CN112961913A

CN112961913A - Application of lncRNA in diagnosis and treatment of recurrent abortion

Info

Publication number: CN112961913A
Application number: CN202110162597.XA
Authority: CN
Inventors: 毛海婷; 赵汇; 董娜娜; 李亚丽; 毛慧慧; 冯一民; 李宁; 任含笑
Original assignee: Second Hospital of Shandong University
Current assignee: Second Hospital of Shandong University
Priority date: 2021-02-05
Filing date: 2021-02-05
Publication date: 2021-06-15
Anticipated expiration: 2041-02-05
Also published as: CN112961913B

Abstract

The invention provides application of lncRNA in diagnosis and treatment of recurrent abortion, and belongs to the technical field of crude drug medicine and molecular biology. The invention firstly discovers that the expression quantity of LINC01088 is closely related to recurrent abortion, and the sensitivity and specificity of diagnosis in recurrent abortion are higher, and further research shows that LINC01088 can inhibit the proliferation and invasion of trophoblasts, inhibit the period of the trophoblasts and promote the apoptosis of the trophoblasts, thereby causing abortion. Therefore, the lncRNA such as LINC01088 and the like can be used as a predictive diagnosis marker and a potential treatment target point of recurrent abortion, thereby playing an important role in diagnosis and treatment of recurrent abortion, and having good practical application value.

Description

Application of lncRNA in diagnosis and treatment of recurrent abortion

Technical Field

The invention belongs to the technical field of crude drug medicine and molecular biology, and particularly relates to application of lncRNA in diagnosis and treatment of 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.

According to the provisions of the american society for reproductive medicine, recurrent abortion (RSA) refers to the loss of two and more consecutive embryos. Apart from the clear causes of genetic factors, abnormal uterine anatomy, infection, etc., there is recurrent abortion caused by unknown reasons. Due to the lack of effective clinical treatment measures, RSA has become one of the important causes of infertility at present. Therefore, exploring the etiology and pathogenesis of RSA is the key to preventing and treating RSA.

Long non-coding RNA (lncRNA) is non-coding RNA with a transcript of more than 200nt, and lncRNA does not participate in the process of encoding a protein, but plays an important role in gene transcription, post-transcriptional regulation, and the like. Recent studies have found that lncRNA plays an important role in regulating physiological processes of the body, such as cell proliferation, cell cycle, chromosome remodeling, histone modification, and the like, and in addition, abnormal expression of lncRNA is closely associated with various tumors, cardiovascular diseases, and the like. However, the research on the role of lncRNA in recurrent abortion is still relatively rare, so it is necessary to search for more effective lncRNA as a marker for clinical diagnosis and therapeutic detection of RSA, and provide a basis for diagnosis, treatment and development of related drugs.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of lncRNA in diagnosis and treatment of recurrent abortion. The invention discovers for the first time that the expression level of LINC01088 is closely related to recurrent abortion, and the diagnosis sensitivity and specificity of LINC01088 in recurrent abortion are very high, and further research proves that LINC01088 can inhibit the proliferation and invasion of trophoblasts, inhibit the period of the trophoblasts and promote the apoptosis of the trophoblasts, thereby causing abortion. Therefore, LINC01088 and the like lncRNA have been used as a predictive diagnostic marker and a potential therapeutic target for recurrent abortion, and thus the present invention has been completed.

Specifically, the invention relates to the following technical scheme:

in a first aspect of the invention, the application of lncRNA as a marker in the preparation of a recurrent abortion diagnosis or auxiliary diagnosis product is provided.

Wherein the lncRNA comprises LINC 01088.

According to the invention, the research shows that LINC01088 is highly expressed in villus tissues of patients with recurrent abortion compared with normal pregnancy patients. Further plotting of the receiver operating characteristic curve (ROC curve) and calculation of the area under the curve (AUC) indicate that LINC01088 has a diagnostic effect and that LINC01088 has a high sensitivity and specificity for diagnosis in recurrent abortion.

In a second aspect of the invention, a product is provided, said product comprising the above-mentioned agent for detecting lncRNA, said product being used for the diagnosis or the auxiliary diagnosis of recurrent spontaneous abortion.

The materials for detecting lncRNA include, but are not limited to, materials for detecting the expression level of lncRNA by RT-PCR, real-time quantitative PCR, in-situ hybridization, gene chip and gene sequencing.

The product comprises but is not limited to a primer, a probe, a chip, a nucleic acid membrane strip, a preparation or a kit for detecting the expression level of the lncRNA in a sample to be detected.

The sample to be tested can be a human sample, and more specifically, the sample to be tested comprises villus tissue of a tested person.

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 detection substance for determining the expression level of a test sample from the subject selected from the group consisting of lncRNA described above, and;

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

the specific evaluation process of the evaluation unit in the step ii) comprises the following steps:

(ii) if the expression level of lncRNA in the test sample of the subject is up-regulated compared to a reference, then the subject is or is candidate for a recurrent abortion patient; otherwise, the subject is not, or is not candidate for, a patient with recurrent abortion.

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

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

wherein an upregulation of the expression level in the sample compared to the level in the reference is indicative of or is candidate for a patient with recurrent miscarriage; otherwise, the subject is not, or is not candidate for, a patient with recurrent abortion.

In a fifth aspect of the invention, the lncRNA is provided as a target for application in treatment and/or screening of recurrent abortion drugs.

In a sixth aspect of the invention, there is provided the use of an agent that inhibits the expression level of said lncRNA 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) promoting the progress of the cell cycle of the trophoblast;

(a3) inhibiting apoptosis of trophoblasts;

(a4) promoting migration and/or invasion of trophoblasts;

(a5) inhibiting Arginase-1 expression;

(a6) facilitating the eNOS/NO system;

(a7) inhibition of the p38, JNK MAPK signaling pathway;

(a8) preventing and/or treating recurrent abortion.

