Molecular marker related to screening of spontaneous abortion risk and pregnancy maintenance and application of molecular marker

Abstract

The invention relates to a molecular marker related to screening of spontaneous abortion risk and pregnancy maintenance and application of the molecular marker, and belongs to the technical field of biological medicine. The molecular marker provided by the invention is a BRDT gene and / or a BRDT protein, experiments prove that the BRDT gene is expressed in both early pregnancy and full-month placenta, the molecular marker provided by the invention can be specifically combined with a promoter region of a gestation immune tolerance key molecule HLA-C, and meanwhile, the BRDT gene and / or the BRDT protein can be used for detecting the immune tolerance key molecule HLA-C in early pregnancy and full-month placenta. The expression level of BRDT and HLA-C in a patient with spontaneous abortion is obviously lower than that of a healthy pregnancy sample, which indicates that BRDT can play a role in pregnancy maintenance. The invention not only discloses the expression of BRDT in placenta, but also shows the key effect of BRDT in regulating and controlling the expression of HLA-C. The molecular marker provided by the invention can be used for screening or diagnosing the risk of spontaneous abortion, and provides a new insight for the potential treatment strategy of reproductive biology and pregnancy-related diseases.

Description

Technical Field

[0001] The present invention relates to a molecular marker for screening the risk of spontaneous abortion and pregnancy maintenance and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Spontaneous abortion (SA) is one of the most common pregnancy complications in obstetrics and gynecology. Abortion is harmful to women in many ways, and may have serious effects on both their physical and mental health. Abortion not only causes damage to reproductive organs, endocrine disorders, increases the risk of uterine infection, and damages mental health, but is also closely related to adverse obstetric outcomes. As the number of abortions increases, the risk of perinatal complications also increases.

[0003] The potential mechanism of spontaneous abortion is a complex process involving multiple factors such as the embryo, mother, father, and environment. Among them, embryonic chromosomal abnormalities are the most common cause of spontaneous abortion. Maternal factors, such as endocrine abnormalities, reproductive organ abnormalities, systemic diseases, and immune dysfunction, are also important causes of abortion. In addition, paternal sperm abnormalities, environmental factors, and bad living habits may also lead to the occurrence of abortion. In recent years, placental immunity has received widespread attention as a new perspective in the study of spontaneous abortion. Abnormal placental immune responses, such as failure of immune tolerance, overactivation of inflammatory responses, and immune cell dysfunction, can lead to placental dysfunction and trigger spontaneous abortion. At present, the incidence of spontaneous abortion continues to increase, but the molecular mechanisms of maternal-fetal immune tolerance and immune protection in spontaneous abortion are still unclear, resulting in certain difficulties in the prediction and prevention of spontaneous abortion.

[0004] Therefore, there is an urgent need to develop new molecular markers and molecular targeted drugs for screening the risk of spontaneous abortion and maintaining pregnancy from the perspective of molecular biology, so as to provide new ideas for the clinical diagnosis and treatment of patients with pregnancy-related diseases. Summary of the invention

[0005] In view of some deficiencies in the prior art, the purpose of the present invention is to provide a molecular marker for screening the risk of spontaneous abortion and pregnancy-related and its application. Specifically, the present invention provides a molecular marker BRDT for screening the risk of spontaneous abortion and pregnancy-related and its application in view of the current lack of understanding of the specific molecular mechanism of maintaining normal human pregnancy and regulating immune tolerance during pregnancy, the lack of new molecular targeted drugs for pregnancy-related diseases, and the inability of existing drugs to effectively prevent the occurrence of pregnancy diseases such as spontaneous abortion.

[0006] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0007] The present invention first provides a molecular marker for screening the risk of spontaneous abortion and maintaining pregnancy, wherein the molecular marker includes a BRDT gene and / or a BRDT protein, wherein the nucleotide sequence of the BRDT gene is shown in SEQ ID No: 1, and the amino acid sequence of the BRDT protein is shown in SEQ ID No: 6.

[0008] Wherein, the primers of the molecular marker are shown as SEQ ID No: 2 and SEQ ID No: 3.

[0009] The present invention also provides the use of the BRDT gene and / or BRDT protein as a molecular marker in screening the risk of spontaneous abortion and maintaining pregnancy.

[0010] Furthermore, the expression level of the BRDT gene or BRDT protein is positively correlated with the immune tolerance during pregnancy, and the expression level of the BRDT gene or BRDT protein is negatively correlated with the risk of miscarriage during pregnancy.

[0011] Furthermore, the BRDT gene or BRDT protein is used to promote the expression of HLA-C.

[0012] The present invention also provides a product for improving the immune tolerance during pregnancy and / or reducing the risk of spontaneous abortion during pregnancy, and the product includes a product for promoting the expression of the molecular marker.

[0013] The present invention also provides a primer, which is used for detecting and screening molecular markers related to the risk of spontaneous abortion and pregnancy maintenance. The nucleotide sequences of the primers are shown in SEQ ID No: 2 and SEQ ID No: 3.

