CN114438199A

CN114438199A - Application of BBOX1-AS1 AS diagnostic marker and therapeutic target for recurrent abortion

Info

Publication number: CN114438199A
Application number: CN202210302503.9A
Authority: CN
Inventors: 毛海婷; 李亚丽; 赵汇; 李宁; 董娜娜; 王小玲; 毛慧慧; 冯一民; 李梓汇
Original assignee: Second Hospital of Shandong University
Current assignee: Second Hospital of Shandong University
Priority date: 2022-03-25
Filing date: 2022-03-25
Publication date: 2022-05-06
Anticipated expiration: 2042-03-25
Also published as: CN114438199B

Abstract

The invention provides an application of BBOX1-AS1 AS a diagnostic marker and a therapeutic target for recurrent abortion, belonging to the technical field of crude drug medicine and molecular biology. The invention firstly discovers that the expression quantity of BBOX1-AS1 is closely related to recurrent abortion, and shows that BBOX1-AS1 which is up-regulated in the villus tissue of recurrent abortion regulates the proliferation, migration and invasion of trophoblasts in vitro and participates in activating a P38/JNK MAPK signal pathway. In addition, the technical scheme also researches that BBOX1-AS1 promotes the expression of GADD45A by binding protein hnRNPK, finally causes the change of the function of trophoblasts and promotes the occurrence of recurrent abortion. Finally, the expression of BBOX1-AS1 in the serum of the recurrent abortion patients is increased, which indicates the potential possibility that BBOX1-AS1 is used AS an RPL early diagnosis marker, so that the method has good practical application value.

Description

Application of BBOX1-AS1 AS diagnostic marker and therapeutic target for recurrent abortion

Technical Field

The invention belongs to the technical field of crude drug medicine and molecular biology, and particularly relates to application of BBOX1-AS1 AS a diagnostic marker and a treatment target for recurrent abortion.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Recurrent Pregnancy Loss (RPL) means that 3 and more consecutive fetal losses occur with the same partner before 28 weeks of gestation, with an incidence of 1%, and clinically is usually defined as two or more consecutive pregnancy failures, with an incidence of up to 5%. According to the present research, there are many causes of recurrent abortion, mainly including: parent or embryonic chromosomal abnormalities; genital malformation or cervical insufficiency; an infectious agent; endocrine disorders; immune dysfunction; thrombosis tendency, etc., but the causes of many recurrent abortion are not clear, and the number of cases accounts for about 50% of recurrent abortion, which is clinically called as recurrent abortion of unknown origin. In recent years, people pay more and more attention to reproductive health, but the incidence of RPL is increased year by year, which seriously affects the physical and mental health of pregnant women, the pathogenesis of the current unexplained recurrent abortion is still unclear, no effective prevention and treatment measures are available in clinic, and the clinical problem to be solved is urgent.

The occurrence of recurrent abortion is a complex process. Numerous studies suggest that poorly proliferating trophoblasts, impaired infiltrative capacity, are one of the causes of many pregnancy-associated syndromes in the early stages of pregnancy, where trophoblasts invade the deciduated endometrium, remodeling the uterine spiral artery to increase maternal blood flow to the placental villi, which is critical to waste and nutrient exchange between maternal and fetal blood. Impaired trophoblast function will lead to severe adverse pregnancy outcomes including recurrent abortions of unknown origin, preeclampsia, and the like. Therefore, the research on the trophoblast function can not only provide a basis for the pathogenesis and treatment of recurrent abortion, but also have great significance for the occurrence, development and treatment of diseases.

LncRNA is RNA which is longer than 200nt and does not code protein, has low conservation among species and is widely present in animals and plants. They have long been mistaken for genetic dark material, but recent studies have demonstrated that such RNAs play an important regulatory role in the life-cycle of organisms. Recent studies have shown that lncRNA acts in a cell-type specific or tissue-specific manner, suggesting that lncRNA may be a driver of cell-specific reactions. lncRNA is involved in a variety of biological processes including genomic imprinting, cell proliferation and differentiation, and cell developmental processes. In recent years, studies have reported that LncRNA is associated with many reproductive diseases, including preeclampsia, early ovarian dysfunction, fetal growth restriction, recurrent abortion, etc. But the underlying mechanisms are still not completely understood.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of BBOX1-AS1 AS a diagnostic marker and a therapeutic target for recurrent abortion. The method firstly adopts a high-flux lncRNA expression profile chip to screen lncRNA which is differentially expressed in RPL villus tissue and normal pregnancy villus tissue. Detecting the influence of the difference lncRNA BBOX1-AS1 on the physiological functions of trophoblast proliferation, apoptosis, migration, invasion and the like, and further carrying out related mechanism research. And finally, lncRNA with high expression in placenta tissues is selected as a primary screening target, and is further detected and screened in the plasma of the pregnant women, so that the correlation between the occurrence of the unexplained recurrent abortion and lncRNA as an early diagnosis marker is known. The method is helpful for deeply understanding the pathogenesis of the unexplained recurrent abortion, further perfecting the maternal-fetal immune tolerance mechanism and providing reference for early diagnosis and prevention of the maternal-fetal immune tolerance mechanism.

Specifically, the invention relates to the following technical scheme:

in a first aspect of the invention, the invention provides the use of BBOX1-AS1 AS a marker in the preparation of a diagnostic or an auxiliary diagnostic product for recurrent abortion.

Wherein the BBOX1-AS1 is lncRNASBOX 1 antisense RNA 1(BBOX1-AS 1).

The invention discovers that BBOX1-AS1 is remarkably high expressed in villus tissues of patients with recurrent abortion compared with normal pregnancy through research. Further by detecting BBOX1-AS1 in the blood (serum) of patients with recurrent abortion, whose expression was also elevated, plotting a working characteristic curve (ROC curve) of the subjects and calculating the area under the curve (AUC), BBOX1-AS1 in serum was shown to have higher diagnostic efficacy, AUC was 0.8144.

