Has-miR-3942-5p as premature delivery detection marker and application thereof
By using placental-derived plasma exosome microRNA has-miR-3942-5p as a biomarker, the problem of non-invasive spontaneous premature birth diagnosis is solved, efficient spontaneous premature birth detection is achieved, the pathogenesis of premature birth is improved, and a new method for clinical diagnosis is provided.
Patent Information
- Application Number
- CN202510745442.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-05
- Publication Date
- 2025-08-08
AI Technical Summary
There is a lack of non-invasive and effective diagnosis of spontaneous premature birth in the prior art, especially for the diagnosis of spontaneous premature birth in pregnant women. Current detection methods may stimulate the vaginal wall and cervix, leading to the occurrence of premature birth, and there is no effective biomarker for premature birth prediction and diagnosis.
Has-miR-3942-5p is used as a marker of premature birth. By collecting placental-derived exosome microRNA in pregnant women's blood, using microRNA sequencing and verification technology to lock in the expression level of has-miR-3942-5p, and develop detection kits and drugs for predicting, diagnosing or evaluating spontaneous premature birth.
It provides a new non-invasive diagnosis method, improves the diagnostic accuracy and reliability of spontaneous premature birth, broadens the application of exosomal microRNA in disease markers, improves the pathogenesis of spontaneous premature birth, and provides a theoretical basis for prevention and intervention.
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Figure CN120442783A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and in particular to has-miR-3942-5p as a non-invasive premature birth detection marker and application thereof. Background Art
[0002] Preterm birth, defined as birth after 28 weeks of gestation but before 37 weeks, is the leading cause of perinatal morbidity and mortality. Premature birth has profound effects on the neonatal nervous system and multi-organ development, severely impacting fetal health and even increasing the risk of cardiovascular disease and metabolic syndrome in adulthood. It has become one of the most important global public health issues. Premature birth can be classified into three types: spontaneous preterm birth, premature rupture of membranes (PROM), and therapeutic preterm birth. Spontaneous preterm birth is the most common type, accounting for approximately 45% of premature births, while premature rupture of membranes (PROM) accounts for approximately 25%. According to the World Health Organization, the global incidence of premature births ranges from 5% to 18%, resulting in approximately 15 million premature births each year. With the increasing number of older mothers, the incidence of premature births is increasing.
[0003] The pathogenesis of spontaneous preterm birth remains unclear. A variety of abnormal factors have been reported to contribute to premature labor and lead to preterm birth, including maternal genetic variation, infection of the reproductive system or other maternal organs, decidual aging and bleeding, imbalance in maternal-fetal immune regulation, functional progesterone withdrawal, and psychological factors. For pregnant women without a history of miscarriage, the pathogenesis of spontaneous preterm birth is even more elusive, making clinical diagnosis extremely difficult. Currently, the main method for clinical prediction of spontaneous preterm birth is transvaginal ultrasound measurement of cervical length between 16 and 28 weeks of gestation. A shortened cervical length (<25 mm) indicates an increased risk of preterm birth. However, this method stimulates the vaginal wall and cervix, potentially inducing uterine contractions and even leading to the development of preterm birth. Therefore, guidelines limit this method to prediction and diagnosis in high-risk individuals and do not recommend its use in other pregnant women. Consequently, there is currently no noninvasive and effective diagnostic method for spontaneous preterm birth in pregnant women.
[0004] Exosomes, as messengers of cellular communication, are widely present in most body fluids. Encapsulating components such as lipids, proteins, and nucleic acids derived from host cells, they have become novel non-invasive biomarkers for early disease diagnosis and prognostic assessment, forming the cornerstone of liquid biopsy. Recent studies have shown that exosomes can serve as potential signaling mediators and carriers between the fetus and the mother, extensively participating in maternal-fetal exchange in a paracrine manner. MicroRNAs encapsulated in maternal circulating exosomes play a crucial role in pregnancy progression, serving as diagnostic markers for pregnancy-related diseases and participating in gene regulation within the placental microenvironment. Placental tissue-specific exosomes have been studied as diagnostic markers for preeclampsia and other pregnancy outcomes. Furthermore, recent studies have reported that placental tissue-specific exosomes mediate inflammatory signaling across aging fetal membranes (amnion and chorion) at term, promoting fetal labor behavior. Thus, placental tissue-specific exosomes have broad applications in the diagnosis of pregnancy-related diseases. However, studies have so far failed to identify placental tissue-specific exosomes as diagnostic markers for spontaneous preterm birth.
