Biomarker for evaluating invasion and migration injury of trophoblast cells in preeclampsia

Abstract

The invention belongs to the technical field of biology, discloses a biomarker for evaluating preeclampsia trophoblast cell invasion and migration damage, and particularly discloses application of CCAT2 as the biomarker, and the application comprises the following steps: (a1) preparing a product for diagnosing or evaluating preeclampsia; and / or (a2) screening a product for preeclampsia treatment. The invention discloses the effect of the CCAT2 in PE for the first time, in-vitro function research (evaluation of migration and invasion functions of the CCAT2) is carried out through a trophoblast cell line, potential targets of the CCAT2 are predicted through bioinformatics, and the molecular mechanism of the CCAT2 participating in PE pathogenesis is discussed. The result shows that compared with normal pregnancy placenta tissue, the expression of CCAT2 in PE placenta tissue is reduced, the invasion and migration ability of trophoblast cells can be promoted by overexpression of CCAT2, and the effect is achieved through a CCAT2 / miR-216b-5p / ZBTB8A signal axis.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to biomarkers for evaluating trophoblast cell invasion and migration damage in preeclampsia. Background Art

[0002] Pre-eclampsia (PE) is defined as new-onset hypertension and new-onset end-organ damage (with or without proteinuria) after 20 weeks of pregnancy. Approximately 3% to 5% of pregnant women will experience pregnancy complications, such as fetal growth restriction, stillbirth, and placental abruption. In severe cases, they may further develop into eclampsia or lead to maternal death. Currently, for PE patients, clinical treatment methods can only alleviate related complications. In addition to termination of pregnancy, there is still a lack of effective cure methods. The etiology of pre-eclampsia is complex and the pathogenesis is still unclear. Its pathological process is closely related to superficial placental implantation, maternal-fetal immune imbalance, placental hypoxia, extensive vascular endothelial damage, and genetic background. A large amount of evidence shows that the potential pathophysiology of placental dysfunction is closely related to the clinical manifestations of PE complications. The formation of a normal placenta depends on the moderate proliferation, invasion and migration of trophoblasts. Extravillous trophoblasts (EVT) invade the myometrium and participate in the transformation of spiral arteries, which directly affects the anchoring of chorionic cells, ensuring that sufficient nutrients in the maternal blood enter the fetus, providing a key guarantee for the normal growth and development of the fetus. Therefore, exploring the reasons for the decline in the invasion and migration ability of trophoblasts in PE and finding the key factors for its regulation can provide a strong theoretical basis and experimental basis for the treatment of PE.

[0003] Long non-coding RNAs (lncRNAs) are involved in a variety of biological processes such as cell migration, cell invasion, cell proliferation and apoptosis, and show a variety of functional roles in different human diseases. In recent years, many studies have shown that a large number of long non-coding RNAs that regulate trophoblast invasion and migration are related to the occurrence of PE. lncZBTB39 is upregulated in PE placenta and inhibits the invasion and migration ability of HTR-8 cells. The abnormal expression of lncRNA MG828507 in PE placenta may be one of the key factors in the occurrence of PE. Recent studies have confirmed that lncRNA AFAP1-AS1 inhibits the proliferation and migration of trophoblasts by epigenetically promoting the expression of DUSP5 in PE.

[0004] Colon cancer associated transcript 2 (CCAT2) is a lncRNA located in the 8q24.21 region, which can mediate the transcriptional regulation of colon cancer cell genes, thereby promoting the proliferation and metastasis of colon cancer cells. Studies have found that CCAT2 can promote the proliferation, migration and invasion of various tumor cells. CCAT2 affects the development of osteosarcoma by enhancing the proliferation and invasion of osteosarcoma cells. CCAT2 promotes the proliferation and invasion of prostate cancer cells by regulating the Wnt / β-catenin signaling pathway. CCAT2 is abnormally highly expressed in cholangiocarcinoma tissue and promotes the epithelial-mesenchymal transition of cholangiocarcinoma cells. CCAT2 is a carcinogenic factor for hepatocellular carcinoma and can promote the invasion of cancer cells. These studies show the key role of CCAT2 in regulating cell migration and invasion. In the early stages of pregnancy, trophoblasts have similar biological functions of proliferation, migration and invasion as tumor cells.

[0005] lncRNA can directly regulate miRNA or exert corresponding biological effects through the action mode of competing endogenous RNAs (ceRNAs). CCAT2 interacts with different miRNAs to regulate the proliferation, migration and invasion of tumor cells. Studies have confirmed that CCAT2 positively regulates the expression of FOXC1 by competitive binding with miR-23b-5p, thereby regulating the proliferation and migration of lung adenocarcinoma cells; CCAT2 interacts with miR-34a, inhibits miR-34a, and thus upregulates the expression of FOXM1. FOXM1 activates CCAT2 transcription and promotes the growth of hepatocellular carcinoma.

[0006] miRNA regulates post-transcriptional gene expression by specifically binding to target messenger ribonucleic acid (mRNA), and plays an important role in regulating gene expression, cell cycle and timing of organism development. miR-424-5p inhibits inflammation of endometrial epithelial cells by targeting and binding to IRAK2. miR-424-5p targets KIF23 to inhibit the proliferation, migration and invasion of hepatocellular carcinoma. SOX4 is a downstream target of miR-214-5p and affects the proliferation, migration and invasion of osteosarcoma cells. However, the role and mechanism of CCAT2 in PE have not been reported, and it is not yet known whether it plays a key role in the pathogenesis of PE. Hereby, this application is proposed. Summary of the invention

[0007] The first aspect of the present invention aims to provide the use of CCAT2 as a biomarker.

[0008] The second aspect of the present invention aims to provide use of a reagent for detecting CCAT2 in the preparation of a product for diagnosing or evaluating preeclampsia.