In a second aspect of the invention, a product is provided, said product comprising a substance for detecting the above-mentioned BBOX1-AS1, for use in the diagnosis or assisted diagnosis of recurrent abortion.

In a third aspect of the invention, there is provided a system for diagnosing or aiding in the diagnosis of recurrent spontaneous abortion, said system comprising:

i) an analysis unit, the analysis unit comprising: a test substance selected from the group consisting of BBOX1-AS1 expression levels in a test sample from a subject, and;

ii) an evaluation unit comprising: determining whether the subject has recurrent miscarriage based on the BBOX1-AS1 expression level determined in i);

in a fourth aspect of the invention, there is provided a method of diagnosing or aiding in the diagnosis of recurrent pregnancy loss, the method comprising:

a) isolating a test sample from a subject;

b) detecting the above BBOX1-AS1 expression level in a test sample of the subject, and;

c) comparing the expression level in the sample to the expression level in the reference;

wherein, an upregulation of the level of BBOX1-AS1 expression in the sample AS compared to the level in the reference, is the subject or candidate for a patient with recurrent abortion; conversely, the subject is not, or is not candidate for, a patient with recurrent abortion.

In a fifth aspect of the invention, the invention provides the use of BBOX1-AS1 AS a target for treating recurrent abortion and/or screening drugs related to recurrent abortion.

In a sixth aspect of the invention, there is provided the use of an agent that inhibits the expression level of BBOX1-AS1 in the manufacture of a product;

the function of the product is any one or more of the following:

(a1) promoting proliferation of trophoblast cells;

(a2) inhibiting apoptosis of trophoblasts;

(a3) promoting migration of trophoblasts;

(a4) promoting the invasion of trophoblasts;

(a5) promoting the formation of nourishing tubules;

(a6) inhibition of activation (phosphorylation) of the P38/JNK MAPK signaling pathway;

(a7) inhibits binding to hnRNPK, and thereby inhibits GADD45A expression;

(a8) preventing and/or treating recurrent abortion.

In a seventh aspect of the invention, there is provided a product comprising as active ingredients any one or more of:

(b1) a substance that inhibits the expression level of BBOX1-AS 1;

(b2) a substance which inhibits the expression level of hnRNPK.

The products of the sixth to seventh aspects may be drugs or test reagents, which are available for basic research.

In an eighth aspect of the present invention, there is provided a method for the prevention and/or treatment of recurrent spontaneous abortion, said method comprising: administering to the subject a therapeutically effective dose of the above-described medicament.

The beneficial technical effects of one or more of the above technical solutions are as follows:

the technical scheme firstly discovers that the expression quantity of BBOX1-AS1 is closely related to recurrent abortion, and shows that BBOX1-AS1 which is up-regulated in the villus tissue of recurrent abortion regulates the proliferation, migration and invasion of trophoblasts in vitro and participates in activating a P38/JNK MAPK signaling pathway. In addition, the technical scheme also researches that BBOX1-AS1 promotes the expression of GADD45A by binding protein hnRNPK, finally causes the change of the function of trophoblasts and promotes the occurrence of recurrent abortion. Finally, increased expression of BBOX1-AS1 in serum from patients with recurrent abortion indicated the potential for BBOX1-AS1 AS an early diagnostic marker for RPL.

In conclusion, the BBOX1-AS1 and the molecular mechanism thereof found in the technical scheme are helpful for deeply understanding the pathogenesis of RPL and providing potential markers and therapeutic targets for clinical diagnosis, prevention and treatment of recurrent abortion, so that the method has good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a graph showing the results of screening for differential lncRNA in villus tissues of patients with normal pregnancy and RPL using a high-throughput LncRNA expression profiling chip in the example of the present invention; A. heatmaps from lncRNA expression profiling chips differentially expressed in control (n-3) and RPL patients (n-3). B. The 5 lncrnas with the most significant fold difference were screened from lncRNA expression profiling chip and validated in control (n-20) and RPL patients (n-18) villus tissue.

FIG. 2 is a graph correlating the detection of BBOX1-AS1 in villus tissue and in 3 trophoblast lines in 40 pairs of patients with normal pregnancy and RPL in accordance with the present invention. A: expression of BBOX1-AS1 was detected by qRT-PCR in villus tissue of 40 pairs of normal pregnant and RPL patients. B: expression of BBOX1-AS1 was detected by qRT-PCR in 4 trophoblast lines. C: after overexpression of BBOX1-AS1 in a feeder cell line, the overexpression efficiency was examined. D: after the BBOX1-AS1 was knocked down in the trophoblast lines HTR8/SVneo, JAR, the efficiency of BBOX1-AS1 knocking down was examined.

FIG. 3 is a graph showing that BBOX1-AS1 inhibits trophoblast proliferation and promotes apoptosis in vitro in the examples of the present invention. A. C, E: CCK8 experiment (A), EDU experiment (C) and clone formation experiment (E) test the influence of over-expression of BBOX1-AS1 on the proliferation of trophoblasts. B. D, F: CCK8 (B), EDU (D) and clonogenic (F) tests the influence of BBOX1-AS1 on trophoblast proliferation. G: the influence of the over-expression of BBOX1-AS1 on the apoptosis of trophoblasts is detected by a flow cytometer. H-I: the influence of the over-expression of BBOX1-AS1 on apoptosis-related proteins is detected by a flow cytometer.

FIG. 4 is a graph showing that BBOX1-AS1 inhibits trophoblast migration and invasion in vitro in the present example. A-B: the Transwell assay measures the effect on trophoblast migration and invasion following overexpression or knockdown of BBOX1-AS 1. C-D: BBOX1-AS1 was overexpressed or knocked down, and its effect on the tubular forming ability of HTR-8/SVneo cells was examined. E-F: in vitro culture of villus explants overexpressing or knocking down BBOX1-AS1, and testing the effect on migration distance.

