Kit for detecting DNA m5C demethylase

Abstract

The embodiment of the invention discloses a kit for detecting DNA m5C demethylase, relates to the field of genetic engineering, and can be used for detecting DNA m5C demethylase. The kit comprises a primer for detecting TET3 and an auxiliary material, the primer for detecting the TET3 comprises a sequence 2 and a sequence 3, wherein the nucleotide sequence of the sequence 2 is as shown in Seq NO.2; the nucleotide sequence of the sequence 3 is as shown in Seq NO.3.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to a kit for detecting DNA m5C demethylase. Background Art

[0002] Preeclampsia is a multi-system disease that occurs during pregnancy. It is mainly manifested by hypertension after 20 weeks of pregnancy, which may be accompanied by non-specific symptoms such as proteinuria and edema. Preeclampsia affects about 5-7% of pregnant women worldwide, and can increase the incidence of premature birth, placental abruption, cesarean section and low birth weight. It is one of the main causes of maternal and perinatal death. Current studies believe that preeclampsia is a placental disease. Although the diagnosis and treatment of preeclampsia have improved, delivery of the placenta and termination of pregnancy are still the only effective treatment methods. However, more and more studies have shown that women with a history of preeclampsia have a significantly increased risk of cardiovascular disease, chronic kidney disease, metabolic syndrome and diabetes in the long term compared with women with normal pregnancy. With the rapid development of epigenetics and molecular biology, molecular targeted therapy has gradually become the focus and hotspot of the treatment of preeclampsia. Therefore, it is necessary to deeply explore the molecular mechanism of the occurrence and development of preeclampsia in order to develop highly sensitive and specific diagnostic methods and carry out personalized treatment.

[0003] A large number of recent studies have shown that DNA methylation plays a vital role in maintaining normal cell function, embryonic development, the occurrence and development of tumors and diseases, and is also involved in the pathogenesis of preeclampsia to a certain extent. The dynamic regulation of DNA methylation and demethylation during pregnancy promotes the silencing of trophoblast progenitor cell genes and the activation of cell differentiation genes. This difference in gene expression promotes the differentiation of trophoblast cells from progenitor cells to more mature trophoblast subtypes to maintain normal placental development. DNA demethylase TET can oxidize 5-mC to 5-hmC, reduce the level of DNA methylation, and thus promote gene transcription. The TET family (TET1, TET2 and TET3) plays a vital role in maintaining the pluripotency of embryonic stem cells and promoting the differentiation of trophoblast cells. It is also involved in the occurrence of various diseases including preeclampsia. For example, downregulation of TET2 causes a decrease in the demethylation level of the MMP9 promoter region, inhibiting the proliferation, invasion and migration of trophoblast cells, thereby participating in the pathogenesis of preeclampsia.

[0004] Epigenetic modification refers to the heritable functional changes in gene expression without changing the genomic DNA sequence. DNA methylation modification is one of the main epigenetic modifications. It is catalyzed by DNA methyltransferase to transfer the methyl provided by S-adenosylmethionine (SAM) to the 5th carbon atom of cytosine in CpG dinucleotide to generate 5-methylcytosine (5-mC), thereby inhibiting gene transcription. As pregnancy progresses, the DNA methylation profile of the placenta at different gestational stages is in dynamic change, indicating that DNA methylation plays an important role in regulating placenta-specific gene expression. Although the placental trophoblast cell lineage shows low methylation levels, DNA methylation still plays an important role in the biological behavior of trophoblast cells. Therefore, how to apply methyltransferases in the treatment of preeclampsia has become a topic that needs to be studied. Summary of the invention

[0005] The embodiment of the present invention provides a kit for detecting DNA m5C demethylase, which can detect DNA m5C demethylase.

[0006] To achieve the above object, the embodiments of the present invention adopt the following technical solutions:

[0007] A kit for detecting DNA m5C demethylase, wherein the pharmaceutical composition in the kit comprises: primers and auxiliary materials for detecting TET3;

[0008] The amino acid sequence of DNA m5C demethylase is shown in Seq NO:1.

