Application of circ0005704 and related biological elements thereof in diagnosis and treatment of unexplained recurrent spontaneous abortion

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of circ0005704 and related biological elements thereof in diagnosis and treatment of repeated spontaneous abortion caused by unknown reasons. Researches find that differential expression and autophagy level of ULK1 in trophoblast cells of URSA patients are increased. High-throughput transcriptome sequencing is combined with bioinformatics analysis, and it is found that miR-26a-5p is down-regulated in URSA patients, is responsible for up-regulation of ULK1 and promotes autophagy of trophoblast cells, so that occurrence of URSA is increased. Through a high-throughput transcriptional set screening strategy and dual luciferase reporter gene analysis, it is found that circ0005704 enhances the expression of ULK1 through miR-26a-5p. In a word, the research of the invention shows that the circ0005704 / miR-26a-5p / ULK1 signal axis participates in the pathogenesis of URSA by adjusting the migration of the trophoblast cells, and a new target and scientific evidence are provided for the clinical 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 circ_0005704 and related biological elements 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] Unexplained recurrent spontaneous abortion (URSA) refers to two or more consecutive spontaneous abortions with the same partner before 20 weeks of pregnancy. URSA is the most common complication of pregnancy, with an incidence of up to 5% in women of childbearing age. The risk of recurrent miscarriage increases significantly with the increase in the number of miscarriages. Although researchers around the world have made achievements in studying the pathogenesis of RSA, approximately 50% of RSA cases remain unexplained, except for known pathogenic factors such as genetics, uterine abnormalities, thrombosis, hormonal or metabolic disorders, infection, autoimmune factors, and abnormal sperm parameters. Repeated failed pregnancies cause serious physical harm to patients and bring great pressure to families. Therefore, exploring the pathogenesis, identifying intervention targets and regulatory mechanisms, and clarifying proven effective drugs or methods are urgent issues in the field of reproductive medicine.

[0004] The beginning of a new life begins with the fusion of the egg and sperm. After fertilization, the fertilized egg undergoes several rounds of division and morphogenesis, gradually forming a blastocyst. At this stage, the blastocyst cells have differentiated into two parts: the trophoblast and the inner cell mass, preparing for implantation. The key to successful embryo implantation lies in the synchronous differentiation and development of the trophoblast and endometrial receptivity, as well as the mutual recognition and communication between the blastocyst and the endometrium. Trophoblast cells penetrate the luminal epithelium, pass through the basement membrane, and embed into the matrix of the uterine wall. Trophoblast cells are fetal cells that directly interact with the maternal system and play a vital role in embryo implantation and the formation of a functional placenta. Therefore, the disruption of trophoblast function may lead to a series of adverse pregnancy outcomes, such as malformations, fetal growth restriction, miscarriage, and stillbirth. At present, the role and regulatory mechanism of trophoblast function imbalance in URSA are still unclear. Therefore, elucidating the function and regulatory mechanism of trophoblast cells will provide new targets and insights for the clinical treatment of URSA.

[0005] Autophagy is a major catabolic process in eukaryotic cells, responsible for the degradation and recycling of damaged macromolecules and organelles. It has become a research hotspot in the medical field. Recent studies have shown that autophagy dysfunction is associated with the development of various diseases, such as tumors, neurodegenerative diseases, and metabolic disorders. In addition, it is believed that autophagy may provide new pathways and targets for the prevention and treatment of diseases. Autophagy is mainly a cellular defense mechanism, however, in many cases, excessive autophagic degradation may be harmful. Under specific conditions, when ULK1 is activated, it triggers the activation of the phosphoinositide 3-kinase (PI3K) complex containing Beclin1, initiating the formation of autophagosomes. Therefore, ULK1 is a key regulator of autophagy, and changes in its expression can affect the formation and function of autophagosomes. It has been found that excessive activation of trophoblast autophagy promotes the occurrence of URSA. For example, Cai et al. found that decreased MFN2 expression levels can lead to mitochondrial dysfunction, elevated autophagy, and impaired trophoblast cell function, which may lead to early unexplained miscarriage; Pan et al. revealed that impairment of Shh signaling triggers autophagy, which in turn inhibits trophoblast migration in the setting of recurrent miscarriage. However, the role of ULK1 in regulating trophoblast autophagy in URSA development has not been fully elucidated. Summary of the invention

[0006] In view of the deficiencies of the above-mentioned prior art, the object of the present invention is to provide the application of circ_0005704 / miR-26a-5p in the diagnosis and treatment of unexplained recurrent spontaneous abortion. Specifically, the present invention found through research that the differential expression of ULK1 and the increase in autophagy levels in trophoblast cells of URSA patients. Combining high-throughput transcriptome sequencing with bioinformatics analysis, it was found that miR-26a-5p was downregulated in URSA patients and was responsible for the upregulation of ULK1, promoting trophoblast cell autophagy, thereby increasing the occurrence of URSA. Through high-throughput transcriptome screening strategy and dual luciferase reporter gene analysis, it was found that circ_0005704 enhanced the expression of ULK1 through miR-26a-5p. In summary, the research of the present invention shows that the circ_0005704 / miR-26a-5p / ULK1 signaling axis participates in the pathogenesis of URSA by regulating the migration of trophoblast cells, providing new targets and scientific evidence for the clinical treatment of URSA.

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

[0008] In a first aspect of the present invention, there is provided the use of a substance for detecting the expression level of circ_0005704 and its related biological elements in the preparation of a product for screening, diagnosis, detection, monitoring or prognosis of unexplained recurrent spontaneous abortion.

[0009] Among them, the circ_0005704-related biological elements include miR-26a-5p and ULK1.

[0010] Specifically, the present invention has found through research that circ_0005704 promotes autophagy of trophoblast cells, thereby determining the migration and abortion rate of URSA patients. Overexpression of circ_0005704 eliminates the inhibitory effect of miR-26a-5p on ULK1, resulting in its accumulation and enhanced autophagy, thereby affecting the migration of trophoblast cells. Mechanistically, circ_0005704 acts as a molecular sponge for miR-26a-5p, thereby weakening the binding of miR-26a-5p to the 3'UTR of ULK1 mRNA. Importantly, the upregulation of circ_0005704 is closely related to the downregulation of miR-26a-5p, and is positively correlated with ULK1-mediated autophagy in patients and URSA models. Therefore, the above-mentioned circ_0005704 and its related biological elements can be used as (auxiliary) diagnostic and prognostic markers for unexplained recurrent spontaneous abortion.

