Application of CDH3 in diagnosis of recurrent abortion

Abstract

The invention discloses an application of CDH3 in diagnosis of recurrent abortion. The invention discovers that the expression quantity of the CDH3 is closely related to the recurrent abortion for the first time, and the reduction of the expression quantity can promote the occurrence of the recurrent abortion, which indicates the potential possibility of the CDH3 as an RSA early diagnosis marker. The invention is helpful for deeply knowing pathogenesis of RSA, provides a potential marker and a therapeutic target for clinical diagnosis, prevention and treatment of recurrent abortion, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, the application of CDH3 in the diagnosis of recurrent miscarriage. Background Technology

[0002] Three or more consecutive spontaneous abortions are termed recurrent spontaneous abortion (RSA). Most recurrent abortions occur in early pregnancy, and the majority of these pregnancies terminate at the same gestational age. The clinical presentation and course of recurrent abortion are consistent with common spontaneous abortions. The causes of early pregnancy loss are complex, often including uterine developmental abnormalities, endocrine disorders, and environmental physical and chemical factors. However, approximately 50% of recurrent abortions have no identifiable cause and are clinically classified as unexplained recurrent abortion.

[0003] In recent years, people have paid more and more attention to reproductive health, but the incidence of recurrent spontaneous abortion (RSA) has been rising year by year, seriously affecting the physical and mental health of pregnant women. At present, the pathogenesis of unexplained recurrent miscarriage is still unclear, and there are no effective prevention and treatment measures in clinical practice. It is a clinical problem that urgently needs to be solved.

[0004] CDH3, or cadherin-3, also known as P-cadherin, is an important member of the cadherin family. Clinically, abnormal CDH3 expression is closely related to gastric cancer metastasis, and its overexpression may promote tumor spread by disrupting intercellular connections. In the field of dermatology, CDH3 gene mutations have been confirmed as a cause of congenital pachyonychia type 4B. However, the role of CDH3 in recurrent miscarriage is not yet publicly available. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the present invention aims to provide a novel diagnostic biomarker for recurrent miscarriage—CDH3.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides the use of a substance for detecting the expression level of CDH3 in a sample in the preparation of a diagnostic or assisted diagnostic, predictive or assisted predictive of recurrent miscarriage.

[0007] Furthermore, the sample may include cells, tissues, or bodily fluids.

[0008] Furthermore, the cells include epithelial cells and trophoblast cells.

[0009] Furthermore, the tissue includes decidual tissue, villous tissue, and endometrial tissue.

[0010] Furthermore, the bodily fluids include blood, lymph, urine, vaginal secretions, sweat, breast milk, bile, and interstitial fluid.

[0011] The term "CDH3" as used in this invention is intended to include its fragments, variants (e.g., allele variants), and derivatives. Representative human CDH3 cDNA and human CDH3 protein sequences are well known in the art and are publicly available from the NCBI website. For example, at least one human CDH3 isotype is known: human CDH3, Gene ID: 1001. Nucleic acid and polypeptide sequences of CDH3 orthologs in organisms other than humans are well known.

[0012] The terms "sample to be tested" and "sample" as used in this invention can refer to any biological sample isolated from a subject. The term "sample" includes any biological sample that can be extracted from a subject, whether untreated, treated, diluted, or concentrated. Samples may include, but are not limited to, cells, tissues, organs, or body fluids. Cells include epithelial cells and trophoblast cells. Tissues include decidual tissue, villous tissue, and endometrial tissue. Organs include the placenta or uterus. Body fluids include blood, lymph, urine, vaginal secretions, sweat, breast milk, bile, and interstitial fluid, preferably blood, which may be serum or plasma. Using blood as the sample to be tested reduces trauma to the subject and improves subject adaptation and compliance.

[0013] As used in this invention, the term "diagnosis" refers to the identification or classification of a molecular or pathological state, disease, or symptom. The term "auxiliary diagnosis" refers to methods that assist in clinical determination of the presence or nature of a specific type of symptom or symptom of a disease or symptom. For example, auxiliary diagnostic methods for a disease or symptom may include measuring certain biomarkers in biological samples from an individual.

[0014] Furthermore, the substances used to detect CDH3 expression levels in the sample include substances that detect CDH3 gene expression levels and / or substances that detect CDH3 protein expression levels.

[0015] Furthermore, the substance comprises substances obtained through PCR reaction, RT... Reagents for detecting the presence, absence, and / or amount of CDH3 gene, fragments, and protein in samples using PCR-derived reactions, 3SR amplification, LCR, SDA, NASBA, TMA, SYBR Green, TaqMan probes, molecular beacons, two-hybrid probes, complex probes, ISH, microarrays, Southern blotting, Northern blotting, multianalyte assays, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, immunofluorescence assays, enzyme immunoassays, immunoprecipitation assays, chemiluminescence assays, immunohistochemical assays, dot blot assays, or narrow-line blot assays.

[0016] As used in this invention, "expression" refers to the transcription of a gene to produce RNA transcripts (e.g., mRNA, antisense RNA, siRNA, shRNA, miRNA, etc.), and, if appropriate, the translation of the resulting mRNA transcripts into proteins. Therefore, it is clear from the context that the expression of a coding sequence is produced by the transcription and translation of that coding sequence. Conversely, the expression of a non-coding sequence is produced by the transcription of that non-coding sequence.

[0017] The terms “level of expression” or “expression level” as used in this invention are generally used interchangeably and generally refer to the amount of a biomarker in a sample. “Expression” generally refers to a process by which information (e.g., coding genes and / or epigenetics) is translated into a cellular and operable structure. Therefore, as used herein, “expression” can refer to transcription into a polynucleotide, translation into a polypeptide, or even modifications of polynucleotides and / or polypeptides (e.g., post-translational modifications of polypeptides). Transcribed polynucleotides, translated polypeptides, or fragments of polynucleotides and / or polypeptide modifications (e.g., post-translational modifications of polypeptides) should also be considered as expressed, whether they originate from transcripts produced by alternative splicing or degraded transcripts, or from post-translational processing of polypeptides, such as by proteolysis. “Expressed genes” include those genes that are transcribed into polynucleotides (mRNA) and then translated into polypeptides, as well as those genes that are transcribed into RNA but not translated into polypeptides (e.g., transfer RNA and ribosomal RNA).