[0009] The sequences of the primers for detecting TET3 include sequence 2 and sequence 3, wherein: the sequence 2 is: TET3 F5'-GACGAGAACATCGGCGGCGT-3' (Seq ID.2); the sequence 3 is: TET3R 5'-GTGGCAGCGGTTGGGCTTCT-3' (Seq ID.3). BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0011] Figure 1 A schematic diagram of TET3 upregulation in preeclampsia placental tissue and inhibition of trophoblastic cell migration and invasion provided in an embodiment of the present invention, wherein:

[0012] A The cell categories of preeclampsia and normal control group were clustered after merging the cells using the Shared Nearest Neighbor (SNN) algorithm, and then the Uniform Manifold Approximation and Projection (UMAP) was used for dimensionality reduction visualization. The right figure shows the expression level of TET3 in the four samples.

[0013] B Distribution of TET3 gene in different cell populations in four single cell samples.

[0014] C TET3 is highly expressed in placenta tissue of preeclampsia compared with placenta tissue of normal pregnancy (n=64).

[0015] D. IHC was used to detect the level of TET3 protein in placental tissue. TET3 was highly expressed in placental tissue of preeclampsia.

[0016] E After overexpression of TET3, the migration and invasion abilities of HTR-8 / SVneo and JAR cells were decreased, while after knockdown of TET3, the migration and invasion abilities of cells were increased.

[0017] Figure 2 A schematic diagram of TET3 promoting the expression of KLF13 in trophoblast cells provided in an embodiment of the present invention, wherein:

[0018] A RNA transcriptome sequencing analysis of potential downstream target genes of TET3-mediated HTR-8 / SVneo trophoblasts.

[0019] GO analysis of B cell sequencing.

[0020] CD overexpression of TET3 and knockdown of TET3 verified KLF13 as its potential target gene.

[0021] Figure 3 A schematic diagram of TET3 regulating the expression of KLF13 through epigenetic modification provided in an embodiment of the present invention, wherein:

[0022] A The CpG islands in the promoter region of KLF13 were queried using the UCSC genome browser.

[0023] B Nucleotide sequence of the key transcriptional regulatory region of KLF13. The numbers indicate the position of the nucleotide relative to the transcription start site, and differentially methylated cytosine residues are indicated in red.

[0024] C MethylTarget sequencing analysis showed that the methylation levels of 14 methylation sites in the target CpG island of the KLF13 promoter region in human placental tissue were decreased.

[0025] D MethylTarget sequencing analysis of the methylation levels of CpG sites in the promoter region of the KLF13 gene in trophoblasts after knockdown of TET3.

[0026] E ChIP-qPCR analysis showed that the enrichment level of KLF13 promoter region in trophoblasts decreased after knockdown of TET3.

[0027] Figure 4 A schematic diagram of miR-544 regulating the expression of TET3 and KLF13 through post-transcriptional modification provided in an embodiment of the present invention, wherein:

[0028] AB bioinformatics tools predicted potential binding sites between TET3 or KLF13 3'UTR and miR-544.

[0029] CD GPL23980 and GSE103542 databases suggested that miR-544 was significantly downregulated in placental tissues of preeclampsia.

[0030] E Dual luciferase reporter assay confirmed the interaction of miR-544 with TET3 or KLF13 3'UTR in HEK-293T cells and HTR-8 / SVneo cells.

[0031] G Overexpression of miR-544 inhibited the expression of TET3 and KLF13, while inhibition of miR-544 promoted the expression of TET3 and KLF13.

[0032] Figure 5 A schematic diagram of abnormal expression of TET3 and KLF13 in placental tissues of pregnant mice with preeclampsia and human preeclampsia provided in an embodiment of the present invention, wherein:

[0033] A. Gross appearance of embryos and placentas of pregnant rats injected intraperitoneally with L-NAME or saline.

[0034] B. Trajectory of systolic blood pressure during pregnancy in the control group and L-NAME group. **p indicates systolic blood pressure < 0.001 compared with the systolic blood pressure on the 8th day of pregnancy before intervention.

[0035] C Average weights of placentas and embryos in the control and L-NAME groups.

[0036] DHE staining to observe the developmental characteristics and trophoblast invasion status of the placenta in the control group and L-NAME group

[0037] E Spearman correlation analysis showed that the expressions of KLF13 and TET3 were positively correlated, and miR-544 was negatively correlated with TET3 or KLF13.