In a seventh aspect of the invention, there is provided a product, wherein the active ingredients comprise a substance inhibiting the expression level of said incrna; the function of the product is any one or more of the following:

(a1) promoting proliferation of trophoblast cells;

(a2) promoting the progress of the cell cycle of the trophoblast;

(a3) inhibiting apoptosis of trophoblasts;

(a4) promoting migration and/or invasion of trophoblasts;

(a5) inhibiting Arginase-1 expression;

(a6) facilitating the eNOS/NO system;

(a7) inhibition of the p38, JNK MAPK signaling pathway;

(a8) preventing and/or treating recurrent abortion.

In an eighth aspect of the invention, there is provided the use of an agent that promotes the expression level of said lncRNA in the manufacture of a product.

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

(b1) inhibiting proliferation of trophoblasts;

(b2) inhibiting the progression of the trophoblast cell cycle;

(b3) promoting apoptosis of trophoblasts;

(b4) inhibition of trophoblast and/or invasion;

(b5) promoting Arginase-1 expression;

(b6) suppression of the eNOS/NO system;

(b7) activating p38, JNK MAPK signaling pathway;

(b8) and constructing a recurrent abortion animal model.

In a ninth aspect of the invention, there is provided a product, the active ingredients of which comprise a substance that promotes the expression level of said incrna;

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

(b1) inhibiting proliferation of trophoblasts;

(b2) inhibiting the progression of the trophoblast cell cycle;

(b3) promoting apoptosis of trophoblasts;

(b4) inhibition of trophoblast and/or invasion;

(b5) promoting Arginase-1 expression;

(b6) suppression of the eNOS/NO system;

(b7) activating p38, JNK MAPK signaling pathway;

(b8) and constructing a recurrent abortion animal model.

The product of the sixth to ninth aspects may be a medicament.

The beneficial technical effects of one or more technical schemes are as follows:

the technical scheme firstly discovers that the expression quantity of LINC01088 is closely related to recurrent abortion, wherein the diagnosis sensitivity and specificity of LINC01088 in recurrent abortion are high, and further research shows that LINC01088 can inhibit the proliferation and invasion of trophoblasts, inhibit the period of the trophoblasts and promote the apoptosis of the trophoblasts, thereby causing abortion.

In conclusion, the lncRNA such as LINC01088 and the like is used as a predictive diagnosis marker and a potential treatment target point of recurrent abortion, thereby playing an important role in diagnosis and treatment of recurrent abortion, and having 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 shows the expression of IncRNA in villus tissue of patients with recurrent abortion (RSA) and normal pregnancy (control) according to the example of the present invention. P < 0.05; p < 0.001.

FIG. 2 is a graph showing the diagnostic effect of IncRNA in recurrent pregnancy loss in an example of the present invention.

FIG. 3 is a graph relating to the inhibition of trophoblast proliferation by LINC01088 in embodiments of the present invention. Wherein A is CCK8 experiment, which proves that LINC01088 can inhibit the proliferation of trophoblast after over-expression; b is CCK8 experiment, which proves that LINC01088 knockout promotes the proliferation of trophoblasts; edu C experiment proves that LINC01088 can inhibit the proliferation of trophoblast after over-expression; d is Edu experiment, which proves that the LINC01088 knockout promotes the proliferation of trophoblasts; e is a clone formation experiment which proves that LINC01088 can inhibit the proliferation of trophoblasts after overexpression; f is a clone formation experiment which proves that the LINC01088 knockout promotes the proliferation of trophoblasts.

FIG. 4 is a graph relating to LINC01088 inhibiting the trophoblast cycle and promoting trophoblast apoptosis in an embodiment of the present invention. Wherein, A is LINC01088 capable of inhibiting the progression of cell cycle G1 to S phase after overexpression; b, the nocodazole is used for carrying out cycle synchronization treatment on the trophoblasts, and G1 stage cells of the trophoblasts over-expressed by LINC01088 are obviously increased; c is a cycle-related molecule CDK2 in the trophoblast cell inhibited after LINC01088 overexpression, and CDK4 is expressed at the mRNA level; d is the expression of cycle-related molecules CDK2, CDK4 and cyclin E1 in trophoblasts under protein level after LINC01088 overexpression; e is that LINC01088 overexpression promotes apoptosis of trophoblasts; f is the expression of LINC01088, inhibits Bcl2 and promotes the expression of Bax at the mRNA level; g is the Bcl2 inhibited after LINC01088 overexpression and promotes the expression of Bax and clear caspase-3 at protein level.

FIG. 5 is a graph relating to the inhibition of trophoblast invasion by LINC01088 in an embodiment of the present invention, wherein A is the inhibition of trophoblast invasion and migration after LINC01088 overexpression; and B is the function of promoting invasion and migration of trophoblasts after the LINC01088 knockout is performed.

FIG. 6 is a graph relating to the indirect suppression of the eNOS/NO system by LINC01088 binding to and promoting expression of Arginase-1 in an embodiment of the present invention. Wherein, A is the nuclear and cytoplasmic separation experiment detection that LINC01088 is mainly expressed in the nucleus of a trophoblast cell; b and C are RNA-pull down combined with western-blot experiments to prove that LINC01088 can be combined with Arginase-1; d is that the expression of LINC01088 has no influence on Arginase-1 at mRNA level; e is the expression of Arginase-1 at the protein level promoted after LINC01088 is over-expressed; f is the expression of the level of NOS protein reduced after the overexpression of LINC 01088; g is the reduction of NO content in the grinding fluid of the villus tissue of the patient with recurrent abortion; h is the reduction of NO content in the supernatant of trophoblasts over expressing LINC 01088.

FIG. 7 is a graph relating to the activation of JNK, P38/MAPK signaling pathways by LINC01088 through inhibition of NO production in an embodiment of the present invention, wherein A is the activation of only P38 and JNK signaling pathways in the MAPK family after LINC01088 overexpression in trophoblasts; b is the activation of p38 and JNK signal pathways after LINC01088 overexpression in villus tissues; c is the activation of p38 and JNK signaling pathways in the villus tissue of patients with recurrent abortion.

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.

