Application of miR-126-5p in the diagnosis and treatment of unexplained recurrent spontaneous abortion

Abstract

The present invention belongs to the field of biomedicine and molecular biology technology, and specifically relates to the application of miR‑126‑5p in the diagnosis and treatment of unexplained recurrent spontaneous abortion. Through research, the present invention found that the CASP1 mRNA, protein expression levels and cell pyroptosis pathway in URSA patients were significantly upregulated, and then the whole transcriptome high-throughput sequencing technology was used to screen the differentially expressed miRNAs in the villous trophoblast cells of URSA patients. At the same time, combined with bioinformatics analysis, it was found that miR‑126‑5p was downregulated in URSA patients, and was significantly negatively correlated with CASP1 mRNA expression. It was finally determined that miR‑126‑5p alleviated the occurrence of URSA by targeting the Caspase‑1 / GSDMD signaling pathway to reduce trophoblast cell pyroptosis, which explained the pathogenesis of URSA from the perspective of epigenetic immune regulation, and provided new targets and pathways for the clinical diagnosis and treatment of URSA.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to the application of miR-126-5p in the diagnosis and treatment of unexplained recurrent spontaneous abortion. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Spontaneous abortion that occurs three or more times in a row is called recurrent spontaneous abortion (RSA). The causes of this disease are complex. In addition to known factors such as genetics, anatomy, and infection, about 50% of the causes are unknown, which is called unexplained recurrent spontaneous abortion (URSA). Multiple abortions not only threaten women's reproductive health, but also harm the patient's mental health. Its prevention and treatment are currently a difficult problem that needs to be solved in the medical field.

[0004] Trophoblast cells are key cells for embryo implantation in early pregnancy. Their dysfunction is the key to embryo implantation failure, immune disorders at the maternal-fetal interface, and is an important factor in the onset of URSA. More and more studies have shown that pyroptosis, as an inflammatory programmed cell death, plays a role in pregnancy and pregnancy-related diseases. However, excessive pyroptosis breaks the immune balance at the maternal-fetal interface, releases a large number of inflammatory factors, and causes the maternal-fetal interface to present a high inflammatory state, leading to miscarriage and other pregnancy-related diseases. However, the relationship between trophoblast cell pyroptosis and URSA has not yet been fully clarified.

[0005] MicroRNA (miRNA) is a class of widely existing endogenous small RNAs with a length of about 20 to 24 nucleotides. MiRNAs exert their biological functions by binding to the 3'UTR of target gene mRNA, affecting the stability of target gene mRNA and inhibiting its translation. Studies have reported that miRNAs play a role in a variety of physiological and disease processes, including cell proliferation, apoptosis, pyroptosis, infectious diseases, diabetes and tumors. Studies have shown that miRNAs can participate in the occurrence of URSA, but their role in regulating trophoblast cell pyroptosis in URSA has not been reported. In-depth exploration in this direction will further explain the pathogenesis of URSA and provide new targets and pathways for the clinical diagnosis and treatment of URSA. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide the application of miR-126-5p in the diagnosis and treatment of unexplained recurrent spontaneous abortion. The present invention found through research that the CASP1 mRNA, protein expression levels and cell pyroptosis pathways in URSA patients were significantly upregulated, and then the whole transcriptome high-throughput sequencing technology was used to screen the differentially expressed miRNAs in the villous trophoblast cells of URSA patients. At the same time, combined with bioinformatics analysis, it was found that miR-126-5p was downregulated in URSA patients, and was significantly negatively correlated with CASP1 mRNA expression. It was finally determined that miR-126-5p alleviated the occurrence of URSA by targeting the Caspase-1 / GSDMD signaling pathway to reduce trophoblast cell pyroptosis. Based on the above research results, the present invention is completed.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] The first aspect of the present invention provides the use of a reagent for detecting the expression level of miR-126-5p and related biological elements in the preparation of a product for (assisting) diagnosing whether a subject suffers from unexplained recurrent spontaneous abortion or predicting the risk of a subject suffering from unexplained recurrent spontaneous abortion;

[0009] Wherein, the miR-126-5p related biological elements include at least one of Caspase-1 and GSDMD.

[0010] Furthermore, detecting the expression levels of Caspase-1 and GSDMD includes detecting the total protein and activation level of Caspase-1, the expression levels of GSDMD and its active fragment GSDMD-N, and the expression levels of the corresponding coding genes.

[0011] The products include but are not limited to primers, probes, (gene or protein) chips, nucleic acid membrane strips, detection kits, detection devices or equipment for detecting the expression levels of miR-126-5p and related biological elements in the sample to be tested.

[0012] The sample to be tested is a human or non-human (such as mouse) sample, including but not limited to a subject's blood (such as serum) sample, endometrium, placenta, and trophoblast cells.

[0013] The second aspect of the present invention provides a system for (assisting) diagnosing whether a subject suffers from unexplained recurrent spontaneous abortion or predicting the risk of unexplained recurrent spontaneous abortion in a subject, the system comprising at least:

[0014] An acquisition unit, configured to: acquire the expression level of miR-126-5p and related biological elements of the subject;

[0015] The evaluation unit is configured to evaluate whether the subject suffers from unexplained recurrent spontaneous abortion or predict whether the subject suffers from unexplained recurrent spontaneous abortion according to the expression levels of the subject's miR-126-5p and related biological elements obtained by the acquisition unit.