[0038] Figure 6The diagram shows the mechanism by which the miR-544-TET3-KLF13 axis is involved in the development of preeclampsia by affecting trophoblast cell invasion and migration. DETAILED DESCRIPTION

[0039] The present invention is further described below by way of examples, but the present invention is not limited thereto.

[0040] The TET3 gene is located on human chromosome 2p13.1. The protein encoded by this gene consists of 1795 amino acid residues. Its amino acid sequence is shown in Seq NO.1, that is, sequence 1 is the DNA m5C demethylase that needs to be detected. In the study, it was found that TET3 was upregulated in the placenta of pregnant women with preeclampsia compared with the placental tissue of normal pregnant women. After knocking down or overexpressing TET3, the functions of TET3 related target genes and upstream regulatory molecules in trophoblasts were studied.

[0041] Specifically, an embodiment of the present invention provides a kit for detecting DNA m5C demethylase, wherein the pharmaceutical composition in the kit includes: primers and excipients for detecting TET3.

[0042] In this example, the amino acid sequence of DNA m5C demethylase is shown in Seq NO:1.

[0043] The sequence of the primer for detecting TET3 includes an upstream primer sequence 2 and a downstream primer sequence 3, wherein: the nucleotide sequence of the sequence 2 is: TET3 F 5'-GACGAGAACATCGGCGGCGT-3'; the nucleotide sequence of the sequence 3 is: TET3 R 5'-GTGGCAGCGGTTGGGCTTCT-3'.

[0044] The following examples involve siRNAs that interfere with TET3, and the sequences of siRNAs include sequence 4 and sequence 5, wherein: the sequence 4 is: siTET3-1#sense 5'-CAGCAACUCCUAGAACUGAtt-3' (Seq NO.4); antisense 5'-UCAGUUCUAGGAGUUGCUGga-3' (Seq NO.8); the sequence 5 is: siTET3-2#sense 5'-CGAUUGCGUCGAACAAAUAtt-3' (Seq NO.5), antisense5'-UAUUUGUUCGACGCAAUCGca-3' (Seq NO.9). In use, the concentrations of sequence 4 and sequence 5 were 50 nM respectively.

[0045] Specifically, in the kit, the concentration of the primers for detecting TET3 is 10 mol / L. The following examples involve primer sequences for detecting KLF13, including upstream primer sequence 6 (Seq NO.6) and downstream primer sequence 7 (Seq NO.7), wherein: sequence 6 is: KLF13 F5'-CGGCCTCAGACAAAGGGTC-3'; sequence 7 is: KLF13 R5'-TTCCCGTAAACTTTCTCGCAG-3'.

[0046] Specifically, the pharmaceutical composition also includes various reagents required for the transfection process, that is, the excipients include: lipo3000, Opti-MEM culture medium and PBS phosphate buffered saline. In use, the excipients are 1.5ml of lipo3000, 500ml of Opti-MEM culture medium, and 500ml of PBS phosphate buffered saline. Among them, Lipo3000, a multi-purpose liposome transfection reagent, combined with the most advanced lipid nanoparticle technology, is suitable for the transfection of DNA, RNA and oligonucleotides, and has a high transfection efficiency for most eukaryotic cells. Its unique formula reduces the amount of reagents required while ensuring transfection efficiency and reducing toxicity to cells, and ultimately transfers the TET3 interference sequence into the cell. Opti-MEM culture medium contains HEPES, 2400mg / l sodium bicarbonate, hypoxanthine, thymine, sodium pyruvate, L-glutamine, trace elements, growth factors, and phenol red reduced to 1.1mg / l. As an auxiliary material of transfection reagent, it itself has no harm to cells and is very suitable for cationic liposome transfection such as lipo3000 to achieve the desired purpose. PBS phosphate buffer saline is generally used as a solvent to dissolve and protect reagents. It is the most widely used buffer in biochemical research. Its main components are Na2HPO4, KH2PO4, NaCl and KCl. Since Na2HPO4 and KH2PO4 have secondary dissociation, the buffer pH range is very wide; while NaCl and KCl mainly act to increase the salt ion concentration. Exclude its own influence on the experimental object.