[0014] The present invention also provides the use of the primer in preparing a product for screening the risk of spontaneous abortion and maintaining pregnancy, wherein the product comprises a kit.

[0015] The present invention also provides a product for screening the risk of spontaneous abortion and maintaining pregnancy, and the product comprises the primer.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The present invention first confirms that BRDT gene and / or BRDT protein can be used as a molecular marker for spontaneous abortion risk and pregnancy maintenance. The present invention first uses single-cell RNA sequencing, qPCR, Western Blot and immunohistochemistry and other technologies to discover and confirm the high expression of BRDT protein in placental tissue during pregnancy, filling the gap in BRDT in the field of placental research. Through the analysis of placental samples from patients with spontaneous abortion, it was found that the expression levels of BRDT and HLA-C were significantly reduced, indicating that BRDT is a key regulatory molecule for pregnancy maintenance, and its abnormal expression is closely related to spontaneous abortion. Based on this, the present invention proposes that BRDT gene and / or BRDT protein can be used as a new molecular marker for assessing the risk of spontaneous abortion and pregnancy monitoring, providing a new target for maternal-fetal immune regulation, and providing a scientific basis for the development of prevention and intervention measures for spontaneous abortion, which has important clinical application value and broad market prospects.

[0018] (2) The present invention has discovered for the first time the molecular mechanism by which BRDT regulates HLA-C expression. The present invention has revealed for the first time that BRDT regulates its expression level in the trophoblast cell line JEG3 by directly binding to the HLA-C promoter region, thereby regulating the immune tolerance at the maternal-fetal interface. The present invention has clarified the molecular mechanism by which BRDT regulates HLA-C expression, indicating that HLA-C is an important molecule for maintaining the immune balance during pregnancy, and that the regulatory effect of BRDT is crucial for maintaining pregnancy. It can be used as a new molecular target for immune regulation during pregnancy, and has broad clinical application prospects and market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 BRDT expression analysis in single-cell RNAseq data of placenta / chorionic villus samples.

[0020] Figure 2 The qPCR results of BRDT gene expression in normal full-term placenta.

[0021] Figure 3 The results of Western Blot analysis are for the expression of BRDT protein in the placenta of early pregnancy and normal full-term pregnancy.

[0022] Figure 4 These are the immunohistochemical results of BRDT protein expression in the placenta of early pregnancy and normal full-term pregnancy.

[0023] Figure 5 The effect of BET inhibitor on the expression level of HLA-C gene in trophoblast JEG3 cells.

[0024] Figure 6 These are the test results for the expression levels of the corresponding target genes and HLA-C genes in trophoblast JEG3 cells after treatment with siRNA.

[0025] Figure 7 The effect of JQ1 on the expression level of HLA-C gene in primary EVT cells.

[0026] Figure 8 This is the result of enrichment analysis of some differentially expressed genes in JEG3 cells after treatment with JQ1 through RNA-seq experiment.

[0027] Fig. 9 The figure shows the experimental results of the enrichment efficiency of HLA-C promoter sequence in JEG3 cells.

[0028] Fig.10 The results show the expression levels of BRDT and HLA-C in placental EVT cells of patients with healthy pregnancy and spontaneous abortion (SA).

[0029] Fig.11 This is a correlation analysis diagram between BRDT and HLA-C in placental tissue EVT cells. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the technical solution of the present invention, the preferred embodiments of the present invention are described in detail below, but the following embodiments do not limit the protection scope of the present invention.

[0031] In the embodiments of the present invention, those that are not described in detail are all completed by conventional experimental methods. Those processes involved in the embodiments that are not described in detail are all understandable and easily implementable by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore are not described in detail.

[0032] It should be noted that in the following embodiments:

[0033] (1) The JEG3 cells used were purchased from Saiku Biotechnology. The listed JEG3 cell lines were cultured according to existing technologies. All cell lines were identified by STR and verified by PCR detection kits to exclude mycoplasma contamination. They were cryopreserved in liquid nitrogen and revived for subsequent experiments. JEG3 cells express human chorionic gonadotropin (hCG), human chorionic somatotropin (placental lactogen), and progesterone, which are commonly used for the diagnosis of pregnancy.

[0034] (2) Human placental tissue was obtained from the Reproductive Center of Sun Yat-sen Memorial Hospital, Sun Yat-sen University and has passed ethical review.

[0035] (3) Representative results from repeated biological experiments were selected and presented in the accompanying drawings of the specification. The data were presented as mean±SD or mean±SEM as specified in the drawings. The data were analyzed using GraphPad Prism 10.0 software. The statistical method of t-test was used to compare the mean differences between two or more groups. p<0.05 indicated a statistical difference, p<0.01 indicated a significant statistical difference, *** represented p<0.001, ** represented p<0.01, * represented p<0.05, and ns represented no significant difference.

[0036] Example 1: Expression analysis of BRDT in human placenta samples

[0037] The placentas involved in the embodiments of the present invention were obtained from the Reproductive Center of Sun Yat-sen Memorial Hospital, Sun Yat-sen University, and three groups of human placentas discarded by women with full-term pregnancies were obtained, denoted as: donor 1, donor 2, donor 3, donor 4 and donor 5.

