CN111172161B - Long non-coding RNA and application thereof in diagnosis/treatment of preeclampsia - Google Patents

Abstract

The invention belongs to the field of genetic engineering, and particularly relates to application of PDIA3P1 in preparation of drugs for diagnosing preeclampsia and target spots; down-regulation of PDIA3P1 in the placenta tissue of a preeclampsia pregnant woman is related to the occurrence and development of preeclampsia, low expression level of PDIA3P1 has close relation with pathogenesis of preeclampsia, and by changing the expression of PDIA3P1 to influence proliferation, apoptosis, invasion, migration and the like of trophoblast of the preeclampsia pregnant woman, the expression of PDIA3P1 is enhanced, so that the proliferation, invasion and migration of trophoblast can be promoted.

Description

Long non-coding RNA and application thereof in diagnosis/treatment of preeclampsia

Technical Field

The invention belongs to the field of genetic engineering, and in particular relates to application of long non-coding RNA-PDIA3P1 in diagnosis and preparation of a medicament for treating preeclampsia.

Background

Preeclampsia is one of the most common gestational complications worldwide and is the main cause of death associated with the gestational complications, and world health organization statistics reports indicate that preeclampsia or eclampsia directly leads to about 14% (about 8500) of maternal death worldwide each year, which is one of the most common causes of fetal premature delivery and fetal growth limitation and maternal death. Despite the long-felt progress of current medical treatments, the overall incidence of patients remains high. With rapid development of sequencing technology and molecular biology, gene diagnosis and molecular targeted therapy have become hot spots in preeclampsia treatment. Thus, the study of the molecular mechanisms involved in the development and metastasis of preeclampsia is critical to the formulation of specific diagnostic methods and personalized therapeutic strategies.

Over the last decade, gene expression analysis techniques and bioinformatics based on rapid emergence of high throughput sequencing have driven extensive research in human genomics to discover non-coding RNAs. Only 2% of the human genome is transcribed into proteins, while the vast majority is transcribed into non-coding RNAs, including small ribonucleic acids, long non-coding RNAs (lncRNAs), and pseudogenes. Recently, the role of mirnas in various aspects of cellular processes has been demonstrated, however, functional studies of lncRNAs are not well-defined. The new data of the GENECODE research group in the code program shows thousands of lncRNAs, but only some of them are biologically functional. Interestingly, these lncRNAs are involved in regulating a variety of cellular processes, including recombinant stem cell pluripotency, parental imprinting and diffusion and metastasis of tumor cells, and epigenetic modification and adsorption of miRNAs through chromatin remodeling.

Recently, extensive studies have shown that aberrant lncRNAs expression exert different biological mechanisms involved in the development of a variety of human diseases. For example, lncRNA ROR can promote the promoter region H3K9 demethylation of TESC to participate in tumorigenesis by inhibiting methyltransferase G9A. Meanwhile, AOC4P inhibits epithelial-to-mesenchymal transition (EMT) by binding to vimentin and promoting its degradation, thereby inhibiting hepatocellular carcinoma metastasis. In addition, upregulated SPRY4-IT1 inhibits preeclampsia trophoblast proliferation, migration and angiogenic capacity by binding to HUR. These findings suggest that lncRNAs play a critical role in the development and progression of human disease, particularly preeclampsia. Thus, the discovery of more preeclampsia-related lncRNAs and the study of their biological functions and mechanisms is of great importance for a better understanding of the molecular biology of the development of preeclampsia.

PDIA3P1 is a 2099nt long lncRNA with the sequence shown in sequence 1 and located on human chromosome 1q21.1. We found that PDIA3P1 was down-regulated in the placenta tissue of preeclampsia pregnant women compared to that of normal pregnant women. After overexpression or knockout of PDIA3P1, the effect of PDIA3P1 on the onset and progression of preeclampsia was studied and the function of the relevant target gene of PDIA3P1 in preeclampsia pregnant women's trophoblasts was studied.

Disclosure of Invention

The invention aims to provide an application of PDIA3P1 in diagnosing preeclampsia and preparing a medicament for treating preeclampsia.

The invention relates to a long-chain non-coding RNA, the nucleotide sequence of which is shown as SEQ ID NO. 1.