There are several sequences of lncrnas known in the art, and it is understood that the database accession numbers for each of the lncrnas shown below are human-derived mrnas. However, these database entries also provide the database accession numbers of the respective lncrnas from different sources such as: for example any mammalian, reptile, or avian origin lncrnas, such as those selected from laboratory animals (e.g., mice or rats), domesticated animals (including, for example, guinea pigs, rabbits, horses, donkeys, cattle, sheep, goats, pigs, chickens, camels, cats, dogs, turtles, terrapin, snakes, or lizards), or primates (including chimpanzees, bonobos, and gorillas).

The term "expression level" refers to the amount of a gene product present in vivo or in a sample at a particular time point. The expression level can be measured/quantified/detected, for example, by protein or mRNA expressed by the gene. The expression level can be quantified, for example, as follows: normalizing the amount of the gene product of interest present in the sample with the total amount (total protein or mRNA) of the same type of gene product in the same sample or reference sample (e.g., a sample obtained from the same individual at the same time or a fraction of the same size (weight, volume) of the same sample), or determining the amount of the gene product of interest/defined sample size (weight, volume, etc.). The expression level can be measured or detected by any method known in the art, such as a method for direct detection and quantification of a gene product of interest (e.g., mass spectrometry), or a method for indirect detection and measurement of a gene product of interest that generally works by binding the gene product of interest to one or more different molecules or detection devices (e.g., primers, probes, antibodies, protein scaffolds) specific for the gene product of interest. Also known to the skilled person is the determination of the level of gene copy, which also includes the determination of the absence or presence of one or more fragments (e.g. by nucleic acid probes or primers, such as quantitative PCR, Multiplex ligation-dependent probe amplification (MLPA) PCR).

The terms "indicator" and "marker" are used interchangeably herein and refer to a sign or signal of a condition or to monitor a condition. Such "disorder" refers to a biological state of a cell, tissue or organ, or to a health and/or disease state of an individual. The indicator may be the presence or absence of molecules including, but not limited to, peptides, proteins, and nucleic acids, or may be a change in the level or pattern of expression of such molecules in a cell, or tissue, organ, or individual. The indicator can be a sign of the occurrence, development or presence of a disease in an individual or of further progression of such a disease. The indicator may also be a sign of the risk of developing a disease in the individual.

The term "up-regulation", "increase" or "increase" of the level of an indicator means that the level of such indicator is reduced in a sample compared to a reference.

The term "down-regulation", "reduction" or "decrease" of the level of an indicator refers to a decrease in the level of such indicator in a sample compared to a reference.

In principle, a reference amount can be calculated for a group or cohort of subjects specified by the invention by applying standard statistical methods based on the mean or median of a given lncRNA. In particular, the accuracy of tests such as methods that aim or do not aim at determining events is best described by their recipient-operating characteristics (ROC) (see, inter alia, Zweig1993, Clin. chem.39: 561-. ROC plots are plots of all sensitivity versus specificity pairs obtained by varying the decision threshold over the entire range of data observed. The clinical performance of a diagnostic method depends on its accuracy, i.e., its ability to correctly assign a subject to a certain prognosis or diagnosis. The ROC plot represents the overlap between the two distributions by plotting sensitivity against 1-specificity over the full threshold range suitable for discrimination. On the y-axis is the sensitivity or true positive score, which is defined as the ratio of the number of true positive test results to the sum of the number of true positive and false negative test results. This is also referred to as positive in the presence of the disease or disorder. Which are calculated individually from the affected subgroups. On the x-axis is the false positive score or 1-specificity, which is defined as the ratio of the number of false positive results to the sum of the number of true negatives and the number of false positives. It is an index of specificity and is calculated entirely from unaffected subgroups. Since true and false positive scores are calculated entirely separately, the ROC plot is independent of the prevalence of events in the cohort by using test results from two different subgroups. Each point on the ROC graph represents a sensitivity/-specificity pair corresponding to a particular decision threshold. Tests with perfect discrimination (no overlap in the two result distributions) have ROC plots passing through the upper left corner with a true positive score of 1.0 or 100% (perfect sensitivity) and a false positive score of 0 (perfect specificity). The theoretical plot for the test without discrimination (same distribution of the two sets of results) is a 45 ° diagonal from the bottom left to the top right. Most of the figures fall between these two extremes. If the ROC plot falls well below the 45 ° diagonal, this is easily corrected by reversing the "positive" criterion from "greater than" to "less than" and vice versa. Qualitatively, the closer the graph is to the upper left corner, the higher the overall accuracy of the test. Depending on the desired confidence interval, a threshold can be derived from the ROC curve, allowing a given event to be diagnosed or predicted with the appropriate balance of sensitivity and specificity, respectively. Thus, a reference for the method of the invention may preferably be generated by establishing a ROC for the group and deriving a threshold amount therefrom as described above. Depending on the desired sensitivity and specificity of the diagnostic method, ROC plots allow the derivation of appropriate thresholds. Preferably, the reference amount lies within a range of values representing a sensitivity of at least 75% and a specificity of at least 45%, or a sensitivity of at least 80% and a specificity of at least 40%, or a sensitivity of at least 85% and a specificity of at least 33%, or a sensitivity of at least 90% and a specificity of at least 25%.

The term "kit" as used herein refers to a collection of the above-mentioned components, preferably provided separately or in a single container. The container also preferably contains instructions for carrying out the method of the invention. Examples of these components of the kit and methods of use thereof have been given in the present specification. Preferably, the kit comprises the above components in a ready-to-use formulation. Preferably, the kit may additionally comprise instructions, such as a user's manual for adjusting the components (e.g., the concentration of the detection agent) and for interpreting the results of any assay with respect to the diagnosis provided by the methods of the invention. In particular, such a manual may comprise information for assigning the amount of a determined gene product to a diagnostic type. Details are found elsewhere in this specification. Furthermore, such user manual may provide instructions on the correct use of the kit components for determining the amount of the respective biomarker. The user manual may be provided in paper or electronic form (e.g., stored on a CD or CD ROM). The invention also relates to the use of said kit in any method according to the invention.

