Molecular markers of recurrent spontaneous abortion and diagnostic applications

Abstract

The application discloses a molecular marker for recurrent spontaneous abortion and a diagnostic application thereof, and relates to the technical field of biological medicine.The application discloses application of a JAZF1 gene in preparation of a product for diagnosis or auxiliary diagnosis of abortion.It is found for the first time that the expression amount of JAZF1 is closely related to recurrent spontaneous abortion, which indicates that the expression of JAZF1 is down-regulated in decidua tissue and endometrial tissue of recurrent spontaneous abortion, and indicates potential possibility of JAZF1 as a diagnostic marker for recurrent spontaneous abortion, which is helpful to clinical diagnosis and prevention of recurrent spontaneous abortion.Furthermore, it is proposed for the first time that JAZF1 can promote survival of endometrial stromal cells and normal decidualization, so as to maintain normal pregnancy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a molecular marker for recurrent miscarriage and its diagnostic applications. Background Technology

[0002] Recurrent spontaneous abortion (RSA), also known as recurrent pregnancy loss (RPL), is a highly heterogeneous condition defined as two or more spontaneous abortions, including consecutive biochemical pregnancies (documented by ultrasound or histopathological examination). Besides chromosomal abnormalities, immune system disorders, and uterine malformations, endometrial dysfunction is also considered a significant cause of RSA. Furthermore, recurrent abortion increases the incidence of adverse pregnancy outcomes in subsequent pregnancies, such as preterm birth, premature rupture of membranes, and low birth weight.

[0003] Decidualization of the endometrium is crucial for the establishment and maintenance of pregnancy. During pregnancy establishment and maintenance, decidualization of endometrial stromal cells (EnSCs) refers to the multi-step reprogramming process of EnSCs transforming into secretory decidual stromal cells (DSCs), with the secretion of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) as classic markers of decidualization. The decidualization process not only limits excessive infiltration of trophoblast cells but also directly or indirectly affects the recruitment, distribution, and function of immune cells, spiral artery remodeling, and placental formation through the secretion of growth factors and cytokines. Defects in decidualization can lead to various pregnancy complications, including recurrent implantation failure (RIF), recurrent implantation failure (RIF), and preeclampsia. However, the pathogenesis of endometrial dysfunction in patients with RSA remains unclear.

[0004] Chinese patent CN202210073849.6 discloses the application of LILRB1 in the preparation of a detection product for predicting spontaneous abortion. This invention found that patients with spontaneous abortion exhibit a decreased expression rate of LILRB1 in peripheral blood and decidual NK cells, and that the expression rate of LILRB1 in peripheral blood NK cells is positively correlated with that in decidual NK cells. Using LILRB1-expressing peripheral blood NK cells and LILRB1-expressing decidual NK cells as biomarkers for predicting spontaneous abortion and assessing the local and systemic immune tolerance status of the pregnant woman's uterus allows for early identification of spontaneous abortion.

[0005] Chinese patent CN202210302503.9 discloses the application of BBOX1-AS1 as a diagnostic biomarker and therapeutic target for recurrent miscarriage. This invention is the first to discover a close correlation between BBOX1-AS1 expression and recurrent miscarriage, indicating that BBOX1-AS1 upregulated in chorionic villus tissue of recurrent miscarriage regulates the proliferation, migration, and invasion of trophoblasts in vitro and participates in activating the p38 / JNK MAPK signaling pathway. Furthermore, this invention also investigated that BBOX1-AS1 promotes GADD45A expression by binding to the protein hnRNPK, ultimately causing changes in trophoblast function and promoting the occurrence of recurrent miscarriage. Finally, the elevated expression of BBOX1-AS1 in the serum of patients with recurrent miscarriage suggests the potential of BBOX1-AS1 as an early diagnostic biomarker for recurrent pregnancy loss.

[0006] Numerous studies have found excessive apoptosis in DSCs from patients with early miscarriage and recurrent miscarriage. Current research indicates that acrylamide (ACR) impairs decidualization by inhibiting cyclin D3 and p21, while upregulating apoptosis-related molecules BAX and cleaved-caspase 3, thereby reducing the number of mouse embryos (Yu D., Liu Q., Qiao B., et al. Exposure to acrylamide inhibits uterine decidualization via suppression of cyclin D3 / p21 and apoptosis in mice. J Hazard Mater 388:121785,2020.). Other studies have found that abnormally elevated BAX levels can induce apoptosis, thereby damaging stromal cell decidualization and causing recurrent miscarriage (Al-Rubaye S., Ghaderian SMH, Salehpour S., et al. Aberrant expression of BAX, MEG3, and miR-214-3P genes in recurrent pregnancy loss. Gynecol Endocrinol 37(7):660-664,2021.; Lv X., Cai Z. and Li S. Increased apoptosis rate of human decidual cells and cytotrophoblasts in patients with recurrent spontaneous abortion as a result of abnormal expression of CDKN1A and Bax. Exp Ther Med 12(5):2865-2868,2016.).

[0007] The pathogenesis of recurrent miscarriage is still unclear. Providing a molecular marker for recurrent miscarriage and corresponding prevention and treatment strategies is of great theoretical and clinical significance. Summary of the Invention

[0008] The purpose of this invention is to provide the application of the JAZF1 gene in the preparation of products for miscarriage diagnosis or auxiliary diagnosis. By detecting the expression level of the JAZF1 gene in the test sample, a reference can be provided for the diagnosis and prevention of recurrent miscarriage.

[0009] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0010] On the one hand, this invention provides the application of the JAZF1 gene in the preparation of products for miscarriage diagnosis or auxiliary diagnosis.

[0011] Preferably, the miscarriage is a recurrent miscarriage.

[0012] Preferably, the diagnostic product includes a reagent for detecting the expression level of the JAZF1 gene.

[0013] More preferably, the reagent is used to detect the mRNA expression level or protein expression level of the JAZF1 gene.

[0014] Preferably, the diagnostic product is selected from one or more of the following: reagent kits, gene chips, protein chips, formulations, reagent kit quality control products, and reagent kit standards.

[0015] Preferably, the test sample is selected from one or more of decidual tissue, endometrial tissue, and decidual stromal cells.

[0016] More preferably, the test sample is selected from one or more of decidual tissue and decidual stromal cells.

[0017] Preferably, the JAZF1 gene is differentially expressed in samples from patients with recurrent miscarriage.

[0018] More preferably, compared to normal healthy individuals, patients with recurrent miscarriage show decreased expression of the JAZF1 gene.

[0019] Preferably, the diagnostic products include those for diagnosing recurrent miscarriage using qRT-PCR, immunoassay, Western blot, immunohistochemistry, immunofluorescence, flow cytometry, and single-cell sequencing.

[0020] More preferably, the product for diagnosing recurrent miscarriage by qRT-PCR includes at least one pair of primers that specifically amplify the JAZF1 gene.

[0021] Preferably, the application steps are as follows:

[0022] (1) Detect the expression level of the JAZF1 gene in the sample to be tested;

[0023] (2) Compare the expression level of JAZF1 gene in the test sample with that in the control group;

[0024] (3) Compared with the control group, the expression of JAZF1 gene in the test sample was downregulated, indicating that the subject was a miscarriage patient; otherwise, the subject was not a miscarriage patient.

[0025] In another aspect, the present invention provides the application of reagents for detecting the JAZF1 gene in the preparation of diagnostic or auxiliary diagnostic products for miscarriage.

