Application of transcription factor SP1 or YY1 in regulating expression of RSA risk gene ANXA5 or KDR

Abstract

The invention discloses application of a transcription factor SP1 or YY1 in regulation and control of expression of a recurrent spontaneous abortion risk gene ANXA5 or KDR, the transcription factor SP1 regulates and controls the expression of the ANXA5 gene, and the transcription factor YY1 regulates and controls the expression of the KDR gene. The research shows that the reduction of the transcriptional activity of the genes at the SNP sites of the ANXA5 and KDR starting regions is realized by changing the combination with transcription factors SP1 and YY1. The method is beneficial to deep analysis and understanding of genetic etiology of RSA, so that a theoretical basis is provided for clinical early prevention and treatment and individualized treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to the application of transcription factors SP1 or YY1 in regulating the expression of RSA risk genes ANXA5 or KDR. Background Art

[0002] Recurrent spontaneous abortion (RSA) refers to three or more spontaneous abortions before 20 weeks of pregnancy. It is estimated that RSA affects about 1-5% of women of childbearing age globally, and its incidence is on the rise year by year. So far, several risk factors associated with RAS have been identified, including chromosomal abnormalities, anatomical defects of reproductive organs, endocrine disorders, immune disorders, and thrombotic diseases, etc. However, unexplained recurrent spontaneous abortion (URSA) still accounts for nearly 50% of cases. Repeated abortions can cause endometrial injury, infection, and intrauterine adhesions in patients, and in severe cases, can lead to infertility. In addition to causing serious physical harm to patients, because the cause of URSA is unknown, patients often worry about whether they will have another abortion, and the mental and psychological pressure is relatively high, which is not conducive to family happiness and social harmony. Therefore, it is particularly urgent to explore the molecular mechanism of URSA occurrence.

[0003] The exploration of the correlation between gene single nucleotide polymorphism (SNP) and diseases has gradually attracted the attention of researchers. The exploration of the relationship between gene variation and URSA can be traced back to 1990, mainly starting from genes related to thrombosis. As is well known, the success of pregnancy depends on placental blood circulation. Thrombus clots and placental infarcts in the placenta can both cause adverse pregnancy outcomes. Compared with normal pregnant women, the risk of pregnancy complications in women with anticoagulation defects increases by 3-8 times. Abnormal blood coagulation function is one of the main causes of RSA. Since it is difficult to identify the cause, it is speculated that 55% of URSA cases may be related to it.

[0004] Annexin A5 (ANXA5) is a protein that is highly expressed in normal placenta and has anticoagulant function under physiological conditions. In the presence of calcium ions, ANXA5 binds to phosphatidylserine (PS) that is naturally exposed on the apical surface of placental syncytiotrophoblast cells, establishing a two-dimensional barrier that interferes with phospholipid-dependent coagulation reactions. In addition, ANXA5 plays an important role in promoting membrane repair, which is crucial for the integrity of a healthy placenta. Under pathological conditions, if there are anti-phospholipid antibodies or anti-annexin antibodies, or even when the ANXA5 expression level is reduced, due to the hypercoagulable state in the intervillous space, the barrier established by ANXA5 will be disrupted, and such situations are considered to be a significant risk factor affecting normal pregnancy outcomes. During implantation and embryonic development, good placental circulation and fetal vascular system are necessary conditions for maintaining pregnancy. Placental angiogenesis affects blood flow exchange between the uterus (maternal placenta) and the umbilical cord (fetal placenta), and is the key to successful fetal development. Vascular endothelial growth factor (VEGF) has been proven to be related to RSA. Kinase insert domain receptor (KDR), as the main signal transduction receptor of vascular endothelial growth factor receptor (VEGFR), can play a role in placental angiogenesis through the VEGF-KDR pathway. KDR is expressed in placental angiogenesis and angiogenic precursor cells and participates in regulating the proliferation and migration of endothelial cells during the process of blood vessel formation. Abnormal expression or function of KDR will inhibit the activity of VEGF, slow down angiogenesis, lead to shallow placental implantation, ischemia and hypoxia, and even miscarriage.