The term "system" as used herein relates to a system of devices comprising at least the above-mentioned devices operatively interconnected to allow a diagnosis to be made. Preferred means for determining the methylation state or amount of a gene product and means for making a comparison are disclosed above in connection with the methods of the invention. How the devices are operatively contacted will depend on the type of device included in the apparatus. For example, in the case of the application of a device for the automated determination of the methylation state or amount of a gene product, the data obtained by the automated operating device can be processed by, for example, a computer program to establish a diagnosis. Preferably, in this case, the apparatus is comprised in a single device. Thus, the device may comprise an analysis unit for determining the methylation state or amount of a gene product in a sample and an evaluation unit for processing the resulting data for diagnosis. Preferred detection devices are disclosed above in connection with embodiments relating to the methods of the present invention. In this case, the devices are effectively connected so that the user of the system combines the results of the determination of the quantities and their diagnostic values together owing to the instructions and explanations given in the manual. In such embodiments the device may be presented as a separate apparatus and preferably packaged together as a kit. Those skilled in the art will know how to contact the device without further inventive skill. Preferred devices are those that can be applied without the specific knowledge of a skilled clinician, such as test strips or electronic devices that only require loading of a sample. The results can be output as parametric diagnostic raw data, preferably given as absolute or relative quantities. It will be appreciated that these data will need to be interpreted by a clinician. However, expert system devices are also contemplated where the output contains processed diagnostic raw data, the interpretation of which does not require a specialized clinician. Further preferred devices comprise an analysis unit/device (e.g. biosensor, array, solid support coupled to a ligand specifically recognizing a polypeptide, plasmon surface resonance device, NMR spectrometer, mass spectrometer, etc.) or an evaluation unit/device as mentioned above according to the method of the invention.

In a specific embodiment of the invention, the method is used for the application of lncRNA as a marker in the preparation of a recurrent abortion diagnosis or auxiliary diagnosis product.

Wherein the lncRNA comprises LINC 01088; the kit can also comprise other ncRNAs (miRNA and/or lncRNA) which can be used for diagnosis or auxiliary diagnosis of recurrent abortion, and the specificity and sensitivity of detection can be improved by the optimized combination of different ncRNAs, so that a clinician can accurately diagnose recurrent abortion and timely adopt a prevention and treatment scheme to provide support, and the risk of poor pregnancy outcome is reduced.

In a further embodiment of the invention, a product is provided, said product comprising the above-mentioned agent for detecting lncRNA, said product being used for the diagnosis or the auxiliary diagnosis of recurrent spontaneous abortion.

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, urine, gastric juice or saliva, preferably blood; the blood may be serum or plasma. The blood is used as a sample to be detected for detection, so that the traumatic property of a subject is small, the compliance of the subject is better, the LINC01088 as a recurrent abortion marker has higher specificity and sensitivity in the blood, and the specificity and sensitivity of the detection are further improved by being matched with other recurrent abortion markers for combined use.

Still further, the product includes, in addition to the lncRNA biomarkers described above, other biomarkers currently suitable for detecting recurrent miscarriage and combinations thereof, including, but not limited to, genetic factor-related biomarkers (e.g., chromosomal translocations, chimeras, deletions, or inversions), uterine anatomy-related biomarkers (e.g., uterine congenital malformations, cervical insufficiency, uterine lumenal adhesions, uterine fibroids, uterine adenomyosis), endocrine factor-related biomarkers (e.g., polycystic ovary syndrome (PCOS), prolactin, uncontrolled diabetes, thyroid disease), infection factor-related biomarkers (e.g., any severe infection that can cause bacteremia or viremia), immune function-related biomarkers (e.g., anti-phospholipid antibodies, anti-nuclear antibodies, anti-DNA antibodies, antibodies against DNA, and combinations thereof, Antisperm antibodies, antithyroid antibodies, increased numbers and activity of Natural Killer (NK) cells, macrophage dysfunction, dendritic cell dysfunction, complement system dysfunction, blocking antibody deficiency, T, B lymphocyte dysfunction, T helper (Th)1/Th2 cytokine dysfunction, etc.), pre-thrombotic state-related biomarkers (e.g., factor V and factor II (prothrombin) gene mutations, protein S deficiency), and other systemic diseases of a subject, etc. By combining with other biomarkers, the interference of other physiological and pathological states is further eliminated, and the sensitivity and specificity of detection are further improved.

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:

ii) an evaluation unit comprising: (ii) determining whether said subject has recurrent miscarriage based on the lncRNA expression level determined in i).

In still another embodiment of the present invention, the test sample includes cells, tissues, organs and body fluids of the 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, urine, gastric juice or saliva, preferably blood; the blood may be serum or plasma. The blood is used as a sample to be detected for detection, so that the traumatic property of a subject is small, the compliance of the subject is better, meanwhile, the LINC01088 as a recurrent abortion marker has higher specificity and sensitivity in the blood, and the specificity and sensitivity of the detection are further improved by matching with other recurrent abortion markers for combined use.

In yet another embodiment of the invention, said lncRNA comprises LINC 01088; the kit can also comprise other ncRNAs (miRNA and/or lncRNA) which can be used for diagnosis or auxiliary diagnosis of recurrent abortion, and the specificity and sensitivity of detection can be improved by the optimized combination of different ncRNAs, so that a clinician can accurately diagnose recurrent abortion and timely adopt a prevention and treatment scheme to provide support, and the risk of poor pregnancy outcome is reduced.

In still another embodiment of the present invention, the above-mentioned detection substances include, but are not limited to, substances for detecting the expression level of lncRNA by RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing.