[0026] Preferably, the reagent is used to detect the mRNA expression level or protein expression level of the JAZF1 gene.

[0027] In another aspect, the present invention provides a product for diagnosing or assisting in the diagnosis of miscarriage, the product comprising a reagent for detecting the expression level of the JAZF1 gene in a sample to be tested.

[0028] Preferably, the reagents are selected from probe sets, primer sets, or gene chips.

[0029] Preferably, the product is selected from one or more of the following: reagent kits, gene chips, protein chips, formulations, reagent kit quality control products, and reagent kit standards.

[0030] In some embodiments of the present invention, the product is a kit, which includes primer pairs for amplifying the JAZF1 gene, PCR buffer, dNTPs, a reverse transcription system, and RNA extraction reagents.

[0031] Preferably, the reverse transcription system includes a reverse transcription reaction solution, reverse transcriptase, and an RNase inhibitor.

[0032] Preferably, the RNA extraction reagent includes Trizol, chloroform, isopropanol, and ethanol.

[0033] Preferably, the test sample is selected from one or more of decidual tissue, endometrial tissue, and decidual stromal cells.

[0034] More preferably, the test sample is selected from one or more of decidual tissue and decidual stromal cells.

[0035] In another aspect, the present invention provides the use of substances that enhance the content or activity of the JAZF1 gene in the preparation of medicaments for treating or preventing miscarriage.

[0036] Preferably, the miscarriage is a recurrent miscarriage.

[0037] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0038] More preferably, the pharmaceutically acceptable carrier is selected from one or more excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, and lubricants.

[0039] More preferably, the drug dosage form is selected from one or more of the following: powder, tablet, pill, granule, capsule, solution, emulsion, suspension, and oil.

[0040] Preferably, the function of the application is any one or more of the following:

[0041] (1) Promotes the proliferation of endometrial stromal cells;

[0042] (2) Inhibits apoptosis of endometrial stromal cells;

[0043] (3) Maintain the normal decidualization of endometrial stromal cells.

[0044] The beneficial effects of this invention are as follows:

[0045] (1) This invention is the first to discover that the expression level of JAZF1 is closely related to recurrent miscarriage, indicating that JAZF1 expression is downregulated in decidual and endometrial tissues of recurrent miscarriage, suggesting the potential of JAZF1 as a diagnostic marker for recurrent miscarriage, which can help in the clinical diagnosis and prevention of recurrent miscarriage.

[0046] (2) This invention is the first to propose that JAZF1 can promote the survival of endometrial stromal cells and normal decidualization, thereby maintaining normal pregnancy. Attached Figure Description

[0047] Figure 1A The feature plot shows the mRNA expression level of JAZF1 in DS cells.

[0048] Figure 1B The figure shows the results of scRNA-seq analysis of JAZF1 expression in decidual stromal cells from RSA patients (n=6) and normal early pregnancies with matched gestational ages (n=5) using the boxPlot function in the Seurat single-cell sequencing toolkit.

[0049] Figure 1C The figure shows the results of scRNA-seq analysis of JAZF1 expression in DS1, DS2, and DS3.

[0050] Figure 2A Figure showing the expression of JAZF1 in decidual tissue detected by qRT-PCR.

[0051] Figure 2B The figure shows the expression of JAZF1 in decidual tissue as detected by Western blot.

[0052] Figure 2C Figure showing the expression of JAZF1 in primary decidual stromal cells as detected by qRT-PCR.

[0053] Figure 2D Figure showing the expression of JAZF1 in primary decidual stromal cells as detected by Western blot.

[0054] Figure 3A Figure showing the expression of JAZF1 in decidual tissue as detected by immunohistochemistry.

[0055] Figure 3B A statistical graph showing the expression of JAZF1 in decidual tissue as detected by immunohistochemistry.

[0056] Figure 3C Figure showing the expression of JAZF1 in decidual tissue as detected by immunofluorescence.

[0057] Figure 4A To detect the protein levels of FOXO1, IGFBP1, and JAZF1 in HESCs that have been induced to decidualize using Western blot.

[0058] Figure 4B A statistical graph showing the protein levels of FOXO1, IGFBP1, and JAZF1 in HESCs that have been induced to decidualize using Western blot analysis.

[0059] Figure 4C A graph showing the expression level of JAZF1 in HESCs that have been induced to decidualize, as detected by immunofluorescence.

[0060] Figure 5A This image shows the expression levels of JAZF1 in the proliferative and early secretory phases of the endometrium in the RSA and normal groups, as detected by immunohistochemistry.

[0061] Figure 5B The expression of JAZF1 in endometrial tissue during the proliferative and secretory phases was determined using microarray data (GDS2052) stored in the gene expression database.

[0062] Figure 5C To analyze the expression pattern of JAZF1 using 10× single-cell sequencing transcriptome sequencing (scRNA-seq) of endometrial tissue at different time points in a public database, the data were obtained from microarray data (GSE111976) stored in the gene expression library.

[0063] Figure 6A The graph shows the JAZF1 expression levels after siRNA knockdown, knockout, and overexpression treatment, as detected by qRT-PCR.

[0064] Figure 6B To detect JAZF1 expression after siRNA knockdown, knockout, and overexpression treatment using Western blot.

[0065] Figure 6C This is a schematic diagram of the structure of the human JAZF1 gene and its CRISPR / Cas9 targeted mutant form (KO).

[0066] Figure 7A This is a graph showing the expression levels of apoptosis-related genes and decidualization-related genes after qRT-PCR detection of JAZF1 knockdown and knockout.

[0067] Figure 7B Western blot was used to detect the expression levels of apoptosis-related genes and decidualization-related genes after JAZF1 knockdown and knockout.

[0068] Figure 7C A statistical graph showing the expression levels of apoptosis-related genes and decidualization-related genes after Western blot analysis of JAZF1 knockdown and knockout.

[0069] Figure 7D A graph showing the level of apoptosis in cells after knockdown, knockout, and overexpression of JAZF1, as detected by flow cytometry.

[0070] Figure 7E A statistical graph showing the apoptosis levels of cells after knockdown, knockout, and overexpression of JAZF1, as detected by flow cytometry.

[0071] Figure 7F To detect the cell status after CCK8 knockdown, knockout, and overexpression of JAZF1.

[0072] Figure 8A The expression levels of BAX, BCL2, IGFBP1 and PRL after JAZF1 overexpression were detected by qRT-PCR.

[0073] Figure 8B A statistical graph showing the protein expression levels of BCL2, cyt c, and IGFBP1 after JAZF1 overexpression, as detected by Western blot.

[0074] Figure 8C The graph shows the protein expression levels of BCL2, cyt c, and IGFBP1 after JAZF1 overexpression, as detected by Western blot.

[0075] Figure 8D A statistical graph showing the protein expression levels of BAX and cleaved-caspase 3 after JAZF1 overexpression, as detected by Western blot.

[0076] Figure 9A The expression and statistical diagram of the invasive ability of decidualized hESCs after JAZF1 knockdown on HTR-8 / SVneo cells.

[0077] Figure 9B The expression and statistical diagrams show the ability of decidualized hESCs overexpressing JAZF1 to invade HTR-8 / SVneo cells.

[0078] Figure 10A Transcriptional levels of BAX (left) and BCL2 (right) in RSA and control groups were analyzed using scRNA-seq.

[0079] Figure 10B The pathways enriched by decidual stromal cell subsets 1 (DS1), 2 (DS2), and 3 (DS3) were analyzed for GO.