[0018] A second aspect of the present invention provides a product for diagnosing or assisting in the diagnosis of recurrent miscarriage.

[0019] Furthermore, the product includes a substance for detecting CDH3 expression levels.

[0020] Furthermore, the substance includes substances for detecting CDH3 gene expression levels and / or substances for detecting CDH3 protein expression levels.

[0021] Furthermore, the products include reagents, kits, test strips, nucleic acid membrane strips, chips, primers, and probes.

[0022] Furthermore, the test strip includes a chromatography test strip.

[0023] Furthermore, the kit also includes a container, a positive control, a negative control, a buffer, an auxiliary agent, a solvent, and an instruction manual with instructions on how to use the kit for detection and how to interpret the results.

[0024] Furthermore, the kit includes a gene detection kit and a protein detection kit.

[0025] Furthermore, the gene detection kit includes primers, reagents, or chips for detecting the transcriptional level of the CCL26 gene.

[0026] Furthermore, the protein detection kit includes reagents, a chip, antibodies, and ligands for detecting the protein level of CCL26 expression.

[0027] Furthermore, the reagents include those used in Western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, Octelloni immunodiffusion, rocket electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip detection.

[0028] Furthermore, the subjects of the test include humans or non-human mammals.

[0029] The term "reagent" as used in this invention includes compounds that induce desired pharmacological and / or physiological effects. The term also encompasses pharmaceutically acceptable and pharmacologically active components of those compounds specifically mentioned herein, including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs, etc. When using the above term, it should be understood that this includes the active agent itself, as well as pharmaceutically acceptable and pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. The term "reagent" should not be interpreted narrowly but should be extended to small molecules, protein molecules such as peptides, polypeptides, and proteins, and compositions comprising them, as well as genetic molecules such as RNA, DNA, their analogues and chemical analogs, and cellular agents.

[0030] The probes described in this invention can be prepared by chemical synthesis, by properly designing them with reference to known information using methods well known to those skilled in the art, and by preparing them by chemical synthesis, or by preparing a gene containing the desired nucleic acid sequence from biological material and amplifying it using primers designed for amplifying the desired nucleic acid sequence.

[0031] The primers described in this invention can be prepared by chemical synthesis, appropriately designed using methods well known to those skilled in the art and with reference to known information, and prepared by chemical synthesis.

[0032] In some implementations, the product may also include other biomarkers and combinations thereof currently suitable for detecting recurrent miscarriage.

[0033] A third aspect of this invention provides the application of CDH3 in constructing computer models or systems for diagnosing or assisting in the diagnosis, prediction or assisting in the prediction of recurrent miscarriage.

[0034] Furthermore, the computer model includes a memory.

[0035] Furthermore, the memory is used to store the executed computer program.

[0036] In some specific implementations, the computer program is used to control the processor to perform operations of different modules.

[0037] The term "computer model" as used in this invention refers to a mathematical model that associates the expression level of the biomarker CDH3 with the risk or likelihood of recurrent miscarriage. Any suitable mathematical method described in the prior art can be used to associate the biomarker combination with the disease. The logarithmic function used to associate the biomarker combination with the disease preferably employs an algorithm developed and obtained by applying statistical methods. Suitable statistical methods include discriminant analysis (DA) (i.e., linear, quadratic, regular DA), kernel methods (i.e., SVM), and nonparametric methods (i.e., k-means analysis). Nearest neighbor classifiers, PLS (partial least squares), tree-based methods (i.e., logistic regression, CART, random forest, boost / bagging), generalized linear models (i.e., logarithmic regression), principal component analysis (i.e., SIMCA), generalized superposition models, fuzzy logic-based methods, and methods based on neural networks and genetic algorithms. Skilled personnel will have no problem selecting appropriate statistical methods to evaluate the biomarker combination of the present invention and thereby obtaining suitable mathematical algorithms. In the implementation scheme, the statistical methods used to obtain the mathematical algorithms used in assessing depression are selected from DA (i.e., linear, quadratic, regular discriminant analysis), Kernel methods (i.e., SVM), nonparametric methods (i.e., k... Nearest Neighbor Classifier, PLS (Partial Least Squares), tree-based methods (i.e., logistic regression, CART, random forest, boosting methods), or generalized linear models (i.e., logistic regression).

[0038] The fourth aspect of this invention provides the use of CDH3 in screening candidate drugs for the treatment of recurrent miscarriage.

[0039] Furthermore, the method for screening candidate drugs for treating recurrent miscarriage includes: 1) Treat systems expressing and / or containing CDH3 with candidate substances; set up parallel controls without candidate substance treatment; 2) After completing step 1), detect the expression level of CDH3 in the system; if the expression level of CDH3 in the system treated with the candidate substance is significantly increased compared with the parallel control, the candidate substance can be used as a candidate drug for recurrent miscarriage.

[0040] In some embodiments, the system may be a cellular system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

[0041] The cells in the cell system can be trophoblasts or epithelial cells; the tissues in the tissue system can be decidual tissue, villous tissue, or endometrial tissue; the organs in the organ system can be the placenta or uterus; and the animals in the animal system can be mammals, including mice, rats, guinea pigs, rabbits, monkeys, chimpanzees, and humans.

[0042] The fifth aspect of this invention provides a medical auxiliary diagnostic system for early recurrent miscarriage.

[0043] Furthermore, the system includes a result determination module for comparing the processed value of detecting the expression level of the CDH3 gene or protein with a set value.

[0044] Furthermore, the system also includes a processing module for detecting the gene or protein expression level of CDH3 in the sample to be tested and obtaining a processed value.

[0045] Furthermore, the diagnostic system includes an output module for outputting the obtained diagnostic results.

[0046] Furthermore, the result determination module's evaluation process includes: if the CDH3 expression level in the subject's test sample is downregulated compared to the set value, then the subject is or is a candidate for recurrent miscarriage; otherwise, the subject is not or is not a candidate for recurrent miscarriage.

[0047] Furthermore, the setpoint can be the CDH3 expression value in samples from normal healthy subjects who do not have recurrent miscarriage-related symptoms and have no abnormal physiological or pathological findings. The setpoint can also be the CDH3 expression value in samples from the same subject before the onset of symptoms or disease, or before a diagnosis of recurrent miscarriage. The setpoint can be the CDH3 expression value in standardized samples.