[0038] The inclusion and exclusion criteria for placenta samples were as follows:

[0039] Inclusion criteria: Macroscopic observation of the placenta: no obvious signs of infection.

[0040] Exclusion criteria: ① Those who underwent assisted reproductive technology to conceive; ② The mother had a known maternal disease that may affect abortion, such as severe infection, high fever disease, severe anemia or heart failure, TORCH (an abbreviation for a group of pathogens, including T for Toxoplasma gondii, O for Others (mainly including Treponema pallidum, etc.), R for Rubella virus, C for Cytomegalovirus, H for Herpes simplex virus (Herpessimplex virus, etc.) virus) infection, etc.; ③ maternal genital abnormalities, such as uterine malformation, intrauterine adhesions, etc.; ④ maternal endocrine abnormalities, such as hyperlactinemia, polycystic ovary syndrome, hypothyroidism, poor blood sugar control in diabetes, etc.; ⑤ maternal strong stress and bad habits, such as severe physical and psychological adverse stimulation during pregnancy (surgery, direct impact on the abdomen or anxiety, fear and other mental trauma) and excessive smoking, alcoholism, etc.; ⑤ maternal history of exposure to adverse toxins and drugs; ⑥ intrauterine infection during pregnancy; ⑦ clear maternal immune dysfunction, such as antiphospholipid syndrome, systemic lupus erythematosus, positive antinuclear antibodies, etc.

[0041] (1) Single-cell RNAseq analysis of BRDT gene expression in placenta samples:

[0042] Single-cell RNAseq data with accession number GSE89497 were retrieved from publicly available data sets catalogued in the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ), and the single-cell RNAseq data included single-cell transcriptome profile data of stromal cells and trophoblastic cells at 8 and 24 weeks of human placental gestation. The single-cell RNA-seq data were read by RStudio, and genes with an expression level of "0" in the original data were filtered out. The weighted truncated mean (TMM) normalization method of the logarithmic expression ratio was adopted by the "edgeR" analysis package, and the sample data were analyzed for expression, and violin plots were drawn for the BRDT gene expression levels of trophoblastic cells at 8 weeks (denoted as EVT_8w) and 24 weeks (denoted as EVT_24w) of placental gestation.

[0043] The results are as follows Figure 1 As shown in the figure, the expression level analysis of the BRDT gene in single-cell RNA-seq data found that the BRDT gene was expressed in the placental tissue, and the relative expression level of the BRDT gene in the placental tissue in the early pregnancy (8 weeks) was higher than that in the placental tissue in the second pregnancy (24 weeks).

[0044] The nucleotide sequence of the BRDT gene is shown in SEQ ID No: 1:

[0045] SEQ ID No: 1:

[0046]

[0047] (2) qPCR verification of BRDT expression in placenta samples:

[0048] The collected placental tissue was cut into small pieces and then added to Triozol in a grinder for tissue grinding. After centrifugation, the supernatant was added with chloroform to extract RNA, precipitated with isopropanol, washed with 75% ethanol several times, and then dissolved with nuclease-free water. The quantitatively extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from Nanjing Novozyme Biotechnology Co., Ltd.), and cDNA was used as a template for real-time fluorescence quantitative PCR experiments. According to the instructions of 2×Universal SYBR GreenFastqPCR Mix, 20 μL qPCR system was prepared and qPCR was performed to detect the expression level of BRDT, and GAPDH was used as an internal reference gene.

[0049] RT-qPCR preparation system:

[0050] Components volume 2×ChamQ Universal SYBR qPCR Master Mix 10.0μL Primer 1 (10 μM) 0.4μL Primer 2 (10 μM) 0.4μL Template cDNA 2.0μL <![CDATA[ddH 2 The]]> 7.2μL

[0051] RT-qPCR reaction procedure:

[0052]

[0053] The primer sequences used for RT-qPCR detection (i.e. Primer1 and Primer2 in the configuration system) are:

[0054] BRDT:

[0055] F:5'CACCCAGCGCAACAGAAAAA' (SEQ ID No: 2),

[0056] R:5'CCTTTTTGTAACTTGGGCCGC' (SEQ ID No: 3);

[0057] GAPDH (NCBI accession number: Gene ID: 2597):

[0058] F:5'GAAGGTGAAGGTCGGAGT' (SEQ ID No: 4),

[0059] R: 5'GAAGATGGGTGATGGGATTTC' (SEQ ID No: 5).

[0060] The qPCR results of BRDT gene expression in normal full-term pregnancy placenta are as follows Figure 2As shown in the figure, it can be seen that the BRDT gene is expressed in normal full-term placental tissue. Although the expression levels of BRDT in each group of placentas vary greatly due to individual differences, this result still verifies that BRDT is expressed in placental tissue.