[0080] Figure 11A Statistical graph showing the expression levels of apoptosis-related genes BAX and BCL2 in decidual tissue detected by qRT-PCR.

[0081] Figure 11B A graph showing the expression levels of apoptosis-related genes BCL2, BAX, cleaved-caspase3, and cyt c in decidual tissue as detected by Western blot.

[0082] Figure 11C A statistical graph showing the expression levels of apoptosis-related genes BCL2, BAX, cleaved-caspase3, and cyt c in decidual tissue as detected by Western blot.

[0083] Figure 11D Western blot was used to detect the expression levels of apoptosis-related genes BCL2, BAX, cleaved-caspase3, and cyt c in primary decidual stromal cells.

[0084] Figure 11E Western blot was used to detect the expression levels of apoptosis-related genes BCL2, BAX, cle-caspase3, and cyt c in primary decidual stromal cells.

[0085] Figure 11F To detect the expression levels of apoptosis-related genes BCL2, BAX, cleaved-caspase3, and cyt c in decidual tissue using immunohistochemistry.

[0086] Figure 12 A graph showing the overall apoptosis level in decidual tissue as detected by the TUNEL assay.

[0087] Figure 13A Correlation analysis of BCL2 and JAZF1 in decidual tissue.

[0088] Figure 13B Correlation analysis of cleaved-caspase 3 and JAZF1 in decidual tissue.

[0089] Figure 13CCorrelation analysis of BAX and JAZF1 in decidual tissue.

[0090] Figure 13D Correlation analysis of cyt c and JAZF1 in decidual tissue.

[0091] Figure 14A This is a graph showing the molecular diagnostic specificity analysis results of JAZF1 in the two groups of patients in Example 1.

[0092] Figure 14B JAZF1 expression was detected by Western blot in four population pairs other than those in Example 1, including the normal group and the recurrent miscarriage group.

[0093] Figure 14C The graph shows the JAZF1 expression statistics in the normal group and the recurrent miscarriage group in four population pairs other than those in Example 1.

[0094] Figure 14D The graph shows the molecular diagnostic specificity analysis results of JAZF1 in four pairs of decidual tissue samples other than those in Example 1.

[0095] In the above figure, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Detailed Implementation

[0096] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0097] In this invention, siRNA is a small interfering RNA that interferes with the expression of the JAZF1 gene.

[0098] Example 1: Expression and localization of JAZF1 in decidual tissue of recurrent miscarriage

[0099] (I) Using a 10× single-cell RNA sequencing (scRNA-seq) dataset, including decidual cells from 6 RSA and 5 gestationally matched normal induced abortions (6-8 weeks gestation), JAZF1 was found to be significantly downregulated in all decidual stromal cells of the RSA group. Figures 1A-1C ).

[0100] (II) To further validate our scRNA-seq results, we used qRT-PCR and Western blotting to detect the expression level of JAZF1 in decidual tissue and extracted primary decidual stromal cells from patients with recurrent miscarriage and selective abortion.

[0101] Experimental steps:

[0102] 1. Real-time quantitative PCR (RT-qPCR)

[0103] (1) RNA extraction from decidual tissue

[0104] 1) Pre-cool isopropanol, 75% ethanol, and centrifuge.

[0105] 2) Weigh 30mg of decidual tissue into a mortar, add liquid nitrogen and grind until fine powder is formed. Transfer the powder to a sterile 1.5mL EP tube without enzymes using a spoon, add 1mL of Trizol reagent, vortex, and let stand at room temperature for 5min.

[0106] 3) Add 0.2 mL of chloroform, mix well, and let stand at room temperature for 5 minutes to allow it to separate into layers naturally;

[0107] 4) Centrifuge (centrifuge parameters: speed 12000g / min, temperature 4℃, time 15min). After centrifugation, the sample will be separated into 3 layers. Transfer the upper aqueous phase to another sterile 1.5mL EP tube without enzymes.

[0108] 5) Add an equal volume of pre-cooled isopropanol to the aqueous phase, mix well, and let stand on ice for 10 minutes.

[0109] 6) Centrifuge. After centrifugation, a white precipitate will be seen at the bottom of the EP tube. Discard the supernatant.

[0110] 7) Add 1 mL of 75% ethanol to the EP tube to allow the precipitate to float in the 75% ethanol;

[0111] 8) Centrifuge (centrifuge parameters: speed 7500 rpm / min, temperature 4℃, time 12min), discard the supernatant and repeat steps 7 and 8 once;

[0112] 9) Discard the supernatant and let it stand at room temperature. When the precipitate at the bottom changes from white to transparent, it is ready for drying.

[0113] 10) Add an appropriate volume of DEPC water according to the size of the precipitate to dissolve the RNA;

[0114] 11) Heat the RNA in a 37°C metal bath to promote RNA dissolution;

[0115] 12) Use a Nanodrop 2000 spectrophotometer to detect RNA samples with an A260 / A280 ratio between 1.8 and 2.0, record their RNA concentration, and proceed to the next step of reverse transcription experiment.

[0116] (2) Reverse transcription to synthesize cDNA (all the following processes are performed on ice)

[0117] ①Step 1: Remove genomic DNA (10μL reaction system)

[0118] Table 1.

[0119]

[0120]

[0121] After mixing, incubate at 42°C for 2 minutes, then store at 4°C or proceed to the next step.

[0122] ② Second step: Reverse transcription (20μL reaction system)

[0123] Table 2.

[0124]

[0125] After mixing, thaw at 37°C for 15 min, then at 85°C for 5 sec. Add 180 μL of DEPC to dilute 10 times and store at -80°C.

[0126] (3) Real-time quantitative PCR (qRT-PCR)

[0127] ① Using TaKaRa TB Premix Ex Taq TM (10μL reaction system);

[0128] Table 3.

[0129]

[0130] ②Reaction conditions: 95℃ for 30 seconds; (95℃ for 5 seconds, 60℃ for 30 seconds), 40 cycles. After amplification, obtain the corresponding CT values.

[0131] ③β-actin is used as an internal reference gene, 2 -△△CT Analyze the data.

[0132] All primers used in this study were synthesized by Guangzhou Funeng Co., Ltd. or Shanghai Jierui Co., Ltd., and their sequences are shown in the table below:

[0133] Table 4.

[0134]

[0135] 2. Protein extraction and concentration determination

[0136] (1) Extraction of total protein from decidual tissue

[0137] ① One day before the experiment, sterilize the experimental equipment at high temperature and pre-cool the centrifuge to 4℃;

[0138] ②Prepare a cell lysis buffer, protease inhibitor (PIC), and phosphatase inhibitor (PHI) in a volume ratio of 100:1:1. Mix the three components thoroughly according to the above ratio and place them on ice.

[0139] ③ Weigh 35mg of decidual tissue and place it in a grinding mortar. Add liquid nitrogen and grind it into a fine powder. Transfer the powder to an EP tube with a spoon, add 350μL of the mixed lysis buffer, and vortex to mix it thoroughly.

[0140] ④ Place the ice on the ice and swirl it once every 10 minutes, for a total of 3 times;

[0141] ⑤ After 30 minutes, centrifuge (centrifuge parameters: speed 14000 rpm / min, temperature 4℃, time 30 minutes), and transfer the supernatant to another enzyme-free and sterile 1.5 mL EP tube. During this process, be careful to avoid aspirating the bottom precipitate.