[0047] The following actual experimental studies show the specific application effects of the kit for detecting DNA m5C demethylase in this embodiment:

[0048] (1) Tissue collection: Placental tissues were collected from 64 pregnant women diagnosed with preeclampsia and normal pregnant women without any underlying diseases who underwent cesarean section at Jiangsu Provincial People's Hospital and Jiangsu Provincial Maternal and Child Health Hospital from 2020 to 2022. Clinical characteristics were recorded, including maternal age, smoking history, gestational age, systolic and diastolic blood pressure, proteinuria, and fetal weight. The following treatments were performed according to different experimental purposes: 1) The tissue blocks were fixed in a 50ml centrifuge tube containing 4% paraformaldehyde for subsequent immunohistochemical detection; 2) The tissue blocks were directly stored in cryovials and placed in liquid nitrogen for transport to the laboratory for tissue RNA and protein extraction. This study was approved by the Ethics Committee of Nanjing Medical University and written informed consent was obtained from all patients.

[0049] (2) Cell line culture:

[0050] Trophoblast cells (HTR-8 / SVneo, JAR) and human embryonic kidney cells (HEK-293T) were purchased from the ATCC cell bank in the United States. The culture conditions of HTR-8 / SVneo and JAR were as follows: RPIM-1640 medium containing 10% FBS and 1% double antibody, cultured in a 37°C constant temperature incubator with 90% humidity and 5% CO2;

[0051] HEK-293T cells were cultured in MEM medium containing 5% FBS and 1% double antibody. The complete medium was replaced every 2-3 days according to the cell growth status, and the cells were subcultured for subsequent experiments when the cell confluence reached 80%-90%.

[0052] (3) RNA extraction and quantitative PCR analysis:

[0053] According to the instructions for use of the reagent, total RNA was isolated using Trizol reagent. The reverse transcription reaction was performed using the RT reverse transcription kit (Nanjing Novogene Biotech Co., Ltd.). 1 μg of total RNA was reverse transcribed in a final volume of 20 μl. Result analysis: The specificity of the primers and the amplification efficiency were analyzed. The specificity of the primers was determined based on the melting curve. The CT value was obtained based on the amplification curve, and the relative expression of the amplified product was calculated: after correction by GAPDH, the expression was expressed as 2 -ΔΔCT Calculation: ΔΔCT = (CT value of target gene in experimental group - CT value of internal reference gene in experimental group) - (CT value of target gene in control group - CT value of internal reference gene in control group). Three replicate wells were set for each sample, and the average value was taken to determine the mRNA level expression of the gene.

[0054] (4) Plasmid construction:

[0055] The human full-length TET3 cDNA was synthesized and inserted into the eukaryotic expression vector pCDNA 3.1 to construct a TET3 overexpression vector plasmid. The plasmid was then transformed into Escherichia coli, and the bacteria were shaken, cultured, and single clones were selected for culture and amplification.

[0056] (5) Cell transfection:

[0057] TET3 interference sequence and interference control sequence (si-NC) were purchased from Guangzhou Ruibo Company. After the cells were revived and passaged three times, the cells were digested when the growth density was about 80%, and 2×105 cells / well were counted and plated into six-well plates for culture. When the cell confluence reached 50%-60%, transfection was performed. siRNA and TET3 overexpression plasmid were taken out of the -20℃ refrigerator and placed on ice to melt; Lipo3000 was taken out of the 4℃ refrigerator for use. 10μl Lipo3000 was added to a 1.5ml EP tube containing 250μl Opti-MEM serum-free medium; siRNA (10μl) and plasmid (2ng) were added to a 1.5ml EP tube containing 250μl Opti-MEM. Gently blow each tube to mix, and incubate at room temperature for 5 minutes; gently mix the incubated Lipo3000 with the above dilutions, and incubate at room temperature for 15 minutes; discard the old culture medium, add 1.5ml OPTI-MEM serum-free culture medium to each well, until the final system is about 2ml, add the incubated mixed solution to the well, shake it gently and return it to the incubator; replace it with complete culture medium after transfection for 6-8 hours and continue to culture. Collect cells 24-48 hours after transfection, extract total RNA and total protein, and use qRT-PCR and Western Blot to verify transfection efficiency or other subsequent experiments.