FIG. 5 shows that BBOX1-AS1 regulates MAPK signaling pathway and promotes GADD45A gene expression in the present invention. A: qRT-PCR examined the effect of BBOX1-AS1 on the expression of the adjacent gene BBOX 1. B-E: performing RNA-seq on HTR-8/SVneo cells overexpressing BBOX1-AS1 and control cells thereof; wherein, B: volcanic chart, C: heat map, D: go assay, E: KEGG analysis. F: western blot detects that overexpression of BBOX1-AS1 activates the P38/JNK MAPK signal channel. G-H: and verifying the partial target gene obtained by the RNA-seq by qRT-PCR. I: western blot was used to examine the effect of over-expression of BBOX1-AS1 on the expression of GADD45A protein. J: qRT-PCR detection of expression K of GADD45A in villus tissue: western blot was used to detect the expression of P38/JNK MAPK signaling pathway and GADD45A in villus tissue from patients with normal pregnancy and RPL. Expression of BBOX1-AS1 and GADD45A mRNA in villus tissue was positively correlated. M: western blot detects the effect of knocking down GADD45A on P38/JNK MAPK signal channel. N: western blot was used to examine the effect of knocking down GADD45A and simultaneously over-expressing BBOX1-AS1 on P38/JNK MAPK signaling pathway.

FIG. 6 is a graph relating to BBOX1-AS1 in the present example to the regulation of trophoblast proliferation, invasion and tube formation by GADD 45A. A: the colony formation experiment examined the proliferation capacity of the transfected pcDNA3.1+ sictrl, BBOX1-AS1+ sictrl, pcDNA3.1+ GADD45A, BBOX1-AS1+ GADD45A in HTR-8/SVneo and JAR cells. B: the Transwell experiment examined the migration and invasion ability of the four transfected plasmids in HTR-8/SVneo and JAR cells. C: the villus explants were transfected with the four plasmids for 24h, then transferred to Matrigel for further culture, and the distance of migration of the villus explant tips was measured under an optical microscope at 24h and 48h, respectively. Each experiment was used to evaluate nine explants from three independent placentas. D: representative images and statistics of tube formation experiments with the 4 plasmid-treated HTR-8/SVneo cells described above.

FIG. 7 shows specific binding of BBOX1-AS1 to hnRNPK in an example of the present invention. A-B: fluorescence In Situ Hybridization (FISH) assay (A) and nuclear and cytoplasmic isolation assay (B) detect the location of BBOX1-AS1 in trophoblasts. C-D: RNA pulldown experiments, combined with Mass Spectrometry (MS), identified BBOX1-AS1 endogenous binding protein E: protein silver staining experiments detect proteins pulled down by RNA pull down. F: RIP experiments confirmed that BBOX1-AS1 was specifically enriched in hnRNPK immunoprecipitates in HTR-8/SVneo cells. Knockdown of hnRNPK in trophoblasts, detection of GADD45A changes in expression at the mRNA level (G-H) and at the protein level (H).

FIG. 8 is a graph showing that BBOX1-AS1 in combination with hnRNPK promotes GADD45A mRNA stability in the present example. A: qRT-PCR level detection BBOX1-AS1 expression levels were changed after knockdown of hnRNPK. B-C: qRT-PCR and WB detected changes in hnRNPK expression after trophoblast HTR-8/SVneo, JAR over-expressed BBOX1-AS 1. D: the dual-luciferase reporter gene experiment detects whether the fluorescence intensity of the over-expressed BBOX1-AS1 and the control group is different. E: HTR-8/SVneo, JAR trophoblasts over-expressing BBOX1-AS1 were induced by Cycloheximide (CHX) for different action times (0, 2, 4, 8h), and WB was used to detect the degradation degree of GADD45A protein. F: at different times (0, 4, 8, 12h) of treatment of trophoblasts overexpressing BBOX1-AS1 and knockdown hnRNPK with actinomycin D (ActD), qRT-PCR measures the degradation rate of GADD45A mRNA.

FIG. 9 is a graph showing that knockdown of hnRNPK may partially reverse the inhibition of BBOX1-AS1 on trophoblast proliferation, migration, and invasion in accordance with an embodiment of the present invention. A-B: knock-down of hnRNPK may partially reverse the inhibitory effect of BBOX1-AS1 on trophoblast migration and invasion. C: knock-down of hnRNPK may partially reverse the inhibitory effect of BBOX1-AS1 on trophoblast proliferation.

FIG. 10 is a graph showing the detection of BBOX1-AS1 expression in serum from RPL patients in accordance with an embodiment of the present invention. A: qRT-PCR detects BBOX1-AS1 expression in the serum of RPL patients (N25) and normal early pregnant women (N25). B: ROC curve analysis chart.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. The experimental procedures, if specific conditions are not indicated in the following detailed description, are generally in accordance with conventional procedures and conditions of molecular biology within the skill of the art, which are fully explained in the literature. See, e.g., Sambrook et al, "molecular cloning: the techniques and conditions described in the laboratory Manual, or according to the manufacturer's recommendations.

The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.

In a typical embodiment of the invention, the application of BBOX1-AS1 AS a marker in the preparation of a recurrent abortion diagnosis or auxiliary diagnosis product is used.

Wherein the BBOX1-AS1 is lncRNA BBOX1 antisense RNA 1(BBOX1-AS 1). Of course, the BBOX1-AS1 of the present invention can also be combined with other ncrnas (miRNA and/or lncRNA) that can be used AS a diagnosis or an auxiliary diagnosis of recurrent abortion, so that clinicians can more easily and accurately diagnose recurrent abortion, thereby reducing the risk of poor pregnancy outcome.

The invention discovers that BBOX1-AS1 is remarkably high expressed in villus tissues of patients with recurrent abortion compared with normal pregnancy through research. Further by detecting BBOX1-AS1 in the blood (serum) of patients with recurrent abortion, whose expression was also elevated, the working characteristic curve (ROC curve) of the subjects was plotted and the area under the curve (AUC) was calculated, it was shown that BBOX1-AS1 in serum had higher diagnostic efficacy, and AUC was 0.8144.

