Application of DCTPP1 in inspection of abortion kit

Abstract

The invention provides an application of DCTPP1 in detection of an auditing abortion kit, relates to the technical field of target spot diagnosis, and particularly provides an application of DCTPP1 as a target spot in preparation of an auditing abortion detection product, and an experimental result shows that DCTPP1 is one of key genes for auditing abortion. Specifically, after the DCTPP1 is knocked down from a human embryo trophoblast cell line HTR8 / SVneo, compared with a control group, the reduction of the DCTPP1 can cause more cell oxidation stress increase, and cell viability is reduced 24 hours later, and finally cell apoptosis occurs; in the mechanism, the DCTPP1 interacts with the RNA binding protein AUF1, and the reduction of the DCTPP1 causes the increase of oxidative stress of human embryo trophoblastic cells through the AUF1, so that apoptosis is caused. Experimental results in the application show that DCTPP1 is one of key genes for auditing abortion, and cell apoptosis can be finally caused by destroying the function of DCTPP1 in embryonic trophoblast cells through technologies such as transfection of specific siRNA and the like. In addition, the DCTPP1 knocked-down embryo trophoblast cells can be subjected to apoptosis, and the DCTPP1 knocked-down embryo trophoblast cells are one of useful genes for detecting and auditing abortion.

Description

Technical Field

[0001] The present invention relates to the technical field of diagnostic target points, and in particular to application of DCTPP1 in missed abortion detection kits. Background Art

[0002] Missed abortion (MA), also known as overdue abortion, is one of the three special types of abortion. It refers to the embryo or fetus in the uterine cavity that has died but has not been expelled from the body in time and is retained in the uterus. The dead embryonic tissue releases a large amount of thromboplastin. Long-term retention in the uterine cavity can cause abnormal coagulation function in pregnant women and even disseminated intravascular coagulation, leading to severe bleeding. If not treated in time, it may cause maternal coagulation disorders, peritonitis, septic shock and other complications, and even threaten the safety of the mother's life in severe cases. There are many causes of missed abortion. Maternal immune dysfunction, adverse environmental factors, chromosomal abnormalities, abnormal embryonic development, single gene genetic diseases and uniparental disomy are all possible causes of missed abortion. The embryo often has varying degrees of adhesion to the inner wall of the uterus, which increases the difficulty of surgical curettage and leads to more complications.

[0003] Due to the significant impact of missed abortion on the mother, routine physical examinations, pregnancy tests, progesterone measurements, etc. are difficult to effectively diagnose missed abortion early. Clinical scholars have been looking for indicators independent of conventional methods for the early diagnosis of missed abortion, with the aim of enabling patients with missed abortion to receive timely diagnosis and treatment. Summary of the invention

[0004] The purpose of the present invention is to solve the technical problem that there is a lack of independent conventional method indicators for missed abortion in the prior art.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] Application of DCTPP1 as a target in the preparation of missed abortion detection products.

[0007] Preferably, the detection product is a detection reagent or a detection kit.

[0008] Preferably, the detection product screens for missed abortion by detecting the expression level of DCTPP1 in embryonic trophoblast cells.

[0009] The present application also provides the use of DCTPP1 as a target in the preparation of drugs for the prevention and early treatment of missed abortion.

[0010] Preferably, the drug achieves the prevention and early treatment of missed abortion by increasing the expression of DCTPP1.

[0011] The present application also provides a missed abortion detection product, which is a detection reagent or a detection kit. The detection product screens for missed abortion by detecting the expression level of DCTPP1 in embryonic trophoblast cells.

[0012] The present application also provides a drug for preventing and treating missed abortion at an early stage, and for increasing the expression of DCTPP1.

[0013] The present application also provides a method for constructing a DCTPP1 knockdown human embryonic trophoblast cell model, comprising the following steps:

[0014] S1: Design and synthesize siRNA to knock down the expression of DCTPP1;

[0015] S2: The knockdown siRNA is transferred into the embryonic trophoblast cells via liposomes;

[0016] S3: RT-PCR and WB confirmed that the knockdown efficiency was greater than 70%, indicating successful knockdown.