[0061] (3) Verify the expression of BRDT protein in placenta samples using Western Blot and immunohistochemistry:

[0062] First, Western Blot was used to verify the expression of BRDT in placenta samples:

[0063] The collected placental tissues were cut into small pieces and then added to RIPA tissue / cell lysis buffer (Solarbio, R0020), protease inhibitor mixture (CW2200S, Kangwei Century) and phosphatase inhibitor mixture (CW2383S, Kangwei Century) in a grinder for tissue grinding. After centrifugation and supernatant, loading buffer was added and placed in a 95℃ metal bath for 10 minutes. The obtained samples were transferred to the membrane after SDS-PAGE electrophoresis, blocked, incubated with primary antibody at 4℃ overnight, incubated with secondary antibody at room temperature for 1 hour, washed with TBST buffer, and exposed. At the same time, the above steps were repeated with trophoblast JEG3 as a control to verify whether BRDT protein was expressed in JEG3, and the universally expressed HSP70 protein was used as the internal reference protein.

[0064] In the determination of BRDT protein, the primary antibody was BRDT antibody (purchased from Santa Cruz Biotechnology), and the secondary antibody was m-IgG1 BP-HRP (purchased from Santa Cruz Biotechnology).

[0065] In the determination of HSP70 protein, the primary antibody was HSP70 antibody (purchased from Santa Cruz Biotechnology), and the secondary antibody was m-IgG1 BP-HRP (purchased from Santa Cruz Biotechnology).

[0066] TBST was prepared by adding TBS buffer (Guangzhou Yongjin Biotechnology Co., Ltd., YJ0008) to a final concentration of 0.1% Tween20 (asegene, 43379).

[0067] Western Blot results Figure 3 As shown, HSP70 protein was used as the internal reference protein, and the results showed that BRDT protein was expressed in the collected early pregnancy and normal full-term placental tissues.

[0068] Immunohistochemistry to verify the expression of BRDT in placenta samples:

[0069] The collected placental tissue samples were fixed, embedded, sliced, dewaxed, hydrated, antigen retrieved, inactivated, blocked, incubated with primary antibodies, incubated with secondary antibodies, developed (DAB staining), counterstained, and sealed for observation. The primary antibody BRDT antibody was purchased from AVIVA, and the secondary antibody was purchased from MXB Biotechnologies.

[0070] The results of immunohistochemistry experiments were Figure 4 As shown, the brown area in the figure indicates positive, which means that BRDT protein is expressed in the collected early pregnancy and normal full-term pregnancy placental tissues.

[0071] This example mainly first retrieves single-cell RNAseq data with accession number GSE89497 from known publicly available data sets by analyzing them, and then verifies by qPCR, Western Blot, and immunohistochemistry that the BRDT gene and BRDT protein are expressed in placental tissue during pregnancy, indicating that BRDT is involved in the pregnancy process and has the potential to serve as a molecular marker related to pregnancy maintenance.

[0072] The amino acid sequence of BRDT protein is shown in SEQ ID No: 6:

[0073] SEQ ID No: 6:

[0074] MSLPSRQTAIIVNPPPPEYINTKKNGRLTNQLQYLQKVVLKDLWKHSFSWPFQRPVDAVKLKLPDYYTIIKNPMDLNTIKKRLENKYYAKASECIEDFNTMFSNCYLYNKPGDDIVLMAQALEKLFMQKLSQMPQEEQVVGVKERIKKGTQQNIAVSSAKEKSSPSPATEKVFKQQEIPSVFPKTSISPLNVVQGASVNSSQTAAQVTKGVKRKADTTTPATSAVKASSEFSPTFT EKSVALPPIKENMPKVNLPDSQQQYNVVETVKVTEQLRHCSEILKEMLAKKHFSYAWPFYNPVDVNALGLHNYYDVVKNPMDLGTIKEKMDNQEYKDAYSFAADVRLMFMNCYKYNPPDHEVVTMARMLQDVFETHFSKIEPVESMPLCYIKDTETTTGRENTNEASSEGNSSDSEDERVKRLAKLQEQLKAVHQQLQVLSQVPRFLKKKEKSKEKKKEKVNNSNENPRK MCEQMRLKEKSKRNQPKKRKQQFIGLKSEDEDNAKPMNYDEKRQLSNLINKLPGDKLGRVHVHIIQSREPSLSNSNPDEIEIDFETLKASTLRELEKYVSACLRKRPLKPPAKKIMMSKEELHSQKKQELEKRLLDVNNQLNSRKRQTKSDKTQPSKAVENVSRLSESSSSSSSSESESSSSDLSSSDSSEMFPKFTEVKPNDSPSKHEHVKKMKNECILPEGRTGVTQIGYCV QDTTSANTTLVHQTTPSHVMPPNHHQLAFNYQELEHLQTVKNISPLQILPPSGDSEQLSNGITVMHPSGDSDTTMLESECQAPVQKDIKIKNADSWKSLKGPVKPSGVMKSSDELFNQFRKAAIEKEVKARTQELIRKHLEQNTKELKASQENQRDLGNGLTVESFSNKIQNKCSGEEQKEHPQSSEAQDKSKLWLLKDRDLARPKEQERRRREAMVGTIDMLQSDIMTMFENNFD