In a further embodiment of the invention, a product is provided, comprising the above-mentioned substance for detecting BBOX1-AS1, for use in the diagnosis or for the auxiliary diagnosis of recurrent abortion.

Among them, substances for detecting BBOX1-AS1 include, but are not limited to, substances for detecting the expression level of BBOX1-AS1 by RT-PCR, gene chip, in situ hybridization and gene sequencing.

The product includes but is not limited to a primer, a probe, a chip, a reagent or a kit for detecting the expression level of the BBOX1-AS1 in a sample to be detected.

The sample to be detected can be a human sample and a non-human sample, and more particularly, the sample to be detected comprises cells, tissues, organs and body fluid of a subject;

wherein the cell may be a feeder cell;

the tissue may be a villous tissue;

the organ may be a placenta or uterus;

the body fluid may be blood, lymph fluid, etc., preferably blood; the blood may be serum or plasma. The blood is used as a sample to be detected for detection, so that the trauma of a subject is reduced, the adaptability and the compliance of the subject are better, meanwhile, the LINC01088 as a recurrent abortion marker has higher specificity and sensitivity in the blood (serum), and can be used together with other recurrent abortion markers to further improve the specificity and the sensitivity of the detection.

Still further, the product may comprise, in addition to the above-mentioned BBOX1-AS1 biomarkers, other biomarkers and combinations thereof currently suitable for detecting recurrent miscarriage.

In yet another embodiment of the present invention, there is provided a system for diagnosing or aiding in the diagnosis of recurrent spontaneous abortion, said system comprising:

i) an analysis unit, the analysis unit comprising: a test substance for determining the expression level of the above-mentioned BBOX1-AS1 in a test sample of a subject, and;

ii) an evaluation unit comprising: determining whether the subject has recurrent pregnancy based on the expression level of BBOX1-AS1 determined in i).

In yet another embodiment of the present invention, the test sample may include cells, tissues, organs and body fluids of the subject;

the cell may be a feeder cell; the tissue may be a villous tissue; the organ may be a placenta or uterus;

the body fluid may be blood, lymph fluid, etc., preferably blood; the blood may be serum or plasma. The blood is used AS a sample to be detected for detection, so that the trauma of a subject is small, the safety is high, the adaptability and the compliance of the subject are better, meanwhile, the BBOX1-AS1 AS the recurrent abortion marker has higher specificity and sensitivity in the blood (serum), and meanwhile, the sensitivity and the specificity of the detection can be further improved by matching with the combined use of other recurrent abortion markers.

It should be noted that the BBOX1-AS1 of the present invention can also be combined with other ncrnas (miRNA and/or lncRNA) that can be used for diagnosis or auxiliary diagnosis of recurrent abortion, and through the optimized combination of different ncrnas, it helps to improve the specificity and sensitivity of detection, thereby accurately diagnosing recurrent abortion for clinicians and reducing the risk of adverse pregnancy outcome.

In yet another embodiment of the present invention, the detection substances described above include, but are not limited to, substances for detecting the expression level of BBOX1-AS1 in RT-PCR, gene chip, in situ hybridization, and gene sequencing.

In another embodiment of the present invention, the specific evaluation flow of the evaluation unit includes:

(ii) an upregulation of BBOX1-AS1 expression level in a test sample of said subject compared to a reference, then said subject is or is candidate for being a patient with recurrent abortion; conversely, the subject is not, or is not candidate for, a patient with recurrent abortion.

Where a "reference" can be a suitable control sample, e.g., a sample from a normal healthy subject that is free of symptoms associated with recurrent abortion and free of abnormal physiological and pathological findings, a reference can also be a sample from the same subject prior to exhibiting symptoms or disease symptoms or prior to diagnosing recurrent abortion. The reference may be a standardised sample.

In yet another embodiment of the present invention, there is provided a method of diagnosing or aiding in the diagnosis of recurrent pregnancy loss, said method comprising:

a) isolating a test sample from a subject;

b) detecting the expression level of the lncRNA in a sample to be tested of the subject, and;

(ii) an upregulation of expression levels in the sample compared to levels in a reference, then the subject is or is candidate for a recurrent abortion patient; conversely, the subject is not, or is not candidate for, a patient with recurrent abortion.

In another embodiment of the present invention, the BBOX1-AS1 is provided AS a target for use in treating and/or screening recurrent abortion.

In another embodiment of the present invention, the medicament for recurrent abortion is a medicament for preventing and/or treating recurrent abortion.

In another embodiment of the present invention, the method for screening drugs for recurrent abortion comprises:

1) treating a system expressing and/or comprising BBOX1-AS1 with a candidate substance; setting a parallel control without candidate substance treatment;

2) detecting the expression level of BBOX1-AS1 in the system after the step 1) is finished; if the expression level of BBOX1-AS1 is significantly reduced in a system treated with a candidate substance AS compared to a parallel control, the candidate substance can be used AS a candidate drug for recurrent abortion.

In yet another embodiment of the present invention, the system may be a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

The cells in the cell system may be feeder cells; the tissue in the tissue system may be villous tissue; the organ in the organ system may be placenta, uterus; the animal in the animal system may be a mammal, including a mouse, rat, guinea pig, rabbit, monkey, orangutan, human, and the like.

In yet another embodiment of the invention, there is provided the use of an agent that inhibits the expression level of BBOX1-AS1 in the manufacture of a product;

the function of the product is any one or more of the following:

(a1) promoting proliferation of trophoblast cells;

(a2) inhibiting apoptosis of trophoblasts;

(a3) promoting migration of trophoblasts;

(a4) promoting the invasion of trophoblasts;

(a5) promoting the formation of nourishing tubules;

(a7) inhibits binding to hnRNPK, and thereby inhibits GADD45A expression;

(a8) preventing and/or treating recurrent abortion.

In yet another embodiment of the invention, there is provided a product, the product comprising active ingredients including any one or more of:

(b1) a substance that inhibits the expression level of BBOX1-AS 1;

(b2) a substance which inhibits the expression level of hnRNPK.