[0009] The third aspect of the present invention aims to provide the use of CCAT2 as a target in the preparation of a drug for treating and / or preventing preeclampsia.

[0010] The fourth aspect of the present invention aims to provide the application of ZBTB8A promoter.

[0011] The fifth aspect of the present invention aims to provide a product.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] For the same disease, there may be changes in the expression of many related genes, different diseases will also cause changes in the expression of the same gene, and any disease or pathological state will cause changes in many genes. For disease markers, it is necessary to have the characteristics of specificity and high sensitivity in detecting a certain disease, and to be able to detect the disease state very effectively, otherwise it will cause false positives or false negatives in the results. The inventors have found that the CCAT2rs6983267G allele is associated with a reduced susceptibility to recurrent miscarriage. Trophoblast dysfunction (weak cell invasion ability) is a key factor leading to recurrent miscarriage and preeclampsia. In addition, by detecting the expression of CCAT2 in PE placenta, it was found that compared with the placenta of the normal pregnancy group, the expression of CCAT2 in the placental tissue of PE patients was significantly decreased, but the role and mechanism of down-regulated CCAT2 in PE have not been reported. The present invention adopts a bioinformatics prediction method to predict the target miRNA that CCAT2 potentially binds to, and further studies its regulatory mechanism. Through the bioinformatics database ENCORI and miR-barbase, the target genes were predicted and the specific molecular mechanism of the CCAT2 / miRNA / mRNA signal axis in the pathogenesis of PE was verified by intersecting with the sequencing results of the previous overexpression of CCAT2 in HTR-8 cells. Further in vitro functional studies were conducted using trophoblast cell lines (HTR-8 / JEG-3) (to evaluate their migration and invasion functions), focusing on the role of CCAT2 in the pathogenesis of PE. The potential targets of CCAT2 were predicted by bioinformatics to explore the molecular mechanism of CCAT2 involvement in the pathogenesis of PE. The present invention provides new insights into the key role of CCAT2 in the pathogenesis of PE, and provides a theoretical basis and experimental basis for the prediction, diagnosis, and treatment of PE.

[0014] The first aspect of the present invention provides a use of CCAT2 as a biomarker, the use comprising:

[0015] (a1) preparing a product for the diagnosis or assessment of pre-eclampsia; and / or

[0016] (a2) Screening of products for the treatment of preeclampsia.

[0017] In some embodiments of the present invention, the product includes at least one of a drug, a reagent, a kit, a test paper, a system and a chip.

[0018] In some embodiments of the invention, the diagnosing or assessing preeclampsia includes diagnosing or assessing trophoblast cell invasion and migration impairment in preeclampsia.

[0019] A second aspect of the present invention provides use of a reagent for detecting CCAT2 in the preparation of a product for diagnosing or evaluating preeclampsia.

[0020] In some embodiments of the present invention, the substance for detecting CCAT2 comprises a substance for quantitatively detecting CCAT2.

[0021] In some embodiments of the present invention, the substance includes a substance for detecting CCAT2 at the gene or protein level.

[0022] In some embodiments of the present invention, the substance for detecting CCAT2 comprises a substance for detecting CCAT2 by enzyme-linked immunosorbent assay, immunofluorescence, radioimmunoassay, immunocoprecipitation, immunoblotting, high performance liquid chromatography, capillary gel electrophoresis, near infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunoassay, fluorescent immunochromatography, surface plasmon resonance, PCR, immuno-PCR or biotin-avidin technology.

[0023] In some embodiments of the present invention, the substance for detecting CCAT2 is selected from: a substance specific to CCAT2, a probe specific to CCAT2, a gene chip, and a PCR primer.

[0024] In some embodiments of the present invention, the substance specific to CCAT2 is any one of (b1) to (b3):

[0025] (b1) an antibody that specifically binds to CCAT2;

[0026] (b2) a ligand protein or polypeptide that specifically binds to CCAT2;

[0027] (b3) Non-protein compounds that specifically recognize CCAT2.

[0028] In some embodiments of the present invention, the antibody comprises at least one of a polyclonal antibody, a monoclonal antibody, a single-chain antibody, a functional antibody fragment, an antibody Fab region, a nanobody, a chimeric antibody, and a multispecific antibody.

[0029] In some embodiments of the present invention, the product includes but is not limited to a reagent, a kit, a test paper, a system or a chip.

[0030] In some embodiments of the present invention, the test sample of the product is selected from at least one of the blood, tissues, cells, and excrement of the subject to be tested.

[0031] The third aspect of the present invention provides the use of a ZBTB8A promoter in at least one of (b1) to (b3):

[0032] (b1) preparing a medicament for treating and / or preventing preeclampsia;

[0033] (b2) promoting proliferation, migration and / or invasion of trophoblast cells;

[0034] (b3) preparing a drug for promoting the proliferation, migration and / or invasion of trophoblast cells.

[0035] In some embodiments of the present invention, the ZBTB8A promoter includes at least one of a substance that promotes ZBTB8A activity, a substance that increases the content of ZBTB8A, a substance that overexpresses ZBTB8A, and a substance that promotes ZBTB8A expression.

[0036] In some embodiments of the present invention, the ZBTB8A promoter includes a miR-216b-5p inhibitor and / or a CCAT2 promoter.

[0037] In some embodiments of the present invention, the miR-216b-5p inhibitor includes at least one of a substance that inhibits the activity of miR-216b-5p, a substance that degrades miR-216b-5p, a substance that reduces the expression level of miR-216b-5p, and a substance that knocks out / knocks down the expression of miR-216b-5p.