[0075] Example 2: Gene regulation by BET inhibitors and siRNA in trophoblast JEG3 or primary extravillous trophoblast (EVT) cells and their relationship with reproduction

[0076] Human leukocyte antigen (HLA) is related to the immune system. The primary extravillous trophoblast cells (referred to as primary EVT cells) involved in this embodiment play an important role in maintaining pregnancy, and JEG3 is a trophoblast cell line. Both cells express the HLA-C gene, which is related to maternal-fetal immune tolerance. This embodiment explores the relationship between the expression of BRDT and the expression of HLA-C by inhibiting or knocking down the expression of BRDT in JEG3 or EVT cells. Since BET inhibitors mainly inhibit BRDT and BRD4 in the protein family containing bromodomains, related experiments on BRD4 are performed simultaneously in the experiment to confirm whether BET inhibitors regulate the expression of HLA-C by inhibiting BRDT or BRD4; this embodiment also conducts related experiments on the HLA-B gene adjacent to the HLA-C locus to confirm whether BRDT will only specifically bind to the HLA-C promoter region.

[0077] (1) JEG3 cells were treated with BET inhibitors (IBET726 and OTX015 were purchased from Cayman Chemical; JQ1 was purchased from BPS Bioscience), and the expression level of HLA-C in the cells was detected by RT-qPCR:

[0078] JEG3 cells in the logarithmic growth phase were inoculated into 6-well culture plates and incubated at 37°C and 5% CO 2 After the cells adhered to the culture medium, BET inhibitors (IBET726, JQ1 or OTX015) were added to each group, and an equal amount of DMSO was added to the control group for 48 hours.

[0079] Figure 5 The results of the test of HLA-C gene expression level in trophoblast JEG3 cells treated with BET inhibitors show that BET inhibitors can inhibit the gene transcription expression of HLA-C in JEG3 cells and are potential drugs for interfering with HLA-C expression, **p<0.01, ***p<0.001, n=3. The difference between the JQ1-treated group and the control group was the most significant, with a p value of less than 0.001, indicating that the probability of the difference being caused by chance is very low, so JQ1 was selected for the subsequent experiments.

[0080] (2) JEG3 cells were treated with siBRD4 or siBRDT, and the expression levels of BRD4 or BRDT in the cells were detected by RT-qPCR (the configuration system and reaction procedure of RT-qPCR were the same as those in Example 1), as well as the expression level of HLA-C in the cells:

[0081] Before transfection, 2 × 10 5 JEG3 cells were plated on 6-well plates and grown for one day. When the cell confluence reached 30% to 40%, BRD4-specific siRNA (siBRD4_1: Cat. No.: 4427038, Assay ID: s23901; siBRD4_2: Cat. No.: 4427038, Assay ID: s23902; siBRD4_3: Cat. No.: 4427038, Assay ID: s23903), BRDT-specific siRNAs (siBRDT_1: Cat. No.: 4427037, Assay ID: s2086; siBRDT_2: Cat. No.: 4427037, Assay ID: s2087; siBRDT_3: Cat. No.: 4427037, Assay ID: s2088), negative control siRNA (Cat No.: 4390843) and lipofectamine RNAiMAX reagent (Cat. Cells were transfected with siRNA (Genentech, Inc., No.: 13778030, Life technologies, USA) and grown at 37°C. Cells were harvested 48 hours after transfection for RNA extraction. All siRNAs were purchased from Thermo Fisher Scientific.

[0082] The detection primer sequences of BRD4 RT-qPCR are as follows:

[0083] F:5'GCTGTCTTCAGACCCTCCAG' (SEQ ID No: 7),

[0084] R: 5'GGCATGCACTCTACCACTGA' (SEQ ID No: 8).

[0085] Figure 6 These are the test results of the expression levels of the corresponding target genes (BRD4 or BRDT) and the HLA-C gene in the trophoblast cells JEG3 cells after siRNA treatment. The results showed that knockdown of BRDT could inhibit the gene transcription expression of HLA-C in JEG3 cells, while knockdown of BRD4 had no effect on the expression of HLA-C in JEG3 cells, indicating that BRDT is involved in the regulation of the expression of the HLA-C gene in JEG3 cells.

[0086] (3) Primary EVT cells were treated with JQ1, and the expression level of HLA-C in cells was detected by RT-qPCR:

[0087] The inclusion criteria for placental tissue were as follows: it was obtained from pregnant women who chose to terminate their pregnancy at a gestational age of <12 weeks without complications and infection.

[0088] The exclusion criteria for placental tissue were as follows: including but not limited to premature induction of labor, cesarean section, or termination of pregnancy due to pregnancy complications, twin or multiple births, IVF, HIV+, maternal hypertension, gestational diabetes, maternal autoimmune diseases, etc.