Application of long-chain non-coding RNA in preparing a medicament for treating preeclampsia;

use of long non-coding RNA in the preparation of a reagent for diagnosing preeclampsia;

a primer for detecting RP11-7K24.3, namely: SEQ ID NO. 2R PDIA3P1F ATGGGCCTGTGAAGGTAGTG,SEQ ID NO:3: PDIA3P 1R GTGGCATCCATCTTGGCTAT;

an siRNA that interferes with RP11-7K24.3, namely: SEQ ID NO. 4 si-PDIA3P1 1#CATTAGTGATAAAGATGCCTCTATA,SEQ ID NO:5 si-PDIA3P1 2#GATAACGGAGATGGTATATCATCTTAT;

a kit comprising the primer;

a pharmaceutical composition comprising said long non-coding RNA;

the application of the primer in preparing a reagent for diagnosing preeclampsia;

the application of the pharmaceutical composition in preparing medicines for treating preeclampsia.

The pharmaceutical composition further comprises auxiliary materials. The auxiliary materials comprise: (lip 2000, opti-mem broth, PBS phosphate buffered saline).

The concentration of the kit in the primer is 10mol/L respectively.

Technical proposal

The differential expression of PDIA3P1 in clinical tissues is screened by qPCR, and the expression level of PDIA3P1 in the placenta tissues of the preeclampsia pregnant women is lower than that in the placenta of normal pregnant women. Guess: whether PDIA3P1 is involved in the pathogenesis of preeclampsia diseases.

And then selecting internationally approved normal trophoblasts as experimental study objects, designing an interference sequence of PDIA3P1, and transferring the interference sequence into cells by taking lip2000 as a transfection vector to simulate the pathogenesis of preeclampsia diseases. By detecting the functions of the cells such as proliferation, apoptosis, angiogenic ability, etc. after the transfer of the interfering sequence into the cells. Thus, it is proved that the knocking down of PDIA3P1 expression in normal trophoblast HTR-8/SVneo affects the function of cells and induces or accelerates the onset of preeclampsia diseases. In contrast, the PDIA3P1 plasmid was constructed and the function of PDIA3P1 in trophoblast HTR-8/SVneo was verified in both positive and negative directions.

By transcriptome sequencing, detecting the related downstream target genes of PDIA3P1 possibly involved in cell functions (such as proliferation, apoptosis or angiogenesis), then preliminary discussion of the regulation mechanism of PDIA3P1, finding that PDIA3P1 exists more in the nucleus of trophoblasts by means of FISH and the like, considering that PDIA3P1 can regulate the corresponding target genes at posttranscriptional level, detecting and verifying that PDIA3P1 promotes the expression of the downstream target gene DCN by recruiting JMJD2A by RNA co-immunoprecipitation and DNA co-immunoprecipitation experiments.

The various reagents required for the transfection process,

(1) lip2000, a versatile liposome transfection reagent, is suitable for transfection of DNA, RNA and oligonucleotides, and has high transfection efficiency for most eukaryotic cells. The unique formula enables the PDIA3P1 interference sequence to be directly added into a culture medium, and the transfection efficiency is not affected by the existence of serum, so that the PDIA3P1 interference sequence is transferred into cells.

(2) Opti-mem culture medium containing HEPES,2400mg/L sodium bicarbonate, hypoxanthine, thymine, sodium pyruvate, L-glutamine, microelements, growth factors and phenol red reduced to 1.1mg/L is used as an auxiliary material of transfection reagent, and is not harmful to cells per se, but is better and more effectively transferred into cells to achieve the intended purpose.

(3) The PBS phosphate buffer salt solution (phosphate buffer saline) generally acts as a solvent to solubilize the protecting agent. The buffer solution is the most widely used in biochemical research, and the main components are Na2HPO4, KH2PO4, naCl and KCl, and the pH value range of the buffer solution is very wide because the Na2HPO4 and KH2PO4 have secondary dissociation; whereas NaCl and KCl act primarily to increase salt ion concentration. Eliminating the influence of the self on the experimental object.