In yet another embodiment of the invention, the assay unit further comprises means for detecting other biomarkers currently suitable for detecting recurrent miscarriage, including, but not limited to, genetic factor-related biomarkers (e.g., chromosomal translocations, chimeras, deletions, or inversions), uterine anatomy-related biomarkers (e.g., uterine congenital malformations, cervical insufficiency, uterine cavity adhesions, uterine fibroids, uterine adenomyosis), endocrine factor-related biomarkers (e.g., polycystic ovary syndrome (PCOS), prolactin, uncontrolled diabetes, thyroid disease), infection factor-related biomarkers (e.g., any severe infection that can cause bacteremia or viremia), immune function-related biomarkers (e.g., anti-phospholipid antibodies, anti-nuclear antibodies, anti-DNA antibodies, anti-sperm antibodies, increased amounts and activities of antithyroid antibodies, Natural Killer (NK) cells, abnormal macrophage function, abnormal dendritic cell function, abnormal complement system, blocked antibody deficiency, abnormal T, B lymphocytes, abnormal T helper lymphocyte (Th)1/Th2 cytokines, etc.), biomarkers associated with prothrombotic state (e.g., mutations in factor V and factor II (prothrombin) genes, protein S deficiency), and other systemic diseases of the subject, etc. By combining with other biomarkers, the interference of other physiological and pathological states is further eliminated, and the sensitivity and specificity of detection are further improved.

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

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 can be a labeled sample, e.g., a sample comprising material or data from samples of several healthy subjects who have no symptoms of recurrent miscarriage nor associated physiological and pathological findings.

The system for diagnosing or assisting in diagnosing recurrent spontaneous abortion of the present invention may be a virtual device as long as the functions of the analyzing unit and the evaluating unit are realized. The analysis unit can comprise various detection reagent materials and/or detection instrument equipment and the like; the evaluation unit may be any computing instrument, module or virtual device capable of analyzing and processing the detection result of the analysis unit to obtain the risk evaluation status of the recurrent abortion, for example, various possible detection results and corresponding risk conditions may be formulated into corresponding data charts in advance, and the detection result of the detection unit is compared with the data charts to obtain the risk evaluation result of the recurrent abortion.

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

a) isolating a test sample from a subject;

(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; otherwise, the subject is not, or is not candidate for, a patient with recurrent abortion.

In another embodiment of the invention, the lncRNA is used as a target for treating recurrent abortion and/or screening recurrent abortion medicines.

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 containing said lncRNA with a candidate substance; setting a parallel control without candidate substance treatment;

2) detecting the expression level of the lncRNA in the system after the step 1) is finished; if the expression level of the DNA 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 for a recurrent abortion drug.

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.

In yet another embodiment of the present invention, the cells in the cell system may be feeder cells;

in yet another embodiment of the present invention, the tissue in the tissue system may be a villus tissue;

in yet another embodiment of the present invention, the organ in the organ system may be placenta, uterus;

in another embodiment of the present invention, the animal in the animal system may be a mammal, such as rat, mouse, guinea pig, rabbit, monkey, human, etc.

In yet another embodiment of the invention, there is provided the use of an agent that inhibits the expression level of said lncRNA 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) promoting the progress of the cell cycle of the trophoblast;

(a3) inhibiting apoptosis of trophoblasts;

(a4) promoting migration and/or invasion of trophoblasts;

(a5) inhibiting Arginase-1 expression;

(a6) facilitating the eNOS/NO system;

(a7) inhibition of the p38, JNK MAPK signaling pathway;

(a8) preventing and/or treating recurrent abortion.

In yet another embodiment of the invention, there is provided a product, the active ingredients of which comprise a substance that inhibits the expression level of said incrna; the function of the product is any one or more of the following:

(a1) promoting proliferation of trophoblast cells;

(a2) promoting the progress of the cell cycle of the trophoblast;

(a3) inhibiting apoptosis of trophoblasts;

(a4) promoting migration and/or invasion of trophoblasts;

(a5) inhibiting Arginase-1 expression;

(a6) facilitating the eNOS/NO system;

(a7) inhibition of the p38, JNK MAPK signaling pathway;

(a8) preventing and/or treating recurrent abortion.

In another embodiment of the present invention, the substance for inhibiting the expression level of lncRNA may be an interfering molecule which uses lncRNA as a target sequence and can inhibit the expression of lncRNA, and specifically may include shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid; compound inhibitors may also be included.

In another embodiment of the invention, there is provided the use of an agent that promotes the expression level of said lncRNA in the manufacture of a product.

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

(b1) inhibiting proliferation of trophoblasts;

(b2) inhibiting the progression of the trophoblast cell cycle;

(b3) promoting apoptosis of trophoblasts;

(b4) inhibition of trophoblast and/or invasion;

(b5) promoting Arginase-1 expression;

(b6) suppression of the eNOS/NO system;

(b7) activating p38, JNK MAPK signaling pathway;

(b8) and constructing a recurrent abortion animal model.

In yet another embodiment of the invention, there is provided a product, the active ingredient of which comprises a substance that promotes the expression level of said incrna;

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

(b1) inhibiting proliferation of trophoblasts;

(b2) inhibiting the progression of the trophoblast cell cycle;

(b3) promoting apoptosis of trophoblasts;

(b4) inhibition of trophoblast and/or invasion;

(b5) promoting Arginase-1 expression;

(b6) suppression of the eNOS/NO system;

(b7) activating p38, JNK MAPK signaling pathway;

(b8) and constructing a recurrent abortion animal model.

The substance for promoting the expression level of the lncRNA comprises a substance for up-regulating the expression of the lncRNA and/or promoting the activity of the lncRNA by adopting a gene-specific Mimics technology; a promoter or lentivirus that upregulates the expression of lncRNA; compound accelerators are also included.

In yet another embodiment of the present invention, the product may be a medicament.

The medicament may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a buffer, emulsifier, suspending agent, stabilizer, preservative, excipient, filler, coagulant and blender, surfactant, dispersing agent or antifoaming agent.

The medicament may also include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be a virus, a microcapsule, a liposome, a nanoparticle, or a polymer, and any combination thereof. The delivery vehicle for the pharmaceutically acceptable carrier can be a liposome, biocompatible polymer (including natural and synthetic polymers), lipoprotein, polypeptide, polysaccharide, lipopolysaccharide, artificial viral envelope, inorganic (including metal) particle, and bacterial or viral (e.g., baculovirus, adenovirus and retrovirus), phage, cosmid, or plasmid vector.