[0016] Wherein, the miR-126-5p related biological elements include at least one of Caspase-1 and GSDMD.

[0017] Furthermore, detecting the expression levels of Caspase-1 and GSDMD includes detecting the total protein and activation level of Caspase-1, and the expression levels of GSDMD and its active fragment GSDMD-N; and the expression levels of the corresponding coding genes.

[0018] The third aspect of the present invention provides the use of miR-126-5p and related biological elements as targets in the prevention and treatment of unexplained recurrent spontaneous abortion and / or the screening of unexplained recurrent spontaneous abortion drugs.

[0019] The method for screening drugs for unexplained recurrent spontaneous abortion comprises:

[0020] 1) Treating the system expressing and / or containing miR-126-5p and its related biological elements with the candidate substance; setting up a parallel control without the candidate substance treatment;

[0021] 2) After completing step 1), the expression level of miR-126-5p and its related biological elements in the system is detected; if the expression level of miR-126-5p is significantly increased and / or the expression level of Caspase-1 is significantly decreased in the system treated with the candidate substance compared with the parallel control, the candidate substance can be used as a candidate drug for the prevention and treatment of recurrent implantation failure.

[0022] The system can be a cellular system, a subcellular system, a solution system, a tissue system, an organ system or an animal system.

[0023] The fourth aspect of the present invention provides a substance for promoting the expression of miR-126-5p and / or enhancing its activity, wherein the substance is used in any one or more of the following:

[0024] (a) inhibiting the expression of Caspase-1 and GSDMD or preparing a product that inhibits the expression of Caspase-1 and GSDMD;

[0025] (b) inhibiting trophoblast cell pyroptosis or preparing a product that inhibits trophoblast cell pyroptosis;

[0026] (c) inhibiting the rate of embryo resorption or preparing products that inhibit the rate of embryo resorption;

[0027] (d) Prevention and / or treatment of unexplained recurrent spontaneous abortion.

[0028] In the present invention, the substance that promotes the expression of miR-126-5p and / or enhances its activity may be miR-126-5pmimics.

[0029] The above-mentioned product can be a medicine or an experimental reagent, and the experimental reagent can be used for basic research.

[0030] According to the present invention, when the product is a medicine, the medicine further comprises at least one inactive pharmaceutical ingredient.

[0031] The inactive ingredients of the drug can be carriers, excipients and diluents commonly used in pharmacy. Moreover, according to the usual methods, the drug can be made into oral preparations, external preparations, suppositories and sterile injection solutions in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc.

[0032] The non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0033] Beneficial technical effects of one or more of the above technical solutions:

[0034] The above technical scheme uses whole transcriptome high-throughput sequencing and 4D Label free quantitative proteomics technology to find that CASP1 mRNA, protein expression levels and cell pyroptosis pathways are significantly upregulated in URSA patients. Subsequently, whole transcriptome high-throughput sequencing technology is used to screen differentially expressed miRNAs in villous trophoblast cells of URSA patients. At the same time, combined with bioinformatics analysis, it is found that miR-126-5p is downregulated in URSA patients and is significantly negatively correlated with CASP1 mRNA expression. Further functional and mechanistic studies have shown that miR-126-5p directly binds to CASP1 mRNA 3'UTR through base complementarity and inhibits its translation. Experiments in vitro and in vivo in mice showed that overexpression of miR-126-5p can significantly inhibit the expression of CASP1 mRNA and protein, reduce trophoblast cell pyroptosis and the release of inflammatory factors, and alleviate the occurrence of URSA; while inhibiting the expression of miR-126-5p can relieve its inhibition on the target gene CASP1, promote trophoblast cell pyroptosis and the massive release of inflammatory factors, disrupt the maternal-fetal immune homeostasis, and participate in the occurrence of URSA.

[0035] In summary, miR-126-5p alleviates the occurrence of URSA by reducing trophoblast cell pyroptosis by targeting the Caspase-1 / GSDMD signaling pathway. The above technical solution further explains the pathogenesis of URSA from the perspective of epigenetic immune regulation, and provides new targets and pathways for the clinical diagnosis and treatment of URSA, so it has good potential practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings, which constitute a part of the specification of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0037] Figure 1 The pyroptosis and CASP1 expression of villous tissue cells in URSA patients in the embodiment of the present invention. (A) Compared with normal early pregnant women (NP), GO (Gene Ontology) enrichment analysis of upregulated genes in the whole transcriptome high-throughput sequencing of URSA patients' villous tissue (Fold change>1.5, P<0.05); (B) Heat map of 9 differentially expressed genes in the cell pyroptosis pathway; (C) Expanded clinical sample qRT-PCR to verify the differential gene CASP1 mRNA expression; (D) Compared with normal early pregnant women, GO enrichment of differential proteins in 4D Label free quantitative proteomics analysis of villous tissue of URSA patients (Fold change>1.5, P<0.05); (E) Western blot detection of Caspase-1, Cleaved 1, and Caspase-2 in human villous trophoblast cells Expression of Caspase-1, GSDMD and GSDMD-N proteins; (FG) ELISA detection of IL-1β and IL-18 protein levels in human serum; (HI) Immunofluorescence detection of Caspase-1 and GSDMD protein fluorescence images in human villous trophoblast cells (blue: DAPI; green: Caspase-1 and GSDMD; magnification ×40, scale bar = 50 μm). *** P<0.001, compared with the NP group.

