buffer (0.25 M EDTA, 0.5 M Tris-HCl pH 7.4, 2.5 M NaCl, 5% Na deoxycholate, 5% SDS, and 5% Triton X-100). They were then incubated on ice for 30 minutes with shaking every 10 minutes. Nuclear fractions were collected by centrifugation at 13,000 x g for 30 min at 4°C. Cytoplasmic and nuclear extracts were then boiled in 5× SDS sample buffer for 5 min and subjected to Western blotting as previously described.

<Example 8> Lentivirus-mediated shRNA (short hairpin RNA) production

shRNA for β-catenin (CTNNB1) was pLKO.1-puro vector (MISSION shRNA) and was obtained from Sigma-Aldrich. The clone IDs were TRCN0000314991 for shCTNNB1-1 and TRCN0000314921 for CTNNB1-2.

<Example 9> Zymography analysis process

First, isolated HESCs were grown to 100% confluence in DMEM containing 1g/L glucose, 10% FBS, and 1% P/S, and cultured in serum-free medium for 48 hours. , C-peptide (50 nM) was treated for 24 hours. Cultures were centrifuged (5 min at 4°C) to remove cells and debris and concentrated to 500 μL using an Amicon® Ultra-15 centrifugal filter device (Cat. No. UFC901024, Merck, Darmstadt, Germany). Then, 500 μL of clarified conditioned medium was mixed with 30 μL of 50% gelatin-agarose beads (Cat. No. 6025-10, Adar Biotech, Rehovot, Israel) slurry. Afterwards, it was rotated at 4°C for 3 hours.

The beads thus obtained were washed twice using TBS-B (50mM Tris-HCl, pH 7.5, 150mM NaCl, 5mM CaCl ₂ , 0.02% Brij-35), 60㎕ of 1ХSDS sample buffer was added, and the reaction mixture was Analyzed using 10% SDS-polyacrylamide-0.1% gelatin gel electrophoresis (SDS-PAGE). Gel _was incubated with denaturing solution (2.5% v/v Triton did. After gently stirring for 30 minutes, it was replaced with new developing buffer and incubated at 37°C for 16 hours. Gels were stained with staining solution (0.5% Coomassie blue R-250, 5% methanol, 10% acetic acid in dH ₂ O) for 1 hour and then with destaining solution (10% methanol, 5% acetic acid in dH ₂ O). did. The gelatinolytic active zone appeared as a clear, sharp band on a blue background. The intensity of MMP2/9 was measured by ImageJ. Each MMP9 level (monomer and dimer) was calculated by normalizing to the MMP2 level and then dividing by the total MMP9 level.

<Example 10> Chromosome immunoprecipitation (ChIP)

Cells were treated with 0.75% formaldehyde for 30 min and lysed in ChIP lysis buffer (50mM Tris-HCl, pH 7.4, 1% Igepal CA-630 (Cat. No. I3021, Sigma), 0.25% sodium deoxycholate, 150mM NaCl; 1mM EDTA, 0.1% SDS, 0.5mM dithiothreitol (DTT), 5mM Na-butyrate, protease inhibitor cocktail (catalog no. P8340, Sigma), phosphatase inhibitor cocktail I (catalog no. P2850, Sigma) and phosphatase inhibitor cocktail II ( Cells were destroyed by sonication in the mixed solution (catalog number P5726, Sigma). Then, the nuclei were harvested and destroyed by sonication again.

Lysates were incubated overnight with anti-β-catenin antibody (8480S, Cell Signaling Technology) and precipitated using protein A/G-PLUS-agarose beads (SC-2003, Santa Cruz Biotechnology, Dallas, TX, USA). I ordered it. The resulting beads were washed with ChIP lysis buffer, and the bound DNA was eluted in elution buffer (1% SDS and 100mM NaHCO3).

Finally, RNA and proteins from the eluted solution were removed using RNase and proteinase K, and DNA was further purified using phenol-chloroform extraction. The resulting DNA was used for PCR analysis using specific primers (Table 1).

primer name order MMP9-ChIP-F 5'-TGTCCCCTTTACTGCCCTGA-3' MMP9-ChIP-R 5'-GAAGACTCCCTGAGACTT-3' TIMP1-ChIP-F 5'-CCCATATTTCCTCATCCGTA-3' TIMP1-ChIP-R 5'-CTTACCCAGGATCACAGAG-3' TIMP3-ChIP-F 5'-CCCAGTCATGTCCAACCCA-3' TIMP3-ChIP-R 5'-CAGCTGGCTATGATATATG-3'

<Experimental Example 1> Determination of whether C-peptide promotes cell migration in endometrial stromal cells

Effect of C-peptide on the mobility of human endometrial stromal cells (HESC) that are differentiated with 8-Br-cAMP and decidualized and undifferentiated endometrial stromal cells that are not decidualized To confirm, wound recovery scratch analysis was performed according to the presence or absence of C-peptide.