Tissue collection

We collected 30 pairs of placenta tissue of pregnant women diagnosed with preeclampsia and placenta tissue of normal pregnant women without any underlying disease, which were subjected to caesarean section surgery in the national hospitals of Jiangsu province, and the young health care homes of Jiangsu province, 2017 through 2018. And records clinical characteristics: including age of pregnant woman, history of smoking, week number of pregnancy, systolic blood pressure, diastolic blood pressure, proteinuria and fetal weight. Tissue samples were all collected with liquid nitrogen at the first time or stored at-80 ℃ until RNA extraction. The study was approved by the ethical committee of the university of south Beijing medical science. Written informed consent was obtained for all patients.

Cell lines

Trophoblasts (HTR-8/SVneo) were selected from Canadian university of Queen. HTR-8/SVneo cells were cultured in RPMI 1640 medium containing 5% fetal bovine serum, 100U/ml penicillin and 100mg/ml streptomycin. The culture was routinely performed in a 37℃incubator with 5% CO 2. Fresh medium was changed every 2-3 days and passaged when cell confluence reached 80% -90%. All cell lines were verified by DNA analysis of short tandem repeats.

RNA extraction and quantitative PCR analysis

Total RNA was isolated with Trizol reagent according to the instructions of the reagent. The reverse transcription reaction was performed using TaKaRa Prime Script kit (TaKaRa, dalian, china). Reverse transcription kit reverse transcription was performed on 0.8. Mu.g total RNA with a final volume of 20. Mu.l. Analysis of results: analyzing the specificity and amplification efficiency of the primer, and judging the reaction specificity of the primer according to the dissolution curve. And (3) obtaining a Ct value according to the amplification curve, and analyzing the relative expression quantity of the target gene by adopting a relative quantity method and an internal reference GAPDH. The calculation formula is as follows: 2 (-DELTACt), DELTA Ct=Ct gene-Ct control.

Plasmid construction

Synthesizing human full-length PDIA3P1 cDNA, inserting it into eukaryotic expression vector pCDNA3.1, constructing PDIA3P1 over-expression vector plasmid, then transforming the above-mentioned plasmid into colibacillus, shaking, culturing, selecting monoclonal bacterium, culturing and amplifying.

Cell transfection

Plasmid vectors (pCDNA3.1-PDIA 3P1, si-PDIA3P1 and empty plasmid) for transfection were all extracted with endotoxin-removing plasmid extraction kit (DNA midi prep kit, qiagen). The interfering sequences of PDIA3P1 and the disorder control (si-NC) were all purchased from Invitrogen (Invitrogen, CA, USA). Planting cells HTR-8/SVneo on a 6-hole culture plate according to 2X 105 cells per hole, sucking the original culture medium before transfection by 6 h after the cells are attached to the wall, and changing the culture medium into a double-antibody-free culture medium; diluting 10 μl of liposome into 240 μl of OPTI-MEM, gently stirring, mixing, and incubating at room temperature for 5min; 100pmol of siRNA, si-NC or 3ug plasmid vector are respectively diluted in 250 mu L of OPTI-MEM, blown and evenly mixed, and incubated for 5min at room temperature; mixing the incubated liposome with siRNA or plasmid diluent, and gently blowing and mixing. Incubation was continued for 20min at room temperature; the mixture was uniformly dropped into a 6-well plate to which 1.5 mL of OPTI-MEM was added, followed by gentle mixing. After continuous culture at 37℃in a 5% CO2 incubator for 6 h, the complete medium was changed. 36 h after transfection, the RNA or protein is collected from the cells for real-time quantitative RT-PCR or immunoblot analysis.

Cell proliferation activity assay

MTT assay treated cells were seeded at 2000-3000 cells per well in 96 well plates. After 80% of the cells adhere, the cells are synchronized to 12 h, and the original culture medium is discarded. Each sample was provided with 6 duplicate wells, with a total reaction volume of 200 μl per well. Mu.l of MTT reaction (5 mg/ml in PBS) was added to each well and incubated at 37℃for 4 h in the absence of light. The supernatant was discarded, 150. Mu.l of Dimethylsulfoxide (DMSO) was added to each well, and the mixture was shaken for 10 min, and the absorbance at 490 and nm wavelengths was measured by a microplate reader.

Clone formation experiments, treated cells were seeded at 600, 800, 1000 cells per well in 6 well plates. 2ml of complete medium is added to each well, the culture is carried out for 10 to 14 days, the treatment is carried out, 4% paraformaldehyde is added after the complete medium is discarded for 30 minutes, and crystal violet is added after the paraformaldehyde is discarded for dyeing for 2 hours. Finally, the solution was rinsed with flowing PBS. Air-drying and photographing.