[0011] The substances for detecting the expression levels of circ_0005704 and its related biological elements may include reagents for detecting the expression levels of circ_0005704, miR-26a-5p and ULK1 encoding genes based on real-time fluorescence quantitative PCR, in situ hybridization, gene chips and gene sequencing, and / or reagents for detecting the expression levels of ULK1 protein based on immunodetection methods.

[0012] The products include, but are not limited to, primers, probes, (gene or protein) chips, nucleic acid membrane strips, detection kits, detection devices and detection equipment for detecting the expression levels of circ_0005704 and its related biological elements, which are not specifically limited here.

[0013] A second aspect of the present invention provides a system for screening, diagnosing, detecting, monitoring or prognosticating unexplained recurrent spontaneous abortion, the system comprising:

[0014] An acquisition unit is configured to: acquire the expression level of circ_0005704 and its related biological elements in the test sample of the subject;

[0015] An evaluation unit is configured to evaluate and determine the disease condition of the subject according to the subject circ_0005704 and its related biological elements obtained by the acquisition unit.

[0016] Among them, the circ_0005704-related biological elements include miR-26a-5p and ULK1.

[0017] The sample to be tested can be a blood sample (including peripheral blood mononuclear cells) and a chorionic villus tissue sample of the subject.

[0018] The third aspect of the present invention provides the use of circ_0005704 and related biological elements as targets in the preparation and / or screening of drugs for unexplained recurrent spontaneous abortion.

[0019] Specifically, the effects of candidate drugs on circ_0005704 and its related biological elements before and after use can be used to determine whether the candidate drugs can be used to prevent or treat unexplained recurrent spontaneous abortion.

[0020] Specifically, methods for screening drugs for preventing or treating unexplained recurrent spontaneous abortion include:

[0021] 1) Treat the system expressing and / or containing circ_0005704 and its related biological elements with the candidate substance; set up a parallel control without the candidate substance treatment;

[0022] 2) After completing step 1), detecting the expression level of circ_0005704 and its related biological elements in the system; if the expression amount of circ_0005704 and its related biological elements in the system treated with the candidate substance is significantly downregulated or upregulated compared with the parallel control, the candidate substance can be used as a candidate drug for preventing or treating unexplained recurrent spontaneous abortion.

[0023] Among them, the circ_0005704-related biological elements include miR-26a-5p and ULK1.

[0024] More specifically, the step 2) is as follows: if the expression of circ_0005704 is significantly downregulated, the expression of miR-26a-5p is significantly upregulated and / or the expression of ULK1 is significantly downregulated in the system treated with the candidate substance, then the candidate substance can be used as a candidate drug for preventing or treating unexplained recurrent spontaneous abortion.

[0025] The system may be a cell system, a subcellular system, a solution system, a tissue system, an organ system or an animal system, and is not specifically limited herein.

[0026] In a fourth aspect of the present invention, a substance for inhibiting the expression of circ_0005704 or reducing its activity is provided for use in any one or more of the following:

[0027] a) promoting the expression of miR-26a-5p or preparing a product that promotes the expression of miR-26a-5p;

[0028] b) inhibiting ULK1 expression or preparing a product that inhibits ULK1 expression;

[0029] c) promoting trophoblast cell migration or preparing a product that promotes trophoblast cell migration;

[0030] d) inhibiting autophagy of trophoblast cells or preparing a product for inhibiting autophagy of trophoblast cells;

[0031] g) Products for the prevention and / or treatment of unexplained recurrent spontaneous abortion.

[0032] Among them, the substance that inhibits the expression of circ_0005704 or reduces its activity can include but is not limited to RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA (SEQ ID NO.7-8), shRNA against circ_0005704, substances for implementing lentiviral infection or gene knockout, and is not specifically limited here.

[0033] The product can be a drug or an experimental reagent for non-medical use, and the experimental reagent can be used for basic research. For example, the product can be used to regulate the migration and autophagy of trophoblast cells in vitro, so as to prepare a trophoblast cell-related biological model, thereby providing convenient conditions for the mechanism research of diseases such as unexplained recurrent spontaneous abortion.

[0034] According to the present invention, when the product is a medicine, the medicine further comprises at least one inactive pharmaceutical ingredient.

[0035] The inactive pharmaceutical ingredients may be carriers, excipients, diluents, etc. commonly used in pharmacy. Meanwhile, the inactive pharmaceutical ingredients such as carriers, excipients, diluents, etc. that may be included are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0036] The medicine of the present invention can be applied to the body by known means. For example, it can be delivered to the tissue of interest by intravenous systemic delivery or local injection. It can be applied optionally via intravenous, percutaneous, intranasal, mucosal or other delivery methods. Such application can be carried out via single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be applied in the present invention can vary depending on various factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0037] The drug administration subjects can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys and gorillas, and are not specifically limited here.

[0038] Beneficial technical effects of one or more of the above technical solutions:

[0039] The above technical schemes indicate that circ_0005704 promotes autophagy of trophoblast cells, thereby determining the migration and abortion rate of URSA patients. Overexpression of circ_0005704 abolishes the inhibitory effect of miR-26a-5p on ULK1, leading to its accumulation and enhanced autophagy, which affects the migration of trophoblast cells. Mechanistically, circ_0005704 acts as a molecular sponge for miR-26a-5p, thereby attenuating the binding of miR-26a-5p to the 3'UTR of ULK1 mRNA. Importantly, the upregulation of circ_0005704 is closely associated with the downregulation of miR-26a-5p and is positively correlated with ULK1-mediated autophagy in patients and URSA models. In summary, the above technical schemes reveal the regulatory axis of ULK1-mediated autophagy to promote the pathogenesis of URSA caused by circ_0005704 and miR-26a-5p dysregulation, and therefore have good potential for practical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings in the specification, which constitute a part 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.