The medicament can also be combined with other medicaments for preventing and/or treating recurrent abortion, and other prophylactic and/or therapeutic compounds can be administered simultaneously with the main active ingredient, even in the same composition.

The medicament may also be administered separately to other prophylactic and/or therapeutic compounds, in a separate composition or in a different dosage form than the main active ingredient. Some of the doses of the main ingredient may be administered simultaneously with other therapeutic compounds, while other doses may be administered separately. The dosage of the agents of the invention may be adjusted during the course of treatment depending on the severity of the symptoms, the frequency of recurrence and the physiological response of the treatment regimen.

The medicament of the present invention can be administered into the body by a known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Optionally via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

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

First, experiment method

1. Culture of feeder cell lines

The feeder cell lines HTR-8/SVneo (HTR8), JAR and HEK293T cells were cultured separatelyCulturing in DMEM/F12 medium, RPMI-1640 medium and DMEM medium containing 10% FBS at 37 deg.C and 5% CO₂Culturing in an environmental cell culture box. Depending on the growth rate of the different cells, the cells are passaged approximately every 1-2 days. Doubling time was recorded for each cell line and a doubling curve was plotted.

2. Collection of clinical specimens

25 villus tissue specimens (6-10 weeks) from eastern university secondary hospital income recurrent abortion patients in 2017 to 2019, and 23 villus tissue specimens (6-10 weeks) of normal pregnant patients. The inclusion standard of patients with recurrent abortion is 2 or more continuous abortions, and the clear causes of gene defects, infection, gynecological inflammation, tumor and the like are excluded. Total RNA, protein and tissue grinding fluid of a fresh tissue specimen are extracted for later experiments. The study was approved by the ethical committee of the second hospital, university of Shandong. Each subject gave informed consent.

3. Transfection of feeder cells

And (3) carrying out cell transfection on the trophoblasts with good growth states, respectively transfecting over-expression plasmids of LINC01088, and collecting total RNA or protein of the extracted cells after 24 hours of transfection.

4. Establishment of LINC01088 knockout cell line

Sgrnas (single guide RNAs) against LINC01088 were designed and synthesized and constructed into the lentiviral vector pLenti CRISPR V2(Hu6-sgRNAEF1a-CAS9-puro), respectively. The 293T cells are inoculated in a 10cm cell culture dish, 300 mu l of buffer solution is mixed with 10 mu g of sgRNA, 10 mu g of psPAX2 and 4 mu g of pMD2.G uniformly, then mixed with 30 mu l of jetPRIME transfection reagent uniformly, added into the cells, after 48 hours, cell culture supernatant rich in virus is collected, immediately used for 8000 revolutions by a4 ℃ centrifuge, centrifuged for 20 minutes, taken out, subpackaged and frozen at-80 ℃. After 12-16 hours of feeder cell inoculation, 7mL of fresh medium and 50. mu.g of Polybrene, as well as 3mL of virus solution, were added and 24 hours after infection, the fresh medium was replaced. 48 hours after infection, 8. mu.g/mL puromycin was added and the medium was replaced every other day (with 8. mu.g/mL puromycin). After 7 days of puromycin screening, a stable expression cell line with LINC01088 knockout was obtained.

5. Fluorescent quantitative PCR

Extracting total RNA in transfected trophoblast or fresh villus tissue specimen, performing reverse transcription on the extracted total RNA by using a kit for removing genome by Toyo Boseki, and performing fluorescence quantitative PCR amplification on cDNA obtained by reverse transcription. In combination with 2^-△△CtAfter analysis, statistical analysis was performed on the data.

6. CCK8 detection

After 24 hours of transfection of the feeder cells, the feeder cells were seeded in a 96-well plate in a number of 2000 cells per well, and 10. mu.L of CCK8 working solution was added to the culture system. Placing at 37 ℃ and 5% CO₂Incubation in the cell incubator was continued for 1 h. And detecting the absorbance value of 450nm by using a microplate reader.

7. EdU detection

24 hours after transfection of feeder cells, feeder cells were seeded in 96-well plates in a number of 2000 cells per well, 10. mu.M EdU reagent was added to the culture system, and cell staining was performed according to the kit procedure after 4 hours. After the completion of the staining, the number of stained cells was observed by a fluorescence microscope.

8. Clone formation assay

After 24 hours of trophoblast transfection, trophoblasts were seeded in 6-well plates in 1000 cells per well, the solution was changed every 3 days, after 7 days of culture, the cells were fixed with methanol and stained with 0.1% crystal violet stain, photographed for subsequent analysis.

9. Cell migration and invasion detection

The culture medium and the machine-made glue are mixed according to the ratio of 8: 1, 50. mu.l per well was added to a Transwell chamber and incubated at 37 ℃ for at least 2 hours to allow the mechanical gel to dry until use. After 24 hours of transfection of trophoblasts, 100. mu.l of the cells were transfected with 5X 10⁴Each feeder cell was seeded in a Transwell chamber, and 600. mu.l of complete medium was added to the lower chamber, and the culture was continued for 24 hours. After removal of the cell, the cells were fixed in methanol and stained with 0.1% crystal violet stain and photographed after observation by an electron microscope.

10. RNA pull-down assay

lncRNA LINC01088 was transcribed in vitro and lncRNA biotinylated. The labeled lncRNA LINC01088 was subjected to RNA-protein pull-down experiments. Cracking trophoblast HTR8 to obtain cell protein, incubating the freshly obtained HTR8 protein, lncRNA LINC01088 labeled by biotin and magnetic beads together, collecting the protein combined with the magnetic beads after incubation is finished, and performing protein mass spectrometry and western-blot detection verification on the protein.

11. Statistical analysis

Statistical analysis GraphPad Prism 5 software was used and data are expressed as mean ± standard deviation. The differences between groups were statistically analyzed by Student's t test, One-way ANOVA and Two-way ANOVA, and P <0.05 is statistically significant.

The sgRNA and primers used for qRT-PCR were used in the examples as shown in tables 1 and 2 below.