Flow cytometry

Apoptosis assay HTR-8/SVneo cells after 48 hours of transfection were collected by pancreatin digestion and subsequently stained with Annexin V-FITC fluorescent probe and Propidium Iodide (PI) according to FITC Annexin V apoptosis assay kit (BD) and instructions for its use. Flow cytometer detection and analysis.

Cell cycle detection PI staining was performed using the CycleTESTTM PLUS DNA kit (BD) according to the instructions followed by FACScan analysis.

Subcellular structure localization (nuclear mass separation)

The nuclei and cytoplasm of HTR-8/SVneo cells were isolated using the PARIS kit (Life Technologies, USA) according to the instructions for use. The distribution of PD1A3P1, GAPDH and U1 in the cytoplasm and nucleus was examined using qPCR method. GAPDH is a cytoplasmic reference and U1 is a nuclear reference. Expression of PDIA3P1, GAPDH and U1 in the cytoplasm and nucleus was presented as a percentage of total RNA.

In situ hybridization technique (FISH)

According to the characteristics of PDIA3P1 gene transcript, designing a corresponding probe (synthesized by Shanghai Bogu biotechnology company), planting HTR-8/SVneo cells in a 6-well plate containing 15mm climbing plates, after the cells reach about 80%, discarding the culture medium, washing twice with PBS, adding 2ml of methanol for fixing for 30min, sending a sample, carrying out the following treatment by Shanghai Bogu biotechnology company, selecting an inverted silver light microscope for photographing, qualitatively detecting the subdosition of PDIA3P1 in the cells, and further verifying the nuclear mass separation experimental result.

RNA-seq (transcriptome sequencing)

Cells were seeded in six well plates, and after cells had grown to about 80%, they were subjected to 10ul of lip2000 si-PDIA3P1 and si-NC treatment, and after 48 hours, cells were collected by Trizol treatment, sampled, and subjected to Beijing gene detection mechanism, and Illumina was selected for subsequent experiments to obtain and process corresponding data.

RIP-qPCR experiments

Scraping the trophoblasts in the oversized dish, and lysing with RIP lysis buffer; incubating the whole cell extract with magnetic beads of JMJD2A antibody at 4 ℃ for 8 hours, wherein mIgG is selected as a control; after washing the beads with various wash buffers, the complexes were incubated with 0.1% SDS/0.5mg/ml proteinase K (30 min at 55 ℃) to remove proteins; the purified RNA was subjected to qRT-PCR analysis to demonstrate that PDIA3P1 can bind to JMJD2A antibody.

ChIP-qPCR

Analysis detects that JMJD2A binds to a promoter region of DCN gene, and apparent regulation of DCN expression is achieved. Briefly, 550. Mu.l of 37% paraformaldehyde was added to a petri dish containing 20mL of medium; incubating at room temperature on a shaking table; adding 10X glycine to the culture dish; shaking on a shaking table and incubating at room temperature; placing the culture dish on ice for standby; sucking the culture medium; washing cells with 20ml of PBS; centrifuging; the supernatant was removed. Incubate on ice for 15min, gently vortex cell lysates every 5min; centrifuging; 0.5ml of nuclear lysate resuspended cells; ultrasonic treatment on ice and centrifugation; the supernatant was dispensed into EP tubes every 50. Mu.l; preparing IP reaction, and adding 450 μl of a solution buffer into each reaction tube; adding the antibody and 20 μl of the uniformly mixed magnetic beads into the reaction solution, and incubating at 4deg.C overnight; washing the magnetic bead-antibody, binding the antibody-target protein-DNA complex, and precipitating with buffer vortex; washing the precipitated complex to remove some nonspecific binding; eluting to obtain an enriched target protein-DNA complex; uncrosslinking and purifying the enriched DNA-fragments; qPCR analysis of DCN promoter region fragment. The desired cDNA was obtained for the next qRT-PCR analysis.

Western blotting (Western blotting)

The denatured cellular protein lysate was added to a pre-prepared 10% denatured polyacrylamide gel (SDS-PAGE) well to isolate the protein in the sample. Subsequently transferred to NC membranes and incubated with specific antibodies. Finally, the photo-resist film is exposed by an ECL luminous hydraulic plate. GAPDH antibody was used as a control and DCN antibody was purchased from protentech company.