[0038] In some embodiments of the present invention, the miR-216b-5p inhibitor is at least one of (c1) to (c5):

[0039] (c1) sgRNA, siRNA, dsRNA, miRNA, ribozyme or shRNA targeting miR-216b-5p;

[0040] (c2) a nucleic acid molecule encoding the sgRNA, siRNA, dsRNA, miRNA, ribozyme or shRNA targeting miR-216b-5p described in (c1);

[0041] (c3) an expression cassette, vector, transgenic cell line or microorganism comprising the nucleic acid molecule described in (c1);

[0042] (c4) Small molecule drugs targeting miR-216b-5p;

[0043] (c5) CCAT2 promoter.

[0044] In some embodiments of the present invention, the ZBTB8A promoter includes an overexpression vector containing a ZBTB8A gene sequence or a virus prepared by packaging the overexpression vector.

[0045] In some embodiments of the present invention, the CCAT2 promoter includes at least one of a substance that promotes CCAT2 activity, a substance that increases CCAT2 content, a substance that overexpresses CCAT2, and a substance that promotes CCAT2 expression.

[0046] In some embodiments of the present invention, the CCAT2 promoter includes an overexpression vector containing a CCAT2 gene sequence or a virus packaged and prepared by the overexpression vector.

[0047] In some embodiments of the present invention, the trophoblast cells include but are not limited to human immortalized trophoblast cell lines and human choriocarcinoma cells.

[0048] The fourth aspect of the present invention provides a product, comprising the ZBTB8A promoter, miR-216b-5p inhibitor or CCAT2 promoter described in the third aspect of the present invention.

[0049] In some embodiments of the present invention, the functions of the product are (d1) and / or (d2):

[0050] (d1) treatment and / or prevention of preeclampsia;

[0051] (d2) Promote the proliferation, migration and / or invasion of trophoblast cells.

[0052] In some embodiments of the present invention, the trophoblast cells include but are not limited to human immortalized trophoblast cell lines and human choriocarcinoma cells.

[0053] The beneficial effects of the present invention are:

[0054] The present invention discloses for the first time the role of CCAT2 in PE, detects the expression of CCAT2 in PE placenta, and finds that compared with the placenta of the normal pregnancy group, the expression of CCAT2 in the placental tissue of PE patients is significantly decreased. In vitro functional studies (assessing its migration and invasion functions) were carried out using trophoblast cell lines (HTR-8 / JEG-3), and potential targets of CCAT2 were predicted by bioinformatics to explore the molecular mechanism of CCAT2 involvement in the pathogenesis of PE. It shows that overexpression of CCAT2 in PE placenta promotes the invasion and migration ability of trophoblast cells, and plays a role through the CCAT2 / miR-216b-5p / ZBTB8A signaling axis. The present invention provides new insights into the key role of CCAT2 in the pathogenesis of PE, and provides a theoretical basis and experimental basis for the prediction, diagnosis and treatment of PE. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Figure 2 is the correlation analysis between CCAT2 and clinically relevant phenotypes; A is the expression level of CCAT2 in placental tissue; B is the correlation analysis result between CCAT2 expression and maternal systolic blood pressure; C is the correlation analysis result between CCAT2 expression and maternal diastolic blood pressure; D is the correlation analysis result between CCAT2 expression and neonatal weight; in the figure, * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001.

[0056] Figure 2 CCAT2 promotes the migration and invasion of trophoblast cells; A and B are the overexpression efficiencies of CCAT2 in HTR-8 (A) and JEG-3 cells (B), respectively; C, E, and F are the overexpression of CCAT2 in HTR-8 cells that promotes cell migration (C, E) and invasion (F); D, E, and F are the overexpression of CCAT2 in JEG-3 cells that promotes cell migration (D, E) and invasion (F); in the figure, * represents p<0.05, ** represents p<0.01, *** represents p<0.001, **** represents p<0.0001, the scale bars for C and D are 500μm, and the scale bars for E and F are 250μm.

[0057] Figure 3The effects of CCAT2, miR-216b-5p and ZBTB8A on the proliferation ability of trophoblast cells; A and B show that overexpression of CCAT2 in HTR-8 (A) and JEG-3 (B) trophoblast cells can promote cell proliferation; C and D show that overexpression of miR-216b-5p in HTR-8 (C) and JEG-3 (D) trophoblast cells can inhibit cell proliferation; E and F show that knockdown of miR-216b-5p in HTR-8 (E) and JEG-3 (F) trophoblast mother cells promotes cell proliferation; G and H show that overexpression of ZBTB8A in HTR-8 (G) and JEG-3 (H) trophoblast cells promotes cell proliferation; in the figure, * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001.

[0058] Figure 4 The effects of CCAT2, miR-216b-5p and ZBTB8A on the trophoblast cell cycle; A and B show that overexpression of CCAT2 in HTR-8 (A) and JEG-3 (B) trophoblast cells will reduce the proportion of G1 phase and increase the proportion of S phase; C and D show that overexpression of miR-216b-5p in HTR-8 (D) and JEG-3 (C) trophoblast cells will increase the proportion of G1 phase and reduce the proportion of S phase; E and F show that knocking down miR-216b-5p will reduce the proportion of G1 phase and increase the proportion of S phase in HTR-8 (F) and JEG-3 (E) trophoblast cells; G and H show that overexpression of ZBTB8A in HTR-8 (G) and JEG-3 (H) trophoblast cells will reduce the proportion of G1 phase and increase the proportion of S phase.

[0059] Figure 5 CCAT2 downregulated the expression of miR-216b-5p; A is the miRNA that may have binding sites with CCAT2 predicted by multiple bioinformatics websites; B is the expression of the target miRNA detected by qPCR in HTR-8 overexpressing CCAT2; C is the increased expression of miR-216b-5p in PE placenta compared with the control group; D is the binding site of miR-216b-5p and CCAT2 predicted by bioinformatics; E is the direct binding effect of CCAT2 and miR-216b-5p; in the figure, ns represents no significant difference, * represents p<0.05, ** represents p<0.01, and *** represents p<0.001.