The function of the product is any one or more of the following:

(a1) promoting proliferation of trophoblast cells;

(a2) inhibiting apoptosis of trophoblasts;

(a3) promoting migration of trophoblasts;

(a4) promoting the invasion of trophoblasts;

(a5) promoting the formation of nourishing tubules;

(a7) inhibits binding to hnRNPK, and thereby inhibits GADD45A expression;

(a8) preventing and/or treating recurrent abortion.

In another embodiment of the present invention, the substance inhibiting the expression level of BBOX1-AS1 can be an interfering molecule targeting BBOX1-AS1 and capable of inhibiting the expression of BBOX1-AS1, and specifically can include shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming the shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid; of course, compound inhibitors may also be included.

In another embodiment of the present invention, the product can be a drug or a test reagent, which can be used for basic research.

When the product is a medicament, the medicament may further comprise one or more pharmaceutically or dietetically acceptable auxiliary materials. The adjuvants can be solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, pills and suppositories. Liquid form preparations include solutions, suspensions and emulsions. In addition, it can be made into small water injection for injection, lyophilized powder for injection, infusion solution or infusion solution.

In yet another embodiment of the present invention, there is provided a method for preventing and/or treating recurrent spontaneous abortion, said method comprising: administering to the subject a therapeutically effective dose of the above-described medicament.

The subject refers to an animal that has been the subject of treatment, observation or experiment, and can be a human and non-human mammal, such as a mouse, rat, guinea pig, rabbit, dog, monkey, orangutan, and the like, and most preferably a human. By "therapeutically effective amount" is meant an amount of active compound or pharmaceutical agent, including a compound of the present invention, that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other medical professional, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated.

It will be appreciated that the optimum dosage and interval for administration of the active ingredients of the invention will be determined by the nature and external conditions, such as the form, route and site of administration and the particular mammal being treated, and that such optimum dosage may be determined by conventional techniques. The optimal course of treatment, i.e., the daily dosage of the compound over the nominal time period, can be determined by methods known in the art.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples

Materials and methods

1.1LncRNA expression profiling chip

Total RNA was extracted from villus tissue of 3 control groups (normal early pregnant women) and 3 RPL patients using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Use of

ND-1000 measures RNA concentration and purity, and adopts formaldehyde denatured agar gel electrophoresis to detect RNA quality. The samples were examined using Arraystar Human LncRNA Microarray V5.0(GPL26963) (Arraystar, USA)LncRNA expression profiles were determined. Kangcheng organisms (Shanghai, China) performed microarray scanning and data analysis. The thresholds for gene up-and down-regulation are fold change, respectively>1.5 and p value<0.05。

1.2 sample information

Villous tissue samples were collected from 41 normal pregnant women (20-28 years) and 39 RPL patients (20-30 years) who received an induced abortion in obstetrics and gynecology at the second hospital of Shandong university from 9 months to 2021 months in 2019. These cases of RPL underwent 2 consecutive abortions of unknown cause. Cases of chromosomal abnormalities, uterine anatomical abnormalities, infections, immune factors and malignancies were excluded. The control group consisted of randomly selected women who terminated pregnancy for non-medical reasons between 5 and 10 weeks of pregnancy. These villus specimens were immediately stored at-150 ℃ and then used for RNA and protein extraction. The study was approved by the ethical committee of the second hospital, university of Shandong, and all patients provided written informed consent.

1.3 cell culture

Human trophoblast cell lines (HTR8/SVneo, JAR) were purchased from the cell bank of Chinese academy of sciences. HTR8/SVneo was cultured in Dulbecco's Modified Eagle Medium (DMEM)/F12, JAR cells were supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, Octland, New Zealand), 1% penicillin/streptomycin (Life Technologies/Gibco). All cells were maintained at 37 ℃ and 5% CO₂In a humid environment.

1.4 transfection of cells

Plasmids (pcDNA3.1, pcDNA3.1-BBOX1-AS1, pcDNA3.1-GADD45A) were constructed from GenePharma (Shanghai, China). BBOX1-AS1 Smart Silenecer is a mixture of 3 siRNAs and 3 ASOs designed and synthesized by Ruibo (Guangzhou, China), three different specific siRNAs for GADD45A and hnRNPK and scrambled si-NC purchased from GenePharma (Shanghai, China). Trophoblasts HTR-8/SVneo and JAR were cultured in 6-well plates (2X 10)⁵Individual cells/well). Cells were transfected with plasmids according to the manufacturer's protocol

(Polyplus, France) or use of transfected siRNA

(Ployplus, France). All siRNA sequences are listed in (table 1).

TABLE 1 siRNA sequences

1.5 RNA extraction and qRT-PCR

Total RNA was isolated from villus tissue or cultured cells using Trizol (Invitrogen, Carlsbad, Calif., USA) or RNAfast200(Fastagen, Shanghai, China) according to the manufacturer's protocol, and reverse-transcribed into cDNA using a reverse transcription kit (TOYOBO Life Science, Osaka, Japan), and real-time quantitative PCR was performed using SYBRGreen Master Mix (TOYOBO Life Science, Osaka, Japan) and specific primers. Relative RNA levels of the genes were calculated using the 2- Δ Ct method. Normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and converted to fold change. All experiments were performed in 3 independent replicates.

1.6 cell proliferation assay

Transfected HTR8/SVneo or JAR cells were seeded in a 96-well plate (3000 cells/well) and 10. mu.l/well of CCK8 buffer (Bestbio, Shanghai, China) was added to the medium for 1 hour, and cell viability was assessed according to absorbance at 450nm measured every 24 hours by SpectraMax i3X (Molecular Devices, USA). Each spot was replicated 5 times and all experiments were performed in 3 independent replicates.