[0041] Figure 1 Figure 1 shows that autophagy is upregulated and associated with the pathogenesis of URSA in the present invention; (A) The accumulation of autophagosomes in the villous tissue of NP subjects and URSA patients was detected by electron microscopy (scale bar, 50 μm). (B) The expression of ULK1, Beclin1, ATG7, ATG13, LC3, and p62 protein levels in the villous tissue of NP subjects and URSA patients (n=30) was determined by Western blotting. (C) Representative IF staining images of ULK1 in the villous tissue of NP subjects and URSA patients (scale bar, 50 μm). (D) qRT-PCR analysis of ULK1 expression in the villous tissue of NP subjects (n=30) and URSA patients (n=10). (E) The diagnostic value of ULK1 in the villous tissue of NP subjects and URSA patients was evaluated by ROC curve (n=30). (FG) The URSA mouse model was established, and the day of vaginal plug discovery was designated as day 0.5 of pregnancy. Each female mouse was intravenously injected with DMSO, CQ, and SBI-0206965 at a dose of 25 mg / kg / day through the tail vein, and the mice were killed on day 11.5 of pregnancy. The embryo absorption rate of the four groups of treated URSA mice was calculated, and the arrows indicated the absorbed embryos (n=5). (H) ULK1, LC3, and P62 protein levels were measured in the placenta of pregnant mice (n=5). Values ​​are listed as mean ± SD. *P<0.05, ****P<0.0001, ns: not statistically significant.

[0042] Figure 2 Figure 1 shows that miR-26a-5p is negatively correlated with ULK1 expression in URSA patients in the present invention; (A) Microarray analysis determined that the expression of 9 miRNAs was reduced in PBMCs of NP subjects and URSA patients (P<0.05, fold change>1.5). Red dots indicate increased expression, while green dots indicate decreased expression (n=3). (B) Venn diagram showing microRNAs predicted by Target Scan, miRDB, DIANA, and Chip (P<0.05, fold change>1.5). (C) qRT-PCR analysis of miR-26a-5p expression in villous tissue of NP subjects and URSA patients (n=30). (D) FISH observation of the location and expression level of miR-26a-5p in villous tissue (miR-26a-5p, red; DAPI, blue; scale bar=50μm). (E) The diagnostic value of miR-26a-5p for URSA was evaluated by ROC curve (n=30). (F) Pearson correlation analysis (n=30) was used to analyze the correlation between miR-26a-5p and ULK1 mRNA in URSA villus tissue. Values ​​are presented as mean ± SD. ****P < 0.0001.

[0043] Figure 3 Figure 2 shows that miR-26a-5p targets ULK1 to negatively regulate autophagy in trophoblast cells in an embodiment of the present invention; (A) Schematic diagram of the putative binding sequence of miR-26a-5p in ULK1 mRNA 3'UTR, and luciferase activity of wild-type (WT) and mutant (MUT) constructs. (B) Luciferase activity was determined by co-transfecting vectors (ULK1 3'UTR-WT, MUT1, MUT2, MUT1+MUT2) with negative control (NC) and miR-26a-5p mimics into 293T cells. (C-D) qRT-PCR analysis of miR-26a-5p and ULK1 mRNA expression in HTR-8 / Svneo cells after transfection with NC, miR-26a-5p mimics, INC, and miR-26a-5-p inhibitors. (E) ULK1, LC3, and P62 protein levels were measured in HTR-8 / Svneo cells after transfection with NC, miR-26a-5p mimics, INC, and miR-26a-5g inhibitor. Values ​​are presented as mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001; ns: not statistically significant.

[0044] Figure 4circ_0005704 interacts with miR-26a-5p expression and is negatively correlated in the embodiment of the present invention; (AB) Schematic diagram showing 24 predicted potential circ-RNAs that may target miR-26a-5p screened by miRanda v3.3a and PBMCs of NP subjects and URSA patients determined by microarray analysis (P<0.05, fold change>1.5). Red dots indicate increased expression, while green dots indicate decreased expression (n=3). (C) RNA pull-down assay detected by qRT-PCR shows the binding of circ-RNA to miR-26a-5p by Bio probe. (D) Relative expression of GAPDH, U6, circ_0005704 and circ_0064954 in the nucleus and cytoplasm, respectively. (E) RIP analysis detected by qRT-PCR showed that circ_0005704, which was downregulated by AGO2, was enriched in miR-26a-5p mimics treated with HTR-8 / Svneo. (F) RNA pull-down assay detected by qRT-PCR showed that miR-26a-5p was enriched in HTR-8 / Svneo by circ_0005704 probe. (G) qRT-PCR analysis detected the expression of circ_0005704 in trophoblast cells after RNase R treatment. (H) qRT-PCR detected the expression of circ_0005704 in NP group and URSA patients' villus tissue (n=30). (I) The diagnostic value of circ_0005704 for URSA was evaluated by ROC curve (n=30). (J) Pearson correlation analysis (n=30) was used to analyze the correlation between miR-26a-5p and circ_0005704 in villus tissue. (K) Schematic diagram of the miR-26a-5p binding sequence in the circ_0005704 sequence predicted by bioinformatics software, and the wild type (WT) and mutant (MUT) were constructed to determine the luciferase activity. (L) Luciferase activity was determined by co-transfecting 293T cells with vectors (circ_0005704-WT, MUT) and negative control (NC) and miR-26a-5p mimics. Values ​​are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ns: not statistically significant.