[0038] Figure 2The effect of Caspase-1-mediated pyroptosis on abortion in pregnant mice in the embodiments of the present invention. (A) Representative images of uterine embryo absorption in each group of mice after URSA mice were treated with Caspase-1 inhibitor (VX-765) or pyroptosis inhibitor (DSF), and arrows indicate embryo absorption points; (B) Statistical graph of embryo absorption rate in each group of mice (n=5); (C) Western blot detection of Caspase-1, Cleaved Caspase-1, GSDMD and GSDMD-N protein expression in placental trophoblast cells in each group of mice; (D-E) ELISA detection of IL-1β and IL-18 protein expression in serum of each group of mice; (F) Representative images of uterine embryo absorption in each group of mice in normal pregnant mice (NP) overexpressing Caspase-1 and overexpressing Caspase-1 and adding DSF, and arrows indicate embryo absorption points; (G) Statistical graph of embryo absorption rate in each group of mice (n=5); (H) Western blot detection of Caspase-1, Cleaved Caspase-1, GSDMD and GSDMD-N protein expression in placental trophoblast cells in each group of mice (IJ) ELISA was used to detect the expression of Caspase-1, GSDMD and GSDMD-N proteins; (IJ) ELISA was used to detect the expression of IL-1β and IL-18 proteins in the serum of mice in each group. ** P<0.01, *** P<0.001, ns means no statistical difference.

[0039] Figure 3 Screening of differentially regulated miRNAs for CASP1 in the embodiments of the present invention. (A) Venn diagram of the intersection of down-regulated miRNAs (Fold Change>2) in human villous tissue by whole transcriptome high-throughput sequencing and miRNAs predicted to regulate CASP1 by Targetscan7.2; (B) qRT-PCR expanded clinical samples to verify the expression changes of miR-126-5p, miR-181c-5p and miR-181d-5p in human villous tissue trophoblast cells; (C) Correlation analysis between CASP1 mRNA and miR-126-5p in human villous tissue trophoblast cells; (D) ROC curve analysis of the diagnostic value of miR-126-5p for URSA; (E) Localization and expression of miR-126-5p in human villous tissue trophoblast cells by fluorescence in situ hybridization (red: miR-126-5p; blue: DAPI; magnification ×40, scale bar = 50μm). *** P<0.001, ns means no statistical difference compared with the NP group.

[0040] Figure 4The effect of regulating miR-126-5p on CASP1 expression and cell pyroptosis in the embodiments of the present invention. (A) qRT-PCR detection of miR-126-5p level changes in HTR-8 / SVneo after up-regulation or down-regulation of miR-126-5p; (B) qRT-PCR detection of CASP1 mRNA level changes in HTR-8 / SVneo after up-regulation or down-regulation of miR-126-5p; (C) Western blot detection of Caspase-1, Cleaved mitochondria, and leukocytes in HTR-8 / SVneo after up-regulation or down-regulation of miR-126-5p. Expression of Caspase-1, GSDMD and GSDMD-N proteins; (D) Immunofluorescence detection of changes in CASP1 protein levels in HTR-8 / SVneo after upregulation or downregulation of miR-126-5p (blue: DAPI; green: Caspase-1; magnification ×20, scale bar = 50 μm); (EF) ELISA detection of IL-1β and IL-18 protein expressions in the supernatant of HTR8 / SVneo cells after upregulation or downregulation of miR-126-5p; (G) Microscopic observation of pyroptosis images of HTR-8 / SVneo cells after upregulation or downregulation of miR-126-5p, arrows indicate cells undergoing pyroptosis (magnification ×20, scale bar = 50 μm). ** P<0.01, *** P<0.001, ns means no statistical difference.

[0041] Figure 5 The targeted regulatory relationship between miR-126-5p and CASP1 in the embodiment of the present invention. (A) Schematic diagram of CASP1 mRNA 3'UTR shows the potential binding sites and constructed mutant sequences of miR-126-5p and CASP1 mRNA 3'UTR; (BC) Dual luciferase reporter gene experiment verifies the effect of miR-126-5p mimics on the luciferase activity of WT-pmirGLO-CASP1 3'UTR and Mut-pmirGLO-CASP1 3'UTR. *** P<0.001, compared with the NC group, WT represents wild type; MUT represents mutant.

[0042] Figure 6In the present invention, overexpression of miR-126-5p alleviates URSA mouse embryo absorption by downregulating Casp1-mediated cell pyroptosis. (A) Representative images of uterine embryo absorption in each group of mice with upregulated miR-126-5p expression or upregulated miR-126-5p expression and upregulated Casp1 expression, with arrows indicating embryo absorption points; (B) Statistical graph of embryo absorption rate of each group of mice (n=5); (C) qRT-PCR detection of miR-126-5p expression in mouse placental trophoblast cells in each group; (D) Western blot detection of Caspase-1, Cleaved Caspase-1, GSDMD, GSDMD-N protein expression levels in placental trophoblast cells in each group; (EF) ELISA detection of IL-1β and IL-18 protein expression levels in the serum of each group of mice. ** P<0.01, *** P < 0.001, **** P<0.0001.