[0048] As used in this invention, the terms "patient," "subject," "test subject," or "individual" refer to any subject requiring treatment or prevention, particularly those with recurrent miscarriage, and even more particularly mammalian subjects. Suitable vertebrates falling within the scope of this invention include, but are not limited to, any member of the subphylum Chordata, including primates (e.g., humans, monkeys, and apes, and including monkey species (e.g., genus Macaca, such as macaques like Macacafascicularis, and / or Macaca mulatta)) and baboons (Papio... Marmosets (from the genus Callithrix), squirrel monkeys (from the genus Saimiri) and tamarins (from the genus Saguinus), as well as species of orangutans, such as chimpanzees (Pantroglodytes), rodents (e.g., mice, rats, guinea pigs), rabbits (e.g., rabbits, hares), cattle (e.g., cattle), sheep (e.g., sheep), goats (e.g., goats), pigs (e.g., pigs), horses (e.g., horses), dogs (e.g., dogs), felines (e.g., cats), birds (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars, etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.) and fish.

[0049] The sixth aspect of this invention provides the use of a CDH3 promoter in the preparation of a medicament for the prevention and / or treatment of recurrent miscarriage.

[0050] Furthermore, the CDH3 promoter includes substances that can increase the activity of CDH3 protein, improve the stability of CDH3 gene or protein, upregulate CDH3 protein expression, increase the effective action time of CDH3 protein, or promote the transcription and translation of CDH3 gene.

[0051] Advantages and beneficial effects of the present invention: This invention is the first to discover a strong correlation between CDH3 expression and recurrent miscarriage (RSA), with decreased CDH3 expression promoting the occurrence of RSA, suggesting its potential as an early diagnostic biomarker for RSA. This invention contributes to a deeper understanding of the pathogenesis of RSA and will also help provide potential biomarkers and therapeutic targets for the clinical diagnosis, prevention, and treatment of RSA, thus possessing significant application value. Attached Figure Description

[0052] Figure 1 The transcriptomic features of the RSA decidua were displayed; among them... Figure 1A shows the volcano plot of differential molecules in the decidua of RSA and Control based on the GSE113790 dataset (|log2FC|>1, P<0.05). Figure 1 B shows the biological processes involved in RSA decidua based on the GSE113790 dataset and GO enrichment analysis. Figure 1 C shows the volcano plot of differential molecules in the decidua of RSA and Control based on the GSE161969 dataset (|log2FC|>1, P<0.05). Figure 1 D shows the biological processes involved in RSA decidua based on the GSE161969 dataset and GO enrichment analysis. Figure 1 E shows the volcano plot of differential molecules in the decidua of RSA and Control based on the GSE178535 dataset (|log2FC|>1, P<0.05). Figure 1 F shows the biological processes involved in RSA decidua based on the GSE178535 dataset and GO enrichment analysis. Figure 2 This shows the changing trends of adhesion molecules in the RSA decidua; among them... Figure 2 A shows the fold difference in expression levels of the decidual selectin adhesion molecules SELP, SELL, and SELE in RSA compared to the Control group; Figure 2 B shows the fold difference in expression levels of immunoglobulin superfamily adhesion molecules ICAM1, VCAM1, and JAM3; Figure 2 C shows the fold change in expression levels of cadherins CDH1, CDH2, and CDH3; Log2FC: log2 (fold change); SELP: P-selectin; SELE: E-selectin; SELL: L-selectin; ICAM1: intercellular adhesion molecule; VCAM1: vascular cell adhesion molecule; JAM3: junctional adhesion molecule; Figure 3 The data showed the intersection of RSA decidual up- and down-regulated molecules; among them... Figure 3 A shows downregulated molecular intersection; Figure 3 B shows an upregulation of molecular intersection; Figure 4 The expression levels of CDH3 in the decidual tissues of RSA and normal mice GD4, GD5, and GD6 were shown. Figure 5 This study demonstrated the expression level of CDH3 in the endometrium during the mid-secretive phase and its predictive efficacy for miscarriage; among which... Figure 5 A shows the CDH3 expression level in the mid-secretive endometrium of women with live births and miscarriages before pregnancy; Figure 5 B shows the ROC analysis of mid-secretive endometrial CDH3 levels to predict the outcome of this pregnancy. Figure 6 The results showed decreased CDH3 expression in the decidua of RSA; among which... Figure 6 A shows the localization and semi-quantitative detection of CDH3 expression in the decidua of the Control and RSA groups by immunohistochemistry (IHC), scale bar = 50 μm; Figure 6 B shows the levels of CDH3 protein in the decidua of the Control and RSA groups using Western blotting; n=6 for each group, and all data were statistically analyzed using t-tests. *P<0.05, and all data are expressed as mean±SEM. Figure 7 The RSA villous tissue showed decreased CDH3 expression; among which... Figure 7 A shows the localization and semi-quantitative detection of CDH3 in the villi of the Control and RSA groups by IHC. The left scale bar is 200 μm and the right scale bar is 50 μm. Figure 7 B shows the protein levels of CDH3 in the chorionic villi of the Control and RSA groups detected by Western blotting; n=6 in each group, and t-tests were used for statistical analysis. *P<0.05, **P<0.001, and all data are expressed as mean±SEM. Figure 8 The study showed differential expression of CDH3 in the endometrium of non-pregnant women and the decidua of pregnant women; among them... Figure 8 A shows the tissue and cellular localization of CDH3 in the endometrium of non-pregnant women and the decidua of pregnant women, using CK-19 (epithelial cell marker) labeled cells and heterologous fluorescence double staining. Scale bar = 50 μm. Figure 8 B shows the use of vimentin to label stromal cells, and heterologous fluorescence double staining to detect the tissue and cellular localization of CDH3 in the endometrium of non-pregnant women and the decidua of pregnant women, scale bar = 50 μm; Figure 8 C shows the IHC method for further detection of CDH3 in the tissue and cell localization of the endometrium of non-pregnant women and the decidua of pregnant women, scale bar = 50 μm; Figure 9 The expression of CDH3 in primary decidual cells was shown; among them... Figure 9 A shows the identification of primary cell components using CK-19 and vimentin as marker proteins, scale bar = 50 μm; Figure 9 B shows the co-localization of CK-19 and CDH3, and heterologous fluorescence detection of CDH3 cell localization, scale bar = 50 μm; Figure 9 C shows the co-localization of vimentin and CDH3, and heterologous fluorescence is used to detect the cell localization of CDH3. Scale bar = 50 μm. Figure 10 This section evaluates the effectiveness of lentiviral transfection in knocking down CHD3 in cells; among other things... Figure 10A shows that h-CDH3 gene expression level was detected by qRT-PCR with NC as calibration and h-ACTB as internal control. Figure 10 B shows that the PCR product size is correct and there are no extraneous bands, indicating that there is no nonspecific amplification; Figure 10 C shows the Western blotting verification of CDH3 protein expression after knockdown; Figure 11 The study showed that CDH3 knockdown impaired endometrial proliferation and migration; among which... Figure 11 A shows the results of CCK8 assay for the viability of RL95-2, LV-NC, and shCDH3 cells; Figure 11 B shows the EdU assay for the proliferation capacity of RL95-2, LV-NC, and shCDH3 cells, scale bar = 50 μm; Figure 11 C shows the scratch assay to detect the migration ability of RL95-2, LV-NC, and shCDH3 cells; KD: knockdown. Figure 12 The results showed that abnormal CDH3 expression in endometrial epithelial cells affected trophoblast migration; among which... Figure 12 A shows a schematic diagram of co-culturing RL95-2, LV-NC, and shCDH3 cell supernatants with HTR-8 / SVneo; Figure 12 B shows the migration capability of HTR-8 / SVneo as detected by Tmanswell; Figure 12 C shows the scratch test to detect the migration ability of HTR-8 / SVneo; Figure 13 The results show the establishment of a mouse model of miscarriage by locally and gently pushing Anti-CDH3 into the uterine horn before embryo implantation; among them... Figure 13 A shows a mouse model of miscarriage constructed by injecting Anti-CDH3 into the uterine cornua and a schematic diagram of specimen collection. Figure 13 B shows the actual procedure of injecting the mouse uterine horn; Figure 13 C shows the differences in uterine morphology and appearance in EI0.5 mice after injection of different concentrations of Anti-CDH3 into the uterine horn; Figure 13 D shows the morphology of the heart, liver, spleen, lungs, and kidneys of mice after intrauterine injection of PBS and 5 mg / Kg Anti-CDH3, as detected by HE. Scale bar = 100 μm. Figure 14 This image shows the anatomical and morphological changes in Anti-CDH3-induced abortion in mice. The top layer image shows an anatomical diagram of missed abortion with HE staining, the middle layer image shows an anatomical diagram of biochemical pregnancy with HE staining, and the bottom layer image shows a normal pregnancy. D: Decidua, OT: Organized tissue, BC: Blood clot, P: Placenta, LZ: Labyrinthine zone. Figure 15 The results showed that Anti-CDH3 specifically binds to CDH3, inhibiting Caspase-L activation and leading to miscarriage; among which... Figure 15 A shows HE staining and CDH3 IHC detection of aborted uterus and normal pregnant uterus, scale bar = 500 μm (HE and left IHC), scale bar = 100 μm (right IHC); Figure 15 B shows the expression of CDH3, Pro-CASP1, and Cleaved-CASP1 in the decidua of aborted uterus and normal pregnancy uterus, as detected by Western blotting. Data analysis was performed using t-tests. *P<0.05, **P<0.01, ***P<0.001. Data are expressed as mean+SEM. D: decidua, OT: organized tissue, BC: blood clot, P: placenta, LZ: labyrinthine zone. Detailed Implementation