[0142] (2) Protein concentration determination using the BCA method

[0143] The protein concentration of the sample was determined according to the operating instructions of the BCA protein quantification kit (purchased from ThermoFisher Scientific, catalog number 23227);

[0144] (3) Protein aliquoting and storage

[0145] ① Calculate the required sample volume, and add 1 / 3 of the volume of 4× sample buffer, then mix thoroughly;

[0146] ②In a 100℃ metal bath for 10 minutes, shake thoroughly to mix, centrifuge, and then store at -80℃.

[0147] (4) SDS-PAGE gel preparation

[0148] The concentration of the concentrated gel used in this embodiment is 6%.

[0149] (5) Electrophoresis: Protein electrophoresis was performed at a constant current of 24mA / plate at room temperature. Electrophoresis was stopped when bromophenol blue reached the bottom of the gel.

[0150] (6) Transfer: Electrotransfer instrument (constant voltage 100V), the whole process is carried out at 4℃. After activating the PVDF membrane with methanol for 30s, wash with double distilled water for 3min, then transfer the PVDF membrane and electrophoresis gel to 1× transfer buffer and equilibrate for 10min. Prepare the clamping plate: negative electrode - 2 sheets of filter paper - electrophoresis gel - PVDF membrane - 3 sheets of filter paper - positive electrode.

[0151] (7) Blocking: After the transfer is completed, the PVDF membrane is placed in the blocking solution and slowly shaken, and incubated at room temperature for 1 hour.

[0152] (8) Primary antibody incubation: Rinse the residual blocking solution with PBS, add the diluted primary antibody, shake slowly on a shaker, and incubate overnight at 4°C. The primary antibody dilution ratio is shown in Table 5 below.

[0153] Table 5.

[0154]

[0155] (9) Washing the membrane: Wash the PVDF membrane with PBS for 10 min × 3 times.

[0156] (10) Secondary antibody incubation: Add the diluted secondary antibody, place it on a shaker and shake slowly, incubate at room temperature for 1 hour.

[0157] (11) Washing the membrane: Wash the PVDF membrane with PBS for 10 min × 3 times.

[0158] (12) Developing and photographing.

[0159] (13) Results processing: The protein bands in the Western blot were quantitatively analyzed using ImageJ software and standardized using the expression level of the internal reference gene β-actin.

[0160] 3. Extraction of primary decidual stromal cells

[0161] ① Wash the decidual tissue with sterile PBS solution 3-4 times, and then cut the decidual tissue into a paste;

[0162] ② Transfer the decidual tissue to a 50mL centrifuge tube, add digestion solution, the ratio of tissue volume to digestion solution volume is 1:1, place in a 37℃ water bath for 1.5h, and shake to mix once every 20min;

[0163] ③ Add an equal volume of 2% complete culture medium to stop digestion, filter (100μm), filter (40μm);

[0164] ④ Centrifuge (centrifuge parameters: speed 800 rpm / min, time 5 min) and remove the supernatant;

[0165] ⑤ Add 4 mL of erythrocyte lysis buffer and incubate at room temperature for 10 min;

[0166] ⑥ Centrifuge (800 rpm / min, 5 min), remove supernatant, resuspend cells in 10% complete culture medium, seed a portion of cells into 4-well plates for immunofluorescence detection of the purity of primary uterine decidual stromal cells; seed the remainder into 10 cm culture dishes and incubate overnight in a 5% CO2, 37°C cell incubator.

[0167] ⑦ After culturing for 24 hours, collect cells from a 10cm culture dish, add Trizol, and proceed to the next experiment.

[0168] Experimental results:

[0169] qRT-PCR ( Figure 2A ), Western blot ( Figure 2B The results all indicated that the mRNA and protein levels of JAZF1 were significantly downregulated in RSA decidual tissue. Consistent with this, from... Figure 2C and Figure 2D As can be seen, the mRNA and protein levels of JAZF1 in primary decidual stromal cells isolated from RSA decidua were lower than those in the normal group.

[0170] (III) Immunohistochemistry and immunofluorescence detection of the location and relative expression level of JAZF1 in decidual tissue.

[0171] 1. Immunohistochemical technique

[0172] 1) Sectioning: Paraffin-embedded continuous sections of decidual tissue were 4 μm thick and spread in warm water at 42℃;

[0173] 2) Baking: Decidual tissue sections were placed in a 65℃ constant temperature electric baking oven and baked for 30 minutes;

[0174] 3) Dewaxing: Decidual tissue sections were dewaxed in xylene for 10 min*3 times, in anhydrous ethanol for 10 min*2 times, in 95% ethanol for 5 min*2 times, in 85% ethanol for 5 min*2 times, in 75% ethanol for 5 min*2 times, and then washed with running water.

[0175] 4) Antigen retrieval: First, place the slide in the retrieval solution and put it in a pressure cooker. Heat at 1600W until the valve is opened, then reduce the pressure to 900W and continue for 15 minutes. Remove the retrieval box and let it cool.

[0176] 5) Rinse with PBS to remove residual antigen retrieval solution;

[0177] 6) Place the slide in 3% H2O2 solution and incubate at room temperature in the dark for 10 min, then wash with PBST for 5 min each time, 3 times.

[0178] 7) Shake off the liquid, add 5% BSA blocking solution, and incubate at room temperature for 60 min;

[0179] 8) Remove the blocking solution, add primary antibody (enough to completely cover the tissue), and incubate overnight at 4°C;

[0180] 9) Wash with PBST for 5 min × 3 times;

[0181] 10) Shake off the liquid, add secondary antibody, incubate at room temperature for 60 min, and wash with PBST for 5 min × 3 times;

[0182] 11) Shake off the liquid, add DAB colorimetric solution, and then stop the reaction in double-distilled water;

[0183] 12) Restaining and blueing: Hematoxylin was applied for 1 minute, and the color development was stopped immediately with tap water to avoid the nucleus color becoming too dark.

[0184] 13) Immerse the glass slide in 75% ethanol for 3 min × 1 time, 85% ethanol for 3 min × 1 time, 95% ethanol for 3 min × 2 times, anhydrous ethanol for 3 min × 2 times, and xylene for 3 min × 2 times in sequence.

[0185] 14) Add neutral resin to seal the sheet and place it in a fume hood to dry;

[0186] 15) Immunohistochemical image analysis: The slides were photographed by another person. Five 400× fields of view were randomly selected and quantitative analysis was performed using the IHC Profiler plugin of ImageJ software. The scores corresponding to negative, weak positive, positive and strong positive were 1, 2, 3 and 4 points, respectively. The average value of the five fields of view was calculated.

[0187] 2. Immunofluorescence detection experimental steps:

[0188] 1)-10) are consistent with the steps of immunohistochemistry;

[0189] 11) Shake off the liquid, gently add 1 μg / ml of nuclear dye diamidinylphenylindole (DAPI), stain at room temperature for 10 minutes, and wash with 1×PBST for 5 minutes;

[0190] 12) Using a pipette, add 50 μL of mounting medium to prevent fluorescence quenching onto the tissue on the slide (if the tissue block is large, add more mounting medium as needed; too much will easily create air bubbles and affect the mounting effect). Store the mounted slide at 4°C in the dark and photograph it under a fluorescence microscope as soon as possible.

[0191] Experimental results: Immunohistochemical results are as follows Figures 3A-3B As shown, the immunofluorescence results are as follows: Figure 3CAs shown, JAZF1 is widely expressed in the decidual tissue of normal induced abortion and RSA, mainly located in the cell nucleus. Furthermore, its expression in both the cytoplasm and nucleus of RSA decidual stromal cells is lower than that in the gestational age-matched normal group. These results indicate that JAZF1 expression is significantly downregulated in the decidual stromal cells of RSA patients.