[0005] Previous studies have shown that ANXA5 and KDR gene mutations are closely related to RSA. For example, Literature 1 reported mutant ANXA5 (c.949G>C; p.G317R), KDR (c.2440G>A; p.D814N); Literature 2 reported mutations in the KDR gene: KDR-604T→C (rs2071559), 1192G→A (rs2305948), and 1719A→T (rs1870377); Literature 3 reported mutations in ANXA5: rs112782763, rs28717001, rs28651243, and rs113588187.

[0006] Literature 1: Identification of genetic polymorphisms in unexplained recurrent spontaneous abortion based on whole exome sequencing; Jiang-Tao Mou, Shi-Xing Huang; Ann Transl Med. 2022 May; 10(10):603. doi: 10.21037 / atm-22-2179.

[0007] Literature 2: Association of kinase insert domain-containing receptor (KDR) gene polymorphisms with idiopathic recurrent spontaneous abortion in Korean women; Hyungchul Rah, Young Joo Jeon,; Fertil Steril. 2013 Mar 1; 99(3):753-760.e8. doi: 10.1016 / j.fertnstert.2012.10.038.

[0008] Literature 3: Investigation of the Annexin A5 M2 haplotype in 500 white European couples who have experienced recurrent spontaneous abortion; Charalambos Demetriou, Sayeda Abu-Amero; Reprod Biomed Online. 2015 Nov; 31(5):681-8. doi: 10.1016 / j.rbmo.2015.07.004. Summary of the Invention

[0009] The object of the present invention is to provide an application of transcription factor SP1 or YY1 in regulating the expression of RSA risk genes ANXA5 or KDR in view of the above problems.

[0010] In order to achieve its object, the technical solution adopted by the present invention is as follows:

[0011] The first aspect of the present invention provides the use of transcription factors in the preparation of reagents for regulating the expression of genes related to the risk of recurrent spontaneous abortion, wherein the transcription factors are SP1 or YY1, the genes related to the risk of recurrent spontaneous abortion are human annexin ANXA5 gene or vascular endothelial growth factor type II receptor KDR gene, the transcription factor SP1 regulates the expression of the ANXA5 gene, and the transcription factor YY1 regulates the expression of the KDR gene.

[0012] The transcription factor SP1 binds to the promoter region of the ANXA5 gene, and the transcription factor YY1 binds to the promoter region of the KDR gene.

[0013] Furthermore, the transcription factor SP1 binds to the SNP mutation site in the promoter region of the ANXA5 gene that causes recurrent spontaneous abortion, and the transcription factor YY1 binds to the SNP mutation site in the promoter region of the KDR gene that causes recurrent spontaneous abortion.

[0014] Among them, the transcription factor SP1 positively regulates the ANXA5 gene, and the transcription factor YY1 positively regulates the KDR gene.

[0015] The second aspect of the present invention provides the use of agents that regulate the activity and / or expression of the transcription factor SP1 or YY1 in the preparation of reagents for regulating the expression of genes related to the risk of recurrent spontaneous abortion, wherein the genes related to the risk of recurrent spontaneous abortion are human annexin ANXA5 gene or vascular endothelial growth factor type II receptor KDR gene. Agents that regulate the activity and / or expression of the transcription factor SP1 regulate the expression of the ANXA5 gene, and agents that regulate the activity and / or expression of the transcription factor YY1 regulate the expression of the KDR gene.

[0016] In the above application technical solutions, agents that promote the activity and / or expression of the transcription factor SP1 promote the expression of the ANXA5 gene, and agents that inhibit the activity and / or expression of the transcription factor SP1 inhibit the expression of the ANXA5 gene; agents that promote the activity and / or expression of the transcription factor YY1 promote the expression of the KDR gene, and agents that inhibit the activity and / or expression of the transcription factor YY1 inhibit the expression of the KDR gene.

[0017] In the above application technical solutions, the agent that inhibits the activity and / or expression of the transcription factor SP1 is siRNA of SP1, and the agent that inhibits the activity and / or expression of the transcription factor YY1 is siRNA of SP1.

[0018] Preferably, the nucleotide sequence of the siRNA of SP1 is: 5’-GCUGGUGGUGAUGGAAUACAU-3’;

[0019] The nucleotide sequence of the siRNA of YY1 is: 5’-GACGACGACUACAUUGAACAA-3’.