[0053] The present invention is further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. Reagents or instruments used that do not specify the manufacturer are all commercially available conventional products.

[0054] Example 1: Screening of molecular markers for RSA decidual membrane Given the importance of normal decidua structure and function for maintaining pregnancy, and the close relationship between its pathological changes and the occurrence and development of recurrent spontaneous abscess (RSA), our study first systematically analyzed the gene expression profiles of decidua tissue in RSA and normal pregnancy (Control) based on transcriptomics. This revealed the molecular changes at the transcriptional level in RSA decidua tissue at a holistic level, and identified genes with significant differential expression. These results will lay the foundation for subsequent experiments to pinpoint key factors in the pathogenesis of RSA.

[0055] Following strict inclusion and exclusion criteria, bulk RNA-seq sequencing data of RSA decidua tissue from three independent studies were obtained from public database systems. These studies were derived from datasets GSE113790 (Dataset1), GSE161969 (Dataset2), and GSE178535 (Dataset3), respectively. Differential expression analysis was performed on the RNA-seq data of RSA and Control decidua. The results showed that, compared to Control decidua, RSA decidua exhibited widespread and significant molecular expression abnormalities. In the three independent datasets, the number of upregulated genes was 1064, 964, and 364, respectively, while the number of downregulated genes was 682, 734, and 293, respectively (|log2FC|>1, P<0.05). Figure 1 A, C, E). GO enrichment analysis revealed that these disordered molecules are widely involved in key biological processes such as maintaining cellular homeostasis and regulating immune inflammation (P<0.05), and their cellular function regulation encompasses basic cellular activities such as cell proliferation, differentiation, and migration. Figure 1 (B, D, F). Further comparison revealed that cell adhesion and its related functional activities may have played a stable and important role in the pathological process of RSA.

[0056] Given the role of cell adhesion molecules (CAMs) in cell adhesion, we further investigated their expression in the decidua of the RSA. Based on their structure and function, CAMs can be categorized into selectins, integrins, cadherins, the immunoglobulin superfamily, and other cell adhesion molecules. Selectins include P-selectin, E-selectin, and L-selectin; the immunoglobulin superfamily includes intercellular adhesion molecules, vascular cell adhesion molecules, and junctional adhesion molecules; cadherins include E-cadherin (CDH1 / E-cadherin), N-cadherin (CDH2 / N-cadherin), and P-cadherin (CDH3 / P-cadherin). Since integrins often interact with selectins, the immunoglobulin superfamily, and the cadherin family, we did not analyze them here. Instead, we analyzed the transcriptional data of selectins, cadherins, and the immunoglobulin superfamily. The comparative results showed that the expression levels of the above-mentioned adhesion molecules in the RSA decidua were significantly disordered. In different datasets, the change trends of most adhesion molecules were unstable, with only CDH3 molecules showing a stable and significantly downregulated trend in the RSA decidua. Figure 2 ).