[0192] (iv) In order to explore the potential function of JAZF1 in decidualization, we induced decidualization in human endometrial stromal cells (HESCs) in vitro and detected changes in JAZF1 mRNA and protein levels, as well as the expression of decidualization-related markers IGFBP1, PRL, and FOXO1.

[0193] The in vitro induction of decidualization of human endometrial stromal cell lines includes the following steps: Cell density reaches 50% under a light microscope, followed by in vitro decidualization induction treatment: Control group: 2% complete culture medium; Experimental group: decidualization induction medium (2% complete culture medium: 4mM medroxyprogesterone acetate solution: cyclic adenosine monophosphate solution, volume ratio 1000:1:10), 2 ml / well. Cell morphology changes are observed daily under a light microscope. The decidualization induction medium is changed every 2 days. Cells are collected 6 days after decidualization for the next experimental step.

[0194] Experimental results:

[0195] The results of Western blot are as follows Figures 4A-4B As shown, the immunofluorescence results are as follows: Figure 4C As shown, the experimental results indicate that with the progression of decidualization, the expression of decidualization-related markers IGFBP1, PRL and FOXO1 increases, and the expression of JAZF1 also gradually increases.

[0196] (V) To verify the results of the above in vitro experiments, we used IHC to detect the expression of JAZF1 in human endometrium during the proliferative and early secretory phases. The immunohistochemical results of human endometrium during the proliferative and early secretory phases are as follows: Figures 5A-5B As shown, the results suggest that the abundance of JAZF1 protein is higher in the early secretory phase of the endometrium than in the proliferative phase, especially in stromal cells.

[0197] By reviewing single-cell sequencing data of endometrial tissue from public databases, we analyzed the expression of JAZF1 in endometrial tissue and stromal cells at different stages. Endometrial public dataset scRNA-seq data is available as follows: Figure 5C The results showed that, compared with the proliferative phase, JAZF1 was upregulated in the endometrium during the early secretory phase, and its expression was also upregulated in stromal cells.

[0198] Immunohistochemical results indicated that JAZF1 expression was downregulated in the proliferative phase and early secretory phase of the endometrium in patients with recurrent spontaneous abscess (RSA). These results suggest that JAZF1 is a potential regulator of decidualization establishment and development.

[0199] Example 2: Effects of JAZF1 knockout on hESC survival and decidualization

[0200] (I) Effective knockout and overexpression of JAZF1 in HESCs

[0201] To investigate the significance of JAZF1 in endometrial stromal cells, this invention utilized siRNA knockdown (siJAZF1), CRISPR / Cas9 knockout (KO-JAZF1), and lentiviral overexpression of JAZF1 (OE-JAZF1). After 48 hours of culture, cells were collected to assess the efficiency of JAZF1 knockdown, knockout, and overexpression. Subsequently, the DNA fragments from PCR-derived stable KO cells with effective knockout efficiency were subjected to Sanger sequencing to determine whether the sgRNA targeted the coding region of JAZF1.

[0202] 1. siRNA transfection:

[0203] JAZF1-siRNA was designed and synthesized by China's Gemma Biotechnology Co., Ltd. Transfection was performed using Lipofectamine RNA iMAX, with the following specific steps:

[0204] ① The day before transfection, seed cells at an appropriate density into 6-well plates to achieve a confluence rate of 40-50% at the time of transfection, and incubate overnight with complete culture medium.

[0205] ② Remove the old culture medium 60 minutes before transfection and add 1.5 mL of fresh, antibiotic-free culture medium.

[0206] ③ For each transfected well, prepare a mixture of siRNA and Lipofectamine RNAiMAX.

[0207] 1) Add 100 pmol (5 μL) of siRNA to 250 μL of serum-free growth medium and mix well.

[0208] 2) Before using Lipofectamine RNA iMAX, mix it well and then add 5 μL of Lipofectamine RNA iMAX to 250 μL of growth medium for dilution. Incubate at room temperature for 5 min.

[0209] 3) Mix the diluted siRNA and Lipofectamine RNA iMAX and incubate at room temperature for 20 min.

[0210] ④ Add the mixture into the cells, with a final volume of 2 mL per well, and gently shake to mix.

[0211] ⑤ Culture the cells at 37℃ in a 5% CO2 incubator for 6 hours.

[0212] ⑥ Replace the culture medium with one 10% serum and continue cell culture for 48-72 hours until gene knockout experiments are performed.

[0213] The siRNA sequences used in this study are shown in the table below:

[0214] Table 6.

[0215]

[0216] 2. Establish JAZF1 knockout cell lines

[0217] The JAZF1 KO cell line was established using CRISPR / Cas9 technology. The specific steps are as follows:

[0218] (1) Find the coding region sequence of JAZF1 on NCBI.

[0219] (2) Copy the sequence into https: / / portals.broadinstitute.org / gpp / public / and design sgRNA.

[0220] (3) Verify sgRNA and select 100% specific sequences for synthesis.

[0221] (4) Lentiviral vector enzyme digestion system:

[0222] Table 7.

[0223]

[0224]

[0225] 5 μg of lentiviral CRISPR plasmid was digested and dephosphorylated with BsmBI and reacted at 37°C for 30 min.

[0226] (5) Perform agarose gel electrophoresis on the enzyme-digested plasmid and use a gel recovery kit to recover the enzyme-digested plasmid. The specific gel recovery steps are as above.

[0227] (6) Preparation of phosphorylated and annealed sgRNA system:

[0228] Table 8.

[0229]

[0230] (7) Annealing PCR system:

[0231] Table 9.

[0232]

[0233] (8) Dilute the primers that have been annealed in step (7) into sterile water at a ratio of 1:100.

[0234] (9) Ligation system of annealed sgRNA and enzyme-digested plasmid vector:

[0235] Table 10.

[0236]

[0237] 16℃, overnight.

[0238] (10) Transformation

[0239] 1) Thaw a tube of DH5-alpha competent E. coli cells on ice until the last ice crystal disappears. Gently transfer 50 μL of cells into a transformation tube on ice.

[0240] 2) Add 5 μL of the plasmid DNA ligated the day before to the cell mixture and mix.

[0241] 3) Place the mixture on ice for 30 minutes. Do not vortex.

[0242] 4) Heat at 42°C for 30 seconds to activate competent cells. Place on ice for 3 minutes.

[0243] 5) Add 950 μL of room temperature LB liquid medium to the competent cells in step 4).

[0244] 6) Shake at 37°C for 60 minutes, or shake vigorously (250 rpm) or rotate.

[0245] 7) Before coating, centrifuge the bacterial solution from 6) at 3500 rpm for 5 min, remove 800 μL of supernatant, and mix the remaining bacterial solution with a pipette tip.

[0246] 8) Spread 100 μL of the bacterial culture dilution onto the selection plate and incubate overnight at 37°C.

[0247] 9) Pick a single, larger bacterium and add it to the culture medium, then add 1000× ampicillin.

[0248] 10) Take 4 mL of bacterial culture for mini-prep, sequence the plasmid, and confirm whether the sequence inserted in the plasmid is the designed sgRNA.

[0249] (11) Place the three bacterial cultures with correct sequencing sequences into 100 mL of LB liquid medium, add 100 μL of 1000× ampicillin, and place them in a constant temperature shaker at 37°C and 200 rpm overnight.