[0058] (6) Cell migration and invasion assay:

[0059] Dilute the matrix gel 1:6 to coat the upper chamber surface of the bottom membrane of the Transwell chamber, and incubate in the incubator for 6 hours to allow the matrix gel to fully solidify. 24-48 hours after cell transfection, trypsinize the cells, resuspend the cells in serum-free culture medium and count them, and dilute the single cell suspension to 2×105 cells / ml. Add 600μl of RPMI-1640 culture medium containing 10% FBS to the lower chamber, and carefully transfer the Transwell chamber to a 24-well plate with tweezers; add 200μl of cell suspension to the upper chamber and culture it in the incubator. After 24-48 hours of culture, take out the chamber according to the time gradient, fix the lower chamber with 4% paraformaldehyde for 30 minutes; aspirate and discard paraformaldehyde, and stain with crystal violet for 15-20 minutes; rinse with PBS 3 times, and gently wipe the uninvaded cells in the chamber with a cotton swab. Observe and take pictures and count under an inverted microscope. The Transwell chamber used for migration experiments does not need to be covered with matrix gel, and the rest of the steps are the same as above.

[0060] (7) RNA-seq (transcriptome sequencing):

[0061] The cells were seeded in a six-well plate, and when the cells grew to 80%, they were transfected with 10ul Lipo3000 with siTET3 and siCon. After 48 hours, the cells were collected and treated with Trizol, and the samples were sent to Shanghai Tianhao Biotechnology Co., Ltd., which selected Illumina for subsequent experiments to obtain and process the corresponding data.

[0062] (8) Western blotting:

[0063] The denatured cell protein lysate was added to the pre-prepared 10% denaturing polyacrylamide gel (SDS-PAGE) sample well to separate the protein in the sample. Then it was transferred to the NC membrane and incubated with specific antibodies. Finally, it was exposed with ECL luminescent film. GAPDH antibody was used as a control, TET3 antibody was purchased from GeneTex, USA, and KLF13 antibody was purchased from Proteintech, USA.

[0064] (9) MethylTarget methylation sequencing:

[0065] Cell samples: cells were collected using Trizol 48 hours after transfection with siTET3 and siCon; placental tissue samples: placental tissues of pregnant women with preeclampsia and normal pregnancy were selected and commissioned to Shanghai Tianhao Biotechnology Co., Ltd. The genomic DNA was converted by bisulfite, amplified by multiple PCR, and high-throughput sequencing was performed after adding specific tag sequences, and the corresponding data were processed and analyzed.

[0066] (10) Chromatin immunoprecipitation (ChIP-qPCR):

[0067] After trophoblast cells were transfected with siTET3 and siCon, the protein-DNA complexes were cross-linked with formaldehyde and the cells were lysed to release the cellular components. The chromatin was fragmented by ultrasound, followed by chromatin immunoprecipitation using specific antibodies, and the protein-DNA complexes were further purified and eluted. The separated chromatin fragments containing the target protein were decrosslinked and the DNA was purified. The purified DNA products were quantified by qPCR.

[0068] (11) Luciferase reporter gene experiment:

[0069] Complementary DNA fragments containing wild-type and mutant TET3 3'UTR and KLF13 3'UTR were ligated into pgl3 basic vector (Promega). Wild-type or mutant 3'UTR of TET3 and KLF13 vectors were co-transfected with miR-544 mimics and miR-NC in 24-well plates in trophoblast cells (HTR-8 / SVneo) and human embryonic kidney cells (HEK-293T). After 48 hours of transfection, the luciferase activity of each group was evaluated using a dual luciferase kit (Promega). Renilla luciferase was used as a control.

[0070] (12) Construction of a pregnant mouse model of preeclampsia:

[0071] 8-week-old male and female SD rats were caged together and the pregnancy was confirmed, which was recorded as day 0.5. They were then randomly divided into two groups: a control group and an N-nitro-L-arginine methyl ester (N'-Nitro-L-arginine-methyl ester hydrochloride, L-NAME) modeling group. Pregnant rats in the L-NAME modeling group were given 125 mg / kg / d of L-NAME by intraperitoneal injection every day from day 9.5 to day 15.5 of pregnancy; the normal control group was given an equal amount of sterile saline. The systolic blood pressure of the two groups of rats was measured by the tail cuff method on days 8.5, 10.5, 12.5, 14.5 and 16.5 of pregnancy. After day 18.5 of pregnancy, the pregnant rats were euthanized, and the fetuses and placentas were dissected out, dried, weighed, and photographed. The placental tissue was taken and fixed in 4% formaldehyde for subsequent IHC and HE staining.