[0057] Given the potential differences in molecular expression characteristics among the three independent datasets, to further identify key molecules stably dysregulated in the RSA decidua, we calculated the intersection of the differentially upregulated and downregulated genes. The results showed that there were three stably upregulated genes (…). Figure 3 A), there is one gene that is stably downregulated ( Figure 3 B). Molecules stably upregulated in the decidua of RSA include INMT, FTH1P10, and NPHP3-ACAD11, while only CDH3 is stably downregulated (Table 1 and...). Figure 3 ).

[0058] Table 1. Dysregulation of selected molecules in the decidua of RSA

[0059] Furthermore, based on transcriptomic data (dataset GSE169544) of the endometrial / decidual lesion RSA mouse model (PARP-1 and PARP-2 double knockout), the dynamic trends and differences in the selected molecules in the early pregnancy decidua of the RSA and Control groups were verified. This model has been proven to be a stable miscarriage model and is mainly associated with endometrial / decidual lesions.

[0060] The results showed that INMT was significant, but its expression trend was opposite to that of human decidua; NPHP3-ACAD11 was not detected, possibly due to its low expression level; CDH3 was stably downregulated, consistent with the results of human decidua (Table 2).

[0061] Overall, CDH3 expression showed an upregulation trend after embryo implantation. Compared with the control group, CDH3 expression in the RSA decidua was significantly reduced, and the difference increased significantly with pregnancy progression. Figure 4 ).

[0062] After mouse embryo implantation, the morphology and function of the uterine decidua change. PARP-1 and PARP-2 are important factors affecting decidual function, which directly affects the success rate of embryo implantation. Knockout of PARP-1 and PARP-2 genes (endometrial / decidual lesions) significantly reduced CDH3 expression in the uterine decidua. We hypothesize that CDH3 may affect pregnancy outcomes by influencing decidual regulatory function.

[0063] Table 2. Dysregulation of selected molecules in the decidua of RSA mice

[0064] The decidua is derived from the endometrium. This process begins with embryo implantation in mice and in the mid-secretory phase in humans. Therefore, based on mid-secretory endometrial transcriptome data (dataset GSE180485), we further evaluated CDH3 expression and divided women into two groups according to subsequent pregnancy outcomes (miscarriage or live birth). Results showed that women who ultimately miscarried had lower CDH3 expression in their pre-pregnancy mid-secretory endometrium than the live birth group. Figure 5 A), using pre-pregnancy endometrial CDH3 as a variable to predict the likelihood of miscarriage in this pregnancy, the AUC reached 67% ( Figure 5 (B) We hypothesize that endometrial CDH3 dysregulation during the pre-pregnancy secretory phase may have adversely affected the pregnancy outcome. In summary, we have preliminarily completed molecular screening of the RSA decidua / endometrium and identified CDH3 as a potential key molecule at the RSA maternal-fetal interface.

[0065] Example 2: Validation of CDH3 molecular characteristics at the maternal-fetal interface based on clinical samples The transcription results showed that CDH3, the endometrial / decidual adhesion molecule, was significantly associated with pregnancy outcomes in both human and mouse models. Further, using women who requested abortion due to unplanned pregnancy (Control group) and women with recurrent spontaneous abortion (RSA group) as subjects, decidual and chorionic villus samples were collected from both groups after abortion at 7-10 weeks of gestation, following strict inclusion and exclusion criteria, to validate the expression characteristics of CDH3 at the protein level. Inclusion criteria: ① Women aged 20-35 years; ② Women who underwent laparoscopic surgery for uterine and adnexal masses at the Women and Children's Hospital Affiliated to Chongqing Medical University; Exclusion criteria: ① Women with endocrine-functioning ovarian cysts; ② Women with a history of infection or infectious diseases within the past six months, such as TORCH, mycoplasma, chlamydia, gonorrhea, tuberculosis, etc.; ③ Women with uterine tumors, uterine malformations, or other gynecological diseases; ④ Women with other medical or surgical diseases. Samples were obtained from Chongqing Women and Children's Health Hospital / Women and Children's Hospital Affiliated to Chongqing Medical University.

[0066] IHC was used to locate and semi-quantitatively detect CDH3 expression in decidual tissues from two groups. Results showed that CDH3 was widely expressed in decidual epithelial cells and stromal cells. Further comparative analysis revealed that CDH3 expression in the decidual tissue of the RSA group showed a significant downregulation trend compared to the Control group. Careful observation of CDH3-positive cells in both groups revealed that the decidual stromal cells and glandular epithelial cells in the Control group exhibited a denser distribution, with a significantly increased cell number compared to the RSA group, and the cells were more tightly arranged, presenting an orderly and compact tissue structure. Figure 6A). The fundamental function of CDH3 adhesion molecules is to stabilize cell adhesion, maintain cell polarization and tissue structure, and promote cell proliferation and migration. Therefore, we believe that the cell loosening and disorder caused by the downregulation of CDH3 expression in the RSA decidua is likely related to abnormal cell-cell adhesion, abnormal cell proliferation, and abnormal polarity.

[0067] To more accurately indicate the difference in CDH3 expression levels between the Control and RSA groups, we further used Western blotting to detect CDH3 protein levels. The results showed that CDH3 expression was significantly downregulated in the RSA group compared to the Control group. Figure 6 B).

[0068] CDH3 exhibits highly specific binding patterns with other cells, achieving this binding through its extracellular domain. During pregnancy, the invasion of the decidua by trophoblasts is a crucial and complex process. Given the binding characteristics of CDH3, is it closely related to the establishment of a stable connection between trophoblasts and decidual cells? To verify this hypothesis, we further examined CDH3 expression in villous tissues from the Control and RSA groups, hoping to indirectly reveal the role of CDH3 in the decidual cell-trophoblast connection process through differential CDH3 expression in villous tissues.