In the absence of C-peptide treatment, decidualized cells moved faster than non-decidualized cells (Figure 1a).

Looking at Figure 1a, treatment with C-peptide increases cell mobility in cells that have not decidualized, further reducing the cell-free area and increasing the number of migrating cells. However, for decidualized cells, the cell mobility did not change depending on the presence or absence of C-peptide treatment, so there was no change in the cell-free area and the number of moving cells. Through this, it was confirmed that treatment with C-peptide increased the mobility of non-decidualized cells, but did not change the mobility of decidualized cells.

To confirm the difference in cell mobility depending on whether decidualization occurred, a transwell assay was additionally performed (Figure 1b).

As a result, treatment with C-peptide significantly increased the mobility of cells that were not decidualized, but did not increase the mobility of cells that were decidualized (Figure 1b).

Similar to the results in Figure 1B, the invasiveness of non-decidualized cells was improved on collagen-coated Transwell filters (Figure 1C).

Additionally, a wound-recovery scratch assay was performed to determine the effect of C-peptide on cell motility in HESCs isolated from three different human samples. All three different HESCs isolated from different people showed a decrease in the cell-free area and an increase in the number of migrating cells by treatment with C-peptide, so the increase in HESC mobility by C-peptide was observed in HESCs isolated from tissues. It was confirmed that this was a common phenomenon (Figure 1d).

Here, to further investigate whether C-peptide regulates the migration and invasion of the elixir film, transwell analysis and invasion assay were performed on JEG-3 cells.

C-peptide improved the mobility of JEG-3 cells (Figure 1e) and increased the number of invaded JEG-3 cells on collagen-coated transwell filters (Figure 1f).

These results confirmed that C-peptide promotes the migration of non-decidualized endometrial stromal cells (HESC) and JEG-3 cells.

<Experimental Example 2> Determination of whether C-peptide controls β-catenin phosphorylation and nuclear localization in an Akt or protein phosphatase-dependent manner

To analyze the underlying mechanism of C-peptide-enhanced mobility of decidualized endometrial stromal cells (HESCs), we analyzed the effects of C-peptide on ERK1/2 and Akt in both decidualized and decidualized HESCs. did.

C-peptide treatment significantly enhanced the phosphorylation of Akt (Ser 473) but not ERK1/2 in undifferentiated HESCs (Figure 2a).

Additionally, C-peptide increased the phosphorylation of β-catenin at Ser 552 and decreased it at Ser 33/37/41. However, phosphorylation of Akt and β-catenin remained unchanged in decidualized endometrial stromal cells (HESCs) ( Fig. 2A ).

Consistent with these results, the mRNA level of G-protein coupled receptor 146 (GPR146), a putative C-peptide receptor, was higher in non-decidualized HESCs than in decidualized HESCs. As a result, it was confirmed that pre-differentiation ESCs showed high reactivity to C-peptide due to the high expression level of GPR146 (Figure 2b).

Meanwhile, pretreatment with an Akt inhibitor (Akti) reduces β-catenin phosphorylation at Ser 552 (Figure 2c), indicating that Akt plays a role in assisting phosphorylation of β-catenin at Ser 552.

Next, to identify the protein phosphatase (PP) responsible for dephosphorylation of β-catenin in C-peptide treated cells, the role of C-peptide in the interaction between β-catenin and PP2A was investigated. An experiment was conducted to confirm the effect.

PR55α, a regulatory subunit of PP2A, is known to specifically bind to β-catenin and regulate PP2A-mediated β-catenin dephosphorylation.

C-peptide treatment reduced the interaction between β-catenin and PP2A (Figure 2d), which suggests that PP2A is not involved in the dephosphorylation of β-catenin at Ser 33/37/41 induced by C-peptide treatment. indicates no.

On the other hand, OA, a PP inhibitor, restored the phosphorylation of β-catenin Ser 33/37/41, which was reduced by C-peptide treatment (Figure 2e). Consistently, C-peptide treatment did not change phosphorylation at β-catenin Ser 33/37/41 in PP1A-deficient cells, but in control scramble shRNA-infected cells, C-peptide treatment did not alter phosphorylation at β-catenin Ser 33/37/41. decreased phosphorylation (Figure 2f).

C-peptide increased the amount of β-catenin in the nucleus without decreasing the amount of β-catenin in the cytoplasm (Figure 3a).