[0020] The third aspect of the present invention provides the application of a transcription factor regulator in the preparation of a drug for treating recurrent spontaneous abortion, wherein the transcription factor is SP1 or YY1, the regulator promotes the activity and / or expression of the transcription factor SP1 or YY1, and the recurrent spontaneous abortion is caused by SNP mutations in the risk genes ANXA5 gene or KDR gene.

[0021] In the above application technical solution, the regulator promotes the activity and / or expression of the transcription factor SP1 or YY1, thereby promoting the binding of SP1 to the promoter region of the ANXA5 gene or promoting the binding of YY1 to the promoter region of the KDR gene, and improving the transcriptional activity of the ANXA5 gene or KDR gene.

[0022] The beneficial effects of the present invention are:

[0023] Based on the association between the SNP sites in the promoters of the risk genes ANXA5 and KDR and URSA, the regulatory relationship between the transcription factors SP1 or YY1 and the RSA risk genes was discovered: the fragment containing the SNP site in the ANXA5 promoter region binds to the transcription factor SP1, the fragment containing the SNP site in the KDR promoter region binds to YY1, and the change in the SNP site will affect the binding rate between the promoter region and the transcription factor, thereby leading to a change in gene transcriptional activity; the silencing of SP1 and YY1 is positively correlated with the expression of ANXA5 and KDR, the gene and protein expression levels of ANXA5 decrease with the silencing of SP1, and the gene and protein expression levels of KDR decrease with the knockdown of YY1; the research of the present invention shows that the SNP sites in the promoters of ANXA5 and KDR reduce the transcriptional activity of the genes by changing the binding to the transcription factors SP1 and YY1.

[0024] The present invention is conducive to in-depth analysis and understanding of the genetic etiology of RSA, thereby providing a theoretical basis for early clinical prevention and treatment and individualized treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shows the binding sites of the transcription factor SP1 predicted by PROMO to the ANXA5 promoter fragment (A) and the binding sites of the transcription factor YY1 to the KDR promoter fragment (B).

[0026] Figure 2 Is the electrophoresis pattern of amplifying the target fragment with biotin-labeled primers. ①-⑥ are biotin-labeled - ANXA5 - wild type, full mutation, single mutation, biotin-labeled - KDR - wild type, full mutation, single mutation in sequence.

[0027] Figure 3 It was as follows: (A) Silver staining results after ANXA5 probe pulled down HTR-8 nuclear proteins: M: 130 kDa protein marker; ①: Protein pulled down by ANXA5-wild type probe; ②: Protein pulled down by ANXA5-full mutation probe; ③: Protein pulled down by ANXA5-single mutation probe; ④: Input HTR-8 nuclear proteins; ⑤: Blank control without probe. (B) Silver staining results after KDR probe pulled down HUVEC nuclear proteins: M: 130 kDa protein marker, ①: Protein pulled down by KDR-wild type probe; ②: Protein pulled down by KDR-full mutation probe; ③: Protein pulled down by KDR-single mutation probe; ④: Input HUVEC nuclear proteins; ⑤: Blank control without probe.

[0028] Figure 4 It showed: (A) WB results of proteins pulled down by ANXA5 promoter region fragments: ①②③: Input of HTR-8 nuclear proteins; ④: Protein pulled down by ANXA5 wild type fragment; ⑤: Protein pulled down by ANXA5 full mutation fragment; ⑥: Protein pulled down by ANXA5 single mutation (SNP5) fragment; ⑦: Control without DNA probe (pulled down after incubating naked magnetic beads with HTR-8 nuclear proteins). (B) ①②③: Input of HUVEC nuclear proteins; ④: Protein pulled down by KDR wild type fragment; ⑤: Protein pulled down by KDR full mutation fragment; ⑥: Protein pulled down by KDR single mutation (SNP3) fragment; ⑦: Control without DNA probe (pulled down after incubating naked magnetic beads with HUVEC nuclear proteins).

[0029] Figure 5 It showed the effects of siRNA silencing of SP1 (A) and YY1 (B).