[0069] We collected chorionic villus tissue from the same patient corresponding to the decidua of the Control group and RSA group, and used IHC to locate and semi-quantitatively detect CDH3 in the chorionic villus tissue. Comparative analysis revealed that, compared to the Control group, CDH3 expression in the chorionic villus tissue of the RSA group showed a significant downregulation trend. Figure 7 A).

[0070] Western blot results further confirmed that CDH3 expression was significantly downregulated in the RSA group compared to the control group. This expression trend was consistent with previous findings in decidual tissue. Figure 7 B).

[0071] The above studies show that CDH3 expression in the decidua and chorionic villi of RSA patients is significantly reduced. We speculate that CDH3 may be an important pathogenic factor at the maternal-fetal interface in RSA patients.

[0072] Example 3: CDH3 expression characteristics of endometrium / decidua before and after pregnancy To better understand the function of CDH3 molecules and their role in RSA, we first investigated the cellular localization of CDH3 in the endometrium during non-pregnancy and the decidua during pregnancy.

[0073] Endometrial tissue from non-pregnant women and decidua from pregnant women were collected and paraffin sections were prepared. Epithelial cells were labeled with the epithelial cell marker protein CK-19. Heterologous fluorescence double staining was used to detect the localization of CDH3 in the decidual tissue. The results showed that CDH3 and CK-19 were co-localized in non-pregnant women, indicating that CDH3 was mainly expressed in the epithelial cells of the endometrium during non-pregnancy. After pregnancy, in addition to co-localization with CK-19, CDH3 was also widely expressed in the stromal cells surrounding the endometrial epithelial cells. Figure 8 A).

[0074] Next, using Vimentin as a stromal cell marker, we employed heterologous fluorescence double staining to co-localize CDH3 with Vimentin. We also found that in the endometrium of non-pregnant women, CDH3 was mainly expressed in glandular epithelial cells, and that CDH3 was also widely expressed in stromal cells after pregnancy. Figure 8 B).

[0075] To further verify the accuracy and reliability of the results, immunohistochemistry was used to detect CDH3 in the endometrium of non-pregnant women and the decidua of pregnant women. The results were consistent with those of immunofluorescence, indicating that in non-pregnant women, CDH3 was mainly expressed in endometrial epithelial cells; after pregnancy, CDH3 was widely expressed in epithelial cells and stromal cells. Figure 8 C). Our study shows that CDH3 expression differs between the endometrium of non-pregnant women and the decidua of pregnant women at the cellular and tissue levels. Based on the structural characteristics of CDH3 molecules and their relatively specific intermolecular binding mode, we hypothesize that the widespread expression of CDH3 molecules in decidual cells after pregnancy facilitates trophoblast invasion, implantation, and stable anchoring of cells in the decidual tissue.

[0076] In summary, this study found that CDH3 expression at the RSA maternal-fetal interface was significantly reduced. Before implantation, CDH3 was expressed only in endometrial epithelial cells. Endometrial epithelial cells are the initial maternal cells for embryo localization, recognition, and adhesion, representing the first step towards successful implantation and healthy development. Therefore, we believe that the expression of CDH3 in endometrial epithelial cells is a key gateway factor for successful embryo implantation and normal development, and has significant research value.

[0077] Example 4: CDH3 expression characteristics in primary decidual cells After tissue identification, we further examined the expression of CDH3 in primary decidual cells, laying the foundation for subsequent research on the effect of CDH3 in endometrial cells on RSA.

[0078] We extracted decidua from women with normal early pregnancies who underwent abortion and cultured primary cells using the tissue block adherence method. We identified cellular components using the epithelial cell marker protein CK-19 and the stromal cell marker protein Vimentin. The results showed that stromal cells and epithelial cells were the main components of the primary cells. Figure 9A).

[0079] After primary cells were crawled onto slides, heterologous fluorescence double staining was used to co-localize CDH3 and CK-19. Figure 9 B), Vimentin colocalization ( Figure 9 C), it was found that CDH3 was also expressed in primary stromal cells and epithelial cells. Immunofluorescence analysis revealed that CDH3 expression was stronger in epithelial cells than in stromal cells (C). Figure 9 B). CK-19 is a membrane protein, and we found that CK-19 and CDH3 can completely colocalize ( Figure 9 B).

[0080] Therefore, we draw the following conclusions: First, CDH3 is a membrane protein, mainly expressed on the cell membrane. Second, CDH3 expression is stronger in epithelial cells than in mesenchymal cells.

[0081] Example 5: Functional experiments further demonstrated that CDH3 is a biomarker for predicting recurrent miscarriage. First, a CDH3 knockdown cell line was constructed. After successful construction, the knockdown efficiency was verified using qRT-PCR and Western blotting. The method for constructing the CDH3 knockdown cell line is as follows: (1) Lethal concentration screening: ① Cell seeding: After digesting and resuspending RL95-2 cells, seed them into 24-well plates and incubate at 37°C. ② Preparation of puromycin solutions of different concentrations: 2.5 μg / mL puromycin was prepared into concentrations of 0, 0.5, 1.0, 1.5, 2.0, and 2.5 μg / mL using complete culture medium (DMEM / F12 + 100 U / ml penicillin + 100 μg / ml Streptomycin + 0.005 mg / ml insulin) and cultured completely. ③ Cell culture: When the cell density reaches 50%, remove the complete culture medium and add complete culture medium with puromycin concentrations of 0, 0.5, 1.0, 1.5, 2.0, and 2.5 μg / mL respectively. Culture for more than 120 hours. If the medium needs to be changed, each well should still be added with complete culture medium containing the same concentration of puromycin. ④ Selecting the puromycin concentration: Select the optimal puromycin concentration that can completely kill the cells. In this experiment, under good cell conditions, 0.5 μg / mL of puromycin was the optimal concentration.

[0082] (2) Detection of stable mutant strains: Cell culture: RL95-2 cells were cultured in complete medium (DMEM / F12 + 100U / ml penicillin + 100μg / ml Streptomycin + 0.005mg / ml insulin) in a 37℃, 5%CO2 saturated humidity incubator.