Data processing

Experimental data were analyzed using SPSS17.0 software and expressed as mean ± standard error of three experiments, and differences between groups were checked using two-tailed Student's T, rank sum test and chi-square test. Subsequent reuse of p <0.05 in single factor analysis was a multifactorial analysis.

Compared with the prior art, the invention has the technical effects that:

we have found a novel lncRNA PDIA3P1 and demonstrated down-regulation of its expression in preeclampsia tissues. Down-regulated PDIA3P1 expression is intimately involved in preeclampsia pathogenesis. Inhibiting cell proliferation and angiogenesis after knocking down PDIA3P1, and promoting apoptosis of trophoblasts. Furthermore, we believe that knockdown of PDIA3P 1-mediated trophoblast growth inhibition is dependent to some extent on DCN expression. Here, we demonstrate for the first time that PDIA3P1 performs a related function in human trophoblasts by inhibiting expression of trophoblast DCN. lncRNAs regulate expression of a target gene by various mechanisms, such as recruitment of chromatin-regulating enzymes to the target gene and cis-or trans-regulated transcription, as scaffold binding to related molecular origins or adsorption of miRNAs. In this study, we found that PDIA3P1 recruited JMJD2A mediated key regulators of the trophoblast DCN up-regulated network. After inhibition of JMJD2A, DCN expression was up-regulated, qPCR and immunoblotting showed. Taken together, these findings demonstrate that down-regulated PDIA3P1 expression can impair trophoblast biological function by recruiting JMJD 2A-mediated JDP2 expression.

Our study found for the first time that placental tissue and cellular PDIA3P1 expression was down-regulated in preeclampsia pregnant women. The placenta trophoblast HTR-8/SVneo knockdown PDIA3P1 expression in preeclampsia pregnant women shows a cell inhibition function, and the downregulation of PDIA3P1 shows the effects of inhibiting cell proliferation and angiogenesis and promoting apoptosis increase. Furthermore, it is possible to provide a device for the treatment of a disease. Our findings further enrich preeclampsia pathogenesis and facilitate lncRNA-directed diagnosis and treatment.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.

FIG. 1, down-expression of PDIA3P1 in placental tissue of preeclampsia pregnant women

1A-1BPDIA3P1 was down-regulated in the placental tissue (n=30) expression of preeclampsia pregnant women.

1C the relative expression of PDIA3P1 was examined in four trophoblast cell lines and HTR-8/SVneo was found to be the highest and JEG-3 was found to be the lowest.

FIG. 2, effect of PDIA3P1 on the angiogenic ability of HTR-8/SVneo cells

2A-2D knockdown of PDIA3P1 inhibits the angiogenic capacity of trophoblast HTR-8/SVneo.

FIG. 3 detection of potential downstream target genes involved in PDIA3P1 mediated feeder cell growth by sequencing

3A RNA transcriptome sequencing was performed after HTR-8/SVneo trophoblasts interfered with PDIA3P 1.

3B carries out various bioinformatics technical analyses including GO enrichment analysis and COG function classification on the sequencing result, and the downstream differential gene functions are mainly as follows: cell migration-related genes, apoptosis-related genes, extracellular matrix-related genes, and proliferation-related genes.

After 3C-3D knocking down PDIA3P1, the DCN is verified to be a potential target gene by RT-PCR and a western blotting method.

Fig. 4, PDIA3P1, regulates DCN expression by recruiting JMJD 2A.

4A-4BFIsh and nucleoplasm isolation experiments detected the position of PDIA3P1 in HTR-8/SVneo.

The 4C RIP experiment shows that the association degree of the PDIA3P1 and JMJD2A protein is highest, and the up-regulation multiple is highest.

After 4D-4E interfered with JMJD2A, DCN was up-regulated at the gene and protein level.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the invention.

The experimental procedures described in the examples, without reference to specific conditions, are essentially carried out according to the conditions and methods described in Sambrook, J et al, code of molecular cloning, laboratory Manual,3rd edition Huang Peitang et al, science Press 2002.8, or according to the conditions and methods recommended by the materials provider, other techniques not described in detail corresponding to standard procedures well known to those skilled in the art.