[0072] (13) Data processing:

[0073] SPSS17.0 software was used to perform statistical analysis on the experimental data. The measurement data were expressed as mean ± standard error. The t test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups. The Spearman rank correlation test was used for correlation analysis. P<0.01 (**), P<0.05 (*) indicated that the difference was statistically significant.

[0074] The experimental methods not described in detail in the embodiments are basically in accordance with the conditions and methods described in "Molecular Cloning: A Laboratory Manual, Fourth Edition" compiled by Michael R. Green and Joseph Sambrook (Molecular Cloning: A Laboratory Manual, Fourth Edition. Translated by He Fuchu. Science Press. 2017) or the conditions and methods recommended in the material specification. Other techniques not described in detail correspond to standard methods well known to those skilled in the art. The cell lines and experimental reagents involved in this application can be purchased and can be replaced by other suitable reagents. Combined with the design of the above-mentioned embodiment and related experimental studies, the practical use of the kit for detecting DNA m5C demethylase in this embodiment at least includes:

[0075] Example 1

[0076] Detection of TET3 expression in tissues and cells

[0077] Take 0.1g tissue, cut it into pieces with ophthalmic scissors, put it in a sterile enzyme-free EP tube containing 1ml Trizol, add magnetic beads, and grind it on ice in a homogenizer for 2-3min. Centrifuge at 4℃12000rpm for 5min, take the supernatant to a new EP tube; add 200μl chloroform, shake for 15s, and let it stand at room temperature for 3-5min. Centrifuge at 4℃12000rpm for 15min, and the liquid in the tube can be seen to be divided into 3 layers, namely the colorless upper aqueous phase containing total RNA, the white middle layer containing DNA and protein, and the pink lower layer containing organic matter. Carefully pipette 500μl of the upper aqueous phase into a new EP tube, add an equal volume of isopropanol, and let it stand at 4℃ for 30min. Centrifuge at 4℃12000rpm for 15min, discard the supernatant, add 1ml 75% ethanol (pre-cooled RNA free water and anhydrous ethanol are prepared on the spot) and mix well. Centrifuge at 7500rpm for 5min at 4℃, discard the supernatant, and air dry at room temperature for 5-10min to evaporate the RNA particles and the ethanol remaining in the centrifuge tube. Add 10-20μl RNA-free water to the EP tube to dissolve the RNA, mix gently, and measure the RNA purity and concentration.

[0078] The purity and concentration of RNA were determined using the NanoDrop2000 ultra-micro spectrophotometer. Before the determination, zero the solution with DEPC water, mix the RNA solution thoroughly and measure the concentration, measure the absorbance at wavelengths of 260nm and 280nm and calculate the A260 / A280 ratio to determine the RNA purity. 1.8-2.1 meets the standard (too high indicates RNA degradation, too low indicates possible contamination with impurities such as proteins). RNA was used for subsequent experiments or stored in a -80°C refrigerator for future use (try to avoid repeated freezing and thawing of RNA to reduce its degradation).

[0079] Real-time quantitative PCR

[0080] Placental tissue specimens from preeclampsia and normal pregnant women, total RNA from HTR-8 / SVneo cells, and reverse transcription reaction were performed using the RT reverse transcription kit (Nanjing Novogene Biotech Co., Ltd.). The reverse transcription reaction system is as follows:

[0081] 5×HiScript III qRT SuperMix 2μl

[0082] RNA 1ng

[0083] RNase-free ddH2O to 10 μl

[0084] The reverse transcription reaction conditions were as follows: 37°C for 15 min, 85°C for 5 sec.

[0085] The synthesized cDNA can be used immediately for qPCR reactions and can be stored at 4°C for a short period of time or at -20°C for a long period of time. Repeated freezing and thawing of cDNA should be avoided.

[0086] qPCR ApplicationsRoche 480 fluorescence quantitative system. cDNA samples were amplified using the three-step PCR standard procedure. Reaction system:

[0087]

[0088] Reaction conditions:

[0089]

[0090] Result analysis: Analyze the specificity of primers and amplification efficiency. Determine the specificity of primers based on the melting curve. Obtain the CT value based on the amplification curve and calculate the relative expression of the amplified product: Calculate with 2-ΔΔCT after correction by GAPDH, ΔΔCT = (CT value of target gene in experimental group - CT value of reference gene in experimental group) - (CT value of target gene in control group - CT value of reference gene in control group). Set up 3 replicate wells for each sample and take the average value to determine the mRNA level expression of the gene.