[0005] Therefore, it is of great social significance to conduct in-depth research on the pathogenesis of spontaneous preterm birth in pregnant women and develop an effective new strategy for diagnosing spontaneous preterm birth based on this. Summary of the Invention
[0006] To this end, the present invention provides has-miR-3942-5p as a marker for premature birth detection and its application to solve the related technical problems existing in the prior art.
[0007] In order to achieve the above object, the present invention provides the following technical solutions:
[0008] According to a first aspect of the present invention, hsa-miR-3942-5p is used as a marker for detecting premature birth, wherein the sequence of hsa-miR-3942-5p is as SEQ ID NO.1.
[0009] Furthermore, has-miR-3942-5p is used as a marker for premature birth detection in predicting, diagnosing or evaluating the risk of spontaneous premature birth.
[0010] Furthermore, has-miR-3942-5p is used as a marker for detecting premature birth in the preparation of a detection kit for predicting, diagnosing or evaluating the risk of spontaneous premature birth.
[0011] Furthermore, the has-miR-3942-5p is derived from pregnant women's blood, plasma, serum, cerebrospinal fluid, urine, vaginal drainage fluid, placental biopsy specimens, uterine fluid or amniotic fluid samples.
[0012] Furthermore, the has-miR-3942-5p is a placenta-derived maternal plasma exosome microRNA or serum exosome microRNA.
[0013] Furthermore, the sample is derived from a pregnant woman with a gestational age of 28-31 weeks.
[0014] According to a second aspect of the present invention, has-miR-3942-5p is used as a premature birth detection marker in the preparation of spontaneous premature birth diagnosis and prognosis drugs.
[0015] Furthermore, the dosage form of the drug is any oral dosage form of granules, decoction, tablets or capsules.
[0016] Furthermore, the medicine is added with one or more excipients selected from microcrystalline cellulose, powdered cellulose, mannitol, starch, lactose, gelatin, methylcellulose, dextrin, pregelatinized starch, micropowdered silica, hydroxypropyl methylcellulose, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, polyethylene glycol, xylitol, lactitol, glucose, glycine, mannitol, tartaric acid, silicon dioxide, calcium stearate or magnesium stearate.
[0017] Furthermore, the drug also includes a pharmaceutically acceptable carrier.
[0018] The present invention has the following advantages:
[0019] This application collects plasma exosomes EXO from placenta of pregnant women with full-term and spontaneous preterm birth. PD Perform microRNA sequencing, analyze the microRNA sequencing results, and select spontaneous premature EXO PD The expression level of differentially upregulated microRNAs was verified and EXO was locked PD The diagnostic potential of has-miR-3942-5p for spontaneous preterm birth was tested in clinical specimens. This application is the first to use placental-derived plasma exosomal microRNA as a biomarker for the diagnosis of spontaneous preterm birth, providing a novel clinical diagnostic approach for spontaneous preterm birth and broadening the application of exosomal microRNA as a disease marker. Elucidation of the mechanism of action of has-miR-3942-5p in exosomes, including its role in disrupting the homeostasis of the placental immune microenvironment, will enhance theoretical understanding of the pathogenesis of spontaneous preterm birth and provide a theoretical basis for its prevention and intervention. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.
[0021] The structures, proportions, sizes, etc. illustrated in this specification are intended only to complement the contents disclosed herein and to facilitate understanding and reading by persons familiar with the art. They are not intended to limit the conditions under which the present invention may be implemented and therefore have no substantive technical significance. Any structural modifications, changes in proportions, or adjustments in sizes, without affecting the efficacy and objectives of the present invention, shall still fall within the scope of the technical contents disclosed herein.