[0030] Figure 6 It showed the effects of siRNA silencing of SP1 (A) and YY1 (B) at different transfection times.

[0031] Figure 7 It showed the changes in A5 expression level after silencing SP1 (A) and the changes in KDR expression level after silencing YY1 (B).

[0032] Figure 8 It showed the changes in A5 expression level after silencing SP1 at different transfection times (A) and the changes in KDR expression level after silencing YY1 at different transfection times (B).

[0033] Figure 9 It showed the changes in ANXA5 gene protein expression after silencing SP1: ① HTR-8 nuclear proteins; ② siRNA NC control; ③ siRNA SP1 knockdown.

[0034] Figure 10Shows the change in the protein expression of the KDR gene after silencing YY1: ① Nuclear protein of HUVEC; ② siRNA NC control; ③ siRNA YY1 knockdown. Detailed implementation manners

[0035] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0036] The experimental methods in the following embodiments are all conventional methods unless otherwise specified.

[0037] Example 1: Bioinformatics prediction of transcription factors that may bind to the promoter regions of A5 and KDR genes

[0038] 1 Transcription factor prediction

[0039] Download the sequences of ANXA5 (NCBI Gene ID: 308) and KDR gene (NCBI Gene ID: 3791) in NCBI

[0040] -200 to 2000 bp sequences are used as the promoter region, and the error tolerance rate is set to 5% in the PROMO database and Jaspar, and the species is human for the transcription factor prediction of the target gene.

[0041] 2 Prediction results

[0042] Figure 1 Shows the binding sites of the transcription factor SP1 predicted by PROMO to the ANXA5 promoter fragment (A) and the binding sites of the transcription factor YY1 to the KDR promoter fragment (B).

[0043] The binding sites of the transcription factor SP1 predicted by JASPA to the ANXA5 promoter fragment are shown in Table 1, and the binding sites of the transcription factor YY1 predicted by JASPA to the KDR promoter fragment are shown in Table 2.

[0044] Table 1 Binding sites of the transcription factor SP1 predicted by JASPA to the ANXA5 promoter fragment

[0045]

[0046] Table 2 Binding sites of the transcription factor YY1 predicted by JASPA to the KDR promoter fragment

[0047]

[0048] Transcription factors play an irreplaceable role in gene transcriptional regulation. Mutations in the SNP sites of the ANXA5 and KDR genes reduce gene transcriptional activity. Therefore, we believe that variations in SNP sites can change the binding of gene promoters to transcription factors, thereby affecting gene transcriptional activity. Through bioinformatics analysis, we predicted that the transcription factor SP1 may bind to the promoter region of ANXA5, and the transcription factor YY1 may bind to the promoter region of KDR. Next, we will use these transcription factors that may bind to the promoter regions of ANXA5 and KDR to explore the role of the SNP sites of the ANXA5 and KDR genes in the formation of URSA.

[0049] Example 2: DNA-pull-down verification of promoter-binding proteins

[0050] 1 Main experimental reagents and sources

[0051]

[0052] 2 Experimental methods

[0053] 2.1 Preparation methods of main reagents

[0054] (1) Rinsing buffer: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl.

[0055] (2) Binding buffer: 50 mM Tris, 150 mM NaCl, 0.1% - 0.5% Tween 20, pH 7.5.

[0056] (3) Western Blot electrophoresis buffer: 3.03 g of Tris-base, 14.4 g of Glycine, 1 g of SDS, made up to 1 L with ddH2O and stored at room temperature.

[0057] (4) Western Blot transfer buffer: 3.03 g of Tris-base, 14.4 g of Glycine, dissolved in 600 ml of ddH2O and then added with 200 mL of methanol, made up to 1 L. Methanol should be added freshly when in use and stored at room temperature.

[0058] (5) Preparation of TBST: 8 g of NaCl, 2.42 g of Tris-base, 1 mL of Tween-20, made up to 1 L with ddH2O and stored at room temperature.

[0059] (7) Western Blot blocking solution: 5 g of skim milk powder was fully dissolved in 100 mL of 1×TBST and stored at 4°C.