[0043] Figure 7 Inhibition of miR-126-5p expression in the present invention aggravates URSA mouse embryo absorption by upregulating Casp1-mediated cell pyroptosis. (A) Representative images of uterine embryo absorption in each group of mice with inhibition of miR-126-5p expression or inhibition of miR-126-5p expression and addition of VX-765, arrows indicate embryo absorption points; (B) Statistical graph of embryo absorption rate of each group of mice (n=5); (C) qRT-PCR detection of miR-126-5p expression in mouse placental trophoblast cells in each group; (D) Western blot detection of Caspase-1, Cleaved Caspase-1, GSDMD, GSDMD-N protein expression levels in placental trophoblast cells in each group; (EF) ELISA detection of IL-1β and IL-18 protein expression levels in the serum of each group of mice. ** P<0.01, *** P<0.001.

[0044] Figure 8 It is a schematic diagram of the URSA generating principle of the present invention. DETAILED DESCRIPTION

[0045] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0046] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in the present invention, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0047] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the reagent company; the reagents and consumables used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0048] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods for the specific conditions in the following examples are usually carried out under normal conditions.

[0049] Example

[0050] 1 Materials and methods

[0051] 1.1 Research subjects

[0052] Thirty URSA patients and 30 NP healthy control subjects who visited the Department of Obstetrics and Gynecology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine from August 2021 to August 2022 were collected. All enrolled URSA patients experienced at least three consecutive spontaneous early miscarriages from 6 to 10 weeks of gestation. NP healthy controls were normal early pregnant women who voluntarily terminated their pregnancy without spontaneous early miscarriage. Among these patients, we excluded individuals with infection, endocrine or metabolic disorders, anatomical abnormalities, autoimmune diseases, and chromosomal abnormalities of the father or mother. This study was approved by the Ethics Committee of Shandong University of Traditional Chinese Medicine, and all participants signed informed consent. URSA patients had an age of (33.23±1.00), number of miscarriages (2.57±0.09), and weeks of miscarriage (7.20±0.21); NP patients had an age of (30.10±1.03), number of miscarriages 0, and weeks of artificial abortion (7.87±0.29).

[0053] 1.2 Sample collection

[0054] Venous blood was collected from URSA patients after abortion and NP women before abortion using conventional blood collection tubes (Becton Dickinson), and serum was collected after ultracentrifugation and stored at -80°C. All patients underwent negative pressure aspiration of chorionic villus tissue during artificial abortion, and the collected chorionic villus tissue was immediately placed in a culture dish containing pre-cooled 1× PBS or saline, and the blood stains on the surface of the villi were quickly washed off, immediately separated, and quickly frozen in liquid nitrogen for subsequent qRT-PCR and Western blot detection or fixed in 4% paraformaldehyde and embedded in paraffin for immunofluorescence (IF) and fluorescence in situ hybridization (FISH) analysis.

[0055] 1.3 Whole transcriptome high-throughput sequencing

[0056] Chorionic villus tissues were collected from 3 URSA patients and 3 NP women, and 6 clinical samples were analyzed by high-throughput sequencing of the whole transfer group by Shanghai Ouyi Biomedical Technology Co., Ltd. The method was briefly described as follows: total RNA was extracted by TRIzol method, libraries were constructed by TruSeq Stranded Total RNA and Ribo Zero Gold, libraries were sequenced by Illumina HiSeq X Ten platform, and sequencing reads were analyzed in human genome (GRCh38) using HISAT2. In order to screen differential mRNAs, differentially expressed mRNAs and miRNAs were screened for subsequent studies based on P≤0.05 and Fold change≥1.5.

[0057] 1.4 4D Label-free Quantitative Proteomics

[0058] Chorionic villus tissues from 3 URSA patients and 3 NP women were collected. Protein quantitative detection and analysis of 6 clinical samples were performed by Hangzhou Jingjie Biological Co., Ltd. using 4DLabel free technology. The 6 samples were ground into powder using liquid nitrogen, and then 4 times the volume of lysis buffer (1% SDS, 1% protease inhibitor) was added and ultrasonic lysed. The BCA kit was used for protein concentration determination. After passing the quality inspection, equal amounts of sample protein were enzymatically hydrolyzed. First, the samples were washed and precipitated with acetone, and then dried and added with TEAB at a final concentration of 200mM. After ultrasonically dispersed, trypsin was added at a ratio of 1:50 (protease: protein, m / m) and enzymatically hydrolyzed overnight. The next day, dithiothreitol (DTT) was added at 56℃ for reduction for 30min; then iodoacetamide (IAA) was added and incubated at room temperature in the dark for 15min. The enzymatic peptides were dissolved in liquid chromatography mobile phase A and separated using the NanoElute ultra-high performance liquid system. After separation, they were injected into the Capillary ion source for ionization and then analyzed using the timesTOF Pro2 mass spectrometer. By comparing the signal intensity of the corresponding peptides in different samples, the corresponding proteins were relatively quantified and standardized. For the standardized data, according to the screening criteria of P ≤ 0.05 and Fold change ≥ 1.5, the differentially expressed proteins that met the conditions were retained for subsequent research.