[0060] Figure 6The direct binding of CCAT2 and miR-216b-5p affects the migration and invasion of trophoblast cells; A is the overexpression / knockout efficiency of miR-216b-5p in HTR-8 / JEG-3 cells; B and C are that overexpression of miR-216b-5p in HTR-8 (B) and JEG-3 cells (C) reduces cell migration; D and E are that knockout of miR-216b-5p in HTR-8 (D) and JEG-3 cells (E) enhances cell migration; F is that overexpression of miR-216b-5p in HTR-8 and JEG-3 cells reduces Cell migration; G: Knockout of miR-216b-5p in HTR-8 and JEG-3 cells enhances cell migration; H: Overexpression of miR-216b-5p in HTR-8 and JEG-3 cells reduces cell invasion ability; I: Knockout of miR-216b-5p in HTR-8 and JEG-3 cells enhances cell invasion ability; In the figure, ns represents no significant difference, * represents p<0.05, ** represents p<0.01, **** represents p<0.0001, the scale bars of B, C, D and E are 500μm, and the scale bars of F, G, H and I are 250μm.

[0061] Figure 7 The effects of CCAT2 and miR-216b-5p on the migration and invasion of trophoblast cells; A and B show that overexpression of miR-216b-5p in HTR-8 (A) and JEG-3 cells (B) can inhibit the promotion of CCAT2 on cell migration ability; C and D show that overexpression of miR-216b-5p in HTR-8 and JEG-3 cells can inhibit the promotion of CCAT2 on cell invasion ability; in the figure, ns represents no significant difference, * represents p<0.05, ** represents p<0.01, **** represents p<0.0001, the scale bar for A and B is 500μm, and the scale bar for C and D is 250μm.

[0062] Figure 8is the target binding of miR-216b-5p and ZBTB8A; A is the intersection result of the bioinformatics database and the RNA-seq result of overexpressing CCAT2; B is the log2Foldchange sorting result; C is a heat map; D is the expression of ZBTB8A detected after overexpressing CCAT2, overexpressing and knocking out miR-216b-5p in HTR-8 cells; E is the expression of ZBTB8A detected after knocking out miR-216b-5p in HTR-8 cells; F~H are the expressions of ELK4 (F), ITGB8 (G) and ZBTB8A (H) in the placenta of PE patients, respectively; I is the binding site of miR-216b-5p and ZBTB8A predicted by bioinformatics; J is the binding site of HTR-8 The results of dual fluorescence reporter gene detection were performed in order to verify the binding of miR-216b-5p to ZBTB8A; K is the result of protein level detection, HTR-8 overexpression of miR-216b-5p will downregulate the expression of ZBTB8A, L is the result of protein level detection, HTR-8 knockout of miR-216b-5p will upregulate the expression of ZBTB8A; M is HTR-8 overexpression of miR-216b-5p weakened the promotion effect of upstream CCAT2 on ZBTB8A protein expression; N is in the placenta tissue, compared with the normal pregnancy group, the protein expression of ZBTB8A in the PE group was significantly reduced; in the figure, ns represents no significant difference, * represents p<0.05, ** represents p<0.01, **** represents p<0.0001.

[0063] Fig. 9 Figure 1 shows that ZBTB8A promotes trophoblast cell migration and invasion; A and B show the overexpression efficiency of ZBTB8A in HTR-8 (A) and JEG-3 cells (B). Figures C to F show that overexpression of ZBTB8A in HTR-8 (C) and JEG-3 cells (D) enhances cell migration (E) and invasiveness (F); G shows that overexpression of ZBTB8A in HTR-8 cells alleviates the inhibitory effect of overexpression of miR-216b-5p on migration ability; in the figure, ns means no significant difference, * means p<0.05, *** means p<0.001, **** means p<0.0001, and the scale bars in C, D and G are 500μm, and the scale bars in E and F are 250μm.

[0064] Fig.10Figure 3 is the effect of ZBTB8A on the migration and invasion of trophoblast cells; A and B are the results of scratch test and dual fluorescence reporter gene detection, respectively, and the results show that overexpression of ZBTB8A in JEG-3 cells alleviated the inhibitory effect of overexpression of miR-216b-5p on migration ability; C is the overexpression of ZBTB8A in JEG-3 cells alleviated the inhibitory effect of overexpression of miR-216b-5p on invasion ability; in the figure, * represents p<0.05, *** represents p<0.001, **** represents p<0.0001, the scale bar in A is 500μm, and the scale bars in B and C are 250μm. DETAILED DESCRIPTION

[0065] The present invention is further described in detail below through specific examples.

[0066] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0067] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0068] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0069] Example 1

[0070] Materials and methods

[0071] 1. Research subjects and specimen collection

[0072] Fifty normal pregnancies (control group) and 45 preeclampsia patients (PE group) were recruited from Guangzhou Women and Children's Medical Center. The diagnostic inclusion criteria included: pregnant women in the normal pregnancy group had no pregnancy complications, no history of miscarriage during pregnancy, and chose cesarean section for delivery; preeclampsia group: no history of previous / chronic hypertension, when the patient was in bed rest after the first 20 weeks of pregnancy, showed a systolic blood pressure greater than or equal to 140 mmHg and (or) a diastolic blood pressure greater than or equal to 90 mmHg. With or without severe proteinuria (urine protein greater than or equal to 0.3 g / 24 h), with (without) any of the following: (1) thrombocytopenia (platelets <100×10 9 / L). (2) Liver function impairment (serum transaminase level is more than 2 times the normal value). (3) Renal function impairment (serum creatinine level is greater than 1.1 mg / dl) or more than 2 times the normal value). (4) Pulmonary edema. After obtaining written informed consent from the enrolled patients, placental tissue was collected according to the protocol approved by the Guangzhou Women and Children Ethics Committee. Ethics number: Approval No.