[0083] Transfection with lentivirus: ① Cell seeding: After digesting and resuspending healthy cells, seed them into 6-well plates and incubate at 37°C. ② Transfection with lentivirus: Lentiviral virus was mixed with complete culture medium at a ratio of 1:10, the original cell culture was removed, diluted lentivirus complete culture medium was added, and Polybrene was added at 5 μg / mL. A blank control group was set up at the same time. The cells were incubated at 37℃ for 24 h, and the transfection status was observed. The details of the lentivirus are shown in Table 3 below, where NC is the negative control group; K9358, K9359, K9360, and K9361 are the lentivirus numbers of different interfering fragments of the target gene CDH3. ③ Culture and passage cells: Remove the complete culture medium containing lentivirus, add the complete culture medium without virus, incubate at 37℃ for 72h, when the cells have proliferated to about 90% confluence, digest and passage to 6mm dishes. ④ Screening of successfully transfected cells: When the cells have grown to about 50% in a 6 mm culture dish, add puromycin at a final concentration of 0.5-0.5 μg / mL and maintain for at least 120 h. Collect some samples for PCR detection, and continue to passage and amplify the remaining samples.

[0084] Table 3 Interference Sequences

[0085] (3) Detection of target gene expression in stable screening strains: RNA extraction: Collect the sample from step (2) ④ above, add 500 μL Trizol lysis buffer, lyse thoroughly by pipetting and shaking, add 0.1 ml chloroform, shake vigorously for 20 s, and place at room temperature for 1 min. Centrifuge at 12,000 g for 15 min at 4℃, transfer the aqueous phase to another new RNase-free EP tube, add an equal volume of 100% ethanol, add the mixture to a mini-spin centrifuge column with a 2 ml collection tube, centrifuge at 8,000 g at room temperature for 15 s, discard the lower layer liquid, add 700 μL Wash Buffer to the centrifuge column, centrifuge at 8,000 g at room temperature for 15 s, discard the liquid; wash the centrifuge column twice with 500 μL Wash Buffer; transfer the centrifuge column to another new enzyme-free EP tube, add 30 μL DEPC water, centrifuge at 10,000 g for 3 min at 4℃ to elute RNA; Primer designs are shown in Table 4, reverse transcription systems are shown in Table 5, and reaction procedures are shown in Table 6.

[0086] Table 4 qPCR primer information

[0087] Table 5 Reverse Transcription System

[0088] Table 6 PCR reaction procedure

[0089] Experimental Results: Using untreated cells (NC) as calibration and h-AXTB as an internal control, the expression level of the h-CDH3 gene was detected by qRT-PCR. The results are as follows: Figure 10 As shown in Figure A, the expression level of CDH3 in the CDH3 knockdown cell line was significantly lower than that in the control group. PCR verification showed that the PCR product size was correct and there were no extraneous bands, indicating no nonspecific amplification. Figure 10 B). Furthermore, Western blotting was used to verify the expression of CDH3 protein after knockdown, and the results showed that the expression level of the knockdown group was significantly reduced (B). Figure 10 These results indicate that the CDH3 knockdown cell line was successfully constructed.

[0090] Subsequently, we verified the effect of CDH3 knockdown on endometrial cell proliferation and migration in CDH3 knockdown cell lines. Cell viability was assessed using CCK8 assay, and the results are as follows: Figure 11 As shown in Figure A, cell viability significantly decreased after CDH3 knockdown at 48 h. The effects of CDH3 knockdown on cell proliferation and migration were then examined using EdU and cell scratch assays, with results shown below. Figure 11 As shown in B and C, knocking down CDH3 significantly reduced both cell proliferation and migration abilities.

[0091] Furthermore, we investigated the effect of CDH3 knockdown on the migration of HTR-8 / SVneo trophoblast cells. The results are as follows: Figure 12 As shown, knocking down CDH3 significantly reduced the migration ability of trophoblast cells.

[0092] Furthermore, we established a miscarriage mouse model by injecting Anti-CDH3 into the uterine cornua. The process for establishing the ICR mouse model by local intrauterine injection of Anti-CDH3 (PF-03732010) is as follows: Prepare the mice: Purchase 5 male mice with ICR 8 weeks of age and 40 female mice with ICR 8 weeks of age, and feed them for 3 days; Cage combination: Female mice: Male mice are caged in a 2:1 ratio. Check for vaginal plugs the next morning. If vaginal plugs are found, it is considered E0.5. Grouping: Each mouse was injected intrauterinely with 30 μl of PBS and the drug, with Anti-CDH3 concentrations of 0.1 mg / Kg, 0.5 mg / Kg, 1 mg / Kg, and 5 mg / Kg, respectively; Cornual injection: At E3.5, anesthetize mice by intraperitoneal injection of pentobarbital at 40-60 mg / kg body weight. After successful anesthesia, shave the hair off the right abdomen of the mouse with a shaving razor and disinfect the abdominal skin with alcohol. Cut the abdominal skin and muscle inward and downward along the upper edge of the hind leg on the right side of the abdomen to expose the right uterine horn of the mouse. Gently lift the uterine horn on this side with ophthalmic forceps, draw up Anti-CDH3, and insert the needle of the microsyringe into the uterine cavity at a 30-45° angle at the uterine horn, about 2-3 mm. After feeling the air drop, slowly inject the solution, observing that the fluid flows slowly along the uterine cavity and that there is no edema in the serosal layer. The injection dose is 30 μl. After injection, remove the injection needle. The incision was sutured and the mice were fed again: the abdominal wall incision was sutured layer by layer with 4-0 sterile absorbable sutures. The general condition of the mice was observed. After the mice recovered from anesthesia, they were placed in cages and fed again.

[0093] Experimental results are as follows Figure 13 As shown, the CDH3 level in the endometrium of patients with miscarriage was abnormal before embryo implantation. Therefore, different concentrations of Anti-CDH3 (0.1 mg / Kg, 0.5 mg / Kg, 1 mg / Kg, 5 mg / Kg, diluted with PBS) and 30 μl of PBS were slowly injected into the uterine cavity through the uterine horn one day before embryo implantation (E3.5). Figure 13 B), mice were euthanized after anesthesia on day E10.5, and their uteruses were collected. Figure 13 A). We found that different concentrations of Anti-CDH3 had varying degrees of impact on pregnancy; 0.1 mg / kg of Anti-CDH3 could lead to miscarriage. Figure 13 C (shown in black box), but its miscarriage phenotype was not as stable or significant as that of the 0.5 mg / Kg group. In the 0.5 mg / Kg group, embryonic tissue abortion and organization occurred, with corresponding significant uterine enlargement and hematoma (…). Figure 13 C, shown in the red box). Intrauterine injection of 1 mg / Kg or 5 mg / Kg of Anti-CDH3 may result in failure of embryo implantation in the uterine cavity on the injection side. Figure 13 (C, shown in the green box) 5 mg / Kg of Anti-CDH3 appears to have also affected the contralateral uterus, potentially leading to abnormal embryonic development in the contralateral uterine cavity. Figure 13 C, indicated by the white arrow).