[0045] Figure 5In the present invention, the control vector plasmid, circ_0005704 plasmid or circ_0005704 plasmid and miR-26a-5p mimics were transfected into HTR-8 / Svneo cells, and circ_0005704 overexpression promoted autophagy through the miR-26a-5p / ULK1 axis. The NP mouse model was constructed, and the vaginal plug of the mouse was checked every morning. The day when the vaginal plug was visible was designated as day 0.5 of pregnancy. On days 0.5, 3.5, 6.5, and 9.5, circ_0005704 plasmids at a dose of 320 ng / g / d were injected into female mice through the tail vein using the in vivo transfection reagent EntransterTM (Engreen, Beijing, China), or a mixture of circ_0005704 plasmids at a dose of 320 ng / g / d and 10 nmol / d miR-26a-5p mimics was injected into female mice through the tail vein, and the mice were killed on day 11.5 of pregnancy. (AC) Relative expression of circ_0005704, miR-26a-5p, and ULK1 mRNA in HTR-8 / Svneo cells. (D) The expression of ULK1, LC3, and P62 protein levels was measured in HTR-8 / Svneo cells. (E) Representative micrographs show HTR8 / Svneo cells fixed and stained on the bottom side of the invasion membrane. (F) The expression levels of MMP9, E-cadherin, N-cadherin, and vimentin in HTR-8 / Svneo cells were determined. (GH) The embryo absorption rate of the three groups of URSA mice was calculated, and the arrows indicate the absorbed embryos (n=5). (I) The expression of ULK1, LC3, and P62 protein levels was evaluated in the placenta of NP-pregnant mice. (J) The expression of MMP9, E-cadherin, N-cadherin, and vimentin in the placenta of NP-pregnant mice was determined. The values ​​are presented as mean ± SD. ***P < 0.001, ****P < 0.0001.

[0046] Figure 6circ_0005704 knockdown inhibits autophagy and alleviates embryo absorption through miR-26a-5p / ULK1 in the present invention; HTR-8 / Svneo cells were transfected with si-NC, si-circ_0005704, si-circ_0005704 and miR-26a-5p inhibitor. The URSA mouse model was constructed, and the vaginal plug of the mouse was checked every morning. The day when the vaginal plug was visible was designated as day 0.5 of pregnancy. URSA mice were injected with 10 nmol si-NC, 10 nmol si-circ_0005704 or 10 nmol si-circ_0005704 and 10 nmol miR-26a-5p inhibitor mixture via tail vein injection on days 0.5, 3.5, 6.5 and 9.5 using the in vivo transfection reagent EntransterTM (Engreen, Beijing, China), and the mice were killed on day 11.5 of pregnancy. (AC) Relative expression of circ_0005704, miR-26a-5p, and ULK1 mRNA in HTR-8 / Svneo cells. (D) Expression of ULK1, LC3, and P62 protein levels were measured in HTR-8 / Svneo cells. (E) Representative micrographs show HTR8 / Svneo cells fixed and stained on the bottom side of the invasion membrane. (F) The expression levels of MMP9, E-cadherin, N-cadherin and vimentin in HTR-8 / Svneo cells were determined. (GH) The embryo absorption rate of the three groups of URSA mice was calculated, and the arrows indicate the absorbed embryos (n=5). (I) The expression of ULK1, LC3, and P62 protein levels was measured in the placenta of URSA pregnant mice. (J) The expression of MMP9, E-cadherin, N-cadherin and vimentin in the placenta of URSA pregnant mice was determined. The values ​​are listed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. DETAILED DESCRIPTION

[0047] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. 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 application belongs.

[0048] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0049] 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.

[0050] The present invention is further explained by the following examples, but it does not constitute a limitation of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The test methods in the following examples are generally carried out under conventional conditions. The nucleotide sequences used in the examples are shown in the following table:

[0051]

[0052]

[0053] Example

[0054] 1. Materials and Methods

[0055] 1.1 Patients

[0056] From September 2019 to December 2023, we recruited 30 URSA patients and 30 normal pregnant (NP) women from the Reproductive Medicine Center of the 960th Hospital and Qianfoshan Hospital for the study. In this study, all risk factors with clear etiology were excluded, such as chromosomal abnormalities, maternal reproductive tract abnormalities, endocrine abnormalities, immune dysfunction, reproductive tract infection, cervical insufficiency, and thrombosis. Written consent and confidentiality agreement were obtained for the collection of all clinical specimens.

[0057] 1.2 Specimen collection

[0058] Peripheral blood and chorionic villus tissue were obtained from URSA patients and NP women, respectively. Peripheral blood was collected using blood collection tubes containing ethylenediaminetetraacetic acid (EDTA) (Becton Dickinson), and peripheral blood mononuclear cells (PBMCs) were subsequently separated by Ficoll density gradient centrifugation. The separated plasma was frozen and stored at -80°C, and PBMCs were used for further studies. Regarding chorionic villus tissue collection, pregnancy was terminated by uterine dilatation and aspiration, fresh chorionic villus tissue was obtained, and part of the tissue was fixed in 4% formaldehyde for subsequent immunofluorescence and FISH experiments. The remaining chorionic villus tissue was frozen and stored at -80°C for Western blot (WB) and polymerase chain reaction (PCR) analysis.

[0059] 1.3 Transmission electron microscopy

[0060] Fresh human villus tissue was cut into pieces with a volume of about 1 mm 3 of small pieces and fixed them in 2.5% glutaraldehyde at room temperature. Then, the fixed tissue was rinsed with phosphate-buffered saline (PBS) and placed in 1% osmium acid, which was further fixed at 4°C for 2 hours. The tissue was dehydrated, embedded and polymerized. Fourth, the embedded tissue was sectioned at a thickness of 70nm. Fifth, the prepared sections were stained with 3% uranyl acetate saturated alcohol solution for 8 minutes and then stained with 2.7% lead acetate solution for 8 hours. The distribution and number of autophagosomes were observed by transmission electron microscopy.

[0061] 1.4 Immunofluorescence (IF)

[0062] Immunofluorescence was used to detect the expression and distribution of ULK1 in NP and URSA villus tissues. First, fresh villus tissues were fixed with 4% paraformaldehyde at room temperature and then prepared into 5 μm paraffin sections. Second, the sections were dried in a slide dryer at 65 °C for 60 min. Third, the sections were dehydrated in gradient alcohol and antigen retrieval was performed for 20 min. Fourth, endogenous peroxidase was removed and the sections were blocked with serum for 30 min. Fifth, the sections were incubated with ULK1 (8054, CST) at 4 °C overnight. The next day, FITC mouse anti-rabbit antibody was used to bind to the primary antibody. Finally, the cell nuclei were stained with 6-diamidino-2-phenylindole (DAPI), and images were captured using a microscope (BZ-X800LE, Keyence, Japan).