[2022] No. 172A01.

[0073] Collection plan: Specimen collection should be completed within 15 minutes immediately after the fetus is delivered. Use scissors to cut a 1×1×1cm piece of the placenta leaflet in the central area of ​​the maternal surface of the placenta. 3 The tissue was fixed in 10% formalin, and then three pieces of the same size as soybeans were cut, rinsed with saline, placed in RNase-free cryotubes, immediately frozen in liquid nitrogen, and stored at -80°C for further use. When collecting, avoid placental calcification points, hematomas, and infarction sites.

[0074] 2. Cell Culture

[0075] HTR-8 / SVneo (human immortalized trophoblast cell line) was obtained from the American Type Cμlture Collection (ATCC) and cultured in an incubator (37°C, 5% CO2) using a culture medium mixture (RPMI-1640 medium + 10% FBS + 1% double antibody).

[0076] JEG-3 (human choriocarcinoma cell) was obtained from the American Type Culture Collection (ATCC) and cultured in an incubator (37°C, 5% CO2) using a culture medium mixture (MEM medium + 10% FBS + 1% double antibody).

[0077] 3. Adenovirus infection

[0078] The night before, an appropriate amount of HTR-8 / JEG-3 cells were evenly spread in a 6-well plate. The next day, the cells adhered to the wall and the cell density reached 60% to 70%. They were gently rinsed with PBS 1 to 2 times and replaced with medium without dual antibodies. According to the recommended virus amount in the instructions, Ad-CCAT2 and Ad-GFP adenoviruses (Genechem Technology, China) were added dropwise from the edge to the medium. The medium was slowly shaken back and forth to mix. The medium was changed 24 hours after infection. The fluorescence was observed under a fluorescence microscope 48 hours after infection, and the cells were collected, RNA was extracted, and the overexpression effect was detected by qPCR.

[0079] 4. Lentiviral Transfection

[0080] The night before, an appropriate amount of HTR-8 / JEG-3 cells were evenly plated in a 6-well plate. The next day, the cells adhered to the wall and the cell density reached 60% to 70%. They were gently rinsed with PBS 1 to 2 times and replaced with dual-antibody-free culture medium. According to the recommended virus amount in the instructions, Lv-miR-216b-5p, Lv-NC, Lv-sh-miR-216b-5p, and Lv-sh-NC lentiviruses (Vigene Biosciences, China) were added dropwise from the edge to the culture medium and slowly shaken back and forth to mix. The medium was changed 24 hours after infection. The fluorescence was observed under a fluorescence microscope 48 hours after infection, and the cells were collected, RNA was extracted, and the expression of miR-216b-5p was detected by qPCR.

[0081] 5. Plasmid Transfection

[0082] pCMV-ZBTB8A targeting human ZBTB8A and negative control were synthesized by Miaoling Biotechnology Co., Ltd. (China). The cells were seeded in a 6-well plate the night before, and the cell density reached 60% to 70%. Advanced DNA RNA high-efficiency transfection reagent (Zeta Life, China) was used, and the transfection reagent: plasmid DNA = 1:1 (4μL: 4μg) was mixed according to the instructions for the 6-well plate transfection reagent recommended in the instructions, and then incubated at room temperature for 15min. Finally, the mixture was added to the wells, and the culture plate was placed in an incubator for culture (37°C, 5% CO2). After 48h of culture, the next step of detection was performed.

[0083] 6. Real-time Quantitative PCR

[0084] Total RNA was extracted using TRIzol reagent (9109, Takara, Japan), cDNA was synthesized using PrimeScriptTM RT kit (RR036A, Takara, Japan), and fluorescent real-time quantitative PCR was performed using TB Green Premix Ex Taq IIqPCR Quantification Kit (RR820A, Takara, Japan) on an Applied Biosystems 7900 Real-Time PCR System (ThermoScientific, USA).

[0085] 7. Western blot analysis

[0086] SDS buffer (BioFroxx, Suzhou, China) was mixed with protease inhibitors (Boster, Wuhan, China). Total protein was obtained from tissues or cells, and the lysate was centrifuged at 12000 rpm for 10 min at 4 °C, and then the supernatant was collected. The protein concentration was detected using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein were transferred to PVDF membranes (Millipore, USA) by electrophoresis. The reaction was carried out for 20 min with a rapid sealing solution (Servicebio, Wuhan, China). After the reaction, the corresponding band regions were cut, and the corresponding primary antibodies of ZBTB8A (Proteintech, Wuhan, China), β-catenin (Proteintech, Wuhan, China) and GAPDH (Proteintech, Wuhan, China) were incubated at 4 °C overnight. The membrane was then incubated with the secondary antibody for 1 h at room temperature, and the bands were finally developed using an ECL colorimetric reagent (Vazyme, China).

[0087] 8. Cell Counting Kit-8 (CCK-8) Experiment

[0088] 2500 cells were seeded in 96-well plates one day in advance, and CCK-8 reagent (DOJINDO, Japan) was added according to the manufacturer's instructions and incubated in an incubator (37°C, 5% CO2) for 2 h. The cell proliferation potential at different time points (0 h, 24 h, 48 h, and 72 h) was evaluated by measuring the absorbance at a wavelength of 450 nm.