[0022] Figure 1 For EXO in this invention PD Collection and identification diagram.
[0023] A.EXO PD A. Schematic diagram of the collection method; B. Western blot analysis of the expression of exosome marker proteins PLAP, CD63, TSG101, and CD81; C. Transmission electron microscopy analysis of the morphology and integrity of exosomes;
[0024] Among them, TB: full-term birth, sPTB: spontaneous preterm birth.
[0025] Figure 2 For EXO in this invention PD Diagram of surface placental marker identification.
[0026] A. Flow cytometry detection of collected EXOs PD Surface exosome markers (CD63) and placental markers (PLAP); B. Transmission electron microscopy identification of EXO PD Surface placental marker expression and localization;
[0027] Among them, TB: full-term birth, sPTB: spontaneous preterm birth.
[0028] Figure 3 For EXO in this invention PD MicroRNA sequencing differential analysis diagram.
[0029] A. EXO in pregnant women with full-term delivery and spontaneous preterm delivery PD Differential microRNA heat map; B. EXO in pregnant women with spontaneous preterm birth PD Heat map of upregulated microRNAs; C. EXO in pregnant women with spontaneous preterm birth PDVolcano plot analysis of upregulated microRNAs;
[0030] Among them, TB: full-term birth, sPTB: spontaneous preterm birth.
[0031] Figure 4 EXO with diagnostic potential in the present invention PD Relative expression levels of microRNAs in plasma of pregnant women with spontaneous preterm birth.
[0032] A. Spontaneous preterm birth in pregnant women with EXO PD The top ten upregulated microRNAs in the sequencing results; BF.Q-PCR detection of top 5 upregulated microRNAs in plasma EXO of pregnant women with spontaneous preterm delivery PD The relative expression levels in
[0033] TB: term birth, sPTB: spontaneous preterm birth, n=10, *p<0.05, Mann-Whitney Rank Sum Test.
[0034] Figure 5 For EXO in this invention PD Diagram showing the validation of the diagnostic value of has-miR-3942-5p in diagnosing spontaneous preterm birth.
[0035] AQ-PCR detection of has-miR-3942-5p in plasma EXO of pregnant women with spontaneous preterm delivery PD B. ROC curve evaluation of the relative expression levels of EXO PD The diagnostic value of has-miR-3942-5p for spontaneous preterm birth;
[0036] TB: term birth, sPTB: spontaneous preterm birth, n=30, ***p<0.001, Mann-Whitney RankSum Test.
[0037] Figure 6 This is the has-miR-3942-5p related pathway enrichment analysis and gene regulatory network prediction diagram in the present invention.
[0038] A. GO and KEGG enrichment analysis of has-miR-3942-5p related pathways; B. Targetscan database predicted has-miR-3942-5p gene regulatory network. DETAILED DESCRIPTION
[0039] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.
[0040] According to the first aspect of the present invention, hsa-miR-3942-5p is used as a marker for detecting premature birth, wherein the hsa-miR-3942-5p sequence is as SEQ ID NO.1, and the specific nucleotide sequence is: AAGCAAUACUGUUACCUGAAAU.
[0041] Furthermore, has-miR-3942-5p is used as a marker for premature birth detection in predicting, diagnosing or evaluating the risk of spontaneous premature birth.
[0042] Furthermore, has-miR-3942-5p is used as a marker for detecting premature birth in the preparation of a detection kit for predicting, diagnosing or evaluating the risk of spontaneous premature birth.
[0043] Furthermore, has-miR-3942-5p is derived from maternal blood, plasma, serum, cerebrospinal fluid, urine, vaginal drainage fluid, placental biopsy specimens, uterine fluid or amniotic fluid samples.
[0044] Furthermore, has-miR-3942-5p is a placenta-derived maternal plasma exosome microRNA or serum exosome microRNA.
[0045] Furthermore, samples were obtained from primiparas with a gestational age of 28-31 weeks.