However, pretreatment with Akti and OA decreased the nuclear translocation of β-catenin (Figures 3b and 3c). These results suggest that C-peptide regulates the phosphorylation of β-catenin in an Akt- or PP-dependent manner, thereby increasing the phosphorylation of β-catenin. It indicates that it promotes nuclear translocation.

<Experimental Example 3> Determination of whether β-catenin, Akt, and PP1 are essential for C-peptide-induced cell migration in human endometrial stromal cells (HESC).

To determine the role of Akt and PP1 in C-peptide-induced cell migration, we analyzed the effects of Akt or PP1 inhibitors on C-peptide-treated endometrial stromal cells (HESCs).

It can be seen that cell migration induced by C-peptide is reduced when pretreated with Akti or OA, as cell migration was reduced in both the wound-recovery scratch assay (Figure 4a) and the transwell assay (Figure 4b).

A decrease in PP1 expression was confirmed by transducing shRNA of PPP1C (Figure 4c), and wound-recovery scratch analysis was performed on cells in which PP1 was knocked down. As a result, it was confirmed that knockdown of PP1 significantly reduced cell mobility through an increase in cell-free area (Figure 4c).

In addition, β-catenin knockdown was confirmed by transduction of shRNA (Figure 4d), and scratch analysis and transwell analysis were performed using the cells in which β-catenin was knocked down. The effect on mobility was confirmed. As shown in Figures 4e and 4f, it was confirmed that knockdown of β-catenin reduced mobility.

In addition, it was confirmed that when β-catenin was inhibited by treatment with PNU-74654, a β-catenin inhibitor, the increase in cell mobility caused by C-peptide was also suppressed. In addition, it was confirmed that pretreatment with PNU-74654 blocked the invasion of endometrial stromal cells (HESC) by C-peptide (Figure 4h). These results indicate that Akt, PP1, and β-catenin are important regulators of C-peptide-mediated cell mobility of endometrial stromal cells (HESC).

<Experimental Example 4> Confirm whether C-peptide regulates the expression of MMP9 and TIMP1/3 through β-catenin

Expression and activation of MMPs are required for cell migration through the extracellular matrix (ECM).

As shown in Figures 5a and 5b, C-peptide significantly increased the expression of MMP9, but did not increase the expression of MMP2. Through gelatin zymography analysis to measure the activity of MMP9, it was confirmed that the activity of MMP9 increased. In particular, the activity of MMP9 dimers was significantly increased in the conditioned medium of C-peptide treated cells (Figure 5c).

Based on the results of increased expression of MMP9, the effect of MMP9 inhibitor was confirmed to determine the effect of inhibition of MMP9 activity on the increase in cell mobility by treatment with C-peptide. Pretreatment with an MMP9 inhibitor completely inhibited the cell mobility increased by C-peptide (Figure 5D), indicating that MMP9 plays an important role in C-peptide-induced HESC migration.

In contrast, C-peptide treatment significantly reduced the mRNA expression of TIMP1 and TIMP3, but not that of TIMP2 (Figure 5e). In addition, pretreatment with Akti, OA, and PNU-74654 significantly reduced MMP9 expression and restored TIMP1 and TIMP3 expression in C-peptide-treated endometrial stromal cells (HESC) (Figure 5f).

When β-catenin is translocated to the nucleus, it directly interacts with TCF/LEF to regulate the expression of target genes. Putative binding sites for TCF-1 can be identified in the proximal promoter regions of MMP9, TIMP1, and TIMP3 (Figure 5g).

Through ChIP analysis using a β-catenin antibody, it was confirmed that β-catenin forms a bond in the putative binding region of MMP9, TIMP1, and TIMP3, and that the bond increases by C-peptide treatment, and this bond is β- It decreased when catenin was knocked down (Figure 5g). These results confirmed that C-peptide directly regulates β-catenin and regulates cell migration of endometrial stromal cells (HESC) by regulating the expression of MMP9, TIMP1, and TIMP3.

<110> Gachon University of Industry Academic Cooperation Foundation <120> A method for diagnosing endometrial-related diseases and screening method for endometrial-related diseases using the mechanism of change in the mobility of endometrial stromal cells treated with C-peptide <130> 2021P-06-015 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223>MMP9-ChIP-F <400> 1 tgtccccttt actgccctga 20 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> MMP9-ChIP-R <400> 2 gaagactccc tgagactt 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223>TIMP1-ChIP-F <400> 3 cccatatttc ctcatccgta 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223>TIMP1-ChIP-R <400> 4 cttaccagg atcacagag 19 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TIMP3-ChIP-F <400> 5 cccagtcatg tccaaccca 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TIMP3-ChIP-R <400> 6 cagctggcta tgatatatg 19

Claims (14)