[0060] (8) 10% separating gel: 5 mL of 30% polyacrylamide, 5.7 mL of 1 M Tris (pH 8.8), 150 μL of 10% SDS, 150 μL

[0061] 10% ammonium persulfate, 4.0 mL of double-distilled water, 6 μL of tetramethylethylenediamine, add them into a beaker in sequence and mix well. Prepare and use immediately.

[0062] (9) 5% stacking gel: 1.3 mL of 30% polyacrylamide, 1 mL of 1 M Tris (pH 6.8), 80 μL of 10% SDS, 80 μL

[0063] 10% ammonium persulfate, 5.5 mL of double-distilled water, 8 μL of tetramethylethylenediamine, add them into a beaker in sequence and mix well. Prepare and use immediately.

[0064] 2.2 DNA-pull-down

[0065] 2.2.1 Preparation of biotin-labeled probe

[0066] Let the 5' end of the primer for amplifying the target fragment be synthesized and labeled with biotin by Sangon Biotech, and amplify the target fragment with the biotinylated primer.

[0067] 2.2.2 Cell culture

[0068] Conventional method.

[0069] 2.2.3 Extraction of nuclear proteins

[0070] Extract using the nuclear protein extraction kit from Sangon Biotech.

[0071] 2.2.4 BCA protein quantification

[0072] Detect using the BCA protein quantification kit from Dingguo ChangSheng Biotechnology.

[0073] 2.2.5 DNA-pull-down

[0074] (1) Gently pipette and resuspend the streptavidin magnetic beads, take 50 μL and put it into a centrifuge tube;

[0075] (2) Add 500 μL of washing buffer, resuspend the magnetic beads, let them stand on the magnetic stand for 1 min, aspirate and discard the supernatant, and wash twice;

[0076] (3) Dilute the prepared biotinylated DNA probe with binding buffer to a final concentration of 10 - 25 μg / mL, and place it on ice for later use;

[0077] (4) Add the diluted DNA probe to the pretreated magnetic beads, incubate at room temperature for 2 h, then place on a magnetic stand. After 1 min, when the magnetic beads are adsorbed on one side of the centrifuge tube, carefully discard the supernatant. The remaining is the biotin-labeled DNA probe-magnetic bead complex;

[0078] (5) Add 500 μL of rinsing buffer to the complex, gently resuspend the magnetic beads, let stand on the magnetic stand for 1 min, then carefully aspirate and discard the supernatant, and rinse twice;

[0079] (6) Add 200 μL of the prepared nuclear protein (add the DNA probe of ANXA5 to the nuclear protein of HTR-8 cells, and add the DNA probe of KDR to the nuclear protein of HUVEC cells) to the washed DNA probe-magnetic bead complex, and incubate at 4 °C for 2 h;

[0080] (7) Adsorb on the magnetic stand for 1 min, discard the supernatant. The remaining in the centrifuge tube is the nuclear protein-DNA probe-magnetic bead complex;

[0081] (8) Add 1 mL of rinsing buffer to the complex obtained in the previous step, gently resuspend the magnetic beads with a pipette, let stand on the magnetic stand for 1 min, discard the supernatant, and repeat three times;

[0082] (9) Add 5×SDS-PAGE loading buffer to the washed nuclear protein-DNA probe-magnetic bead complex and mix well, then heat and denature at 100 °C for 10 min;

[0083] (10) After cooling, adsorb the centrifuge tube on the magnetic stand for 1 min and then collect the supernatant to obtain the pulled-down protein, and store it at -80 °C

[0084] for preservation.

[0085] 2.3 SDS-PAGE protein electrophoresis

[0086] Take 10 μL of the protein sample and 5 μL of protein marker and load them into the wells of a 10% SDS-PAGE gel. Add SDS electrophoresis buffer, turn on the power supply, select constant voltage to start electrophoresis. First use 80 V (about 40 min), and when the marker starts to separate, switch to 120 V (about 80 min).

[0087] 2.4 Silver staining

[0088] Use a silver staining kit (Beyotime), and the basic steps are as follows:

[0089] Fix, wash with 30% ethanol, wash with water, sensitize, wash with water twice, silver stain, wash with water, develop color, terminate, wash with water, and store in ultrapure water

[0090] 2.5 Western Blot: Conventional method.