[0059] 1.5 Cell culture and transfection

[0060] 293T cells were obtained from the cell bank of the Chinese Academy of Sciences, and trophoblast cells (HTR-8 / SVneo) were obtained from Purdue, Wuhan. 293T cells and HTR-8 / SVneo cells were cultured in 1640 medium (Bioind, Kibbuiz, Israel) containing 10% fetal bovine serum (Bioind, Kibbuiz, Israel) and 1% penicillin / streptomycin. The cells were cultured in a 37°C, 5% CO2 incubator. Lipofectamine TM 2000 (Invitrogen, Waltham, MA, USA) was used to transfect 10 μl of miR-126-5p mimics, mimics negative control (NC), miR-126-5p inhibitor or inhibitor negative control (INC) (final concentration was 100 nM) into HTR-8 / Svneo cells or 293T cells and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0061] 1.6qRT-PCR

[0062] According to the instructions, TRIzol reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA from mouse placental tissue, human chorionic tissue, and HTR-8 / SVneo. Reverse transcription of miRNA was performed using the miRNA First Strand cDNA Synthesis Kit (Vazyme, Nanjing, China), or reverse transcription of mRNA was performed using the PrimeScript RT Kit (Toyobo, Osaka, Japan) according to the instructions. The primers were synthesized by Shanghai Jima Co., Ltd. using 2 -ΔΔCt The relative expression of RNA was calculated by the method.

[0063] 1.7 Western Blot

[0064] RIPA was used to extract total proteins of chorionic villus tissue, placental tissue and HTR-8 / SVneo, and the protein concentration was determined by the BCA method. The samples were electrophoresed using 12% SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk for 1 hour, incubated with primary antibodies at 4°C overnight, washed three times with PBST, incubated with secondary antibodies at room temperature for 1 hour, washed three times with PBST, and developed with ECL luminescence.

[0065] 1.8 Enzyme-linked immunosorbent assay (ELISA)

[0066] Human ELISA kits were used to detect IL-1β and IL-18 in serum samples of URSA and NP patients and culture supernatants of HTR-8 / SVneo. Mouse ELISA kits were used to detect IL-1β and IL-18 in mouse serum. The experimental methods were referred to the instructions of the ELISA kits. All experiments were repeated at least 3 times.

[0067] 1.9 Immunofluorescence (IF)

[0068] HTR-8 / SVneo cells were cultured at 2×10 4Cells were seeded in 24-well culture plates at a concentration of 500 μg / well and transfected with NC / miR-126-5p mimics / INC / miR-126-5p inhibitor 24 h later. After 24 h, cells were stimulated with homocysteine ​​(Hcy) (Sigma, Shanghai, China) for 8 h, fixed in 4% paraformaldehyde at room temperature for 45 min, incubated with 10% normal goat serum for 1 h, incubated with Caspase-1 antibody (Proteintech, Chicago, USA) or GSDMD (Novus, Colorado, USA) at 4°C overnight, and incubated with secondary anti-FITC goat anti-rabbit IgG (ZSBIO, Beijing, China) for 1.5 h. Cell nuclei were stained with DAPI and photographed and analyzed using a fluorescence microscope (IX73; Olympus, Japan).

[0069] Localization and expression of caspase-1 in chorionic villus tissue of female patients with URSA and NP. Chorionic villus tissue was fixed with 4% paraformaldehyde at room temperature and embedded in paraffin. The embedded tissue was cut into 5 μM paraffin sections, baked at 65°C for 1.5 hours, dewaxed in xylene for 1 hour, dehydrated with graded alcohol, retrieval with citric acid for 20 minutes, sealed with goat serum for 30 minutes, incubated overnight with caspase-1 antibody (Proteintech, Chicago, USA) or GSDMD antibody (Novus, Colorado, USA), stained with DAPI for nuclei, and photographed and analyzed with a fluorescence microscope (IX73; Olympus, Japan).

[0070] 1.10 Fluorescence in situ hybridization (FISH)

[0071] The villous tissue was fixed with 4% paraformaldehyde at room temperature and embedded in paraffin. The embedded tissue blocks were cut into 4 μm sections. After dewaxing, protease treatment and denaturation, the paraffin sections were hybridized with Cy3-labeled miR-126-5p probe (Cy3-5'-CGCGTACACAAAAGTAATAATG-3', SEQ ID NO.1) (GenePharma, Shanghai, China) at 37°C overnight, the cell nuclei were stained with DAPI, and the images were taken using a laser scanning confocal fluorescence microscope.

[0072] 1.11 Observation of cell pyroptosis under microscope

[0073] In order to observe the morphology of trophoblast cell pyroptosis, HTR-8 / SVneo cells were seeded in 24-well plates. After 24 hours, miR-126-5p mimics / NC and miR-126-5pinhibitor / INC were transfected. After 12 hours, 40mmol / L hcy was added to induce cell pyroptosis. After 8 hours, the dynamic process of cell pyroptosis was continuously filmed, and static cell images were taken at the same time.