[0089] 9. Trophoblast cell migration and invasion assay

[0090] Cells (HTR-8 / JEG-3) overexpressing CCAT2, overexpressing / knockdown miR-216b-5p, and overexpressing ZBTB8A were seeded in upper wells (8 μm pore size; Corning; Corning, NY, USA) with or without Matrigel coating (invasion assay) and serum-free medium was added to the upper wells and complete medium was added to the lower wells for 24 h at 37°C and 5% CO2. Cells that passed through the membrane filter were fixed with formaldehyde, stained with 0.1% crystal violet, and photographed and counted in five random areas using an inverted microscope.

[0091] 10. Scratch test

[0092] Treated trophoblast cells HTR-8 / JEG-3 were seeded in 6-well plates, and when the cells completely covered the bottom, the monolayer was lined with a gun tip and gently rinsed with PBS. The wound width was evaluated at the specified time points (0h and 24h). The wound closure rate was calculated as follows: wound closure rate = (wound width at 0 hours - wound width at 24 hours) / wound width at 0 hours × 100%.

[0093] 11. Luciferase reporter gene analysis

[0094] The binding sites of CCAT2 and miR-216b-5p, miR-216b-5p and ZBTB8A, and ZBTB8A and β-catenin promoter were predicted on the bioinformatics website. The plasmids for luciferase reporter gene analysis were provided by Miaoling Biotechnology (Shanghai, China).

[0095] ① HTR-8 cells that successfully overexpressed miR-216b-5p were seeded in a 96-well plate the night before, with a cell number of 2×10 4 / well, pPGK-Fluc-CCAT2(human)-hRluc(CCAT2-wt) and pPGK-Fluc-CCAT2(human)-mut-hRluc(CCAT2-mut) were transfected into Lv-NC group and Lv-miR-216b-5p group, and the final groups were Lv-NC+CCAT2-wt, Lv-NC+CCAT2-mut, Lv-miR-216b-5p+CCAT2-wt, Lv-miR-216b-5+CCAT2-mut, and a background group (no transfection plasmid) was set up. The transfection used was Advanced DNA RNA high-efficiency transfection reagent (Zeta Life, China).

[0096] ② Dual luciferase reporter gene detection of miR-216b-5p and ZBTB8A: HTR-8 cells overexpressing miR-216b-5p were seeded in a 96-well plate the night before, with a cell number of 2×10 4 / well, plasmids pPGK-Fluc-ZBTB8A(human)-3UTR-wt-Rluc(ZBTB8A-wt) and pPGK-Fluc-ZBTB8A(human)-3UTR-mut-Rluc(ZBTB8A-mut) were transfected into Lv-NC group and Lv-miR-216b-5p group, and the final groups were Lv-NC+ZBTB8A-wt, Lv-NC+ZBTB8A-mut, Lv-miR-216b-5p+ZBTB8A-wt, Lv-miR-216b-5p+ZBTB8A-mut, and background group (no transfection plasmid) was set. The transfection used was Advanced DNA RNA high-efficiency transfection reagent (Zeta Life, China).

[0097] 48 hours after transfection, the dual luciferase reporter gene detection kit (RG088S, Beyotime, China) was used according to the instructions. First, the Duo-Lite Lucferase (mixed with Duo-LieLuciferase Buffer and Duo-Lite Luciferase Substrate) detection reagent with the same volume as the cell culture to be tested was added and mixed evenly, and placed at room temperature in the dark for 10 minutes to detect the luminescence of firefly luciferase (Firefly). Secondly, the Duo-Lite Stop&Lite (Duo-Lite Stop&Lite Substrate diluted 1:100 to the corresponding volume of Duo-Lite Stop&Lite Buffe) detection reagent with the same volume as the original cell culture to be tested was added and mixed evenly, and placed at room temperature in the dark for 10 minutes to detect the luminescence of Renilla luciferase (Renilla). The final statistical formula for the results is: [(Firefly in the experimental group - background Firefly) / (Renilla in the experimental group - background Renilla)] / [(Firefly in the control group - background Firefly) / (Renilla in the control group - background Renilla)].

[0098] 12. Cell cycle flow cytometry

[0099] (1) Collect the cell culture medium into a centrifuge tube. Use trypsin to digest the cells (about 1 minute), observe the cell morphology under a microscope, and when the cells are round again, add twice the volume of culture medium (with 10% serum) to stop digestion. Gently blow off all the adherent cells and collect them in the same centrifuge tube. Sediment the cells (centrifuge at 1000×g for 3 minutes). Carefully aspirate and discard the supernatant, and retain about 50μL of culture medium (avoid aspirating the cells). Then add 1mL of pre-cooled PBS, gently blow and resuspend the cells, repeat the PBS cell washing process once, carefully aspirate and discard the PBS, and retain the cell pellet. Gently tap the bottom of the centrifuge tube to prevent cells from clumping.

[0100] (2) Cell fixation: Add 1 mL of 75% ethanol (pre-cooled in advance), gently pipette to resuspend the cells, and fix them at -20°C overnight to ensure the staining effect. The next day, precipitate the cells (centrifuge at 1000×g for 3 minutes), add about 1 mL of pre-cooled PBS, and gently pipette to resuspend the cells. After repeating the PBS cell washing process, carefully aspirate and discard the supernatant, leaving about 50 μL of PBS (to avoid aspirating the cells). Gently tap the bottom of the centrifuge tube to prevent the cells from clumping.

[0101] (3) Preparation of propidium iodide staining solution (1 sample): The total volume is 0.535 mL: propidium iodide staining solution (20×) (25 μL), staining buffer (0.5 mL) and RNase A (50×) (10 μL). Prepare an appropriate amount of propidium iodide staining solution based on the number of samples to be tested.

[0102] (4) Staining: Add 0.5 mL of propidium iodide staining solution to each tube of cell sample, fully resuspend the cell pellet, and incubate at room temperature in the dark for 20 minutes. Place the sample on ice and store in the dark. Perform flow cytometry detection immediately after staining.