[0091] 3 Experimental Results

[0092] 3.1 Biotin-labeled Probe Amplification

[0093] The target fragment was amplified with biotin-labeled primers and verified by electrophoresis on 2% agarose gel. The amplified fragments with clear bands, correct positions and sizes, and clean backgrounds were sent to Sangon for sequencing. The results are as Figure 2 , showing that the biotin-labeled ANXA5 (900bp) and KDR (900bp) probes were successfully prepared.

[0094] 3.2 DNA-pull-down Silver Staining

[0095] According to the literature "Analysis of the Association between ANXA5 and KDR Promoter Polymorphisms and the Genetic Risk of Recurrent Miscarriage in Chinese Han Women" (Wang Juan et al., 2016, DOI: 10.13417 / j.gab.034.002314) and "Molecular Mechanism of the Association between Single Nucleotide Polymorphisms in the Promoters of ANXA5 and KDR Genes and Unexplained Recurrent Spontaneous Miscarriage" (Liu Guicen, 2018, Master's Thesis, DOI: CNKI:CDMD:2.1018.864283) reporting ANXA5 and KDR promoter SNP mutations related to recurrent miscarriage, SNP mutations were selected for DNA-pull-down experiments.

[0096] For the gene ANXA5, six sites of full mutant types were selected: SNP1 (-467G>A), SNP2 (-448A>C), SNP3 (-422T>C), SNP4 (-373G>A), SNP5 (-302T>G), and SNP6 (-1C>T), as well as the single mutant type and wild type of only the SNP5 (-302T>G) site. For the gene KDR, six sites of full mutant types were selected: SNP1 (-679G>A), SNP2 (-645C>G), SNP3 (-607T>C), SNP4 (-565T>C), SNP5 (-367T>C), and SNP6 (-271A>G), as well as the single mutant type and wild type of only the SNP3 (-607T>C) site.

[0097] The biotin-labeled DNA probe was co-incubated with nuclear proteins (ANXA5 and HTR-8, KDR and HUVEC), and then streptavidin magnetic beads were added to pull down the proteins bound to the probe. Silver staining was used to detect whether there were differences in the pulled-down proteins. The results are as Figure 3。The proteins pulled down by the wild-type, fully mutated, and single-mutated (SNP5) fragments of the ANXA5 promoter region were significantly more than those of the empty magnetic bead control group and less than those of the HTR-8 nuclear protein input group, indicating that the ANXA5 promoter region fragments bound and pulled down transcription factor proteins, and there were significant differences in the proteins pulled down by the wild-type fragment group, fully mutated fragment group, and single-mutated fragment group of ANXA5 at a protein molecular weight of 100 kDa; there were significant differences in the proteins pulled down by the wild-type fragment group, fully mutated fragment group, and single-mutated (SNP3) fragment group of the KDR promoter region compared with the HUVEC nuclear protein input group and the empty magnetic bead control group, and there were differentially bound proteins of the wild-type, fully mutated, and single-mutated fragments of the KDR promoter region at a protein molecular weight of 75 kDa.

[0098] 3.3 WB verification of transcription factors in the pulled-down proteins

[0099] To verify whether the fragments containing SNP sites in the ANXA5 and KDR promoter regions bind to the predicted transcription factors, WB was performed on the proteins pulled down by DNA-pull-down. The results showed that the transcription factor SP1 ( Figure 4 A) was present in the nuclear proteins (HTR-8) pulled down by the ANXA5 promoter region fragments, and the transcription factor YY1 ( Figure 4 B) was present in the nuclear proteins (HUVEC) pulled down by the KDR promoter region fragments.

[0100] 4 Analysis

[0101] SP1 is the first transcription factor isolated and purified from the promoter of simian virus 40. It belongs to the Sp1 / KLF family, is located in the nucleus, exists and is expressed widely in vivo, and participates in regulating the transcriptional expression of multiple genes. SP1 plays a regulatory role in the occurrence and development of cardiovascular diseases, such as cell apoptosis, inflammation, embolism, etc. In recent years, SP1 has also been found to participate in regulating vascular endothelial injury. We found a sequence on the ANXA5 promoter region that may bind to SP1, suggesting that ANXA5 may be a potential target gene of SP1.