[0046] According to a second aspect of the present invention, has-miR-3942-5p is used as a premature birth detection marker in the preparation of spontaneous premature birth diagnosis and prognosis drugs.
[0047] Furthermore, the dosage form of the drug is any oral dosage form of granules, decoction, tablets or capsules.
[0048] Furthermore, one or more excipients selected from microcrystalline cellulose, powdered cellulose, mannitol, starch, lactose, gelatin, methylcellulose, dextrin, pregelatinized starch, micropowdered silica, hydroxypropyl methylcellulose, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, polyethylene glycol, xylitol, lactitol, glucose, glycine, mannitol, tartaric acid, silicon dioxide, calcium stearate or magnesium stearate are added to the medicine.
[0049] Furthermore, the drug also includes a pharmaceutically acceptable carrier.
[0050] In order to solve the technical problems in the prior art, the effect of has-miR-3942-5p in placenta-derived exosomes on spontaneous preterm birth was studied. This application provides the following research plan.
[0051] (1) Placenta-derived plasma exosomes (EXO) from pregnant women with full-term and spontaneous preterm delivery PD ) collection and identification
[0052] (a) Characteristics of pregnant women enrolled
[0053] Based on the Obstetrics and Gynecology Hospital affiliated to Nankai University, we randomly selected primiparas aged 20-35 years who visited the obstetrics clinic from August 2023 to October 2023 as the research subjects. We ensured that all included subjects had no prior treatment for related diseases, no history of hypertension, no major diseases or family history, and no long-term low-protein and low-calorie malnutrition. Based on the pregnancy outcomes and final diagnoses registered in the electronic medical record system of our hospital, we included 15 full-term pregnant women and 10 spontaneous preterm births. The characteristics of pregnant women and their fetuses are shown in Table 1.
[0054] Table 1.
[0055]
[0056] (b)EXO PD Collection and identification
[0057] EDTA anticoagulated plasma of pregnant women in the early third trimester (average 28 weeks) was selected, PLAP was used as a placental marker, and CD63 was used as an exosome marker. Placental derived plasma exosomes (EXO) were collected from the plasma using immunocapture method. PD )( Figure 1 (As shown in A) Western blot was used to detect the collected EXO PD Expresses exosome markers CD63, CD81 and TSG101, and also expresses PLAP( Figure 1 B) Transmission electron microscopy observation of collected EXOs PD The size is within the range of 50-150nm, the morphology is intact, and there is no membrane damage, which can be used for nucleic acid sequencing inside exosomes ( Figure 1 C).
[0058] (2)EXO PD Identification of surface placental markers
[0059] The placenta is an important organ for the exchange of substances between the fetus and the mother. The use of placenta-derived exosomes is beneficial for improving the detection of spontaneous preterm birth. PD The collected EXO were detected by FITC-labeled CD63 fluorescent antibody and APC-labeled PLAP fluorescent antibody. PD Staining was performed, incubated on ice for 45 min, and EXO was detected by flow cytometry. PD The expression ratio of exosome surface specific marker CD63 and surface placental marker PLAP, as well as the proportion of CD63 and PLAP double-positive exosomes ( Figure 2 ). The results show that: the collected EXO PD The expression ratio of PLAP on the surface is about 88%, the expression ratio of CD63 is about 86%, and the EXOs co-expressing PLAP and CD63 PD About 78% of pregnant women with term and spontaneous preterm births received EXO PD About 78% originates from the placenta.
[0060] (3) EXO in full-term pregnant women and pregnant women with spontaneous preterm delivery PD microRNA sequencing
[0061] EXO was extracted from pregnant women with full-term and spontaneous preterm delivery using the MagaBio plus total RNA purification kit. PD The total RNA was extracted and the quality of the extracted RNA was checked. The qualified samples were subjected to microRNA high-throughput sequencing using Illumina NextSeq 500 ( Figure 3 A), sequencing results showed a total of 75 differentially expressed microRNAs (|log2FC|>1, p≤0.05). In the diagnostic process, low expression indicators cannot be well distinguished from detection failures caused by failure of the test system, which will inevitably lead to false negatives in diagnostic indicators. Overexpressed biomarkers are often selected as diagnostic markers. Therefore, it is planned to target spontaneous preterm EXO PD Upregulated microRNAs ( Figure 3 BC) for the next step of key analysis and verification.