1.7 EdU test

The EdU incorporation assay was used as a complementary method to detect cell proliferation. Transfected HTR8/SVneo and JAR cells were seeded in 96-well plates (3000 cells/well) for 24 hours and detected using the Cell-Light EdU Apollo567 Cell Tracking Kit (RiboBio, Guangzhou, China) according to the manufacturer's instructions, and after 30 minutes of final 100. mu.l Hoechst33342 staining, EdU-positive cells were captured and Cell proliferation rate was measured using a fluorescence microscope (Zeiss, Germany). The experiment was independently repeated 3 times.

1.8 cloning experiments

Transfected HTR-8/SVneo and JAR cells (1000cells/well) were seeded in six-well plates and cultured for 7 days. Cells washed with Phosphate Buffered Saline (PBS) were then fixed with 100% methanol for 30min and air dried with 0.1% crystal violet staining. The efficiency of cell colony formation (over 50 cells) was calculated. At least three independent experiments.

1.9 flow assay for apoptosis

Transfected HTR-8/SVneo and JAR cells were cultured in six-well plates for 24 hours. Cells were digested with pancreatin (Solarbio, Beijing, China) without EDTA (0.25%) and harvested, and stained with annexin V-Fluorescein Isothiocyanate (FITC) and PI (Percp-cy5-5) (BestBio, Shanghai, China) to assess apoptosis. The harvested cells were analyzed by flow cytometry (BD LSRFortessa; BD Biosciences) according to the manufacturer's recommendations. At least three independent experiments.

1.10 Transwell experiment

Transwell chambers (8 μm pore size; Costar) were used for cell migration and invasion assays. For migration assays, HTR-8/SVneo and JAR cells (5X 10)⁴Individual cells/well) were seeded in the upper chamber in 200 μ l serum-free medium. The lower chamber was filled with 600 μ l of medium containing 10% FBS, and after 24 hours of incubation, the upper chamber cells were removed with a cotton swab, the lower membrane surface cells were fixed with methanol, and stained with 0.1% crystal violet solution. For cell invasion assay, cells were plated in the upper chamber of a membrane coated with Matrigel (BD Bioscience, San Jose, CA, USA), the number of cells doubled, the incubation time was 48 hours, and the rest of the procedure was the same as the cell migration experiment. Then, the cells were counted under an optical microscope at a magnification of × 100, and five random fields were selected for photographing. The experiment was independently repeated at least 3 times.

1.11 cultivation of the Fuzz explant

Small tissue sections were obtained from villus tissue in the early normal gestation period (6-10 weeks), small tissue sections were transfected with pcDNA3.1-BBOX1-AS1 plasmid or si-BBOX1-AS1 or si-GADD45A plasmid and cultured for 24 hours and tested for transfection efficiency, then small tissue sections were transferred to a 24-well petri dish coated with matrigel (BD Bioscience, San Jose, CA, USA), cultured for 48 hours in DMEM/F12 medium containing 10% FBS and 1% penicillin/streptomycin, and photographs of explants were taken under an optical microscope at 24h and 48h, respectively, of culture and the migration distance of villus tips was measured.

1.12 tube formation experiment

Matrigel (ice-on-ice) was diluted 1:1 with pre-cooled (DMEM)/F12 medium, 50. mu.l of the diluted Matrigel was added to each well of a 96-well plate, the gel was solidified by incubating in an incubator at 37 ℃ for 1 hour, and HTR-8/SVneo cells (2X 10) of different treatment groups were inoculated⁴And/well), culturing in an incubator at 37 ℃ for 12 hours, observing and collecting images under an inverted microscope, measuring the total length of a tube cavity by using ImageJ software, taking 3-5 different visual fields for each well, taking a photograph, and taking an average value.

1.13 immunoWestern blotting

Total cells and tissues were lysed using RIPA lysis buffer (Beyotime, Beyotime, China) supplemented with protease inhibitors (NCM Biotech, Suzhou, China) and phosphatase inhibitors (NCM Biotech, Suzhou, China). Protein quantification was performed using BCA protein assay kit (Beyotime, shanghai, china). Proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.22mm PVDF membrane (Millipore, USA). Blocking with 5% bovine serum albumin was performed for 2 hours at room temperature, after which the membrane was coated with primary antibody overnight at 4 ℃. Then incubated with a corresponding horseradish peroxidase-conjugated secondary antibody for 1h at room temperature, and the blot was detected using a high-sensitivity ECL chemiluminescence detection kit (Vazyme, Nanjing, China) and an enhanced chemiluminescence analysis system (Bio-Rad, shanghai, China).

1.14 fluorescent in situ hybridization assay of RNA

The subcellular localization of BBOX1-AS1 was examined using a fluorescence in situ hybridization kit (GenePharma, Shanghai, China) according to the manufacturer's instructions, and a Cy 3-labeled BBOX1-AS1 probe was designed and synthesized by gimar (GenePharma, Shanghai, China). Finally, images were generated by confocal microscopy (Zeiss, Germany). For the nucleoplasmic isolation experiments, cytoplasmic and nuclear RNA were isolated and purified using a nucleoplasmic isolation kit (ThermoFisher, # AM1921) according to the manufacturer's instructions.

1.15 RNA pulldown experiment

The biotinylated BBOX1-AS1 full-length fragment was transcribed in vitro (Promega, USA). Pierce was used according to the manufacturer's instructions^TMMagnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific) performed RNA Pull-Down assay, followed by incubation of HTR-8/SVneo whole cell lysate with biotinylated RNA and streptavidin Magnetic beads overnight at 4 deg.C, elution of lncRNA interacting proteins, and Mass Spectrometry (MS) or Western blotting of the Protein mixture (New Life of China, Shanghai, China).

1.16 RIP assay (RNA binding protein immunoprecipitation)

The binding of the target RNA to a particular protein was determined using the Magna RIP RNA binding protein immunoprecipitation kit (Milli pore, USA) according to the manufacturer's instructions. First, HTR-8/SVneo and JAR cells were harvested and lysed with RIP lysis buffer, and the lysates were incubated overnight at 4 ℃ with anti-hnRNPK antibody (santa) or normal immunoglobulin G (IgG, negative control) and magnetic bead suspension. The precipitate was eluted, purified and dissolved in RNase-free water, and then co-immunoprecipitated RNA was extracted for qRT-PCR analysis.