[0103] (5) Flow cytometric detection and analysis: Flow cytometry was used to detect the cells at an excitation wavelength of 488 nm and to detect light scattering. FlowJo software was used for analysis.

[0104] result

[0105] 1. General characteristics of the study subjects and clinical data analysis

[0106] In this experiment, a total of 50 normal pregnant women in the control group and 45 patients in the PE group were collected. The general characteristics of the subjects are shown in Table 1. Among them, there was no statistically significant difference in maternal age (p>0.05). In addition, compared with the control group, PE patients had significantly higher systolic blood pressure (p<0.05). Both systolic blood pressure (p<0.0001) and diastolic blood pressure (p<0.0001) were significantly higher than those in the control group. p<0.0001), neonatal weight (p<0.0001) and gestational age (p<0.0001) and gestational age (p<0.0001) were significantly reduced. In addition, 70.21% of PE patients had proteinuria.

[0107] Table 1 General characteristics of the study subjects

[0108]

[0109]

[0110] Note: The t-test was used for comparison among the groups, p<0.05 was considered statistically significant, and the sample mean was expressed as SD±standard deviation.

[0111] 2. CCAT2 is lowly expressed in PE placentas

[0112] The experimental results showed that the expression of CCAT2 in placental tissue of PE patients was lower than that in the control group ( Figure 1 In addition, a correlation analysis between CCAT2 and clinically relevant characteristics was performed, and the results showed that CCAT2 expression was negatively correlated with maternal systolic and diastolic blood pressure ( Figure 1 B and C), and no significant correlation with neonatal weight ( Figure 1 (middle D).

[0113] 3. Overexpression of CCAT2 in vitro promotes trophoblast cell proliferation, migration and invasion

[0114] In order to evaluate whether CCAT2 affects the function of trophoblast cells, the present invention constructed cell lines (HTR-8 and JEG-3) overexpressing CCAT2 ( Figure 2 The results showed that Ad-CCAT2 significantly enhanced the migration of HTR-8 / JEG-3 cells compared with Ad-NC ( Figure 2 C, D, E) and invasion ( Figure 2 F) ability, indicating that overexpression of CCAT2 can promote trophoblast cell proliferation, migration and invasion. At the same time, it also promotes cell proliferation to a certain extent ( Figure 3 A and B), reduced the G1 phase of the cell cycle and increased the S phase ( Figure 4 A and B).

[0115] 4. CCAT2 targets miR-216b-5p

[0116] LncRNA plays a key role in many diseases through its interaction with miRNA. The present invention uses multiple biological gene prediction websites (LncBook, LncRNA2Target, NPInter, LncACTdb, DIANA-LncBase) to predict potential miRNAs that may bind to CCAT2 and produce crosstalk ( Figure 5 The expression of miRNAs in trophoblastic cell lines overexpressing CCAT2 was further detected, and it was found that the decrease in miR-216b-5p was the most significant ( Figure 5 B), and subsequently found that the expression of miR-216b-5p was increased in PE placentas ( Figure 5 In order to verify the direct interaction between CCAT2 and miR-216b-5p, the present invention predicted the potential binding sites between the two ( Figure 5 D), and constructed a CCAT2 mutant plasmid. Through the dual luciferase experiment, the results showed that the luciferase activity decreased after the binding sequence mutation, which verified that CCAT2 targeted binding to miR-216b-5p ( Figure 5 Middle E).

[0117] 5. Direct binding of CCAT2 to miR-216b-5p affects proliferation, migration and invasion of trophoblast cells

[0118] The present invention constructs a stable trophoblast cell line with overexpression and knockout of miR-216b-5p ( Figure 6Middle A), found that the migration of cells after overexpression of miR-216b-5p ( Figure 6 B, C, F) and invasion ( Figure 6 The ability of H) in the middle layer is reduced, and the cell activity of the trophoblast cells is weakened ( Figure 3 Middle C, D, Figure 4 After knocking out miR-216b-5p, this phenomenon was reversed ( Figure 6 Surprisingly, it was found that when CCAT2 and miR-216b-5p were overexpressed simultaneously in trophoblast cells, the positive effect of overexpression of CCAT2 was suppressed by overexpression of miR-216b-5p ( Figure 7 (A~D).

[0119] 6. CCTA2 regulates ZBTB8A via miR-216b-5p

[0120] In order to explore how CCAT2 affects the function of trophoblast cells, the present invention conducted RNA-seq detection on trophoblast cells overexpressing CCAT2 at GENERGY BIO Technology (Shanghai, China). In addition, the potential binding target genes of miR-216b-5p were predicted through multiple bioinformatics websites, and the data were cross-analyzed ( Figure 8 The results showed that ZBTB8A was among the top 10 genes with the most significant changes ( Figure 8 By qPCR, it was found that ZBTB8A, ITGB8 and ELK4 were target genes with high differences ( Figure 8 D and E). ITGB8 is reported to regulate the invasion and migration of trophoblastic cells and mediate the progression of preeclampsia. The expression of ZBTB8A, ITGB8 and ELK4 in placental tissues of PE patients was further detected. The results showed that there was no significant difference in ELK4 between the control group and the PE group ( Figure 8 Compared with the control group, the expression of ITGB8 showed a downward trend ( Figure 8 G), while the expression of ZBTB8A in PE placenta tissue was significantly decreased ( Figure 8 Therefore, the present invention selects ZBTB8A as the target gene for the next step of research. The binding sequence of miR-216b-5p and ZBTB8A was found on the bioinformatics website. The luciferin activity of the non-mutated group decreased (the binding sequence did not change), which verified that miR-216b-5p can directly target ZBTB8A ( Figure 8I, J). The protein expression of ZBTB8A was detected by overexpressing and knocking out miR-216b-5p in HTR-8 cells. The results showed that in the Lv-miR-216b-5p group, the protein expression of ZBTB8A was significantly decreased compared with that in the Lv-NC group ( Figure 8 K), and this trend was reversed after knocking out miR-216b-5p ( Figure 8 In addition, in the placental tissue, the expression of ZBTB8A protein in the PE group was significantly decreased compared with the normal pregnancy group ( Figure 8 N in the middle). It was also found that overexpression of miR-216b-5p could weaken the promoting effect of upstream CCAT on ZBTB8A protein expression ( Figure 8 Therefore, CCAT2 may regulate ZBTB8A through miR-216b-5p, leading to changes in trophoblast cell function.