[0074] 1.12 Dual luciferase reporter gene assay

[0075] Bioinformatics software predicted that there were potential binding sites between miR-126-5p and CASP1. The wild type (WT) 3'UTR sequence and mutant type (MUT) 3'UTR sequence of CASP1 were cloned into the pmirGLO vector, named WT-pmirGLO-CASP1 3'UTR and MUT-pmirGLO-CASP1 3'UTR, respectively. TM 200, 293T cells were cotransfected with WT or MUT luciferase reporter plasmids and miR-126-5pmimics or NC at a final concentration of 100 nM, and cells were collected 24 h later. The fluorescence intensity was detected using a GloMax 20 / 20 luminometer, and the ratio of firefly luciferase luminescence to renal luciferase luminescence was calculated.

[0076] 1.13 Animal experiments

[0077] CBA / J female mice were mated with BALB / c male mice at a ratio of 2:1 to establish the NP model (CBA / J×BALB / c), and with DBA / 2 male mice at a ratio of 2:1 to establish the URSA model (CBA / J×DBA / 2). The mice were put together at 8 pm every night and separated at 7 am the next morning. The vaginal plugs of the mice were observed, and the day of vaginal plugs was considered the 0.5th day of pregnancy.

[0078] NP were randomly divided into NP+Vehicle group, NP+Flag-mCasp1 group, and NP+Flag-mCasp1+DSF group; URSA mice were randomly divided into URSA+normal saline group, URSA+VX-765 group, URSA+DSF group, URSA+miR-126-5p inhibitor group, URSA+miR-126-5p inhibitor+VX-765 group, URSA+miR-126-5p mimics group, and URSA+miR-126-5p mimics+Flag-mCasp1 group, with 5 mice in each group. All mice were injected with 200ul of VX-765 / DSF at 50mg / Kg / d, 200ul of plasmid at 2.5mg / Kg / d using in vivo transfection reagent, and 10nmol of miR-126-5p mimics and inhibitor per mouse each time. The injections were given every 3 days starting from the 0.5th day of pregnancy. The mice were killed on the 11.5th day of pregnancy to observe the absorption of the embryos.

[0079] 1.14 Mouse embryo absorption

[0080] Fix the mouse's limbs, cut along the midline of the abdominal wall with surgical scissors, remove the mouse uterus and place it in 1×PBS buffer, remove the fat tissue around the uterus, and after trimming, take a photo of the mouse uterus with a mobile phone and keep the mouse placental tissue in preparation for subsequent experiments.

[0081] 1.15 Statistical methods

[0082] SPSS19.0 was used for statistical calculation of the experimental data, and GraphPad Prism Version 7.0 software was used to process the experimental data and images. The experimental results were presented as mean ± standard deviation (mean ± SEM). Paired t-test was used to compare the mean differences between the two groups. One-Way ANOVA was used to compare the mean differences among multiple groups. Pearson correlation analysis was used to analyze the correlation between samples. The difference was statistically significant when P < 0.05.

[0083] Experimental Results

[0084] 2.1 CASP1 and pyroptosis levels are significantly increased in villous trophoblast cells of URSA patients

[0085] To explore the role of trophoblast cell pyroptosis in URSA, we used high-throughput sequencing and 4D label-free quantitative proteomics to sequence differentially expressed genes and proteins in the villus tissues of URSA patients and NP women. GO enrichment analysis of upregulated differentially expressed genes in the whole transfer group showed that the pyroptosis pathway was significantly enriched in the villus tissues of URSA patients ( Figure 1 A). CASP1 is a differentially expressed gene in the pyroptosis pathway. Figure 1 B). By expanding the clinical samples to detect CASP1 mRNA expression, the results showed that compared with NP, CASP1 mRNA expression in URSA patients' villus tissue was significantly increased. In addition, GO enrichment of upregulated proteins by 4D label-free quantitative proteomics was performed, and the cell pyroptosis pathway was also enriched ( Figure 1 D). Western blot results showed that the total protein and activation levels of Caspase-1, GSDMD and its active fragment GSDMD-N protein expression in the villous trophoblast cells of URSA patients were significantly increased ( Figure 1 E). At the same time, the expression of IL-1β and IL-18 in serum was significantly increased ( Figure 1 FG). Immunofluorescence experiments showed that Caspase-1 and GSDMD proteins were mainly localized in the cytoplasm of the villous trophoblast and significantly increased in URSA patients ( Figure 1 HI). The above results suggest that the pyroptosis of villous trophoblast cells caused by increased expression of Caspase-1 is involved in the occurrence of URSA.

[0086] 2.2 Caspase-1 / GSDMD-mediated pyroptosis is involved in the occurrence of URSA