[0091] The primers for TET3 are as follows:

[0092] F Primer 5'-GACGAGAACATCGGCGGCGT-3', see SEQ ID NO: 2, R Primer 5'-GTGGCAGCGGTTGGGCTTCT-3', see SEQ ID NO: 3.

[0093] The experimental results showed that the expression of DNA m5C demethylase TET3 in placental tissue of preeclampsia was upregulated compared with that in normal placental tissue. The content of TET3 in placental tissue of 64 pairs of preeclampsia pregnant women and normal pregnant women was detected by qPCR. The results showed that TET3 was upregulated in placental tissue of 60% (38 / 64) of preeclampsia pregnant women compared with normal tissue (P<0.05)( Figure 1 C). This suggests that TET3 may play an important role in the development, diagnosis and treatment of preeclampsia.

[0094] Table 1 Clinical data of pregnant women with preeclampsia and normal pregnancy

[0095]

[0096] Example 2

[0097] TET3 is involved in trophoblast cell migration and invasion.

[0098] Abnormal trophoblast migration and invasion function leads to uterine spiral artery remodeling disorder, which in turn participates in the occurrence and development of preeclampsia. HTR-8 / SVneo and JAR cell lines were used as research objects, and TET3 interference sequence or TET3 overexpression plasmid was transfected with Lipo3000 to downregulate or upregulate TET3 expression. Transwell experiment was used to study the effect of TET3 knockdown or overexpression on the migration and invasion ability of HTR-8 / SVneo and JAR cells. The results showed that knockdown of TET3 promoted the migration and invasion ability of trophoblast cells compared with the control group cells, and overexpression of TET3 inhibited the migration and invasion ability of trophoblast cells HTR-8 / SVneo compared with the control group cells ( Figure 1 E). It can be seen that the high expression of TET3 affects the migration and invasion ability of normal trophoblastic cells, further affecting the shallow implantation of the placenta and inducing the occurrence of preeclampsia.

[0099] Example 3

[0100] TET3 is involved in the methylation modification of the promoter region of the downstream target gene KLF13.

[0101] To study the role of TET3 in DNA demethylation, HTR-8 / SVneo and JAR cell lines were used as research objects, and TET3 interference sequences were transfected with Lipo3000 to downregulate the expression of TET3. ChIP-qPCR experiments were used to detect whether there was specific binding between TET3 and KLF13 promoter regions.

[0102] (1) Preparation of chromatin: Add 4% formaldehyde to a cell culture dish containing 10 ml of culture medium to make the final concentration of paraformaldehyde 1%, and incubate on a horizontal shaker at 37°C for 10 min; add 10x glycine buffer to quench unreacted formaldehyde, and incubate on a horizontal shaker for 10 min. Carefully aspirate the culture medium, wash the residual liquid on the cell surface with pre-cooled PBS containing protease inhibitors, add 1 ml of pre-cooled PBS containing protease inhibitor cocktail to the culture dish, and use a sterile cell scraper to scrape the cells and transfer them to a new 1.5 ml sterile enzyme-free EP tube. Centrifuge at 4°C and 1000 rpm for 5 min to precipitate the cells. During this period, prepare the cell lysis buffer (add 2.5 μl of cocktail to 500 μl of lysis buffer). Aspirate the supernatant in the EP tube, add the cell lysis buffer prepared in the previous step to resuspend the cells, and incubate on ice for 15 min, vortexing briefly every 5 min. Centrifuge at 4℃1000rpm for 5min, during which time prepare the cell nuclear lysis buffer (add 2.5μl of cocktail to 500μl of Nucleus lysis buffer). Carefully discard the supernatant in the EP tube and add the cell nuclear lysis buffer to resuspend the cells.