[0017] The experimental results of this application show that DCTPP1 is one of the key genes for missed abortion. Specifically, after the human embryonic trophoblast cell line HTR8 / SVneo knocked down DCTPP1, compared with the control group, the reduction of DCTPP1 caused a surge in cell oxidative stress and a decrease in cell viability after 24 hours, which eventually led to apoptosis; Mechanistically, DCTPP1 interacted with the RNA binding protein AUF1, and the reduction of DCTPP1 caused an increase in oxidative stress in human embryonic trophoblast cells through AUF1, leading to the occurrence of apoptosis. The experimental results in this application show that DCTPP1 is one of the key genes for missed abortion, and the destruction of DCTPP1 function in embryonic trophoblast cells by transfection-specific siRNA and other technologies will eventually lead to cell apoptosis. In addition, embryonic trophoblast cells knocked down by DCTPP1 will undergo apoptosis, which is one of the useful genes for detecting missed abortion. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure shows the results of constructing superoxide and lipid peroxide content after DCTPP1 transient transfection knockdown cells in the embodiment of the present invention, wherein (A) RT-PCR verifies the knockdown efficiency of three specific siRNAs of DCTPP1; (B) WB verifies the knockdown efficiency of three specific siRNAs of DCTPP1;

[0019] (C) LPO content determination showed that after knocking down DCTPP1, LPO content increased; (D) Immunofluorescence showed that after knocking down DCTPP1, the DHE staining signal became stronger;

[0020] Figure 2The results of cell proliferation and apoptosis after DCTPP1 knockdown in the embodiments of the present invention are shown in the figure, wherein (A) after DCTPP1 knockdown in HTR8 / SVneo, proliferation was significantly inhibited after 24 hours; (B) immunofluorescence showed that the apoptosis signals of cells with DCTPP1 knockdown increased; (C) statistical data of apoptotic cells;

[0021] Figure 3 The results of RNA-Seq sequencing of cells after DCTPP1 knockdown in the embodiment of the present invention are analyzed, wherein (A) KEGG analysis found that differentially expressed genes were enriched in oxidative stress and apoptosis pathways; (B)

[0022] GO analysis found that differentially expressed genes were enriched in oxidase activity; (C) RT-PCR verified the changes in genes affecting the cell cycle in the treatment group; (D) RT-PCR verified the changes in genes affecting oxidative stress in the treatment group; (E)

[0023] RT-PCR verified the changes in genes affecting apoptosis in the treatment groups;

[0024] Figure 4 The results of mass spectrometry analysis of DCTPP1 binding proteins and interacting proteins in HTR8 / SVneo cells according to the embodiments of the present invention are shown in Figure 1, wherein (A) mass spectrometry analysis found that DCTPP1 binds to AUF1; (B) overexpression of DCTPP1-6xHA and 3xFlag-AUF1 in HTR8 / SVneo cells and forward and reverse pulls with labeled magnetic beads proved that the two proteins interacted with each other;

[0025] Figure 5 The functional analysis diagram of AUF1, a binding protein of DCTPP1, in HTR8 / SVneo cells according to an embodiment of the present invention, wherein (A) WB proves that the specific siRNA knockdown of AUF1 is successful; (B) Immunofluorescence experiment shows that after knocking down AUF1, the DHE staining signal becomes stronger and the data statistics; (C)

[0026] After knocking down AUF1 in HTR8 / SVneo cells, proliferation was significantly inhibited 24 hours later; (D) Immunofluorescence experiment showed that the apoptosis signal of cells knocked down AUF1 increased.

[0027] Figure 6 The analysis diagram of RNA-Seq sequencing results of cells after AUF1 knockdown in the embodiment of the present invention, wherein (A) KEGG analysis found that differential genes were enriched in oxidative stress and apoptosis pathways; (B) GO analysis found that differential genes were enriched in oxidase activity; (C) RT-PCR verified the gene changes in the treatment group that affected the cell cycle; (D) RT-PCR verified the gene changes in the treatment group that affected oxidative stress; (E) RT-PCR verified the gene changes in the treatment group that affected cell apoptosis;

[0028] Figure 7 The expression results of DCTPP1 in the chorionic tissue of patients with normal abortion and missed abortion in the embodiment of the present invention. (A) RT-PCR verified the expression of DCTPP1 in the chorionic tissue of patients with normal abortion and missed abortion (3 cases each); (B) WB verified the expression of DCTPP1 in the chorionic tissue of patients with normal abortion and missed abortion (3 cases each).

[0029] Figure 8 In the embodiment of the present invention, the expression of DCTPP1 is increased to reduce the apoptosis of HTR8 / SVneo cells, wherein (A) WB test of the expression of apoptosis and anti-apoptotic proteins in the three groups of cells after DCTPP1 rescue; (B) statistical results of the ratio of apoptosis and anti-apoptotic proteins in the three groups of cells. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments.