[0063] 1.5 Fluorescence in situ hybridization (FISH)

[0064] FISH experiments were used to detect the localization and expression of miR-26a-5p in the villous tissue of NP and URSA patients, and the results showed that it could clearly distinguish NP from URSA. The preparation of villous tissue sections was the same as that for immunofluorescence. First, the sections were incubated with a diluted solution of proteinase K at 37°C for 20 min. Then, the sections were dehydrated with graded alcohols. After that, they were incubated with denaturants in an oven at 78°C for 8 min. Next, the sections were incubated with Cy3-labeled miR-26a-5p probes in a dark environment at 37°C for 16 h. Finally, the nuclei were stained with DAPI working solution and then incubated in the dark for 10 min. The samples were then observed under a microscope and photographed. All procedures were performed according to the manufacturer's protocol (Genepharma, Shanghai, China).

[0065] 1.6 Microarray analysis

[0066] Total RNA was extracted from PBMC using TRIzol (Invitrogen, Carlsbad, CA, USA). OE Biotech (Shanghai, China) was used for URSA patients (n=3) and control subjects (n=3). Sample labeling, microarray hybridization, and washing were performed according to the manufacturer's standard protocol. In this article, the top 19 downregulated miRNAs with fold change >2 and P<0.05 were selected as candidates for further study. In addition, circular RNAs were also studied in depth to gain a more comprehensive understanding of their mechanism of action.

[0067] 1.7 Cell culture and treatment

[0068] 293T cells and HTR-8 / Svneo of human trophoblast cell line were obtained from the cell bank of Chinese Academy of Sciences. All cells were cultured in 1640 (Bioind, Kibbiz, Israel) containing 10% FBS (Bioind, Kibbiz, Israel) and 1% penicillin / streptomycin. Cells were maintained in an incubator at 37°C with 5% CO2.

[0069] 1.8 Cell transfection

[0070] The proteins containing circ_0005704, vevtor, miR-26a-5p mimics, negative control (NC), small interfering negative control (siNC), si-circ_0005704, miR-26a-5p inhibitor, and inhibitor negative control (INC) were purchased from GenePharma (Shanghai, China). Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) was used for transfection according to the manufacturer's instructions. First, cells were cultured at 1×105 The cells were seeded at a density of 10 cells / mL into 12-well plates. After reaching 50% confluence, they were transfected with Lipofectamine 2000. After 48 hours, the cells were used for further experiments.

[0071] 1.9 Dual luciferase reporter gene assay

[0072] The 3'UTR region of ULK1 with miR-26a-5p binding or mutant sequence and circ_0005704 were cloned into the pmirGLO luciferase vector (Promega, USA) to synthesize wild-type plasmids (ULK1-WT and circ_0005704-WT) or mutant plasmids (ULK1-MUT and circ_0005704-MUT). The above plasmids were transfected into 293T cells with NC mimics or miR-26a-5p using Lipofectamine 2000 (Invitrogen). After 48 hours, luciferase activity was determined by a dual luciferase reporter gene assay kit (Promega, USA) and normalized to the activity of Renilla luciferase.

[0073] 1.10 RNA immunoprecipitation protein (RIP)

[0074] RIP assay was performed using PureBinding RNA Immunoprecipitation Kit (Geneseed, P0102, Guangzhou) according to the manufacturer's instructions. A total of 1 × 10 7 HTR8 sneo cells were cultured and lysed in RIP lysis buffer, and the supernatant was collected after centrifugation at 12000g for 15 minutes. At the same time, protein A and G were incubated with anti-IgG (2729S, Cell Signaling Technology) or anti-AGO2 (ab186733, Abcam) at 4°C for 2 hours and washed with lysis buffer. Then, the supernatant was incubated with protein A and G at 4°C overnight. The immunoprecipitated RNA was extracted, and the expression of miRNA and circRNA was detected by qRT-PCR using specific primers.

[0075] 1.11 RNA pull-down

[0076] Biotin-labeled circ-RNA probes were provided by Invitrogen, and RNA pull-down was performed according to the instructions of the Magnetic RNA Protein Pull-Down Kit (Thermo Fisher Scientific, USA). Briefly, HTR-8 / Svneo cell RNA extracts were incubated with 2 μg of probe and 40 μL of streptavidin beads at 4 °C for 2 h. After centrifugation, RNA complexes were collected, washed and analyzed by qRT-PCR.

[0077] 1.12 Transwell assay

[0078] The pretreated cells were resuspended in serum-free medium to a density of 2 × 10 5 Single cell suspension was prepared and 700 μL of serum-containing medium was added to the lower chamber. After 24 h of culture, the cells that did not migrate in the upper chamber were removed, and the cells that migrated to the outside of the chamber were fixed with 4% paraformaldehyde fixative for 30 minutes and washed three times with PBS for 5 minutes each. Then, the fixed cells were stained with crystal violet for 10 minutes and washed appropriately with PBS. Finally, the number of migrated cells was observed and counted under a microscope and recorded with photos.

[0079] 1.13 Subcellular localization

[0080] RNA was extracted and separated between the nucleus and cytoplasm of HTR-8 / Svneo using a cytoplasmic and nuclear RNA purification kit (Amyjet Scientific, China). First, cells were lysed using a cell lysis buffer, and the supernatant containing the cytoplasm was clearly distinguished from the precipitated nuclei by centrifugation. Then, cytoplasmic RNA and nuclear RNA were purified separately, and the expression levels of circ_0005704 in the nucleus and cytoplasm were determined by qRT-PCR to determine its subcellular localization. U6 and GAPDH were considered as positive controls for the nucleus and cytoplasm, respectively.

[0081] 1.14 RNA isolation and quantitative real-time PCR (qRT-PCR)

[0082] Total RNA was extracted using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. RNA was reverse transcribed using the miRNA First Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) or the PrimeScript RT Kit for mRNA (Toyobo, Osaka, Japan) according to the manufacturer's instructions. qRT-PCR was performed on an Applied Biosystems 7500 instrument (Applied Biosystems, Foster, USA) using SYBR Green (Invitrogen, Waltham, MA, USA) for miRNA and mRNA analysis, with primer sequences shown. For each sample, three replicate wells were set up. By comparing 2 -ΔΔCt Methods Relative RNA quantification was performed. The relative expression levels of miRNAs were normalized to the levels of the internal control U6, while the relative expression of genes was normalized to the expression level of ACTB in each sample.