[0121] 7. miR-216b-5p inhibits the cell cycle, migration and invasion of trophoblast cells by downregulating the expression of ZBTB8A

[0122] The present invention uses a ZBTB8A overexpression plasmid to construct a ZBTB8A overexpression cell line to explore whether ZBTB8A affects the function of trophoblast cells ( Fig. 9 In addition, the cell cycle results showed that ZBTB8A reduced the proportion of G1 phase and increased the proportion of S phase, but the proportion of S+G2 phase was slightly different between the two groups (4H, I). Cell migration and invasion abilities were also significantly enhanced ( Fig. 9 In order to further explain the relationship between miR-216b-5p and ZBTB8A, the present invention simultaneously overexpressed miR-216b-5p and ZBTB8A, and found that the negative effects of overexpression of ZBTB8A on trophoblast cells were reversed ( Fig. 9 Medium G and Fig.10 A to C), indicating that the CCAT2 / miR-216b-5p / ZBTB8A signaling axis affects trophoblast cells, and miR-216b-5p inhibits the cell cycle, migration and invasion of trophoblast cells by downregulating the expression of ZBTB8A.

[0123] In summary, the expression of CCAT2 is reduced in PE placenta, and in vitro experiments have shown that trophoblast cells overexpressing CCAT2 have stronger proliferation, migration and invasion abilities. Subsequent studies have shown that CCAT2 interacts with miR-216b-5p and ultimately targets ZBTB8A. In trophoblast cells, overexpression of miR-216b-5p / overexpression of ZBTB8A reversed the promoting effects of overexpression of CCAT2 / overexpression of miR-216b-5p, respectively. In addition, by detecting the expression of miR-216b-5p and ZBTB8A in PE placental tissue, it was found that miR-216b-5p was expressed at an increased level in PE placental tissue, and ZBTB8A was expressed at a low level in PE placental tissue. The exploration of related mechanisms showed that the CCAT2 / miR-216b-5p / ZBTB8A signaling axis affects the migration and invasion abilities of trophoblast cells, which helps to clarify the pathogenesis of PE.

[0124] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. The use of CCAT2 as a biomarker is characterized by: The applications include: (a1) preparing a product for the diagnosis or assessment of pre-eclampsia; and / or (a2) Screening of products for the treatment of preeclampsia.

2. Use of reagents for detecting CCAT2 in the preparation of products for diagnosing or evaluating preeclampsia.

3. The use according to claim 2, characterized in that: The substances include substances for detecting CCAT2 at the gene or protein level.

4. The use according to claim 3, characterized in that: The substance for detecting CCAT2 includes a substance for detecting CCAT2 by enzyme-linked immunosorbent assay, immunofluorescence, radioimmunoassay, immunocoprecipitation, immunoblotting, high performance liquid chromatography, capillary gel electrophoresis, near infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunoassay, fluorescent immunochromatography, surface plasmon resonance, PCR, immuno-PCR or biotin-avidin technology.

5. The use according to any one of claims 2 to 4, characterized in that: The product includes at least one of a reagent, a test kit, a test paper, a system and a chip.

6. Application of ZBTB8A promoter in at least one of (b1) to (b3): (b1) preparing a medicament for treating and / or preventing preeclampsia; (b2) promoting proliferation, migration and / or invasion of trophoblast cells; (b3) preparing a drug for promoting the proliferation, migration and / or invasion of trophoblast cells.

7. The use according to claim 6, characterized in that: The ZBTB8A promoter includes at least one of a substance that promotes ZBTB8A activity, a substance that increases the content of ZBTB8A, a substance that overexpresses ZBTB8A, and a substance that promotes ZBTB8A expression.

8. The use according to claim 7, characterized in that: The ZBTB8A promoter includes a miR-216b-5p inhibitor and / or a CCAT2 promoter; Preferably, the miR-216b-5p inhibitor includes at least one of a substance that inhibits the activity of miR-216b-5p, a substance that degrades miR-216b-5p, a substance that reduces the expression level of miR-216b-5p, and a substance that knocks out / knocks down the expression of miR-216b-5p; Preferably, the miR-216b-5p inhibitor is at least one of (c1) to (c5): (c1) sgRNA, siRNA, dsRNA, miRNA, ribozyme or shRNA targeting miR-216b-5p; (c2) a nucleic acid molecule encoding the sgRNA, siRNA, dsRNA, miRNA, ribozyme or shRNA targeting miR-216b-5p described in (c1); (c3) an expression cassette, vector, transgenic cell line or microorganism comprising the nucleic acid molecule described in (c1); (c4) Small molecule drugs targeting miR-216b-5p; (c5) CCAT2 promoter.

9. The use according to claim 8, characterized in that: The CCAT2 promoter includes at least one of a substance that promotes CCAT2 activity, a substance that increases CCAT2 content, a substance that overexpresses CCAT2, and a substance that promotes CCAT2 expression.

10. A product comprising the ZBTB8A promoter according to any one of claims 6 to 9, the miR-216b-5p inhibitor according to claim 8, or the CCAT2 promoter according to claim 8 or 9.

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