The material of the invention: the cell lines and media mentioned in this application are commercially available or otherwise publicly available, by way of example only, and are not exclusive of the present invention, and may be replaced with other suitable tools and biological materials, respectively.

Example 1

Detection of expression of PDIA3P1 in tissues and cells

0.1. 0.1 g tissue was taken, either fully ground with liquid nitrogen (powdered) or 1-5X 107 cells were discarded from medium and rinsed 2 times with pre-chilled PBS. Adding Trizol lysate of 1 ml, blowing and mixing with enzyme-free gun head, standing for 5min, and transferring lysate into pre-labeled enzyme-free 1.5 ml centrifuge tube. Centrifuging at 7500 and g at 4deg.C for 5min, adding 1/5 volume of chloroform into supernatant, mixing under reverse condition for 30 s, and standing for 2 min. Centrifuge at 4℃and 12000 and g for 15 min. The solution was separated into three layers (aqueous phase-white precipitate-red organics) and the aqueous phase layer was transferred to a new 1.5 ml centrifuge tube, with minimal aspiration to the white precipitate. Adding equal volume of isopropanol, mixing, and standing for 5-10 min. Centrifuge at 4℃and 12000 and g for 10 min. The supernatant was removed by pipetting, 1 ml of 75% ethanol (as prepared) was added and the RNA pellet was washed. Centrifuge at 4 ℃,7500 g for 5min, discard supernatant. Removing 75% ethanol as much as possible, and air drying at room temperature for about 15 min. RNA precipitate was dissolved with RNase-free water (20-25. Mu.l).

The concentration of RNA was determined by UV absorbance measurement. RNA concentration and purity were determined using an ultraviolet spectrophotometer and zeroed with DEPC water for dissolving RNA prior to measurement. A reading of 1 at 260 nm is 40 ng/. Mu.l, and the ratio of A260/A280 of the RNA solution is used for the detection of RNA purity, with ratios ranging from 1.8 to 2.1 indicating satisfactory. Agarose gel electrophoresis identified the integrity of the RNA. 1% agarose gel was prepared. Agarose was dissolved by heating, cooled and 1. Mu.l ethidium bromide (EB, 10 mg/ml) was added. Shaking, pouring the gel, condensing the gel, placing in an electrophoresis tank, and soaking in 1 xTAE buffer solution for balancing for 10 min for use. And (5) spotting. 5 Xnucleic acid electrophoresis loading buffer was mixed with the samples at 1:4 (v/v) and each sample containing 1. Mu.g of RNA was accurately added to the gel wells. 80 V constant pressure electrophoresis for 50 min. After the electrophoresis was completed, the result was observed on a gel imager.

Tris-acetate (TAE) buffer formulation (1L) 50×:

2M Tris base 242 g

1M acetic acid 57.1 mL glacial acetic acid (17.4M)

100 mM EDTA200 mL 0.5 M EDTA (pH8.0)

Deionized water to 1L

Real-time quantitative PCR

The total RNA of HTR-8/SVneo cells of the placenta tissue of the preeclampsia pregnant woman and the placenta tissue of the normal pregnant woman is used for the reverse transcription reaction by using a TaKaRa PrimeScript kit (Dalianbao bioengineering Co., ltd.). The reverse transcription reaction system is as follows:

5 × PrimeScript Buffer（for Real Time）4 μl

Total RNA (1 μg/μL)1 μl

random or OligadT 2. Mu.l

Primescript RT enzyme Mix1μl

RNase Free dH ₂ O to 20. Mu.l

The reverse transcription reaction conditions were as follows: 37 ℃ for 15min (reverse transcription reaction); 85℃for 5sec (reverse transcriptase deactivation reaction). Designing primer sequences according to gene sequences provided by Genebank,

QPCR uses 7300 PCR system (Applied Biosystems, warrington, UK). The cDNA samples were subjected to the three-part PCR amplification standard procedure. The reaction system:

SYBR Premix Ex Taq2 μl

F primer0.4 μl

R primer0.4 μl

ROX0.4 μl

cDNA1 μl

ddH ₂ O5.8 μl

reaction conditions:

95℃30 s

95℃5 s

60℃34 s

68℃45 s

analysis of results: analyzing the specificity and amplification efficiency of the primer, and judging the reaction specificity of the primer according to the dissolution curve. And (3) obtaining a Ct value according to the amplification curve, and analyzing the relative expression quantity of the target gene by adopting a relative quantity method and an internal reference GAPDH. The calculation formula is as follows: 2 (-DELTACt), DELTA Ct=Ct gene-Ct control.