Table 1 sgRNA sequences

hLINC01088 KO-sgRNA1	5'-CACCGCTCTGCTGCTTCAAGCCTT-3'(SEQ ID NO.1)
		hLINC01088 KO-sgRNA2	5'-CACCGGTCAAAACAGTTGCAACGG-3'(SEQ ID NO.2)
hLINC01088 KO-sgRNA3	5'-CACCGTGTACCCCCTTGGTGGATAG-3'(SEQ ID NO.3)
		hLINC01088 KO-sgRNA4	5'-CACCGACTCTGGCTAACAATAGAGG-3'(SEQ ID NO.4)

TABLE 2 primer pairs for qRT-PCR

Second, experimental results

1. Expression of LncRNA in villus tissue

The expression of LINC01088 in villus tissue of recurrent abortion patients and normal pregnancy patients is detected by fluorescent quantitative PCR method. It was found to be highly expressed in the villus tissue of patients with recurrent abortion (see FIG. 1).

2. Establishing a recurrent abortion IncRNA diagnostic model and evaluating the diagnostic value of the diagnostic model

Based on the above lncRNA expression in villus tissue, a receiver operating characteristic curve (ROC curve) was plotted and the area under the curve (AUC) was calculated, judging the diagnostic role of LINC01088 in recurrent abortion (see FIG. 2). The sensitivity and specificity of the diagnosis in recurrent abortion are very high. Meanwhile, LINC01088 was selected for in vitro cell experimental study to explore its role and function in trophoblasts.

3. LINC01088 inhibits trophoblast proliferation

To clarify the role of LINC01088 in trophoblasts, an overexpression plasmid for LINC01088 (pcDNA3.1-LINC01088) was constructed and transfected into trophoblasts HTR8 and JAR. Meanwhile, HTR8 and LINC01088 knockout stable transgenic cell lines in JAR were constructed. The trophoblasts with high expression of LINC01088 and the trophoblasts with the LINC01088 knockout are tested by CCK8, EdU and clone formation experiments. LINC01088 was found to inhibit the proliferation of trophoblasts (see fig. 3).

4. LINC01088 inhibits trophoblast cell cycle and promotes trophoblast apoptosis

Further examining the effect of LINC01088 on the trophoblast cycle, it was found that the trophoblast cells overexpressing LINC01088 in stage G1 were significantly increased, while the cells in stage S were decreased. To further examine the effect of LINC01088 on the G1/S phase of trophoblasts, cycle-synchronized trophoblasts were transfected 24 hours after transfection of the plasmid with nocodazole, and the G1 phase of trophoblasts transfected with LINC01088 over-expression plasmid was significantly increased. Meanwhile, LINC01088 was found to inhibit the expression of cycle-related molecules CDK2, CDK4 and cyclinE 1. The effect of LINC01088 on trophoblast apoptosis was also examined and the results showed that LINC01088 can promote trophoblast apoptosis, inhibit Bcl2 and promote the expression of Bax, clear caspase3 (see fig. 4).

5. LINC01088 inhibits trophoblast invasion

In order to examine the effect of LINC01088 on trophoblast migration and invasion, cell transwell migration and invasion experiments were performed. As shown in fig. 5, LINC01088 can inhibit the migration and invasion of trophoblasts.

6. LINC01088 binds and promotes Arginase-1 expression, thereby indirectly inhibiting eNOS/NO system

The nucleoplasm separation experiment proves that LINC01088 is mainly expressed in the nucleus, and further RNA pull-down experiment shows that LINC01088 can be combined with Arginase-1. Then the expression of Arginase-1 is detected in the trophoblast, and the Arginase-1 is found to have obviously increased protein level and no obvious change of RNA level in the trophoblast after the over-expression of LINC 01088. Arginase and nitric oxide synthase can competitively bind arginine, wherein the arginase and arginine are combined to generate urea and ornithine, the nitric oxide synthase and arginine are combined to generate nitric oxide, and the nitric oxide plays an important role in promoting the proliferation, invasion and the like of trophoblasts. Therefore, the expression of the trophoblast nitric oxide synthase was examined, and it was found that only endothelial nitric oxide synthase was expressed in trophoblasts, and that the expression level of endothelial nitric oxide synthase was decreased in trophoblasts over-expressed with LINC 01088. At the same time, the content of nitric oxide was also reduced in the supernatant of trophoblasts overexpressing LINC01088, as well as in the villus tissue grinding fluid of patients with recurrent abortion (see FIG. 6).

7. LINC01088 activates JNK, P38/MAPK signaling pathway by inhibiting NO production

In order to explore the signal pathway related to LINC01088, the expression of the signal pathway related to nitric oxide (pathways such as beta-catenin, NF-kB and MAPK) molecules was examined. It was found that only p38 and JNK in the MAPK family were activated after overexpression of LINC 01088.

LINC01088 indirectly inhibits the nitric oxide synthase/nitric oxide system by combining with arginase 1 and promoting the protein level expression of arginase 1, so that the content of nitric oxide in the maternal-fetal interface is reduced, a p38 and JNK MAPK signaling pathway is activated, the proliferation, migration, invasion and the like of trophoblasts are inhibited, and finally abortion is caused.

LINC01088 which is highly expressed in villus tissue in early stage of recurrent abortion is screened, and according to the expression of lncRNA in the villus tissue, a working characteristic curve (ROC curve) of a subject is drawn, and the area under the curve (AUC) is calculated, so that the diagnosis effect of LINC01088 in recurrent abortion is judged. The results show that LINC01088 has higher sensitivity and specificity as a diagnostic marker in recurrent abortion.

LINC01088 was selected for in vitro cell experimental studies to explore its role and function in trophoblasts. LINC01088 is found to inhibit the proliferation and invasion of trophoblasts, inhibit the cycle of trophoblasts and promote the apoptosis of trophoblasts. In mechanism research, LINC01088 can be combined with arginase 1 to further inhibit a nitric oxide synthase/nitric oxide system and reduce the content of nitric oxide in a maternal-fetal interface, so that a p38 and JNK MAPK signal pathway is activated, proliferation, migration, invasion and the like of trophoblasts are further inhibited, and finally abortion is caused.