[0087] In order to clarify the role of Caspase-1-mediated pyroptosis in the development of URSA, we used CBA / J female mice to mate with DBA / 2 male mice to establish the URSA mouse model, and CBA / J female mice to mate with BALB / c male mice to establish the NP mouse model. The pyroptosis inhibitor DSF and Caspase-1 inhibitor VX-765 were injected into URSA mice by tail vein injection to observe the changes in mouse placental trophoblast cell pyroptosis and embryo absorption rate. The results showed that compared with the solvent control group, the embryo absorption rate of mice in the DSF-treated group and the VX-765-treated group was significantly decreased ( Figure 2 AB); the total protein and activation levels of Caspase-1, and the expression of GSDMD and GSDMD-N proteins in the placenta tissue of mice treated with DSF showed no significant changes ( Figure 2 C), but the expression of IL-1β and IL-18 in serum was significantly decreased ( Figure 2DE); the total protein and activation levels of Caspase-1 and the expression of GSDMD-N protein in the placental trophoblast cells of mice treated with VX-765 were significantly decreased ( Figure 2 C), and the expression of IL-1β and IL-18 in serum was also significantly reduced ( Figure 2 DE); The above results show that inhibiting cell pyroptosis can reduce the occurrence of miscarriage in URSA mice. To further confirm the above results, we injected the mouse Caspase-1 overexpression plasmid Flag-mCasp1 and the pyroptosis inhibitor DSF into NP mice through the tail vein to observe the changes in mouse placental trophoblast cell pyroptosis and embryo absorption. The results showed that compared with the solvent control group, the embryo absorption rate of the Flag-mCasp1 group mice was significantly increased; and compared with the Flag-mCasp1 group mice, the embryo absorption rate of the Flag-mCasp1+DSF group mice was significantly decreased ( Figure 2 FG); In addition, the total protein and activation levels of Caspase-1 and the expression of GSDMD-N protein in placental trophoblast cells of mice in the Flag-mCasp1 group were significantly upregulated ( Figure 2 H), and the expression of IL-1β and IL-18 in serum also increased significantly ( Figure 2 IJ); compared with the Flag-mCasp1 group, there were no significant changes in the total protein and activation levels of Caspase-1 and the expression of GSDMD-N protein in the placental trophoblast cells of the Flag-mCasp1+DSF group mice ( Figure 2 H), but the expression of IL-1β and IL-18 in serum was significantly downregulated ( Figure 2 IJ), the above results show that Caspase-1 / GSDMD-mediated cell pyroptosis is involved in the occurrence of recurrent spontaneous abortion.

[0088] 2.3 Screening of miRNAs upstream of CASP1

[0089] To explore the mechanism of regulating CASP1 upstream, we used whole transcriptome high-throughput sequencing technology to detect miRNAs in the villus tissues of 3 NP women and 3 URSA patients, and screened out 19 downregulated miRNAs; at the same time, combined with the bioinformatics software TargetScan screening, we found 3 miRNAs with potential binding sites to CASP1: miR-126-5p, miR-181c-5p and miR-181d-5p ( Figure 3 A). By further expanding the clinical samples for qRT-PCR expression verification, the results showed that compared with the positive NP group, the expression of miR-126-5p in URSA patients was significantly downregulated, while the expression changes of miR-181c-5p and miR-181d-5p were not statistically significant ( Figure 3B). Pearson correlation analysis found that the CASP1 mRNA level in human villous trophoblast cells was significantly negatively correlated with the expression of miR-126-5p (r = -0.5235, P < 0.05) ( Figure 3 C). In order to study the diagnostic significance of miR-126-5p for URSA patients, ROC analysis showed that the area under the ROC curve (AUC) of miR-12a-5p expression was 0.8711 (95% CI: 0.7814-0.9608, P<0.0001). This result showed that the expression changes of miR-126-5p could sensitively distinguish URSA patients from normal early pregnant women ( Figure 3 D). FISH assay revealed that miR-126-5p was mainly localized in the cytoplasm of human chorionic trophoblast and was significantly decreased in URSA patients ( Figure 3 E). The above results suggest that mature miR-126-5p may participate in trophoblast cell pyroptosis and URSA by targeting CASP1 in the cytoplasm of trophoblast cells.

[0090] 2.4miR-126-5p regulates Caspase-1 and affects trophoblast cell pyroptosis

[0091] In order to explore whether miR-126-5p affects trophoblast cell pyroptosis by regulating Caspase-1 expression, we transfected miR-126-5p mimics and inhibitor into HTR-8 / SVneo cells to regulate the expression of miR-126-5p and observed its effects on CASP1 mRNA, protein and cell pyroptosis. The results showed that miR-126-5p mimics could significantly upregulate the expression of miR-126-5p, while miR-126-5p inhibitor significantly inhibited the expression of miR-126-5p ( Figure 4 A); Upregulating miR-126-5p expression can significantly reduce the level of CASP1 mRNA, while the total protein and activation level of Caspase-1 and the expression of GSDMD-N protein are significantly downregulated, and the expression of IL-1β and IL-18 in the cell culture supernatant is also significantly decreased ( Figure 4 BF); inhibition of miR-126-5p expression significantly increased CASP1 mRNA, total Caspase-1 protein and activation levels, GSDMD-N protein expression levels, and significantly upregulated IL-1β and IL-18 expressions in cell culture supernatant ( Figure 4BF). In addition, the microscopic observation of cell pyroptosis showed that after transfection with miR-126-5p mimics, the pyroptosis of HTR-8 / SVneo cells decreased, while after transfection with miR-126-5p inhibitor, the pyroptosis of cells increased significantly ( Figure 4 G). The above results suggest that miR-126-5p can affect trophoblast cell pyroptosis and the release of related inflammatory factors by negatively regulating CASP1 expression.