[0031] Example 1: Construction of transient transfection for DCTPP1 knockdown in HTR8 / SVneo cells

[0032] See also Figure 1 The most efficient siRNA was screened out by RT-PCR and WB, and immunofluorescence and lipid peroxide detection experiments showed that the oxidative stress of DCTPP1 knockdown cells was increased compared with that of the control group cells.

[0033] Example 2: Verification of whether knockdown of DCTPP1 affects proliferation and apoptosis of HTR8 / SVneo

[0034] For verification using CCK8 assay, see Figure 2 , the absorbance value of the CCK8 experiment showed that DCTPP1 knockdown treatment did not affect cell proliferation 24 hours later, probably because only mRNA knockdown was performed at this time. However, at 48 hours, the growth of cells in the knockdown group was significantly inhibited, and the number of cells was reduced compared with 24 hours, indicating that cells underwent apoptosis. Consistent with cell proliferation, the present application demonstrated that after 48 hours of knockdown treatment, the apoptotic cell rate in the knockdown group was significantly increased compared with the control group.

[0035] Example 3: Exploring how DCTPP1 affects HTR8 / SVneo cell viability

[0036] By performing RNA sequencing on the control group and DCTPP1 knockdown cells, it was found that the expression of a series of genes affecting cell cycle, oxidative stress and apoptosis changed significantly after knockdown of DCTPP1. Figure 3 , RT-PCR test verified the same result.

[0037] See also Figure 4 ,Protein spectrum experiments found that DCTPP1 binds to AUF1, and this application also used immunoprecipitation experiments to prove the interaction between DCTPP1 and AUF1.

[0038] Meanwhile, for transient transfection of AUF1 knockdown in HTR8 / SVneo cells, please refer to Figure 5 The successful knockdown of AUF1 was verified by WB, and immunofluorescence and cell proliferation experiments showed that the AUF1 knockdown group had increased oxidative stress and slowed cell proliferation, ultimately leading to increased cell apoptosis.

[0039] Next, see Figure 6 In this application, RNA sequencing was performed on the control group and AUF1 knockdown cells, and it was found that the expression of a series of genes affecting cell cycle, oxidative stress and apoptosis changed significantly after AUF1 knockdown. RT-PCR test verified the same results, which is consistent with the function after DCTPP1 knockdown.

[0040] In addition, the present application used chorionic villus tissues of 3 patients with normal abortion and 3 patients with missed abortion to verify the expression of DCTPP1 at the RNA and protein levels, and found that DCTPP1 was significantly reduced in the chorionic villus tissues of patients with missed abortion.

[0041] Finally, the present application used DCTPP1-6×HA plasmid transfection into DCTPP1 knockdown cells to rescue DCTPP1 knocked down by siRNA. WB test found that the ratio of apoptotic gene protein amount to anti-apoptotic gene protein amount in the rescue group was significantly lower than that in the knockdown group.

[0042] The following are some experimental steps in the above examples:

[0043] Cell culture and transient transfection of siRNA

[0044] The HTR8 / SVneo cells used in the experiment were cultured in DMEM / F12 medium containing 10% fetal bovine serum (FBS) at 37° C. in a 5% carbon dioxide incubator.

[0045] The target sequence of DCTPP1 siRNA is:

[0046] si-DCTPP1-1:CCUCCAUGCUGAGUUUGCUTT,

[0047] si-DCTPP1-2:GCCCUUCAAGAGGAGCUUATT,

[0048] si-DCTPP1-3:GCUCUUCCCGCAAGUAUACTT.

[0049] The target sequence of AUF1 siRNA is: AGACUGCACUCUGAAGUUATT.

[0050] When the cell density was 70%, siRNA was transfected in Opti-MEM reduced serum medium using Lipo3000 reagent according to standard protocols. After 6 hours of transfection, the transfection medium was replaced with complete medium. After 24 hours, cells were collected for RT-PCR and 48 hours for immunoblotting.

[0051] Quantitative RT-PCR

[0052] Total RNA was extracted with TRIzol reagent, and 200 ng of RNA was reverse transcribed using PrimeScript RT MasterMix. Each PCR reaction consisted of 10 μL of TB Green Premix, 7.8 μL of water, 1 μL of cDNA sample, and 1.2 μL of gene-specific primers. Gene expression was detected using a real-time system. The experiment was performed at least 3 times, using 2 -△△Ct The relative gene expression of actin was calculated by this method.

[0053] Western blot analysis

[0054] Total protein was extracted from each group of cells in cell lysis buffer, and the protein concentration was detected using a BCA kit. 30 μg of protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane. After blocking with blocking solution, the membrane was incubated with primary antibodies (DCTPP1 (1:1000), AUF1 (1:1000), Gapdh (1:3000)) at 4°C overnight. After washing, the secondary antibody was incubated at 25°C for 1 hour. The protein bands were observed using the Amersham Typhoon Odyssey infrared imaging system.