In conclusion, LINC01088 can be used as a predictive diagnostic marker for recurrent abortion, can be used as a therapeutic target for recurrent abortion, and plays an important role in diagnosis and treatment of recurrent abortion.

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 changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. 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

Application of <120> lncRNA in diagnosis and treatment of recurrent abortion

<130>

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<170> PatentIn version 3.3

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Claims

The application of lncRNA as a marker in the preparation of a recurrent abortion diagnosis or auxiliary diagnosis product;

wherein the lncRNA comprises LINC 01088.
2. A product for use in the diagnosis or the aided diagnosis of recurrent pregnancy loss, characterised in that it comprises a substance for the detection of lncRNA;

wherein the lncRNA comprises LINC 01088; other ncrnas (miRNA and/or lncRNA) as a diagnosis or aid diagnosis of recurrent abortion are also included;

preferably, the product further comprises a substance suitable for detecting other biomarkers of recurrent abortion, including genetic factor-related biomarkers, endocrine factor-related biomarkers, infection factor-related biomarkers, immune function-related biomarkers, pre-thrombotic status-related biomarkers, and other systemic diseases of the subject;

preferably, the lncRNA detection substance comprises substances for detecting the expression level of lncRNA by RT-PCR, real-time quantitative PCR, in-situ hybridization, gene chip and gene sequencing;

preferably, the product comprises a primer, a probe, a chip, a nucleic acid membrane strip, a preparation or a kit for detecting the expression level of the lncRNA in a sample to be detected;

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

preferably, the cells are trophoblasts;

preferably, the tissue is villous tissue;

preferably, the organ may be a placenta or uterus;

preferably, the body fluid is blood, lymph, urine, gastric juice, or saliva, and more preferably blood.
3. A system for diagnosing or aiding in the diagnosis of recurrent spontaneous abortion, comprising:

i) an analysis unit, the analysis unit comprising: a detection agent for determining the expression level of a test sample from a subject selected from the group consisting of lncRNA, and;

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

preferably, in the analysis unit, the lncRNA comprises LINC 01088; other ncRNAs (miRNA and/or lncRNA) which can be used for diagnosis or auxiliary diagnosis of recurrent abortion are also included;

preferably, the analysis unit further comprises a substance adapted to detect other biomarkers of recurrent abortion, said other biomarkers comprising genetic factor-related biomarkers, endocrine factor-related biomarkers, infection factor-related biomarkers, immune function-related biomarkers, pre-thrombotic status-related biomarkers and other systemic diseases of the subject;

preferably, the specific evaluation flow of the evaluation unit includes:

(ii) if the expression level of lncRNA in the test sample of the subject is up-regulated compared to a reference, then the subject is or is candidate for a recurrent abortion patient; otherwise, the subject is not, or is not candidate for, a patient with recurrent abortion.
4. 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 the expression level of lncRNA in a test sample from said subject, and;

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

preferably, the lncRNA comprises LINC 01088;

preferably, an upregulation of the level of expression in the test sample compared to the level in the reference, then said subject is or is candidate for a recurrent abortion patient; otherwise, the subject is not, or is not candidate for, a patient with recurrent abortion.
5, the application of lncRNA as a target point in treating recurrent abortion and/or screening recurrent abortion medicines;

the lncRNA comprises LINC 01088.
6. The use of claim 5, wherein the method of screening for a drug for recurrent abortion comprises:

1) treating a system expressing and/or containing said lncRNA with a candidate substance; setting a parallel control without candidate substance treatment;

2) detecting the expression level of the lncRNA in the system after the step 1) is finished; if the expression level of the DNA 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 for a recurrent abortion drug.
7. The application of a substance for inhibiting the expression level of lncRNA in preparing products; the function of the product is any one or more of the following:

(a1) promoting proliferation of trophoblast cells;

(a2) promoting the progress of the cell cycle of the trophoblast;

(a3) inhibiting apoptosis of trophoblasts;

(a4) promoting migration and/or invasion of trophoblasts;

(a5) inhibiting Arginase-1 expression;

(a6) facilitating the eNOS/NO system;

(a7) inhibition of the p38, JNK MAPK signaling pathway;

(a8) prevention and/or treatment of recurrent miscarriage;

the lncRNA comprises LINC 01088.
8. A product whose active ingredients comprise a substance that inhibits the expression level of lncRNA; the function of the product is any one or more of the following:

(a1) promoting proliferation of trophoblast cells;

(a2) promoting the progress of the cell cycle of the trophoblast;

(a3) inhibiting apoptosis of trophoblasts;

(a4) promoting migration and/or invasion of trophoblasts;

(a5) inhibiting Arginase-1 expression;

(a6) facilitating the eNOS/NO system;

(a7) inhibition of the p38, JNK MAPK signaling pathway;

(a8) prevention and/or treatment of recurrent miscarriage;

the lncRNA comprises LINC 01088.
9. The application of a substance for promoting the expression level of lncRNA in preparing products; the function of the product is any one or more of the following:

(b1) inhibiting proliferation of trophoblasts;

(b2) inhibiting the progression of the trophoblast cell cycle;

(b3) promoting apoptosis of trophoblasts;

(b4) inhibition of trophoblast and/or invasion;

(b5) promoting Arginase-1 expression;

(b6) suppression of the eNOS/NO system;

(b7) activating p38, JNK MAPK signaling pathway;

(b8) constructing a recurrent abortion animal model;

the lncRNA comprises LINC 01088.
10. A product characterized in that its active ingredients comprise substances that promote the expression level of lncRNA;

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

(b1) inhibiting proliferation of trophoblasts;

(b2) inhibiting the progression of the trophoblast cell cycle;

(b3) promoting apoptosis of trophoblasts;

(b4) inhibition of trophoblast and/or invasion;

(b5) promoting Arginase-1 expression;

(b6) suppression of the eNOS/NO system;

(b7) activating p38, JNK MAPK signaling pathway;

(b8) constructing a recurrent abortion animal model;

the lncRNA comprises LINC 01088.