[0092] 2.5miR-126-5p directly binds to CASP1 mRNA 3'UTR through base complementarity

[0093] In order to clarify the targeted regulatory relationship between miR-126-5p and CASP1, we used the bioinformatics software Targetscan7.2 to predict the binding sites between the two. The results showed that there was a base complementary binding site between miR-126-5p and CASP1 mRNA 3'UTR ( Figure 5 A). Transfection of miR-126-5p mimics in 293T cells significantly reduced the relative luciferase activity of WT-pmirGLO-CASP1 3'UTR, but did not affect the relative luciferase activity of MUT-pmirGLO-CASP1 3'UTR ( Figure 5 BC). The above results indicate that miR-126-5p directly binds to CASP1 mRNA through base complementarity.

[0094] 2.6 Effect of miR-126-5p on mouse pregnancy by regulating Caspase-1 / GSDMD signaling pathway

[0095] To further explore the role of miR-126-5p in regulating trophoblast cell pyroptosis by targeting Caspase-1 in URSA, we regulated the expression of miR-126-5p and Caspase-1 in URSA mice by tail vein injection of miR-126-5p mimics and Caspase-1 overexpression plasmid Flag-mCasp1, and observed the pyroptosis of trophoblast cells and embryo absorption in the placenta of mice. The results showed that compared with the NC group, the embryo absorption rate of mice in the miR-126-5p mimics treatment group was significantly downregulated; and compared with the miR-126-5pmimics group, the embryo absorption rate of mice in the miR-126-5p mimics+Flag-mCasp1 group was significantly upregulated ( Figure 6 AB). At the same time, the expression of miR-126-5p in the placental trophoblast cells of mice treated with miR-126-5p mimics and miR-126-5p mimics+Flag-mCasp1 was significantly increased ( Figure 6 C); compared with the NC group, the total protein and activation levels of Caspase-1 and the expression of GSDMD-N protein in the placental trophoblast cells of the mice in the miR-126-5p mimics group were significantly downregulated, and the expression levels of IL-1β and IL-18 in serum were also significantly decreased; compared with the miR-126-5p mimics group, the total protein and activation levels of Caspase-1 and the expression of GSDMD-N protein in the placental trophoblast cells of the mice in the miR-126-5p mimics+Flag-mCasp1 group were significantly upregulated, and the expression levels of IL-1β and IL-18 in serum were also significantly increased ( Figure 6 DF). The above results indicate that miR-126-5p targets Caspase-1 to negatively regulate trophoblast cell pyroptosis, suggesting that increased expression of miR-126-5p can inhibit the expression of target gene Casp1 mRNA and protein, thereby alleviating trophoblast cell pyroptosis and playing a protective role in pregnancy.

[0096] To further clarify the role of miR-126-5p in the occurrence of URSA by regulating the Caspase-1 / GSDMD signaling axis, we regulated the expression of miR-126-5p and Caspase-1 in URSA mice by tail vein injection of miR-126-5p inhibitor and VX-765, and observed the changes in placental trophoblast cell pyroptosis and embryo absorption rate in mice. Compared with the INC group, the embryo absorption rate of mice in the miR-126-5p inhibitor group was significantly increased; compared with the miR-126-5p inhibitor group, the embryo absorption rate of mice in the miR-126-5p inhibitor+VX-765 group was significantly decreased ( Figure 7 AB). At the same time, the expression of miR-126-5p in the placental trophoblast cells of mice treated with miR-126-5pinhibitor and miR-126-5p inhibitor+VX-765 was significantly downregulated ( Figure 7 C), compared with the INC group, the total protein and activation levels of Caspase-1 and the expression of GSDMD-N protein in the placental trophoblast cells of mice in the miR-126-5p inhibitor group were significantly increased, and the expressions of IL-1β and IL-18 in serum were significantly increased ( Figure 7 DF); compared with the miR-126-5p inhibitor group, miR-126-5p inhibitor+VX-765 treatment could inhibit the total protein and activation level of Caspase-1 and GSDMD-N protein in the miR-126-5p inhibitor group, and the expression of IL-1β and IL-18 in serum was significantly increased ( Figure 7DF). The above results indicate that miR-126-5p targets the Caspase-1 / GSDMD signaling axis to regulate trophoblast cell pyroptosis and participate in the occurrence of URSA.

[0097] In summary, our results showed that miR-126-5p reduction relieved its inhibition on the expression of target gene CASP1, thereby promoting CASP1-mediated trophoblast cell pyroptosis and participating in the occurrence of URSA ( Figure 8 The results showed that miR-126-5p regulates trophoblast cell pyroptosis by targeting the Caspase-1 / GSDMD signaling axis, further explaining the mechanism of URSA from the perspective of epigenetic immune regulation, and providing new targets and pathways for clinical diagnosis and treatment.

[0098] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of miR-126-5p in any one or more of the following: (a) preparing a product for inhibiting the expression of Caspase-1 and GSDMD; (b) preparing a product for inhibiting trophoblast cell pyroptosis; (c) preparing products which inhibit the rate of embryo resorption; (d) Preparation of products for the prevention and / or treatment of unexplained recurrent spontaneous abortion.

2. The use according to claim 1, characterized in that: The product is a drug or an experimental reagent, and the experimental reagent is used for basic research.

3. The use according to claim 2, characterized in that: When the product is a medicine, the medicine further comprises at least one pharmaceutically inactive ingredient.

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