[0055] Reactive oxygen species (ROS) assay

[0056] The generation of ROS was detected based on the fluorescence intensity using a superoxide anion fluorescent probe (DHE). After being taken up by living cells, DHE can be dehydrogenated to produce ethidium bromide under the action of superoxide anions in the cells. Ethidium bromide can bind to RNA or DNA to produce red fluorescence. Each group of cells was seeded in a 24-well plate with a circular cell slide and incubated with a DHE probe in the dark for 30 minutes. After removing the DHE probe, the cells were washed twice with PBS. The cells were then fixed with 4% paraformaldehyde for 30 minutes and stained with Hoechst for 10 minutes. The cells were placed on a slide and examined under a fluorescent laser scanning microscope.

[0057] Lipid peroxide (LPO) content detection

[0058] Oxidative stress is assessed by lipid peroxidation. LPO is heated under acidic conditions to produce malondialdehyde (MDA), which condenses with thiobarbituric acid (TBA) to form a brown-red substance called trimethoate (3,5,5-trimethyloxazole-2,4-dione) with a maximum absorption wavelength of 532nm. The content of LPO in the sample can be determined by colorimetry. Calculate the relative content of LPO according to the instructions.

[0059] Cell proliferation assay

[0060] The cells were seeded in a 96-well plate at a concentration of 5000 cells / 100 μL / well and transfected with DCTPP1 or AUF1 siRNA. At 0h, 24h, and 48h after transfection, the cells were incubated with CCK-8 solution for 2 hours. The OD value at 450nm was detected by an enzyme-labeled instrument.

[0061] TUNEL assay

[0062] The apoptosis rate of cells after knockdown of DCTPP1 and AUF1 was detected by one-step TUNEL apoptosis detection kit, respectively. Fluorescein (FITC)-labeled dUTP can be added to the exposed 3'-OH and exposed to genomic DNA breaks catalyzed by terminal deoxynucleotidyl transferase (TdT). The reaction solution (buffer, dUTP, and TdT enzyme were mixed in a ratio of 50:5:1) and incubated with cells in a humidified chamber at 37°C for 2 hours. The cells were then washed three times with PBS and stained with Hoechst for 10 minutes. Finally, the cells were observed under a fluorescent laser scanning microscope.

[0063] Plasmid construction and transient transfection

[0064] DCTPP1 cDNA was cloned into pcDNA3.1-6×HA vector, and AUF1 cDNA was cloned into p3×FLAG-myc-CMV-24 expression vector. When the cell density was 70%, the vectors were transfected in Opti-MEM medium using Lipo3000 reagent according to the standard protocol. After 6 hours of transfection, the transfection medium was replaced with complete medium. After 24 hours, the cells were collected for IP and immunoblotting.

[0065] Co-immunoprecipitation

[0066] 24h after transfection, cells were lysed in mild RIPA lysis buffer, and protease inhibitors were added before use. After ice bathing for 20min, the cell lysate was centrifuged at high speed, and the supernatant was incubated with anti-HA or FLAG magnetic beads at 4°C overnight. The beads were washed 3 times with buffer (50mM, pH 7.4, 150mM Nacl and 0.1% NP-40), and the bound proteins were eluted with diluted SDS buffer (10mMTris, pH 7.4, 1mM EDTAand1% SDS) and used for immunoblot analysis.

[0067] Transcriptome sequencing

[0068] Total RNA from HTR8 / SVneo cells in the control and treatment groups was isolated using TRIzol. mRNA was enriched from total RNA, and transcriptome libraries were constructed and sequenced using the DNBSEQ high-throughput platform, followed by bioinformatics analysis. RNA sequencing was performed by BGI Genomics Ltd.

[0069] Statistical analysis

[0070] Statistical analysis was performed using a two-tailed unpaired t-test using GraphPad PRISM version 5. All experiments were repeated at least three times, and the data were considered statistically significant when the p value was less than 0.05.

[0071] The following are the experimental materials and sources used in the above experiments of this application:

[0072] Human chorionic trophoblast cells HTR8 / SVneo were purchased from Nanjing KeyGene Biotechnology, catalog number KG557.