1.17 RNA sequencing

Total RNA was extracted from HTR-8/SVneo cells overexpressing BBOX1-AS1 and control cells using the RNeasy Mini Kit (Qiagen, Germany, Cat # 74106). Quality testing (purity and integrity) was performed using an Agilent Bioanalyzer 2100(Agilent Technologies, Santa Clara, CA, US), library construction was performed according to the manufacturer's instructions, and Paired End (PE) sequencing was performed using Illumina NovaSeq 6000(Illumina, usa). And obtaining sequencing data and performing bioinformatics analysis.

1.18 Dual luciferase reporter Gene assay

The GADD45A promoter region was constructed and inserted into pGL3 primary vector downstream of luciferase reporter gene containingThere is a modified coding region for firefly luciferase. The TK vector in the renilla luciferase region was used as a control. Use of

(Polyplus, France) the three plasmids were co-transfected into HTR-8/SVneo and JAR cells. 48 hours after transfection, luciferase signal was detected using the dual luciferase reporter kit (Promega, U.S.A., Cat # E1910) according to the manufacturer's instructions. The test was repeated 3 times.

1.19 data analysis

Statistical analysis was performed by GraphPad Prism 8 software (La Jolla, CA, USA). Each experiment was repeated at least 3 times independently, and the data are presented as mean ± standard deviation. The two groups were compared using Student's t-test or non-parametric Mann-Whitney test, the comparison between the groups was by one-way analysis of variance (ANOVA), and differences were considered significant with p-values less than 0.05.

Second, experimental results

2.1 screening of differential lncRNA in villus tissue of patients with Normal pregnancy and RPL

Firstly, 3 pairs of villus tissues of patients with normal pregnancy and RPL are selected, and high-flux lncRNA expression profiling chips are adopted to screen lncRNA which is differentially expressed. The 5 lncRNAs with the most significant fold difference in the RPL group were selected and verified in villus specimens of normal pregnancy and RPL patients by qRT PCR, and BBOX1-AS1 was found to have significant difference in the RPL patients, so BBOX1-AS1 was selected for subsequent study (see FIG. 1).

2.2 analysis of BBOX1-AS1 expression in RPL patients villus tissue and trophoblast cell lines by qRT-PCR and validation of BBOX1-AS1 overexpression or knockdown efficiency

Expression of BBOX1-AS1 was detected in villus tissue and in 3 trophoblast lines in 40 pairs of normal pregnant and RPL patients. And cell lines overexpressing or knocking down BBOX1-AS1 were constructed in HTR-8/SVneo, JAR two cell lines, respectively (see FIG. 2).

2.3 BBOX1-AS1 inhibits trophoblast proliferation and promotes apoptosis in vitro

Evaluating the influence of over-expression or knocking down BBOX1-AS1 on the biological functions of HTR-8/SVneo and JAR trophoblasts, and finding that over-expression of BBOX1-AS1 can inhibit the proliferation of trophoblasts and promote the apoptosis of the trophoblasts. Knockdown of BBOX1-AS1 resulted in the opposite (see FIG. 3).

2.4 BBOX1-AS1 ability to inhibit trophoblast migration, invasion and tube formation in vitro

The effect of BBOX1-AS1 on HTR-8/SVneo, JAR cell migration, invasion and tubular formation ability was evaluated by Transwell experiments and tube formation experiments. It was found that overexpression of BBOX1-AS1 inhibited trophoblast cell migration, invasion and tube formation ability, whereas knockdown of BBOX1-AS1 had the opposite effect. In addition, transfection of the plasmids pcDNA3.1 or BBOX1-AS1 into villus explants resulted in slower migration of the villus explants transfected with the BBOX1-AS1 plasmid, consistent with the results from the cell lines (see FIG. 4).

2.5 BBOX1-AS1 regulates MAPK signaling pathway and affects GADD45A gene expression

The mechanism of the effect of BBOX1-AS1 on trophoblast function was further explored. When RNA-seq is carried out on HTR-8/SVneo cells over-expressing BBOX1-AS1 and control cells thereof, BBOX1-AS1 is found to regulate a p38/JNK MAPK signaling pathway. Next, the partial genes whose fold difference was significant in RNA-seq were verified, and the results showed that the mRNA expression level of GADD45A was most significantly increased. Finally, whether BBOX1-AS1 modulates MAPK signaling pathway through GADD45A was investigated, and experimental results showed that knockdown GADD45A largely reversed the phosphorylation of P38/JNK MAPK signaling pathway mediated by overexpression of BBOX1-AS 1. These results above indicate that BBOX1-AS1 regulates the MAPK signaling pathway via GADD45A (see fig. 5).

2.6 BBOX1-AS1 regulates trophoblast proliferation, invasion, and tube formation by GADD45A

Knockdown of GADD45A in HTR-8/SVneo, JAR trophoblasts overexpressing BBOX1-AS1, showed that knockdown of GADD45A reversed the inhibitory effect of BBOX1-AS1 on the proliferation, invasion and tubular formation capacity of trophoblasts. Further investigating the effect of knocking down GADD45A on the migratory capacity of villus explants, it was found that knocking down GADD45A reversed the inhibitory effect of BBOX1-AS1 on the migratory capacity of villus explants. In conclusion, BBOX1-AS1 can mediate trophoblast proliferation, invasion and tube-forming ability by modulating GADD45A (see fig. 6).