[0089] Chorionic tissue was scraped from the basement membrane of the obtained placental tissue and digested with trypsin (2 g / L) and EDTA (0.2 g / L) for 8 min at 37°C. Trypsin was quenched with F12 medium containing 1.0 mL of newborn calf serum and 1 mL of 1% penicillin / streptomycin (purchased from Gibco) and filtered through a gauze mesh. The filtrate was layered by density gradient centrifugation through Ficoll (GE Healthcare) (centrifuged at 800 × g for 20 min) and then incubated in a culture dish at 37°C for 20 min to remove macrophages. Non-adherent cells were collected and directly stained with HLA-G flow antibody (Abcam) for flow cytometry fluorescence sorting (Fluorescence activated cell sorting, FACS) to sort out HLA-G+EVT cells, which were recorded as primary EVT cells.

[0090] Cell culture method: About 20K HLA-G+EVT cells (i.e., primary EVT cells) were seeded in a 96-well cell culture plate (Costar) pretreated with 50 μL of 20 ng / mL fibronectin for 45 min, and cultured in DMEM / F12 medium (Gibco) supplemented with 10% (vol / vol) newborn calf serum, insulin-transferrin-selenium-ethanolamine (100×; Gibco, using a final concentration of 1×), 5 ng / mL EGF (Peprotech) and 400 units of human gonadotropin (Sigma).

[0091] The obtained primary EVT cells are named EVT2, EVT6, and EVT7 according to their maternal origin.

[0092] The obtained primary EVT cells were cultured at 37°C and 5% CO 2 After the cells adhered to the wall, 500 nM JQ1 was added to the control group, and the same amount of DMSO was added to the control group for 48, 54 or 72 hours.

[0093] RT-qPCR was used to detect the expression level of HLA-C in cells:

[0094] Total RNA was isolated using RNeasy Mini Kit (catalog no. / ID: 74104, Qiagen, USA) according to the manufacturer's instructions. cDNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (catalog no. 4368814, Applied Biosystems TM , USA) and RNase inhibitor (catalog no.4374966, Life Technologies) were used for reverse transcription, and a total of 1000 ng RNA was used for cDNA synthesis. Gene expression assays and TaqMan TM qRT-PCR was performed using Fast Advanced Master Mix (catalog no. 4444557; Life Technologies). TaqMan assays were performed on a Quantstudio 12k Flex Real-Time PCR System (Thermo Fisher Scientific) or a Quantstudio 6Flex Real-Time PCR System (Thermo Fisher Scientific). The gene information involved in the gene expression assay is as follows (Thermo Fisher Scientific):

[0095] Reference gene: GAPDH, assay ID no.Hs02786624_g1, catalog no.4331182;

[0096] Target gene: HLA-C, assay ID no.Hs03044135_m1, catalog no.4331182.

[0097] Figure 7 The results of the test of HLA-C gene expression level after primary EVT cells were treated with JQ1 show that JQ1 can inhibit the gene transcription expression of HLA-C in primary EVT cells, and the HLA-C reduction effect is more obvious than that of JEG3 cells. It is preliminarily shown that BRDT can regulate the expression of HLA-C, and primary EVT cells are derived from real placental tissue, so the HLA-C reduction effect in primary EVT cells is more obvious than that of JEG3, indicating that it has clinical application significance.

[0098] (4) JEG3 cells were treated with JQ1, and differentially expressed genes were enriched after RNA-seq analysis:

[0099] JEG3 cells in the logarithmic growth phase were inoculated into 6-well culture plates and incubated at 37°C and 5% CO 2 After the cells adhered to the wall, the original culture medium was discarded and fresh culture medium containing 500 nM JQ1 (Cat#27402, BPS Bioscience) or an equivalent volume of DMSO (fresh culture medium was RPMI 1640 medium (Life Technologies) + 10% fetal bovine serum + 1% streptomycin penicillin) was added to culture JEG3 cells for 48 hours; among them, the group containing JQ1 was the experimental group, and the group containing DMSO was the control group.

[0100] The JEG3 cells obtained by the above culture were collected, washed with pre-cooled PBS buffer for 2 to 3 times, and then added with Triozol to grind in a grinder. After centrifugation, the supernatant was added with chloroform to extract RNA, precipitated with isopropanol, washed with 75% ethanol for several times, and then dissolved with nuclease-free water. Then, mRNACapture Beads magnetic beads were added to enrich mRNA. After magnetic bead purification, mRNA was fragmented by high temperature. The fragmented mRNA was used as a template to synthesize the first chain of cDNA in a reverse transcriptase mixed system. While synthesizing the second chain of cDNA, end repair and A tailing were completed. Then, the adapter was connected and the mRNA was synthesized by using DNA Selection Beads magnetic beads are used to purify and select target fragments; PCR library amplification is then performed, and finally Novasek 6000 is used for detection.

[0101] Figure 8 The results of RNA-seq enrichment analysis of some differentially expressed genes in JEG3 cells after treatment with JQ1. The results showed that the differentially expressed genes in JEG3 cells before and after treatment with JQ1 were mainly enriched in three reproductive-related pathways (outlined in red boxes in the figure): reproductive system development, response to steroid hormones, and female pregnancy, indicating that the differentially expressed genes were mainly reproductive-related genes. These pathways are highly correlated, indicating that JQ1 is involved in the regulation of reproductive-related genes in JEG3 cells and plays an important role in the expression of these genes. As a BET inhibitor, JQ1 mainly inhibits the expression of BRDT. The experiments here further prove that JQ1 can indeed inhibit the expression of reproductive-related genes, including the expression of BRDT, indicating that JQ1 is expected to be an immunomodulator during pregnancy.