[0083] 1.15 Western blot analysis

[0084] Cells and villous tissues were lysed in RIPA buffer (Thermo Scientific, Massachusetts, USA) with protease and phosphatase inhibitors (Thermo Scientific, Massachusetts, USA), and proteins were analyzed by Western blotting as described previously. Protein concentration was determined by BCA protein assay kit (ThermoScientific, Massachusetts, USA) according to the manufacturer's instructions. Anti-ULK1 antibody (8054, Cell Signaling Technology), ATG7 antibody (8558s, Cell Signaling Technology), ATG13 antibody (13468s, Cell Signaling Technology), Beclin1 antibody (3495, Cell Signaling Technology), P62 antibody (39749, Cell Signaling Technology), LC3 antibody (4180, Cell Signaling Technology), MMP2 antibody (ab181286, Abcam), MMP9 antibody (ab283575, Abcam), Vimentin antibody (5741S, Cell Signaling Technology), E-cadherin antibody (3195S, Cell Signaling Technology), N-cadherin antibody (13116S, Cell Signaling Technology), GAPDH antibody (ab181603, Abcam) and anti-rabbit IgG secondary antibody (ZF-0311, ZSGB-BIO) were used.

[0085] 1.16 Animal experiments

[0086] Eight-week-old CBA / J female, DBA / 2 male, and BALB / c male mice were purchased from Beijing Biotechnology Co., Ltd. and housed in a specific pathogen-free animal facility. As described previously, URSA model mice were established from CBA / J females and DBA / 2 males, and NP model mice were established from CBA / J females and BALB / c males. Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Basic Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University) and approved by the Institutional Animal Care and Use Committee of the Institute of Basic Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University. Mice were checked for vaginal plugs every morning.

[0087] 1.17 Analysis of Fetal Absorption Rate

[0088] There are significant differences between normal embryos and resorbed embryos. Normal embryos are larger in size, the fetus is fully developed, and the placenta is red in color, while the volume of resorbed embryos is significantly reduced and the placenta is darker in color. In addition, as mentioned above, the number of resorbed embryos and live embryos is calculated by the embryo absorption rate (R) = re / (re+F), where re is the number of resorbed embryos; F is the number of live embryos.

[0089] 1.18 Statistical analysis

[0090] All experiments were repeated at least three times. Values ​​are expressed as mean ± SD. Paired t-test was used to compare data between any two groups with normality and equal variance tests. For multiple comparisons, one-way analysis of variance with Bonferroni post hoc test was used. Data were plotted using GraphPad Prism 8.0.1 software (GraphPad software, Inc., CA, USA). Statistical analysis was performed using SPSS software (version 16.0). Pearson correlation was used for correlation analysis. The diagnostic value was evaluated using the receiver operating characteristic (ROC) curve. P < 0.05 was considered significant.

[0091] 2 Experimental results

[0092] 2.1 Autophagy upregulation and its association with URSA pathogenesis

[0093] To verify whether autophagy is altered in the pathogenesis of URSA, we performed transmission electron microscopy analysis to observe the abundance of autophagy-related particles in the villus tissue of NP and URSA patients. The results showed that the number of autophagosomes in URSA patients was significantly increased compared with the NP group, suggesting that autophagy may play a role in the pathogenesis of URSA ( Figure 1 A). To further examine the involvement of autophagy and decipher the key factors involved in autophagy regulation in URSA, we examined the protein levels of key regulators of autophagy, including ULK1, Beclin1, ATG13, ATG7, p62, and LC3II / I ( Figure 1 B). The results confirmed that autophagy was upregulated in URSA villi, and ULK1 was the only elevated protein among the targets detected, suggesting that ULK1 may be responsible for the enhanced autophagy. We also performed immunofluorescence (IF) assays and verified the protein increase of ULK1 in URSA villi ( Figure 1 C). We then expanded our testing of ULK1 messenger RNA (mRNA) expression in clinical samples of URSA villi and found that ULK1 was upregulated at the mRNA level ( Figure 1 D). ROC analysis showed that the expression of ULK1 could be used as a diagnostic marker for URSA patients ( Figure 1 E).

[0094] To functionally confirm the involvement of ULK1-mediated autophagy in the pathogenesis of URSA, we performed in vivo assays in a mouse URSA model to test whether embryo resorption would be alleviated after autophagy intervention. Chloroquine (CQ) and SBI-0206965 were injected intravenously to inhibit autophagosome-lysosome fusion and ULK1 activity, respectively, and embryo resorption rate was monitored. The study showed that both CQ and SBI-0206965 could significantly reduce embryo resorption rate ( Figure 1 F, G). The inhibitory effects of the two inhibitors on autophagy were verified by Western blotting of the indicated markers in isolated villi of the URSA mouse model ( Figure 1 H). In conclusion, our data suggest that upregulation of autophagy is involved in the pathogenesis of URSA and altered ULK1 transcription levels may be responsible for the elevated autophagy.

[0095] MiR-26a-5p is negatively correlated with ULK1 expression in URSA patients

[0096] Noncoding RNAs, such as microRNAs (miRNAs) and circular RNAs, are important regulators of mRNA expression. To investigate the upstream regulatory mechanisms of ULK1, we performed ChIP sequencing to screen for potential miRNAs targeting ULK1. By crossing the differentially expressed miRNAs with the list of miRNAs retrieved from the targetscan database, 19 miRNA candidates were screened ( Figure 2 A). To further narrow down the range of miRNAs targeting ULK1, we used two additional databases, miRDB and DIANA, and miR-26a-5p was the only miRNA retrieved by all these databases and was the downregulated miRNA from our microarray analysis ( Figure 2 B). Next, we expanded the number of clinical samples and confirmed that miR-26a-5p was downregulated in the villus tissue of URSA patients ( Figure 2 C). In addition, fluorescence in situ hybridization (FISH) analysis also revealed the downregulation of miR-26a-5p ( Figure 2 D). To demonstrate the clinical significance of miR-26a-5p expression in the pathogenesis of URSA, we performed ROC analysis, and the results verified that the expression level of miR-26a-5p in villi can sensitively distinguish URSA patients from NP groups ( Figure 2 E). Correlation analysis clearly showed that miR-26a-5p was negatively correlated with ULK1 expression, which highly suggested that miR-26a-5p might target ULK1 to determine trophoblast function ( Figure 2 F).