2.7 BBOX1-AS1 specific binding to hnRNPK modulates GADD45A

To determine the molecular mechanism by which BBOX1-AS1 modulates GADD45A expression, the localization of BBOX1-AS1 in cells was examined and BBOX1-AS1 was found to be present in both cytoplasm and nucleus. RNA pulldown experiments combined with mass spectrometry identified BBOX1-AS1 endogenous binding proteins, and mass spectrometric peptides of hnRNPK were detected on the sense strand of BBOX1-AS1, while RIP experiments further demonstrated that BBOX1-AS1 was specifically enriched in hnRNPK immunoprecipitates in HTR-8/SVneo cells. qRT-PCR and immunoblotting were used to determine whether sihnRNPK caused changes in GADD45A expression, and as a result, GADD45A was found to have significantly reduced expression at the mRNA and protein levels. These results indicate that hnRNPK mediates BBOX1-AS1 in regulating expression of GADD45A (see figure 7).

2.8 BBOX1-AS1 in combination with hnRNPK promotes GADD45A mRNA stability

The potential molecular mechanism by which BBOX1-AS1 promotes GADD45A expression was further explored. The dual luciferase reporter gene experiments showed that BBOX1-AS1 did not affect the transcriptional level of GADD 45A. Cycloheximide (CHX) experiments found that overexpression of BBOX1-AS1 did not affect cycloheximide-treated GADD45A protein stability.

Actinomycin D (ActD) experiments found that the mRNA stability of GADD45A was enhanced after overexpression of BBOX1-AS1, and that the stability of GADD45A mRNA was reduced after treatment of HTR-8/SVneo, JAR trophoblasts knockdown hnRNPK with actinomycin D. These results indicate that BBOX1-AS1 might enhance the stability of GADD45A mRNA by binding to hnRNPK (see FIG. 8).

2.9 Effect of knockdown of hnRNPK on trophoblast proliferation, migration and invasion

The impact on the proliferative, migratory and invasive capacity of trophoblasts after knockdown of hnRNPK was further evaluated. The results show that knock-down of hnRNPK can partially reverse the inhibitory effect of BBOX1-AS1 on trophoblast proliferation, migration and invasion (see FIG. 9).

2.10 increased expression of BBOX1-AS1 in serum of RPL patients

The serum of RPL patients was further examined for the expression of BBOX1-AS 1. The ROC curve analysis showed that BBOX1-AS1 had higher diagnostic efficacy in serum, with AUC 0.8144 (see fig. 10).

In summary, the present examples show that BBOX1-AS1, which is up-regulated in RPL villus tissue, regulates trophoblast proliferation, migration, and invasion in vitro, and is involved in activating the P38/JNK MAPK signaling pathway. In addition, the invention also researches that BBOX1-AS1 promotes the expression of GADD45A by combining protein hnRNPK, finally causes the change of the function of trophoblasts and promotes the generation of RPL. Finally, the expression of BBOX1-AS1 is increased in RPL serum, which indicates the potential possibility of BBOX1-AS1 serving AS an early diagnosis marker of RPL. The BBOX1-AS1 and the molecular mechanism thereof found in the research are helpful for deeply understanding the pathogenesis of the RPL and are also helpful for providing potential markers and therapeutic targets for clinical diagnosis, prevention and treatment of the RPL.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> secondary Hospital of Shandong university

<120> application of BBOX1-AS1 AS diagnostic marker and therapeutic target for recurrent abortion

<130>

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<170> PatentIn version 3.3

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Claims

Use of BBOX1-AS1 AS a marker in the preparation of a diagnostic or diagnostic aid product for recurrent abortion.
2. A product comprising a substance for detecting the above BBOX1-AS1, for use in the diagnosis or aid in the diagnosis of recurrent abortion.
3. The product of claim 2, wherein the means for detecting BBOX1-AS1 comprises means for detecting the expression level of BBOX1-AS1 by RT-PCR, gene chip, in situ hybridization, and gene sequencing;

the product comprises a primer, a probe, a chip, a reagent or a kit for detecting the expression level of the BBOX1-AS1 in a sample to be detected;

the samples to be tested comprise villus tissue samples and blood samples of the subjects.
4. A system for diagnosing or aiding in the diagnosis of recurrent miscarriage, the system comprising:

i) an analysis unit comprising: a test substance selected from the group consisting of BBOX1-AS1 expression levels in a test sample from a subject, and;

ii) an evaluation unit comprising: determining whether the subject has recurrent abortion based on the expression level of BBOX1-AS1 determined in i).
5. The system of claim 4, wherein said ii) evaluation unit specific evaluation flow comprises:

(ii) an upregulation of BBOX1-AS1 expression level in a test sample of said subject compared to a reference, then said subject is or is candidate for being a patient with recurrent abortion; conversely, the subject is not or is not candidate for a recurrent abortion patient.
6. A method of diagnosing or aiding in the diagnosis of recurrent miscarriage, the method comprising:

a) isolating a test sample from a subject;

b) detecting BBOX1-AS1 expression levels in a test sample of the subject, and;

c) comparing the expression level in the sample to the expression level in the reference;

wherein, an upregulation of the level of BBOX1-AS1 expression in the sample AS compared to the level in the reference, is the subject or candidate for a patient with recurrent abortion; conversely, the subject is not, or is not candidate for, a patient with recurrent abortion.
The application of BBOX1-AS1 AS a target point in treatment and/or screening of drugs related to recurrent abortion.
8. Use of a substance that inhibits the expression level of BBOX1-AS1 in the manufacture of a product;

the function of the product is any one or more of the following:

(a1) promoting proliferation of trophoblast cells;

(a2) inhibiting apoptosis of trophoblasts;

(a3) promoting migration of trophoblasts;

(a4) promoting the invasion of trophoblasts;

(a5) promoting the formation of nourishing tubules;

(a6) inhibition of activation (phosphorylation) of the P38/JNK MAPK signaling pathway;

(a7) inhibits binding to hnRNPK, and thereby inhibits GADD45A expression;

(a8) preventing and/or treating recurrent abortion.
9. A product whose active ingredients include any one or more of:

(b1) a substance that inhibits the expression level of BBOX1-AS 1;

(b2) a substance which inhibits the expression level of hnRNPK.
10. The product of claim 9, wherein the product is a pharmaceutical or laboratory reagent.