[0250] (12) Take 500 μL of bacterial culture, add 500 μL of glycerol, mix and store at -80℃.

[0251] (13) Lentiviral packaging and transfection of HESCs were performed using HEK 293T cells, as follows (10cm culture dish):

[0252] 1) On day 1, inoculate 2.5 × 10⁻⁶ cells per well in 10 mL of culture medium. 6 One HEK 293T cell (morning) or 3 × 10 6 HEK293T cells (afternoon).

[0253] 2) On the second day (morning), prepare a mixture of 3 plasmids in a clean 1.5 mL EP tube:

[0254] Table 11.

[0255]

[0256] Vortex the mixture and incubate for 10 minutes. Add the mixture dropwise to the culture medium of HEK 293T cells.

[0257] 3) Change the medium after 6-8 hours, replacing it with 10% complete culture medium.

[0258] 4) The day before collecting the virus, lay out HESCs in plates at a quantity of 1.5 × 10⁻⁶. 5 .

[0259] 5) First filter the virus supernatant with a 0.44μm filter, then add a 10cm dish of supernatant to one well of a 6-well plate containing hESCs. Change the medium after 24 hours with 10% complete culture medium.

[0260] (17) 72 hours after transfecting the target cells, 1.3 mg / mL Neomycin was used for drug screening for 72 hours.

[0261] (18) After the drug screening was completed, the total JAZF1-KO cells were digested, diluted, and seeded into 96-well plates at a density of 1 cell / well. The cells were collected to extract RNA and DNA.

[0262] (19) Extract RNA to detect JAZF1 knockout efficiency, extract DNA (specific steps as above) and perform PCR amplification (specific steps as above), perform Sanger sequencing on the amplification product to confirm plasmid genotype.

[0263] (20) Once the genotype is confirmed, the corresponding JAZF1-KO cell line can be amplified and the next experiment can be carried out.

[0264] 3. DNA PCR

[0265] (1) Reaction system:

[0266] Table 12.

[0267]

[0268] When amplifying products with a length ≥ 10 kb, use primers with a final concentration of 0.2 μM.

[0269] (2) PCR amplification program:

[0270] Table 13.

[0271]

[0272] (3) Amplification was performed for a total of 30 cycles. After the cycle, agarose gel electrophoresis was run to identify whether the bands were the target gene. Once confirmed, the gene was sent for Sanger sequencing.

[0273] The PCR primer sequences used in this study are as follows:

[0274] Table 14.

[0275]

[0276] 4. Establishment of overexpression cell lines:

[0277] (1) Overexpression PCR primer design: Locate the DNA coding region of the target gene on the NCBI website and open it with snapgene software.

[0278] (2) Select appropriate restriction sites: Select two restriction sites that are present on the plasmid vector but not in the coding region of the target gene, design them and then send them to the company for synthesis.

[0279] (3) Overexpression plasmid vector digestion, the system is as follows:

[0280] Table 15.

[0281]

[0282] (4) Use synthesized primers to PCR the complete CDS region of the target gene. The system is as follows:

[0283] Table 16.

[0284]

[0285] When amplifying products with a length ≥ 10 kb, use primers with a final concentration of 0.2 μM.

[0286] (5) PCR amplification procedure:

[0287] Table 17.

[0288]

[0289] (6) Amplify for a total of 30 cycles. After the amplification is completed, run 1% agarose gel electrophoresis and compare the molecular weight of the DNA marker to determine the molecular weight of the PCR product. This will help determine whether the target gene DNA coding region has been completely PCR-coordinated.

[0290] (7) After successful PCR, the PCR product is recovered by gel extraction.

[0291] (8) All gel recovery products were subjected to enzymatic digestion at 37°C for 60 min. The system is as follows:

[0292] Table 18.

[0293]

[0294]

[0295] (9) Perform 1% agarose gel electrophoresis on the enzyme digestion products and then recover the electrophoretic products from the gel.

[0296] (10) Ligate the enzyme-digested DNA fragments to the enzyme-digested plasmid vector, as follows:

[0297] Table 19.

[0298]

[0299] Ligate overnight at 16°C. The volume ratio of DNA fragment mass to molecular weight to plasmid vector mass to molecular weight in the ligation system is 1:3, thus calculating the required volume of DNA fragment and double-distilled water.

[0300] (11) Convert the coating plate, the steps are the same as before.

[0301] (12) Select single clones, extract plasmids, sequence, extract plasmids in medium quantities, package lentiviruses and screen for drugs as before.

[0302] (13) After the drug screening was completed, a portion of the cells were taken to extract RNA, and the overexpression efficiency was detected by qRT-PCR.

[0303] The overexpression primer sequences used in this study are as follows:

[0304] Table 20.

[0305]

[0306] Experimental results:

[0307] The results of qRT-PCR for siRNA knockdown, CRISPR / Cas9 knockout, and lentiviral overexpression of JAZF1 are as follows: Figure 6A As shown, the Western blot results are as follows: Figure 6B As shown, from Figures 6A-6B As can be seen, compared with the control group, the mRNA and protein levels of JAZF1 in the siRNA and lentivirus treatment groups were significantly reduced, while the expression level of JAZF1 in the overexpression treatment group was significantly upregulated, confirming the effectiveness of knockdown, knockout and overexpression.

[0308] DNA fragments from PCR-derived KO mutants with high knockout efficiency were subjected to Sanger sequencing to determine whether the sgRNA targeted the coding region of JAZF1. The Sanger sequencing results are shown below. Figure 6C As shown in the figure, the results indicate that the edited stable transgenic line has a missing base in the coding region of the DNA, the stop codon is advanced, and gene transcription terminates prematurely, indicating that the stable transgenic line is effective.

[0309] (II) Effects of JAZF1 knockdown on HESCs survival and decidualization

[0310] After treating HESCs with siRNA and KO lentivirus, decidualization was induced in vitro for 6 days with cyclic adenosine monophosphate (cAMP) and medroxyprogesterone acetate (MPA). The induction medium was changed every two days, and the expression of apoptosis and decidualization-related indicators was detected by qRT-PCR and Western blotting.

[0311] Experimental results:

[0312] qRT-PCR results are as follows Figure 7A As shown, the results indicate that knocking down or eliminating JAZF1 upregulated the expression of the pro-apoptotic molecule BAX, while downregulating the anti-apoptotic molecule BCL2. Western blot results are shown below. Figures 7B-7C It also showed that knocking down or eliminating JAZF1 significantly upregulated the protein levels of pro-apoptotic molecules BAX, cyt c, and cleaved-caspase 3, while significantly downregulating the anti-apoptotic molecule BCL2.

[0313] (III) The anti-apoptotic effect of JAZF1 on decidualized HESCs was detected by flow cytometry, and the cell proliferation status was detected by CCK8.

[0314] Experimental steps:

[0315] 1. Flow cytometry combined with Annexin V / PI to detect cell apoptosis

[0316] (1) Induce apoptosis according to experimental requirements. Untreated cell samples serve as negative controls. For flow cytometry analysis, the experimental groups are stained with Annexin V-FITC and PI separately to adjust compensation.

[0317] (2) Collect 1×10 5 -5×10 5 Cells were digested with EDTA-free trypsin. After digestion was terminated, the cells were collected, centrifuged at 1000 rpm and 4°C for 5 min, and the supernatant was discarded.

[0318] (3) Washing cells: Wash cells twice with pre-cooled PBS, centrifuging at 1000 rpm and 4℃ for 5 min each time, and discard the supernatant.