[0102] Example 3: Analysis and verification of promoters regulating HLA-C expression in JEG3 cells by BRDT

[0103] (1) Chromatin immunoprecipitation reaction:

[0104] ChIP PCR / qPCR was performed using Abcam's ChIP kit (ab500) according to the manufacturer's instructions. JEG3 cells were collected (JEG3 cells were treated in the same manner as in Example 2, and were divided into an experimental group treated with JQ1 and a control group treated with DMSO), resuspended in PBS, and cross-linked with 1% formaldehyde. Glycine was added to stop the cross-linking reaction, and the cells were washed twice with ice-cold PBS. The nuclei were isolated and then lysed with ice-cold cell lysis buffer containing protease inhibitors and PMSF. Covaris S220 was used to shear the cross-linked chromatin to 200 to 500 bp fragments, and the resulting fragments were the antibody / chromatin samples. The resulting antibody / chromatin samples were evaluated using TapeStation assay D1000 (Agilent Technologies) to evaluate whether the fragment size of the antibody / chromatin sample was 200 to 500 bp.

[0105] The antibody / chromatin samples were then diluted with ChIP dilution buffer and incubated overnight at 4°C with the following antibodies: IgG1 (Santa Cruz Biotechnology), IgG (Santa Cruz Biotechnology), BRD4 (A301-985A50), BRDT (Santa Cruz Biotechnology), or H3K27Ac (ab4729, Abcam). The antibody / chromatin samples were incubated with protein A magnetic beads (included in the ChIP kit). After incubation, the antibody / chromatin / magnetic beads were obtained, washed, and the DNA was purified using the DNA purification slurry in the ChIP kit.

[0106] (2) PCR verification of the enrichment efficiency of the HLA-C promoter sequence:

[0107] 2 μL of DNA was applied to PCR amplification using Platinum PCR SuperMix High Fidelity (Thermo Fisher Scientific) and analyzed by gel electrophoresis.

[0108] PCR preparation system:

[0109] Components volume <![CDATA[Platinum TM PCR SuperMix High Fidelity]]> 45.0μL Promoter region primers (final concentration 200 nM) 3.0μL DNA template 2.0μL

[0110] PCR reaction procedure:

[0111] step temperature time transsexual 94℃ 30s annealing 55℃ 30s extend 68℃ 1min / kb

[0112] HLA-C promoter region primers:

[0113] F:5'GGCTCCAAGGGCCGTGTCTGCAC' (SEQ ID No: 9)

[0114] R:5'GTTCTGGGGAGAATCTGAGTCC' (SEQ ID No: 10)

[0115] HLA-B promoter region primers:

[0116] F:5'GGCTCCGAGGGCCGCGTCTGCAATG' (SEQ ID No: 11)

[0117] R:5'GTCTGAGGAGACTCTGAGTCC' (SEQ ID No: 12)

[0118] Fig. 9 The test results of the enrichment efficiency of the HLA-C promoter sequence in JEG3 cells show that BRD4 and BRDT can strongly occupy the HLA-C promoter region in JEG3 ( Fig. 9 A). In addition, the BRDT antibody did not immunoprecipitate the proximal promoter of the HLA-B gene adjacent to the HLA-C locus, but BRD4 was still detected in the HLA-B promoter region, indicating that BRDT specifically binds to the HLA-C promoter region ( Fig. 9 C). After JQ1 treatment of JEG3 cells, it can block the binding of BRDT to the HLA-C promoter region in JEG3 cells ( Fig. 9 B). This indicates that BRDT can regulate the expression level of HLA-C by specifically binding to the promoter region of HLA-C, indicating that BRDT is related to the maintenance of pregnancy.

[0119] Example 4: Association between the expression levels of BRDT and HLA-C in clinical placenta samples and healthy pregnant patients and spontaneous abortion patients

[0120] The inclusion and exclusion criteria for placental tissue from healthy pregnant patients were the same as those described in (3) of Example 2.

[0121] The inclusion and exclusion criteria for placental tissue from patients with spontaneous abortion are as follows:

[0122] Inclusion criteria: Women with clinical diagnosis of spontaneous abortion.

[0123] Exclusion criteria include but are not limited to patients with spontaneous abortion due to chromosomal abnormalities, pregnancy complications, twins or multiple births, IVF, HIV+, maternal hypertension, maternal autoimmune diseases, maternal smoking, alcohol intake, etc.

[0124] (1) Immunohistochemistry:

[0125] The placental tissue samples were fixed, embedded, sliced, dewaxed and hydrated, antigen retrieved, inactivated, blocked, incubated with primary antibodies, incubated with secondary antibodies, colored, counterstained, and sealed for observation.