[0097] 2.3miR-26a-5p targets ULK1 to negatively regulate autophagy in trophoblast cells

[0098] To further analyze the interaction between miR-26a-5p and ULK1 mRNA, we analyzed the Targetscan database and found that there were two potential binding sites between miR-26a-5p and ULK1 3' untranslated region (UTR) ( Figure 3 A). We designed a dual luciferase reporter gene assay and subcloned the 3'UTR of ULK1 carrying the mutation site into the pGL3 plasmid respectively. The results showed that miR-26a-5p could bind to the two predicted sites on the 3'UTR of ULK1 ( Figure 3 B). Meanwhile, to investigate the potential biological effects of miR-26a-5p on trophoblast, we altered the expression of miR-26a-5p and measured the expression of ULK1 in HTR-8 / Svneo. The results showed that transfection of miR-26a-5p mimics inhibited the expression of ULK1 in HTR-8 / Svneo, while the inhibitor effectively enhanced its expression ( Figure 3 C, D). These data suggest that miR-26a-5p targets and inhibits ULK1 mRNA expression. Therefore, we further tested whether ULK1-mediated autophagy is affected by miR-26a-5p, and the results consistently showed that miR26a-5p could reduce the abundance of ULK1 protein, thereby inhibiting the level of autophagy as manifested by p62 and LC3II / I protein levels ( Figure 3 E). In conclusion, our data suggest that miR-26a-5p inhibits autophagy in trophoblast cells through ULK1.

[0099] 2.4Circ_0005704 interacts and negatively correlates with miR-26a-5p expression

[0100] It has been demonstrated that circRNAs can compete with specific mRNAs for the same miRNA pool. As more and more circular RNA functions are being identified and the feasibility of circRNA-targeted therapy is increasing, we aimed to screen for circRNAs that may regulate the miR-26a-5p / ULK1 axis to provide new intervention targets for URSA. Therefore, we performed bioinformatics analysis to retrieve all circRNAs with miR-26a-5p binding sites that intersected with the list of dysregulated circRNAs revealed by microarray analysis ( Figure 4 A). Twenty-four upregulated circRNAs were screened in URSA patients, and these circRNAs efficiently regulated the expression of miR26a-5p ( Figure 4B). Therefore, we used a specific miR-26a-5p probe to pull down total RNA from HTR-8 / Svneo lysates and examined the enrichment of all 24 circRNAs by qRT-PCR analysis. The miR-26a-5p probe significantly enriched two circRNAs ( Figure 4 C). The subcellular localization of circRNA is important for its functional mode, so we separated the cytoplasm and nucleus of HTR-8 / Svneo cells and detected the distribution of these two circRNAs. The results showed that circ_0005704 was mainly located in the cytoplasm, while circ_0064954 was mainly present in the nucleus ( Figure 4 D). Given that cytoplasm-localized circRNAs usually function through the ceRNA mechanism, we selected circ_0005704 for further analysis to regulate the miR-26a-5p / ULK1 axis.

[0101] To further validate the interaction between circ_0005704 and miR-26a-5p, we performed RNA immunoprecipitation (RIP) and RNA pull down experiments using Ago2 antibody and circ_0005704 probe, respectively. RIP analysis showed that circ_0005704 could act as an endogenous sponge for miR-26a-5p, and conversely, RNA pull down experiments showed that circ_0005704 could bind to miR-26a-5p ( Figure 4 E, F). To prove that circ_0005704 does exist in the form of circular RNA, we used the abundance of circ_0005704 RNA after RNase R treatment of total RNA from HTR-8 / Svneo. The results showed that circ_0005704 was completely resistant to RNase R digestion compared with the linear transcript of the host gene C20orf94, indicating that circ_0005704 is a stable circular structure ( Figure 4 G). In addition, we correlated the expression of circ_0005704 with that of miR-26a-5p to confirm the functional relationship between the two RNA molecules. By expanding the clinical samples, we found that circ_0005704 was upregulated in the villus tissue of URSA patients ( Figure 4 H), ROC analysis confirmed that the expression of circ_0005704 could significantly distinguish URSA patients from NP groups ( Figure 4 I). Pearson correlation analysis showed that the expression of circ_0005704 and miR-26a-5p was significantly negatively correlated ( Figure 4J). In addition, we analyzed the nucleotide sequence of circ_0005704 that can bind to miR-26a-5p, and we further confirmed this binding by dual luciferase reporter gene analysis ( Figure 4 K, L).

[0102] 2.5Circ_0005704 overexpression promotes autophagy through miR-26a-5p / ULK1 axis

[0103] To establish the functional relationship between circ_0005704 expression and trophoblast cells in vitro, the plasmid overexpressing circ_0005704 was transfected into HTR-8 / Svneo. qRT-PCR analysis further verified the transfection efficiency and the effect of circ_0005704 on the miR-26a-5p / ULK1 axis, that is, circ_0005704 overexpression could inhibit miR-26a-5p and enhance the expression of ULK1 ( Figure 5 AC). Consistently, circ_0005704 overexpression could increase the protein level of ULK1 and significantly promote autophagy of HTR-8 / Svneo ( Figure 5 D). In addition, we also tested whether overexpression of circ_0005704 would affect trophoblast cell function. Our data showed that overexpression of circ_0005704 did not affect proliferation but inhibited migration of HTR-8 / Svneo ( Figure 5 E). The phenotype was further validated by detecting the expression of relevant molecular biomarkers of cell migration, including vimentin, MMP9, E-cadherin, and N-cadherin ( Figure 5 F).