[0319] (4) Cell resuspension: Add 100 μL of 1×Binding Buffer and gently mix until a single cell suspension is formed.

[0320] (5) Cell staining: Add 5 μL Annexin V-FITC and 5 μL PI Staining Solution, mix well; incubate at room temperature in the dark for 10 min; add 400 μL 1× Binding Buffer, mix well. Detect using flow cytometry.

[0321] (6) Flow cytometry analysis: The excitation wavelength for flow cytometry was 488 nm; the green fluorescence of FITC was detected in the FL1 channel; the red fluorescence of PI was detected in the FL2 or FL3 channel. In this invention, FL3 was used, and 10,000 events were collected for each sample. Data analysis was performed using software such as FlowJo, with FL1 as the x-axis and FL3 as the y-axis. The negative and positive boundaries of the two fluorescence parameters were determined based on the FITC and PI fluorescence values, thus defining the cross-gate. In a typical experiment, cells can be divided into three subpopulations: viable cells are double-negative (Annexin V-FITC). ﹣ / PI ﹣ Early apoptotic cells were Annexin V-FITC single positive (Annexin V-FITC) ﹢ / PI ﹣ Late-stage apoptotic cells were double-positive for Annexin V-FITC and PI (Annexin V-FITC). ﹢ / PI ﹢ ).

[0322] 2. Cell proliferation activity detection

[0323] (1) Preparation of standard curve

[0324] 1) Set up four concentration gradients: 1000, 2500, 5000, and 10000 cells / well. After passage, count the cells and dilute them to the above concentrations with culture medium, with 5 replicates per group.

[0325] 2) After 4 hours of cell adhesion, add 10 μL of CCK-8 reagent and incubate for 2 hours, 3 hours, and 4 hours, then measure the OD value. Plot the cell number on the x-axis and the OD value on the y-axis, and incubate for 3 hours.

[0326] (2) Detection of cell proliferation activity

[0327] 1) Add 1*10 to each hole 4 Add cell suspension per 100 μL. Add 100 μL of sterile PBS to the edge well.

[0328] 2) After incubating in the incubator for 24 hours, add 10 μL of CCK8 to each well and mix by pipetting, while keeping the incubator away from light.

[0329] 3) After incubation in the incubator for 4 hours, measure the absorbance of each well at a wavelength of 450 nm using an absorbance microplate reader.

[0330] 4) Data statistics: Take the average value of 5 replicates in each group and compare between groups.

[0331] Experimental results:

[0332] Flow cytometry results as follows Figures 7D-7E As shown, the results indicate that JAZF1 deficiency induces apoptosis, and the CCK8 results ( Figure 7F The study indicated that knockdown and elimination of JAZF1 significantly reduced the proliferative capacity of stromal cells inducing decidualization. Simultaneously, the lack of JAZF1 upregulated the mRNA and protein expression levels of PRL and IGFBP-1, resulting in decidualization defects.

[0333] (iv) After treating HESCs with a lentivirus overexpressing JAZF1, decidualization was induced in vitro for 6 days using cAMP and MPA. Apoptosis and decidualization-related markers were detected by qRT-PCR and Western blotting. Flow cytometry was used to detect apoptosis levels, and CCK8 assays were used to assess cell proliferation. Specific experimental procedures were the same as above.

[0334] qRT-PCR results are as follows Figure 8A The results showed that overexpression of JAZF1 downregulated the level of the apoptosis molecule BAX, upregulated the level of the anti-apoptotic molecule BCL2, and downregulated the levels of decidualizing IGFBP1 and PRL. Western blot results are as follows: Figures 8B-8DThe results indicated that JAZF1 overexpression only downregulated IGFBP1 and cyt c levels and upregulated BCL2, but did not change BAX and cleaved-caspase 3 levels. Flow cytometry and CCK8 results suggested that JAZF1 overexpression downregulated apoptosis in HESCs. Figure 7E ), upregulating cell proliferation capacity ( Figure 7F ).

[0335] In summary, the above results demonstrate that JAZF1 can prevent excessive apoptosis and decidualization defects in HESCs during decidualization.

[0336] Example 3: Effects of JAZF1 knockdown or overexpression on the invasive ability of HTR-8 / SVneo cells.

[0337] Studies have shown that decidual reflex disorders or excessive IGFBP1 levels in decidual cells are associated with miscarriage, premature fetal growth, or spontaneous preterm birth. This invention further evaluated the effect of JAZF1 knockout HESCs on trophoblast cell invasion. First, HESCs were treated with siRNA for 6 hours to induce decidualization, or the OE-JAZF1 stable transgenic line was directly induced. After 6 days of treatment, the supernatant was collected in a transwell chamber, and an appropriate number of HTR-8 / SVneo cells were seeded and co-cultured on top of a matrix gel in the chamber. Transwell assays showed that JAZF1 knockout HESCs inhibited the invasion of HTR-8 / SVneo cells. Figure 9A Overexpression of JAZF1 promotes its invasion. Figure 9B This study demonstrates that the absence of JAZF1 has an adverse effect on the function of decidual stromal cells.

[0338] Example 4: Apoptosis level of decidual stromal cells in recurrent miscarriage

[0339] (1) 10× single-cell sequencing analysis of apoptosis in decidual stromal cells

[0340] Given the pro-apoptotic effect of JAZF1 downregulation observed during in vitro decidualization, this invention further evaluated the apoptosis levels in decidual tissues from RSA and control groups, as well as in primary DSCs. Our previous scRNA-seq analysis of DSCs showed a significant increase in the pro-apoptotic gene BAX in the RSA group, but no significant change in BCL2. Figure 10A Furthermore, GO enrichment analysis of upregulated genes in RSA compared to the control group also indicated that apoptosis and cell death pathways were significantly activated in DS1, DS2, and DS3. Figure 10B )

[0341] (2) Apoptosis of decidual stromal cells in recurrent miscarriage

[0342] To validate the results of single-cell sequencing, qRT-PCR, western blot, and immunohistochemistry were used to detect the expression levels of apoptosis-related genes BAX, cyt c, cleaved-caspase 3, and BCL2 in decidual tissue and primary decidual stromal cells.

[0343] Experimental results:

[0344] qRT-PCR was used to detect the expression levels of apoptosis-related genes in decidual tissue, such as... Figure 11A As shown, Western blot analysis revealed the expression levels of apoptosis-related genes in decidual tissue. Figures 11B-11C As shown, Figures 11A-11C The results showed that the pro-apoptotic genes BAX, cyt c, and cleaved-caspase 3 were significantly upregulated in RSA decidual tissue, while the level of the anti-apoptotic gene BCL2 was downregulated. Furthermore, the same expression pattern was observed in primary decidual stromal cells isolated from RSA decidua. Figures 11D-11E The above results were further verified by immunohistochemical examination. Figure 11F ).

[0345] (3) The overall apoptosis level of decidual tissue was detected by TUNEL staining.

[0346] Experimental steps:

[0347] 1. TUNEL testing:

[0348] (1) Sample pretreatment: Paraffin-embedded tissue sections.

[0349] (2) Positive treatment

[0350] After permeabilization, the samples can be treated with DNase I to prepare a positive control.

[0351] 1) Dilute 10×DNase I Buffer with double-distilled water at a ratio of 1:10 to prepare 1×DNase I Buffer. Prepare 200 μL of 1×DNase I Buffer for each sample.

[0352] 2) Add 100 μL of 1×DNase I Buffer to the permeabilized sample and allow it to equilibrate at room temperature for 5 min.