[0073] DMEM / F12 medium and FBS were purchased from GIBCO. siRNA was designed and purchased from Suzhou Jima Biotechnology Co., Ltd. pcDNA3.1-6×HA and p3×FLAG-myc-CMV-24 plasmids are commercially available expression vectors purchased from Shanghai Sangon Biotechnology Co., Ltd. RNA reverse transcription kit and fluorescent quantitative PCR enzyme were purchased from Japan TaKaRa Biotechnology Co., Ltd. DHE, CCK8 and apoptosis kits were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. LPO kit and Escherichia coli DH5α competent cells were purchased from Beijing Solebao Technology Co., Ltd. Protein immunoblotting precast gels and related reagents were purchased from Nanjing GenScript Biotechnology Co., Ltd. Antibodies against DCTPP1 protein were purchased from Abcam Biotechnology Co., Ltd., UK.

[0074] The transcriptome sequencing technology was completed by Shenzhen BGI Genomics Co., Ltd.

[0075] The experimental reagents not specifically described in the present invention are all conventional reagents in the art, and can be prepared according to conventional methods in the art or purchased from relevant reagent suppliers; the experimental methods not specifically described are all conventional methods in the art, and reference can be made to relevant experimental manuals, such as molecular cloning experimental manuals or instructions of relevant reagent manufacturers.

[0076] Based on the above, this application makes the following conclusions:

[0077] The application of DCTPP1 as a target in the preparation of a missed abortion detection product, wherein the detection product is a detection reagent or a detection kit.

[0078] The present application also provides the use of DCTPP1 as a target in the preparation of a drug for the prevention and early treatment of missed abortion, wherein the drug achieves the prevention and early treatment of missed abortion by increasing the expression of DCTPP1.

[0079] Based on the above content, the present application also provides a missed abortion detection product, which is a detection reagent or a detection kit, and the detection product screens for missed abortion by detecting the expression level of DCTPP1 in embryonic trophoblast cells.

[0080] In addition, the present application also provides a drug for preventing and early treating missed abortion, wherein the drug is used to increase the expression of DCTPP1.

[0081] The present application also provides a method for constructing a DCTPP1 knockdown human embryonic trophoblast cell model, which comprises the following steps:

[0082] S1: Design and synthesize siRNA to knock down the expression of DCTPP1;

[0083] S2: The knockdown siRNA is transferred into the embryonic trophoblast cells via liposomes;

[0084] S3: RT-PCR and WB confirmed that the knockdown efficiency was greater than 70%, indicating successful knockdown.

[0085] In summary, this application has proved through specific experiments that DCTPP1 knockdown can increase the oxidative stress of HTR8 / SVneo cells, inhibit cell proliferation, and ultimately lead to cell apoptosis. Transcriptome sequencing analysis also confirmed that DCTPP1 affects the apoptosis pathway of cells by affecting the oxidative stress of cells; in terms of mechanism, DCTPP1 binds to AUF1, prompting cells to affect cell viability through the oxidative stress pathway. The experimental results in this application show that DCTPP1 is one of the key genes for oxidative stress and apoptosis of embryonic trophoblast cells, and the occurrence of missed abortion can be screened early by detecting the expression of DCTPP1. In addition, DCTPP1 can also be used as a target to prepare drugs for the treatment or prevention of missed abortion.

Claims

1. Application of DCTPP1 as a target in the preparation of missed abortion detection products.

2. The use of DCTPP1 as a target in the preparation of a missed abortion detection product according to claim 1, characterized in that: The detection product is a detection reagent or a detection kit.

3. The use of DCTPP1 as a target in the preparation of a missed abortion detection product according to claim 2, characterized in that: The detection product screens for missed abortion by detecting the expression level of DCTPP1 in embryonic trophoblast cells.

4. Application of DCTPP1 as a target in the preparation of drugs for the prevention and early treatment of missed abortion.

5. The use of DCTPP1 as a target in the preparation of a drug for the prevention and early treatment of missed abortion according to claim 4, characterized in that: The drug achieves the prevention and early treatment of missed abortion by increasing the expression of DCTPP1.

6. A missed abortion detection product, characterized in that: The detection product is a detection reagent or a detection kit, and the detection product screens for missed abortion by detecting the expression level of DCTPP1 in embryonic trophoblast cells.

7. A drug, characterized in that: The medicine is used for preventing and treating missed abortion at an early stage, and the medicine is used for increasing the expression of DCTPP1.

8. A method for constructing a DCTPP1 knockdown human embryonic trophoblast cell model, characterized in that: The following steps are involved: S1: Design and synthesize siRNA to knock down the expression of DCTPP1; S2: The knockdown siRNA is transferred into the embryonic trophoblast cells via liposomes; S3: RT-PCR and WB confirmed that the knockdown efficiency was greater than 70%, indicating successful knockdown.

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