[0126] (2) Immunohistochemistry scoring:

[0127] Two individuals blindly evaluated the IHC staining scores; the scoring system combined the intensity and percentage (staining intensity: no staining for 0 points, weak staining for 1 point, moderate staining for 2 points, strong staining for 3 points; percentage of positive cells: 0%-5% for 0 points, 6%-25% for 1 point, 26%-50% for 2 points, 51%-75% for 3 points, >75% for 4 points) and calculated the comprehensive score by multiplying the scores of the two scoring indicators. The minimum comprehensive score was 0 and the maximum comprehensive score was 12 points.

[0128] Comprehensive score = staining intensity score * positive cell percentage score, the comprehensive score of each group of data is expressed as mean + SD. The significance of the mean values ​​of the two groups was analyzed by t-test. All statistical analyses were performed using GraphPad Prism 10.0 software.

[0129] Fig.10 The results show the expression levels of BRDT and HLA-C in primary EVT cells of placenta from patients with healthy pregnancy (control) and spontaneous abortion (SA). According to the characteristics of EVT cells expressing HLA-G, HLA-G positive areas were identified as EVT cells, and the expression levels of BRDT and HLA-C were observed and scored. The immunohistochemistry (IHC) results showed that in the placenta of spontaneous abortion (n=8, representing data from 8 placentas), the expression levels of BRDT and HLA-C in EVT cells were lower than those in the control group (placental tissue of healthy pregnancy) (n=8, representing data from 8 placentas). *p<0.05, **p<0.01.

[0130] The present invention also analyzed the correlation between BRDT and HLA-C in placental tissue trophoblast cells based on 7 groups of placentas of healthy pregnant patients and 7 groups of placentas of spontaneous abortion patients, a total of 14 groups of placentas (n=14). The analysis results are as follows: Fig.11 As shown, the results showed that in the placental tissue trophoblast cells, the higher the expression level of BRDT, the higher the corresponding expression level of HLA-C.

[0131] The above results indicate that the risk of spontaneous abortion can be screened by detecting the expression level of BRDT in primary EVT cells. At the same time, based on the fact that BRDT can bind to the promoter region of HLA-C, thereby regulating the expression of HLA-C, it indicates that BRDT may play a role in maintaining pregnancy and has clinical application significance.

[0132] In summary, this inventive study reveals a new face of the bromodomain-containing testis-specific protein (BRDT), which traditionally plays a major role in male reproduction, and clarifies its important expression and function in the placenta. Single-cell RNA-seq, qPCR, Western Blot and immunohistochemistry proved that BRDT is expressed in placental tissue. The present study also found the regulatory effect of BRDT on the expression of HLA-C, a key molecule of maternal-fetal immune tolerance. Through ChIP-PCR experiments, it was further proved that BRDT specifically binds to the HLA-C promoter region, and JQ1 can interrupt this binding, leading to the downregulation of HLA-C. And primary EVT is more sensitive to the treatment of JQ1 than JEG3 cells, suggesting the potential application of BET inhibitors in the regulation of gene expression at the maternal-fetal interface. Clinically, it was observed that the levels of BRDT and HLA-C in patients with spontaneous abortion were significantly lower than those in healthy pregnancy samples. This correlation suggests that BRDT may play a role in pregnancy maintenance and highlights its importance in the maternal-fetal interface. In summary, the studies in this application not only reveal the expression of BRDT in the placenta, but also demonstrate its key role in regulating HLA-C expression, providing new insights into reproductive biology and potential therapeutic strategies for pregnancy-related diseases.

Claims

1. A molecular marker for screening the risk of spontaneous abortion and maintaining pregnancy, characterized in that: The molecular marker includes BRDT gene and / or BRDT protein. The nucleotide sequence of the BRDT gene is shown in SEQ ID No: 1, and the amino acid sequence of the BRDT protein is shown in SEQ ID No:

6.

2. The molecular marker according to claim 1, characterized in that The primers for the molecular marker of claim 1 are shown in SEQ ID No: 2 and SEQ ID No:

3.

3. Use of BRDT gene and / or BRDT protein as molecular markers in screening for spontaneous abortion risk and maintaining pregnancy.

4. The use according to claim 3, characterized in that The expression level of the BRDT gene or BRDT protein is positively correlated with the immune tolerance during pregnancy, and the expression level of the BRDT gene or BRDT protein is negatively correlated with the risk of miscarriage during pregnancy.

5. The use according to claim 3, characterized in that: The BRDT gene or BRDT protein is used to promote the expression of HLA-C.

6. A product for improving immune tolerance during pregnancy and / or reducing the risk of spontaneous abortion during pregnancy, characterized in that: The product includes a product that promotes the expression of the molecular marker according to claim 1.

7. A primer for detecting molecular markers related to spontaneous abortion risk and pregnancy maintenance according to claim 1, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID No: 2 and SEQ ID No:

3.

8. Use of the primers according to claim 7 in the preparation of products for screening the risk of spontaneous abortion and maintaining pregnancy, wherein the products include a kit.

9. A product for screening the risk of spontaneous abortion and maintaining pregnancy, characterized in that: The product comprises the primer according to claim 7.

Images