[0062] Specifically, the R software limma package can be used for differential expression analysis; the "clusterProfiler" package in the R language can be used to perform GO / KEGG enrichment analysis on microRNAs with diagnostic potential; and the TargetScan database can be used to predict the target genes of the target microRNA.
[0063] (4) Potential for diagnosing spontaneous preterm birth EXO PD Verification of microRNA expression levels in clinical plasma specimens
[0064] According to the results of microRNA second generation sequencing, the top ten significantly upregulated microRNAs were obtained ( Figure 4 A) The top five overexpressed microRNAs were selected and amplified using specific primers for the target microRNAs, with U6 as the internal reference gene. +6 w EDTA anticoagulated plasma EXO PD Q-PCR validation was performed in Figure 4 BF). The results showed that: PD As a control, plasma samples of pregnant women with spontaneous premature delivery, EXO PD There was no significant difference in the expression levels of hsa-miR-4516, hsa-miR-3138, hsa-miR-4746-5p and hsa-miR-31-5p, while hsa-miR-3942-5p was significantly expressed in EXO2 pregnant women with spontaneous preterm delivery. PD The expression of EXO was significantly upregulated (p<0.05). PD Expression of hsa-miR-3942-5p in plasma EXO of pregnant women with spontaneous preterm delivery PD It is overexpressed in serotonin-positive mice and has the potential to diagnose spontaneous preterm birth.
[0065] (5) Small sample size verification of EXO PD Feasibility of has-miR-3942-5p in diagnosing spontaneous preterm birth
[0066] To further test EXO PD The diagnostic ability of has-miR-3942-5p for spontaneous preterm birth was studied. Pregnant women with spontaneous preterm birth for the first time (n=30) were enrolled at 28-31 gestational age. +6 w EDTA anticoagulated plasma, and EDTA anticoagulated plasma of full-term pregnant women (n=30) matched with their gestational age, and EXO were extracted from them respectively. PD The results showed that compared with age-matched full-term pregnant women, patients with spontaneous preterm birth had higher levels of EXO PD The expression of has-miR3942-5p was significantly increased (p<0.001) ( Figure 5 A).
[0067] According to clinical specimens EXO PD The Q-PCR detection results of hsa-miR-3942-5p were used to draw the receiver operating characteristic (ROC) curve using IBM SPSS Statistics 25.0, and the diagnostic value of EXOPD hsa-miR-3942-5p was determined by the area under the curve (AUC). The ROC curve results showed that the diagnostic value of EXOPD in patients with spontaneous preterm birth was significantly higher than that in patients with spontaneous preterm birth. PDThe AUC value of hsa-miR3942-5p was 0.79, and the cutoff point showed a sensitivity of 66.7% and a specificity of 73.3% ( Figure 5 B) Explain EXO PD has-miR3942-5p has good diagnostic value in spontaneous preterm birth and can be used for clinical auxiliary diagnosis of spontaneous preterm birth.
[0068] (6) Has-miR-3942-5p related pathway enrichment analysis and gene regulatory network prediction
[0069] To further explore the mechanism of action of has-miR-3942-5p in spontaneous preterm birth, GO and KEGG enrichment analysis were performed on has-miR-3942-5p ( Figure 6 A) The results showed that hs-miR-3942-5p was significantly involved in pathways related to endometrial, stroma, and muscle development, including "endomembrane system organization," "positive regulation of extracellular matrix organization," and "muscle organ development." It was also significantly associated with adaptive inflammatory regulation and cytokine activity, including "cytokine-cytokine receptor interaction" and "cytoplasmic stress granules." This suggests that hs-miR-3942-5p plays an important role in the placental immune microenvironment and maternal-fetal immune tolerance.