[0102] YY1 is a member of the GLI-Kruppel family. Initially considered a DNA-binding protein, further research has revealed that YY1 is a specific subunit of the human INO80 chromatin remodeling complex and is involved in regulating many cellular processes. As a transcriptional regulator, YY1 not only has a domain with transcriptional activation function but also a domain with transcriptional repression function, making it a zinc finger protein with dual transcriptional functions. A large number of experimental results show that YY1 can aggregate various transcriptional cofactors, participate in regulating gene expression, thereby promoting or inhibiting transcription, and activate or inhibit the genes it regulates by interfering with binding sites or changes in DNA structure. We found a sequence on the promoter region of KDR that may bind to YY1.

[0103] The results of DNA-pull-down and WB suggest that the fragment of the ANXA5 promoter region containing the SNP site may bind to the transcription factor SP1, and the fragment of the KDR promoter region containing the SNP site may bind to YY1. The change of the SNP site will affect the binding rate between the promoter region and the transcription factor, thus leading to the change of gene transcriptional activity.

[0104] Example 3: Effects of transcription factors SP1 and YY1 on the transcriptional activities of the A5 and KDR promoter regions

[0105] 1 Main experimental reagents and sources

[0106]

[0107] 2 Experimental methods

[0108] 2.1 siRNA synthesis

[0109] siRNA was synthesized by Tsingke Biological, and the synthesized sequences are as follows:

[0110] Gene Sequence SEQ ID NO siRNASP1 5’-GCUGGUGGUGAUGGAAUACAU-3' SEQ ID NO.1 siRNAYY1 5’-GACGACGACUACAUUGAACAA-3' SEQ ID NO.2 siRNANC 5’-GGCUCUAGAAAAGCCUAUGC-3' SEQ ID NO.3

[0111] 2.2 siRNA transfection

[0112] (1) Seed the cells in a 6-well plate.

[0113] (2) Mix the ZETA transfection reagent and siRNA at a ratio of 1:1, and incubate at room temperature for 15 min. The groups are as follows:

[0114] ① 10 uL siRNA SP1 + 10 ul transfection reagent

[0115] ② 10 uL siRNA NC + 10 ul transfection reagent

[0116] ③ 10 uL PBS + 10 ul transfection reagent

[0117] ④ 6 uL siRNA YY1 + 6 ul transfection reagent

[0118] ⑤6 uL siRNA NC + 6 uL transfection reagent

[0119] ⑥6 uL PBS + 6 uL transfection reagent

[0120] (3) Add the incubated siRNA to HTR-8 / HUVEC at 20 uL / 12 uL per well, mix gently, and then place it in the incubator for culture. Change the medium after 24 h.

[0121] 2.3 Cell RNA extraction: Conventional method.

[0122] 2.4 RNA reverse transcription

[0123] Performed using the ABclonal reverse transcription kit.

[0124] 2.5 Fluorescent quantitative PCR

[0125] (1) Design primers as follows:

[0126]

[0127] (2) 10 uL loading system: Template: 100 ng, 2×SYBR Green Master Mix 10 uL, primer 1 uM, supplemented with ddH2O to 10 uL.

[0128] (3) Amplification system: 95°C for 2 min; 95°C for 5 sec, 60°C for 30 s, 40 cycles.

[0129] 2.6 Western Blot: Conventional method.

[0130] 3 Experimental results

[0131] 3.1 siRNA transfection efficiency

[0132] The results of fluorescent quantitative PCR showed that the silencing efficiency of siRNA on transcription factors SP1 and YY1 reached 70% ( Figure 5 ), and the silencing effect was the best 36 h after transfection ( Figure 6 ).

[0133] 3.2 Effects of SP1 and YY1 on ANXA5 and KDR

[0134] The results of fluorescent quantitative PCR showed that after siRNA silenced transcription factors SP1 and YY1, the mRNA expression levels of ANXA5 and KDR both decreased ( Figure 7 ). According to the transfection time gradient, the expression of SP1 was positively correlated with that of ANXA5, and the expression of YY1 was also positively correlated with that of KDR ( Figure 8 ).