[0104] From the in vivo animal experiments, in order to determine whether circ_0005704 promotes embryo absorption through miR-26a-5p, we intravenously injected plasmids overexpressing circ_0005704 with or without miR-26a-5p and found that circ_0057 04 could significantly increase the embryo absorption rate, while overexpression of miR-26a-5p could reduce the embryo absorption rate ( Figure 5 G, H). We then isolated the embryonic villus tissue and measured the levels of autophagy and cell migration by detecting the protein levels of the above molecules. The results showed that overexpression of circ_0005704 not only enhanced autophagy but also inhibited cell migration in the villus tissue, and overexpression of miR-26a-5p could restore the migration of the villus tissue ( Figure 5 I, J). Therefore, we inferred that circ_0005704 might inhibit cell migration through miR-26a-5p / ULK1 and cause abortion by promoting autophagy in trophoblast cells.

[0105] 2.6 Circ_0005704 knockdown inhibits autophagy and alleviates embryonic resorption via miR-26a-5p / ULK1

[0106] To further investigate the functional relationship between circ_0005704 and trophoblast cells, initially, HTR-8 / Svneo were transfected with small interfering RNA (siRNA) targeting circ_0005704. Subsequently, the transfection efficiency of circ_0005704 and the effect on the miR-26a-5p / ULK1 axis were verified by qRT-PCR analysis. The results showed that circ_0005704 knockdown promoted miR-26a-5p and reduced the expression of ULK1 ( Figure 6 AC). In addition, after circ_0005704 knockout, a significant decrease in ULK1 protein level and inhibition of autophagy were observed in HTR-8 / Svneo ( Figure 6 D). In addition, the effects of circ_0005704 knockout on the functional properties of trophoblast cells were investigated. The results showed that although circ_0005704 knockout did not affect cell proliferation, it impeded cell migration in HTR-8 / Svneo ( Figure 6 E). This observation was supported by the evaluation of relevant molecular biomarkers associated with cell migration, including vimentin, MMP9, E-cadherin, and N-cadherin ( Figure 6 F).

[0107] From an in vivo perspective, we further explored the potential mediating role of circ_0005704 in promoting embryo resorption through miR-26a-5p. To address this question, siRNA targeting circ_0005704 was intravenously injected with or without mir-26a-5p inhibitor, and the results showed that circ_0005704 knockdown induced a significant decrease in embryo resorption rate, while mir-26a-5p inhibition offset this effect ( Figure 6 G, H). Chorionic villus tissue was subsequently isolated from the embryos, allowing assessment of autophagy and cell migration-related proteins. The results showed that knockout of circ_0005704 not only inhibited autophagy but also enhanced cell migration in the villus tissue, and inhibition of miR-26a-5p mitigated this effect ( Figure 6 I, J). Based on these findings, we speculated that circ_0005704 knockout might promote cell migration through the miR-26a-5p / ULK1 axis and might prevent abortion by reducing trophoblast cell autophagy.

[0108] In summary, the data provided by the present invention indicate that circ_0005704 promotes autophagy in trophoblast cells, thereby determining migration and abortion rate in URSA patients. Overexpression of circ_0005704 abolishes the inhibitory effect of miR-26a-5p on ULK1, leading to its accumulation and enhanced autophagy, which affects the migration of trophoblast cells. Mechanistically, circ_0005704 acts as a molecular sponge for miR-26a-5p, thereby attenuating the binding of miR-26a-5p to the 3'UTR of ULK1 mRNA. Importantly, the upregulation of circ_0005704 is closely associated with the downregulation of miR-26a-5p and is positively correlated with ULK1-mediated autophagy in patients and URSA models. In summary, these results reveal a regulatory axis of ULK1-mediated autophagy to promote the pathogenesis of URSA caused by circ_0005704 and miR-26a-5p dysregulation.

[0109] Matters not covered by the present invention are known technologies.

[0110] 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. Use of substances for detecting the expression level of circ_0005704 and its related biological elements in the preparation of products for screening, diagnosis, detection, monitoring or prognosis of unexplained recurrent spontaneous abortion.

2. The use according to claim 1, characterized in that: The circ_0005704-related biological elements include miR-26a-5p and ULK1.

3. The use according to claim 1, characterized in that: The substances for detecting the expression levels of circ_0005704 and its related biological elements include reagents for detecting the expression levels of circ_0005704, miR-26a-5p and ULK1 encoding genes based on real-time fluorescence quantitative PCR, in situ hybridization, gene chips and gene sequencing, and / or reagents for detecting the expression levels of ULK1 protein based on immunodetection methods.

4. The use according to claim 1, characterized in that: The products include primers, probes, (gene or protein) chips, nucleic acid membrane strips, detection kits, detection devices and detection equipment for detecting the expression levels of circ_0005704 and its related biological elements.

5. A system for screening, diagnosis, detection, monitoring or prognosis of unexplained recurrent spontaneous abortion, characterized in that: The system comprises: An acquisition unit is configured to: acquire the expression level of circ_0005704 and its related biological elements in the test sample of the subject; An evaluation unit is configured to evaluate and determine the disease condition of the subject according to the subject circ_0005704 and its related biological elements obtained by the acquisition unit.

6. The system according to claim 5, characterized in that The circ_0005704-related biological elements include miR-26a-5p and ULK1.

7. The system according to claim 5, characterized in that The samples to be tested include blood samples (including peripheral blood mononuclear cells) and chorionic villus tissue samples of the subject.

8. Application of circ_0005704 and its related biological elements as targets in the preparation and / or screening of drugs for unexplained recurrent spontaneous abortion; in, The circ_0005704-related biological elements include miR-26a-5p and ULK1.

9. Use of a substance that inhibits the expression of circ_0005704 or reduces its activity in any one or more of the following: a) promoting the expression of miR-26a-5p or preparing a product that promotes the expression of miR-26a-5p; b) inhibiting ULK1 expression or preparing a product that inhibits ULK1 expression; c) promoting trophoblast cell migration or preparing a product that promotes trophoblast cell migration; d) inhibiting autophagy of trophoblast cells or preparing a product for inhibiting autophagy of trophoblast cells; g) Products for the prevention and / or treatment of unexplained recurrent spontaneous abortion.

10. The use according to claim 9, characterized in that: Substances that inhibit the expression of circ_0005704 or reduce its activity include RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, shRNA targeting circ_0005704, and substances for implementing lentiviral infection or gene knockout.

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