[0070] The TargetScan database was used to predict the has-miR-3942-5p gene regulatory network ( Figure 6 B) found that hsa-miR-3942-5p was associated with multiple inflammation-related genes, among which the cumulative weighted score of CXCL16 gene was -0.57, suggesting that CXCL16 may be a potential target of hsa-miR-3942-5p. The CXCL16 / CXCR6 axis has been reported to be an important pathway for maintaining maternal-fetal immune tolerance, and its abnormal expression can lead to pregnancy complications such as trophoblast dysfunction and placental dysplasia. Therefore, it is speculated that EXO PD It is possible that has-miR-3942-5p is transferred through intercellular material exchange, destroying the placental immune microenvironment and inducing spontaneous preterm birth by mediating CXCL16 / CXCR6 axis deficiency.
[0071] It should be noted that the detection techniques such as immune capture technology, Western blot technology, staining technology, electron microscopy, flow cytometry, etc. used in the examples of this application are all commonly used technologies in the prior art. During the specific detection, only slight changes are made to the specific parameters, which does not affect the overall detection effect. The specific detection steps involved will not be repeated here.
[0072] This application collects plasma exosomes EXO from placenta of pregnant women with full-term and spontaneous preterm birth. PD Perform microRNA sequencing, analyze the microRNA sequencing results, and select spontaneous premature EXO PD The expression level of differentially upregulated microRNAs was verified and EXO was locked PD The diagnostic potential of has-miR-3942-5p for spontaneous preterm birth was tested in clinical specimens. This application is the first to use placental-derived plasma exosomal microRNA as a biomarker for the diagnosis of spontaneous preterm birth, providing a novel clinical diagnostic approach for spontaneous preterm birth and broadening the application of exosomal microRNA as a disease marker. Elucidation of the mechanism of action of has-miR-3942-5p in exosomes, including its role in disrupting the homeostasis of the placental immune microenvironment, will enhance theoretical understanding of the pathogenesis of spontaneous preterm birth and provide a theoretical basis for its prevention and intervention.
[0073] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.
Claims
1. The application of has-miR-3942-5p as a marker for premature birth detection is characterized by: The has-miR-3942-5p sequence is as SEQ ID NO.
1.
2. The use according to claim 1, characterized in that The application of has-miR-3942-5p as a marker for premature birth detection in predicting, diagnosing or evaluating the risk of spontaneous preterm birth.
3. The use according to claim 2, characterized in that has-miR-3942-5p is used as a marker for detecting premature birth, and is used in the preparation of a detection kit for predicting, diagnosing or evaluating the risk of spontaneous premature birth.
4. The use according to claim 1, wherein The has-miR-3942-5p is derived from pregnant women's blood, plasma, serum, cerebrospinal fluid, urine, vaginal drainage fluid, placenta biopsy specimens, uterine fluid or amniotic fluid samples.
5. The use according to claim 4, characterized in that The has-miR-3942-5p is a placenta-derived maternal plasma exosome microRNA or serum exosome microRNA.
6. The use according to claim 4, characterized in that The samples were obtained from pregnant women with a gestational age of 28-31 weeks.
7. Application of has-miR-3942-5p as a marker for premature birth detection in the preparation of drugs for the diagnosis and prognosis of spontaneous premature birth.
8. The use according to claim 7, characterized in that The dosage form of the medicine is any oral dosage form of granules, decoction, tablets or capsules.
9. The use according to claim 7, characterized in that The medicine is added with one or more excipients selected from microcrystalline cellulose, powdered cellulose, mannitol, starch, lactose, gelatin, methylcellulose, dextrin, pregelatinized starch, micropowdered silica, hydroxypropyl methylcellulose, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, polyethylene glycol, xylitol, lactitol, glucose, glycine, mannitol, tartaric acid, silicon dioxide, calcium stearate or magnesium stearate.
10. The use according to any one of claims 7 to 9, characterized in that The drug further includes a pharmaceutically acceptable carrier.