[0135] The WB results showed that the protein expressions of ANXA5 and KDR were positively correlated with the protein expressions of transcription factors SP1 and YY1. The protein expression level of ANXA5 decreased with the decrease of SP1( Figure 9 ), and the protein expression level of KDR decreased with the decrease of YY1( Figure 10 ).

[0136] 4 Analysis

[0137] The above experiments showed that the silencing of SP1 and YY1 was positively correlated with the expressions of ANXA5 and KDR. The gene and protein expression levels of ANXA5 decreased with the silencing of SP1, and the gene and protein expression levels of KDR decreased with the knockdown of YY1. The above experiments were repeated three times biologically, and the data were true and reliable. The above results suggested that the decrease of transcriptional activity of the SNP sites in the promoter regions of ANXA5 and KDR might be achieved by changing the binding with transcription factors SP1 and YY1.

Claims

1. Use of a transcription factor in the preparation of a reagent for regulating the expression of genes related to the risk of recurrent spontaneous abortion, characterized in that: The transcription factor is SP1 or YY1, the recurrent spontaneous abortion risk gene is human annexin ANXA5 gene or vascular endothelial growth factor type II receptor KDR gene, the transcription factor SP1 regulates the expression of the ANXA5 gene, and the transcription factor YY1 regulates the expression of the KDR gene.

2. The application according to claim 1, characterized in that: The transcription factor SP1 binds to the promoter region of the ANXA5 gene, and the transcription factor YY1 binds to the promoter region of the KDR gene.

3. The application according to claim 2, wherein: The transcription factor SP1 binds to the SNP mutation site in the promoter region of the ANXA5 gene that causes recurrent spontaneous abortion, and the transcription factor YY1 binds to the SNP mutation site in the promoter region of the KDR gene that causes recurrent spontaneous abortion.

4. The application according to claim 1, wherein: The transcription factor SP1 positively regulates the ANXA5 gene, and the transcription factor YY1 positively regulates the KDR gene.

5. Use of a preparation for regulating the activity and / or expression of transcription factor SP1 or YY1 in the preparation of a reagent for regulating the expression of genes related to the risk of recurrent spontaneous abortion, characterized in that: The recurrent spontaneous abortion risk gene is human annexin ANXA5 gene or vascular endothelial growth factor type II receptor KDR gene, and the preparation that regulates the activity and / or expression of the transcription factor SP1 regulates the expression of the ANXA5 gene, and the preparation that regulates the activity and / or expression of the transcription factor YY1 regulates the expression of the KDR gene.

6. The application according to claim 5, wherein: The preparation that promotes the activity and / or expression of the transcription factor SP1 promotes the expression of the ANXA5 gene, and the preparation that inhibits the activity and / or expression of the transcription factor SP1 inhibits the expression of the ANXA5 gene; the preparation that promotes the activity and / or expression of the transcription factor YY1 promotes the expression of the KDR gene, and the preparation that inhibits the activity and / or expression of the transcription factor YY1 inhibits the expression of the KDR gene.

7. The application according to claim 5, characterized in that: The preparation that inhibits the activity and / or expression of the transcription factor SP1 is siRNA of SP1, and the preparation that inhibits the activity and / or expression of the transcription factor YY1 is siRNA of SP1.

8. The application according to claim 7, wherein: The nucleotide sequence of the siRNA of SP1 is: 5’-GCUGGUGGUGAUGGAAUACAU-3’; The nucleotide sequence of the siRNA of YY1 is: 5’-GACGACGACUACAUUGAACAA-3’.

9. Use of a transcription factor regulator in the preparation of a medicament for treating recurrent spontaneous abortion, characterized in that: The transcription factor is SP1 or YY1, the regulator promotes the activity and / or expression of the transcription factor SP1 or YY1, and the recurrent spontaneous abortion is caused by the SNP mutation of the risk gene ANXA5 gene or KDR gene.

10. The application according to claim 9, wherein: The regulator promotes the activity and / or expression of the transcription factor SP1 or YY1, thereby promoting the binding of SP1 to the promoter region of the ANXA5 gene or promoting the binding of YY1 to the promoter region of the KDR gene, and increasing the transcriptional activity of the ANXA5 gene or KDR gene.

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