1) cultivating non-decidualized endometrial stromal cells from a patient suspected of having an endometrial-related disease and non-decidualized endometrial stromal cells from a healthy person, respectively;
2) treating the endometrial stromal cells of step 1) with C-peptide,
3) measuring the activity of MMP9, GPR146, TIMP1, and TIMP3 proteins, which are substances related to the mobility of endometrial stromal cells, or the expression level of genes encoding these proteins; and
4) Among the substances related to the mobility of endometrial stromal cells in step 3), the activity of MMP9 and GPR146 proteins or the expression level of genes encoding these proteins are increased compared to endometrial stromal cells of healthy people, and TIMP1 and TIMP3 proteins When the activity or expression level of the gene encoding the protein is reduced compared to the endometrial stromal cells of a healthy person, determining that it is an endometrial-related disease; for determining an endometrial stromal-related disease, including As a method of providing information, the endometrial-related diseases include infertility, subfertility, endometriosis, adenomyosis, endometrial cancer, endometrial stromal sarcoma, and endometrial intraepithelial neoplasia. (endometrial intraepithelial neoplasia, choriocarcinoma, endometritis, endometrial ectopia, endometrial glandular hyperplasia, endometrial atrophy, endometrial stromal disease) A method of providing information for determining endometrial stromal-related diseases selected from the group consisting of romatosis, hydatidiform mole, and villous adenoma.
According to paragraph 1,
Patients suspected of having an endometrial-related disease in step 1) have endometrial stromal disease, which is characterized by increased mobility and invasiveness of non-decidualized endometrial stromal cells compared to decidualized endometrial stromal cells. ) A method of providing information for determining related diseases.
According to paragraph 1,
The expression level of the gene in step 3) can be measured using qRT-PCR, competitive RT-PCR, real-time RT-PCR, and RNase protection assay (RPA). A method of providing information for determining endometrial stromal-related diseases, characterized by using one or more of the following methods: , Northern blotting, and DNA chip.
According to paragraph 1,
The protein activity in step 3) can be measured using Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, and Zymo. A method of providing information for the determination of endometrial stromal-related diseases, characterized by using one or more methods of photography, FACS, or protein chip.
delete According to paragraph 2,
The uterus is characterized in that changes in the mobility and invasiveness can be confirmed compared to a normal control group through one or more selected from the group consisting of a transwell assay, an invasion assay, and a wound-healing scratch assay. A method of providing information for determining endometrial stromal-related diseases.
1) Culturing endometrial stromal cells that have not undergone decidualization and then treating them with C-peptide;
2) After treating the test substance with the endometrial stromal cell culture medium treated with C-peptide in step 1), the activity of the proteins MMP9, GPR146, TIMP1 and TIMP3, which are substances related to the mobility of endometrial stromal cells, or the above proteins Measuring the expression level of the gene encoding; and
3) Among the substances related to the mobility of endometrial stromal cells in step 2), the activity of MMP9 and GPR146 proteins or the expression level of genes encoding the proteins increases compared to the untreated control group, and the proteins of TIMP1 and TIMP3 A screening method for a treatment for diseases related to the endometrial stromal, comprising the step of selecting a test substance in which the activity or expression level of the gene encoding the protein decreases compared to an untreated control group, wherein the endometrial stromal-related diseases include infertility, subfertility, endometriosis, adenomyosis, endometrial cancer, endometrial stromal sarcoma, endometrial intraepithelial neoplasia, and choriocarcinoma. (choriocarcinoma), endometritis, endometrial ectopia, endometrial glandular hyperplasia, endometrial atrophy, endometrial tromatosis, hydatidiform mole ), a screening method for a treatment for diseases related to the endometrial stromal, selected from the group consisting of villous adenoma.
In clause 7,
In step 2), endometrial stromal cells are involved in diseases related to the endometrial stromal, characterized in that the mobility and invasiveness of non-decidualized endometrial stromal cells increase compared to decidualized endometrial stromal cells. Methods for screening therapeutics.
delete In clause 7,
The expression level of the gene in step 2) can be measured using qRT-PCR, competitive RT-PCR, real-time RT-PCR, and RNase protection assay (RPA). A screening method for a treatment for endometrial stromal-related diseases, characterized by using one or more of the following methods: , Northern blotting, and DNA chip.
In clause 7,
The protein activity in step 2) can be measured using Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, and Zymo. A screening method for a therapeutic agent for endometrial stromal-related diseases, characterized by using one or more methods among photography, FACS, or protein chip.
delete According to clause 8,
The uterus is characterized in that changes in the mobility and invasiveness can be confirmed compared to the untreated control group through one or more selected from the group consisting of a transwell assay, an invasion assay, and a wound-healing scratch assay. Screening method for treatments for endometrial stromal-related diseases. delete