[0103] (2) Chromatin immunoprecipitation reaction: Ultrasonicate on ice to fragment chromatin, centrifuge at 12000rpm for 10min at 4℃, and aspirate the supernatant into a new EP tube. Take 5μl of the supernatant for agarose gel electrophoresis analysis of the ultrasonic product; divide the supernatant into new sterile enzyme-free EP tubes at 50μl each, and add 450μl Dilution buffer containing 2.5μl protease inhibitors to each system. Take 5μl of the supernatant as input for later use. Add 20μl of completely resuspended protein A / G magnetic beads and immunoprecipitation antibodies: positive control - RNA polymerase antibody, add 1μg antibody to each tube, negative control - normal mouse IgG, add 1μg antibody to each tube, CHIP-level antibodies and IgG of the same species, add 1-10μg antibody to each tube. Incubate overnight on a rotator at 4℃, use a magnetic stand to precipitate the magnetic beads and completely remove the supernatant. The beads were washed and incubated on a rotator for 3-5 min, followed by magnetic separation and careful removal of the supernatant.

[0104] (3) Elution and reverse cross-linking of cross-linked proteins / DNA: Add 100 μl of Elution buffer and 1 μl of proteinase K warmed to room temperature to the tube, incubate in a 62°C metal bath for 2 h, vortex every 15 min; incubate in a 95°C metal bath for 10 min. After cooling to room temperature, separate the magnetic beads using a magnetic stand, and carefully transfer the supernatant to a new EP tube.

[0105] (4) DNA purification: Add 500 μl of Bind reagent A to the above EP tube and vortex to mix; transfer the thoroughly mixed solution to a centrifugal column with a receiving tube and centrifuge at 12,000 rpm for 1 min at room temperature. Discard the filtrate, add 500 μl of Washreagent B to clean the column core, and centrifuge at 12,000 rpm for 1 min at room temperature; discard the filtrate, put the column core back, and centrifuge at 12,000 rpm for 3 min at room temperature; discard the filtrate and put the adsorption column back into a new EP tube. Add 50 μl of Elution buffer C to the adsorption column and centrifuge at 12,000 rpm for 1 min at room temperature. The obtained cDNA is used for qPCR detection.

[0106] The experimental results found that the enrichment level of KLF13 decreased after knocking down TET3, suggesting that TET3 can specifically bind to the KLF13 promoter region and affect its transcription.

[0107] Through clinical sample analysis, it was found that TET3 was highly expressed in the placenta of patients with preeclampsia compared with normal pregnancy placenta. Overexpression of TET3 significantly reduced the migration and invasion ability of trophoblasts. Experimental results suggest that abnormal expression of TET3 significantly affects the biological behavior of trophoblast cells and is a key molecule in the development of preeclampsia. KLF13, a potential downstream target of TET3, was screened by RNA-seq, and it was confirmed for the first time that TET3 exerts biological functions in human trophoblasts by promoting the expression of trophoblast KLF13. miRNAs participate in the occurrence and development of various diseases by binding to the 3'UTR of target mRNAs, promoting mRNA degradation or hindering protein translation.

[0108] This study found that miR-544 is a key molecule that mediates the upregulation of TET3 and KLF13 in trophoblasts. Bioinformatics analysis and dual luciferase reporter experiments verified that miR-544 can bind to TET3 and KLF13 mRNA 3'UTR, inhibit the expression of TET3 and KLF13 at the mRNA and protein levels, thereby affecting the migration and invasion ability of trophoblasts, and participating in the occurrence and progression of preeclampsia. TET3 expression is upregulated in placental tissues and cells of pregnant women with preeclampsia. In trophoblast HTR-8 / SVneo cells, overexpression of TET3 inhibits cell function, and knockdown of TET3 increases cell migration and invasion. In addition, our findings will further reveal the pathogenesis of preeclampsia and promote the diagnosis and treatment of DNA m5C demethylase in preeclampsia.

[0109] Each embodiment in this specification is described in a progressive manner, and the same or similar parts between the embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. The above is only a specific implementation of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A kit for detecting DNA m5C demethylase, characterized in that: The kit includes primers and auxiliary materials for detecting TET3; The primers for detecting TET3 include sequence 2 and sequence 3, wherein the nucleotide sequence of sequence 2 is shown in Seq NO.2; and the nucleotide sequence of sequence 3 is shown in Seq NO.

3.

2. The kit according to claim 1, characterized in that The auxiliary materials include: 2×ChamQ SYBR qPCR Master Mix 5 μl, RNase-free ddH2O 1 μl.

3. The kit according to claim 1, characterized in that The concentrations of sequence 2 and sequence 3 were both 10 μM, and the contents were both 0.4 μl.

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