The primers for PDIA3P1 were as follows:

Primer F 5’- ATGGGCCTGTGAAGGTAGTG-3’，SEQ ID NO：2，

Primer R 5’- GTGGCATCCATCTTGGCTAT-3’， SEQ ID NO：3。

the result shows that the expression of the lncRNA PDIA3P1 in the placenta tissue of the pregnant woman in preeclampsia is down-regulated compared with that in the normal tissue. We used real-time quantitative PCR to detect the expression level of PDIA3P1 in placenta tissue expression of 30 pairs of preeclampsia pregnant women than in normal tissues. The results showed that PDIA3P1 expression was reduced (fold >1.5, P < 0.05) in 60% (12/20) of preeclampsia in the placental tissue of pregnant women compared to normal maternal placental tissue (fig. 1A and 1B). It is suggested that PDIA3P1 may play an important role in diagnosis, development and treatment of preeclampsia diseases.

Example 2

To investigate the effect of PDIA3P1 on the HTR-8/SVneo phenotype of normal trophoblasts.

Firstly, selecting a normal trophoblast HTR-8/SVneo cell line as a study object of the experiment, transfecting a PDIA3P1 interference sequence by using lip2000 as a vector to knock down the expression of PDIA3P1 to simulate the pathogenesis of preeclampsia, and detecting MTT and clone proliferation experiments to find that the interference sequence of PDIA3P1 is transfected in the HTR-8/SVneo cell and the cell growth is inhibited after the expression of PDIA3P1 is knockdown. These data indicate that PDIA3P1 promotes the proliferation of HTR-8/SVneo cells.

Example 3

Effect of PDIA3P1 on apoptosis of placental trophoblast HTR-8/SVneo.

To investigate whether PDIA3P1 affects cell cycle switching on HTR-8/SVneo cell proliferation, the normal trophoblast HTR-8/SVneo cell line was used as a study subject, and lip2000 was used as a vector to transfect PDIA3P1 interfering sequences to knock down expression of PDIA3P1 to mimic the onset of preeclampsia. We performed flow cytometry analysis of whether apoptosis was involved in the inhibition of cell growth induced after PDIA3P1 knockdown. As shown in fig. 2C, early apoptosis (UR) and late apoptosis rate (LR) were higher in PDIA3P1 knockdown HTR-8/SVneo cells than in control cells. It can be seen that PDIA3P1 inhibits trophoblast apoptosis.

Example 4

PDIA3P1 is involved in HTR-8/SVneo cell angiogenic ability.

The angiogenic capacity of trophoblasts is an important aspect of the pathogenesis of preeclampsia. HTR-8/SVneo cell line was used as a subject, and lip2000 was used as a vector to transfect PDIA3P1 interference sequence to down-regulate the expression of PDIA3P 1. The effect on the angiogenic ability of HTR-8/SVneo cells after PDIA3P1 knockdown was studied using an angiogenic ability experiment. It can be seen that low expression of PDIA3P1 affects the angiogenic capacity of normal trophoblasts, further affecting the shallow implantation of the placenta, inducing the onset of preeclampsia diseases.

The numerical values set forth in these examples do not limit the scope of the present invention unless specifically stated otherwise. In all examples shown and described herein, unless otherwise specified, any particular value is to be construed as exemplary only and not as limiting, and thus, other examples of exemplary embodiments may have different values.

SEQUENCE LISTING

<110> Jiangsu province people's hospital (first affiliated hospital of Nanjing medical university)