[0094] In the previous experiment, we found that when the concentration of Anti-CDH3 reached 5 mg / kg, in addition to the embryo on the injected side being affected, the contralateral uterine cavity, which was not injected, also showed a miscarriage phenotype. Could this be due to a systemic reaction caused by the entry of Anti-CDH3 into the bloodstream, leading to an abnormal microenvironment in the contralateral uterine cavity? To verify this hypothesis, we used mice in the PBS group and the 5 mg / kg injection group as research subjects. We performed paraffin sections on the organs with rich blood supply in both groups and observed the morphology of each organ using HE staining. We found no abnormalities in any organ in either group of mice. Therefore, we infer that high concentrations of Anti-CDH3 do not cause abnormalities in the structure of other organs throughout the body. Figure 13 D).

[0095] Furthermore, HE staining of the uterus of different phenotypes in the same mouse revealed that the uterus with significant tissue swelling showed less tissue organization during pregnancy. Figure 14 The organized tissue (OT) is tightly connected to the uterine decidua, a phenotype similar to missed abortion in spontaneous abortion. Uterine swelling is not obvious; through the myometrium, a blood clot appears in the uterine cavity. HE staining results show a clearly demarcated blood clot (BC) within the uterine cavity, along with numerous endometrial glands (…). Figure 14 (Red arrow) No embryonic tissue was observed. Figure 14 (middle), similar to clinical biochemical pregnancy. The embryonic, placental, labyrinthine, and decidual structures of the normal pregnant uterus on the contralateral side were clearly visible ( Figure 14 (bottom row).

[0096] Furthermore, tissues from aborted uteruses and normal pregnant uteruses treated with 0.5 mg / Kg AntiCDH3 were collected. HE staining revealed that the placental tissue on the aborted side was organized, tightly connected to the decidua, and lacked the labyrinthine layer. Figure 15 A). Serial sections were analyzed by IHC on another section, and it was found that CDH3 expression in the decidua of the aborted uterus was significantly reduced compared with that of the normal pregnant uterus. Figure 15 A, lower right). Six mice that received intrauterine injections of 0.5 mg / Kg Anti-CDH3 were selected. Proteins were extracted from the decidual tissue of the injected side, the missed abortion phenotype uterus, and the contralateral normal pregnancy uterus. Western blot analysis revealed that the expression of CDH3, total CASP1, and cleaved-CASP1 in the decidual tissue of the abortion group was significantly lower than that in the contralateral and normal pregnancy uterine cavities. Figure 15 B).

[0097] In summary, this invention provides a biomarker for diagnosing recurrent miscarriage—CDH3, whose decreased expression can promote the occurrence of recurrent miscarriage.

[0098] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. Application of substances that detect CDH3 expression levels in samples in the preparation of diagnostic or auxiliary diagnostic, predictive or auxiliary predictive of recurrent miscarriage.

2. The application according to claim 1, characterized in that, The sample may include cells, tissues, or body fluids; Preferably, the cells include epithelial cells and trophoblast cells; Preferably, the tissue includes decidual tissue, villous tissue, and endometrial tissue; Preferably, the body fluids include blood, lymph, urine, vaginal secretions, sweat, breast milk, bile, and interstitial fluid.

3. The application according to claim 1, characterized in that, The substances used to detect CDH3 expression levels in the sample include substances that detect CDH3 gene expression levels and / or substances that detect CDH3 protein expression levels. Preferably, the substance comprises substances obtained through PCR reaction, RT... Reagents for detecting the presence, absence, and / or amount of CDH3 gene, fragments, and protein in samples using PCR-derived reactions, 3SR amplification, LCR, SDA, NASBA, TMA, SYBR Green, TaqMan probes, molecular beacons, two-hybrid probes, complex probes, ISH, microarrays, Southern blotting, Northern blotting, multianalyte assays, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, immunofluorescence assays, enzyme immunoassays, immunoprecipitation assays, chemiluminescence assays, immunohistochemical assays, dot blot assays, or narrow-line blot assays.

4. A product for diagnosing or assisting in the diagnosis of recurrent miscarriage, characterized in that, The product includes a substance for detecting CDH3 expression levels.

5. The product according to claim 4, characterized in that, The substances include substances for detecting CDH3 gene expression levels and / or substances for detecting CDH3 protein expression levels; Preferably, the product includes reagents, kits, test strips, nucleic acid membrane strips, chips, primers, and probes; Preferably, the kit includes a gene detection kit and a protein detection kit; Preferably, the subject of the test includes a human or a non-human mammal.

6. Application of CDH3 in constructing computer models or systems for diagnosis or assisted diagnosis, prediction or assisted prediction of recurrent miscarriage.

7. Application of CDH3 in screening candidate drugs for the treatment of recurrent miscarriage.

8. A medical auxiliary diagnostic system for early recurrent miscarriage, characterized in that, The system includes a result determination module, which compares the processed value of detecting the expression level of CDH3 gene or protein with a set value. Preferably, the system further includes a processing module for detecting the gene or protein expression level of CDH3 in the sample to be tested and obtaining a processed value; Preferably, the diagnostic system includes an output module for outputting the obtained diagnostic results.

9. Application of CDH3 promoters in the preparation of drugs for the prevention and / or treatment of recurrent miscarriage.

10. The application according to claim 9, characterized in that, The CDH3 promoters include substances that can increase the activity of CDH3 protein, improve the stability of CDH3 gene or protein, upregulate CDH3 protein expression, increase the effective duration of CDH3 protein action, or promote the transcription and translation of CDH3 gene.

Images