[0353] 3) Dilute DNase I (2U / μL) with 1×DNase I Buffer to a final concentration of 20U / mL.

[0354] 4) Gently aspirate excess liquid, add 100 μL of DNase I working solution with a concentration of 20 U / mL, and incubate at room temperature for 10 min.

[0355] 5) Gently aspirate excess liquid and thoroughly rinse the slides / smears 2-3 times in a staining jar containing 1×PBS. Positive control slides must be stained in a separate jar. Residual DNase I on positive control slides / smears may cause false positives in the experimental groups.

[0356] (3) Marking and detection

[0357] 1) Dilute 5×Equilibration Buffer with double-distilled water at a ratio of 1:5 to make 1×Equilibration Buffer.

[0358] 2) Add 100 μL of 1×Equilibration Buffer to completely cover the sample area to be tested, and allow it to equilibrate at room temperature for 10-30 minutes.

[0359] 3) During the equilibration period, prepare the labeling solution according to the table below under light-protected conditions.

[0360] Table 21.

[0361]

[0362] For areas smaller than 5cm 2 The required volume for the reaction is 50 μL. Multiply 50 μL by the number of experimental and positive control reactions to determine the total required volume of TdT incubation buffer. For samples with larger surface areas, the reagent volume can be increased proportionally.

[0363] 4) After equilibration, use absorbent paper to remove 1×Equilibration Buffer, and then add 50 μL TdT incubation buffer to the sample.

[0364] 5) Cut the sealing film to the same size as the tissue or slide and gently cover it over the sample to ensure even distribution of the reagent. Place the slide in a humidified chamber and incubate at 37°C for 60 minutes. Wrap the humidified chamber with aluminum foil to protect it from light.

[0365] 6) Wash twice with fresh 1×PBS solution, incubating at room temperature for 5 min each time. After washing with 1×PBS, the sample can be washed three more times with PBS containing 0.1% Triton X-100 and 5 mg / mL BSA, for 5 min each time.

[0366] 7) Counterstain the sample with 2 μg / mL DAPI solution in the dark, and let it stand at room temperature for 5 min during staining.

[0367] 8) Wash the sample by immersing the slide in 1×PBS solution three times, leaving it at room temperature for 5 minutes each time.

[0368] 9) Add 100 μL of 20% glycerol prepared with 1×PBS to the sample area to keep the sample moist.

[0369] 10) Immediately analyze the sample under a fluorescence microscope. Observe the BrightRed fluorescence at 620±20 nm using a standard fluorescence filter; observe the blue fluorescence of DAPI at 460 nm. If necessary, the sample can be mounted and stored overnight at 4°C in the dark. DAPI stains both apoptotic and non-apoptotic cells blue. BrightRed incorporation produces red fluorescence only in the nuclei of apoptotic cells.

[0370] Experimental results:

[0371] The results of TUNEL staining are as follows: Figure 12 As shown in the figure, the fluorescence intensity of the decidual tissue in the RSA group was higher than that in the control group, indicating that the apoptosis level of the decidual tissue in the RSA group was higher.

[0372] (iv) Analyze the relationship between JAZF1 and the expression levels of apoptosis-related molecules.

[0373] Figure 13A For correlation analysis of BCL2 and JAZF1 in decidual tissue, Figure 13B For correlation analysis of cleaved-caspase 3 and JAZF1 in decidual tissue, Figure 13C Correlation analysis of BAX and JAZF1 in decidual tissue. Figure 13D Correlation analysis of cyt c and JAZF1 in decidual tissue was conducted. We noted a positive correlation between BCL2 expression and JAZF1 expression levels, while the opposite trend was observed in cleaved-caspase 3. No correlation was observed between BAX and cyt c and JAZF1.

[0374] Example 5

[0375] A model for predicting recurrent miscarriage using JAZF1 in the two groups of patients in Example 1 was established using ROC curves. The AUC value, sensitivity, specificity, positive detection rate, negative detection rate, and Youden's index of this prediction method were calculated. The results are shown in Table 22. Figure 14A As shown.

[0376] Table 22 Molecular diagnostic specificity analysis of JAZF1 in the original 10 pairs of decidual tissue samples

[0377]

[0378] The results showed that JAZF1 expression level could predict recurrent miscarriage, with an AUC of 0.79; high sensitivity (0.90); medium specificity (0.60); optimal cutoff value of 0.3654; positive predictive value of 0.90; negative predictive value of 0.60; and Youden index of 0.50.

[0379] Meanwhile, Table 23 and Figures 14B-14C The results showed that, in the four pairs of patients other than those in Example 1, the JAZF1 expression level was significantly different in both the normal group and the recurrent miscarriage group (p<0.05). When the four pairs of patients were diagnosed using the optimal cutoff value, the true positive rate was 0.75, the false positive rate was 0, the true negative rate was 1.00, and the false negative rate was 0.25, indicating that the diagnostic effect was good.

[0380] Table 23 shows the detection rates of four pairs of decidual tissue samples other than those in Example 1.

[0381] protein True positive rate False positive rate True negative rate False negative rate JAZF1 0.75 0 1.00 0.25

[0382] We further utilized ROC curves to establish a model for predicting recurrent miscarriage based on JAZF1 expression in the four pairs of tissues in Example 5, and calculated the AUC value, sensitivity, specificity, and Youden index of this prediction method. The results are shown in Table 24. Figure 14D As shown.

[0383] Table 24. Molecular diagnostic specificity analysis of JAZF1 in four pairs of decidual tissue samples other than those in Example 1.

[0384] protein AUC (95% CI) Sensitivity Specificity Yoden Index JAZF1 0.94(0.76-1.00) 0.75 1.00 0.75

[0385] The results showed that JAZF1 expression level could predict recurrent miscarriage, with an AUC of 0.94, good sensitivity of 0.75, high specificity of 1.00, and Youden index of 0.75.

[0386] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of the JAZF1 gene as a biomarker in the preparation of products for miscarriage diagnosis or auxiliary diagnosis, wherein the miscarriage is recurrent miscarriage.

2. The application according to claim 1, characterized in that, The diagnostic products include those that diagnose miscarriage using qRT-PCR, immunoassay, flow cytometry, and single-cell sequencing.

3. The application according to claim 1, characterized in that, The product is selected from one or more of the following: reagent kits, gene chips, protein chips, and formulations.

4. The application according to claim 1, characterized in that, The application steps are as follows: (1) Detect the expression level of the JAZF1 gene in the sample to be tested; (2) Compare the expression level of the JAZF1 gene in the test sample with that in the control group; (3) Compared with the control group, the JAZF1 gene expression in the test sample was downregulated, indicating that the subject was a miscarriage patient; otherwise, the subject was not a miscarriage patient.

5. The application of reagents for detecting the JAZF1 gene in the preparation of diagnostic or auxiliary diagnostic products for miscarriage, characterized in that, The reagent is used to detect the mRNA or protein expression level of the JAZF1 gene, and the miscarriage is a recurrent miscarriage.

6. The application of substances that enhance the content or activity of the JAZF1 gene in the preparation of drugs for treating or preventing miscarriage, wherein the miscarriage is recurrent miscarriage, and the substance that enhances the content or activity of the JAZF1 gene is decidualization induction culture medium or lentiviral overexpression vector JAZF1. The decidualization induction medium consisted of 2% complete culture medium, 4mM medroxyprogesterone acetate solution, and cyclic adenosine monophosphate solution in a volume ratio of 1000:1:10.

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