<120> a long non-coding RNA and its use in diagnosing/treating preeclampsia

<130> 2020

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 2099

<212> DNA

<213> Homo sapiens

<400> 1

aaactaaatc aaacttgagt atgaaacttc ttttttttta agtgcctaag tcctgaatga 60

caacaaaaga ctgaagaact gacaacgatt agaggacact aaggagactt aacaactaaa 120

tgcaaaatgg gaccctgaat ctgatcctgg aacacaaaaa ctaaattagt agaaaagtgg 180

taaaataggt ggtcgcgcgc ccgaccgccg cagtcccagt cgagccgcga cccttccggc 240

tgcccccacc ccacctcgcc gccatgtgcc tccgccgccc agcgctgttc ccgggcgtgg 300

cgctgcttct cgccgcggcc cgcctcgcgg ctgcctccga cgtgctagga ctcagggacg 360

acaacttgga gagtcgcatc tccgacacgg gctctgcggg cctcatgctc gtcgagttct 420

tcgccccctg gtgtggacac tgcaagagac ttgctcctga gtatgaagct gcagctacca 480

gattaaaagg aatagtccca ttagcaaagg ctgattgcac tgccaacact aacacctgta 540

ataaatatgg agtcagtgga tatccaaccc tgaatatgtt tagagatggt gaagaagcag 600

gtgcttatga tggacctagg actgctgatg gaattgtcag ccacctgaag aagcaggcag 660

gcccagcttc agtgcctctc aggactgagg aagaatttaa gaaattcatt agtgataaag 720

atgcctctat agtaggtttt ttcgatgatt cattcagtga agctcactcc gagttcctaa 780

aagcagccag caacttgagg gataactacc gatttgcaca tacgaatgtt gagtctctgg 840

tgaacgagta tgatgataac ggagatggta tcatcttatt tcgtccttca catctcacta 900

acaagttgga ggacaagact gtggcatata cagtgcaaaa aatgaccagt ggcaaaatta 960

aaaagtttat ccaggaaaac atttttggta tctgccctca catgacagaa gacaataaag 1020

atttgataca gggcaaggac ttacttattg cttactatga tgtggactat gaaaagaatg 1080

ctaaaggttc caactactgg agaaacaggg taatgatggt ggcaaagaaa ttcctggatg 1140

ctgggcacaa actcaacttt gctgtagcta gccgcaaaac ctttagccat gaactttctg 1200

attttggctt ggagagcact gctggagaga ttcctgttgt tgctatcaga actgctaaag 1260

gagagaagtt tgtcatgcag gaggatttct cgcgtgatgg gaatgctctg gagaggttcc 1320

tgcaggatta ctttgatggc aatctgaaga gatacctgaa gtctgaacct atcccagaga 1380

gcaatgatgg gcctgtgaag gtagtggtag cagagaattt tgatgaaata gtgaataatg 1440

aaaataaaga tgtgctgatt gaattttatg ccccttggtg tggtcactgt aagaacctgg 1500

agcccaagta taaagaactt ggcgagaagc tcagcaaaga cctgaatatc gtcatagcca 1560

agatggatgc cacagccaat gatgtgcctt ctccatatga agtcagagtt ttcctaccat 1620

atacttctct ccagccaaca agaagctaaa tccaaagaaa tatgaaggtg gccatgaatt 1680

aagtgatttt attagctatc tacaacgaga agctacaaac ccccctgtaa ttcaagaaga 1740

aaaacccaag aagaagaagg cacaggagga tctctaaagc agtaggcaaa caccactttg 1800

taaaaggact cttccaccag agatgggaaa accactgggg aggactagga cccatatggg 1860

aattattacg tctcagggcc gagaggacag aatggatata atctgaatcc tgttaaattt 1920

tctctaagcc atttcttagc tgcactgtta tggaaatacc aggaccagtt tatgtttgtg 1980

gttttgggaa aaattattgg tgttggggga aatgttgtgg gagtcgggtt gagttggggg 2040

tattttctaa tttttttgtg catttggaac agtgacaata aataagaccc ctttaaact 2099

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

atgggcctgt gaaggtagtg 20

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

gtggcatcca tcttggctat 20

<210> 4

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

cattagtgat aaagatgcct ctata 25

<210> 5

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

gataacggag atggtatcat cttat 25

Claims (3)

1. Application of PDIA3P1 in preparing a medicament for treating preeclampsia, wherein the nucleotide sequence of PDIA3P1 is SEQ ID NO:1.
2. 2. use of a pharmaceutical composition comprising PDIA3P1 as defined in claim 1 for the preparation of a medicament for the treatment of preeclampsia.
3. 3. The use according to claim 2, wherein the composition further comprises adjuvants, said adjuvants comprising lip2000, opti-mem broth, PBS phosphate buffered saline.

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