CN106754914B - A kind of long non-coding RNA and its application in diagnosis/in treatment preeclampsia - Google Patents
A kind of long non-coding RNA and its application in diagnosis/in treatment preeclampsia Download PDFInfo
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- CN106754914B CN106754914B CN201611064872.XA CN201611064872A CN106754914B CN 106754914 B CN106754914 B CN 106754914B CN 201611064872 A CN201611064872 A CN 201611064872A CN 106754914 B CN106754914 B CN 106754914B
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Abstract
The invention belongs to genetic engineering field, in particular to application of the PVT1 in preparation diagnosis preeclampsia and target drug treatment；The downward of PVT1 is related with the occurrence and development of preeclampsia in preeclampsia pregnant women placental tissue, the PVT1 and Attack of Preeclampsia mechanism of low expression level have close relationship, expression by changing PVT1 has an impact the proliferation of preeclampsia pregnant woman's trophocyte, apoptosis, invasion, migration etc., and enhancing PVT1 expression can promote trophoblastic proliferation, invasion and migration.
Description
Technical field
The invention belongs to genetic engineering fields, in particular to long non-coding RNA -- and PVT1 is diagnosing and is preparing treatment eclampsia
Early period drug application.
Background technique
Preeclampsia is one of most common complication of pregnancy 2222 in the whole world, is the main original of Pregnancy Complication associated death
Cause, World Health Organization's statistics report show that preeclampsia or eclampsia directly result in global annual about 14% (about 8500)
Maternal death is one of fetus premature labor and fetal growth restriction and the most common reason of maternal death.Although at present
Medical treatment has obtained significant progress, but patient's overall incidence is still higher.With sequencing technologies and molecular biology
Fast development, gene diagnosis and molecular targeted therapy become a hot issue in preeclampsia treatment.Therefore, research participates in son
The occurrence and development of epilepsy early period and the molecular mechanism of transfer, to formulating specific diagnostic method and personalized therapeutic strategy to closing
It is important.
In past ten years, gene expression analysis technology and bioinformatics based on the high-flux sequence quickly occurred
The research of large-scale human activities environment has been pushed, and then has had found non-coding RNAs.There was only 2% volume in human genome
Code is transcribed into albumen, and the overwhelming majority is transcribed into non-coding RNAs, including small molecule RNA, long non-coding rna
(lncRNAs) and pseudogene.Recently, miRNA has been confirmed in the effect of the various aspects of cellular processes, however,
The functional study of lncRNAs is not very thorough.ENCODE in the works GENECODE study group new data show it is thousands of
LncRNAs, but only some of which has biological function.What is interesting is these lncRNAs participate in adjusting by chromatin remodeling
Control various kinds of cell process, including recombination stem cell versatility, the diffusion and transfer of the marking and tumour cell of parent and apparent
Genetic modification and absorption miRNAs.
Recently, a large amount of studies have shown that exception lncRNAs expression is related to the human diseases of multiplicity.Such as lncRNA
ROR can promote the promoter region H3K9 demethylation of TESC to participate in tumour by inhibiting transmethylase G9A.Meanwhile
AOC4P inhibits Epithelial and stromal conversion (EMT) by the way that its degradation is bound and promoted with vimentin to inhibit hepatocellular carcinoma to turn
It moves.In addition, the SPRY4-IT1 of up-regulation inhibits preeclampsia trophoblastic proliferation, migration and vascularization by binding with HUR
Ability.It is most important that these discoveries show that lncRNAs plays the part of during the occurrence and development of human diseases especially preeclampsia
Role.Thus, it is found that more preeclampsia relevant lncRNAs and their biological function and mechanism are studied, to more preferable
Ground understands that the molecular biology of preeclampsia occurrence and development has important meaning.
PVT1 is the lncRNA that a length is 1957nt, is located at human chromosomal 8q24.21.We have found that PVT1 exists
Expression quantity is lowered in more normal pregnant women placental in preeclampsia pregnant women placental tissue.After being overexpressed or knocking out PVT1, have studied
PVT1 preeclampsia occurrence and development effect and have studied related target base of the PVT1 in preeclampsia pregnant woman's trophocyte
The function of cause.
Summary of the invention
Technical purpose
The object of the present invention is to provide PVT1 in diagnosis preeclampsia and the application in preparation treatment preeclampsia drug.
The present invention relates to a kind of long-chain non-coding RNA, nucleotides sequence is classified as SEQ ID NO:1；Its sequence are as follows: i.e.:
A kind of application of long-chain non-coding RNA in preparation treatment preeclampsia drug；
A kind of application of long non-coding RNA in preparation diagnosis preeclampsia reagent；
A kind of primer detecting PVT1, as shown in SEQ ID NO:4, SEQ ID NO:5；
A kind of siRNA interfering PVT1, as shown in SEQ ID NO:2, SEQ ID NO:3；
A kind of kit, including the primer；
A kind of pharmaceutical composition including the long-chain non-coding RNA；
Application of the primer in preparation diagnosis preeclampsia reagent；
Application of the described pharmaceutical composition in preparation treatment preeclampsia drug.
Described pharmaceutical composition, wherein further including auxiliary material.Auxiliary material includes: (lip2000, Opti-mem culture solution, PBS phosphorus
Acid buffering salting liquid)
The kit, the concentration in the primer is respectively 10mol/L.
Technical solution
Pass through the differential expression of PVT1 in qPCR screening clinical tissue, discovery PVT1 in preeclampsia pregnant women placental tissue
The more normal pregnant women placental of expression quantity in expression quantity it is low.Guess: whether PVT1 takes part in the pathogenic process of preeclampsia disease.
It then selects the normal trophocyte of international endorsement as experimental study object, designs the interference sequence of PVT1, with
Lip2000 is that interference sequence is transferred to the pathogenic process that preeclampsia disorder is simulated after cell by transfection carrier.Pass through detection
The function of cell is as being proliferated after interference sequence is transferred to cell, apoptosis, migration and ability etc..To prove to nourish carefully normal
The expression that low PVT1 is struck in born of the same parents HTR-8/SVneo, affects the function of cell, induction or accelerate the morbidity of eclampsia premenstrual disorders into
Journey.On the contrary, building PVT1 plasmid, function of the positive de-authentication PVT1 in trophocyte HTR-8/SVneo.
It is sequenced by transcript profile, under the correlation of possibility participation cell function (as being proliferated, apoptosis or migration) for detecting PVT1
Target gene is swum, then to the Primary Study of PVT1 regulatory mechanism, finds that PVT1 is more present in nourishing by means such as FISH
In the nucleus of cell, consider that PVT1 may pass through RIP and CHIP experiment detection in the corresponding target gene of transcriptional level control
PVT1 inhibits the expression of downstream target gene TP53INP1 and ANGPTL4 by binding EZH2 albumen.
Various reagents needed for transfection process,
(1) lip2000, a kind of multiduty lipofectamine, suitable for the transfection of DNA, RNA and oligonucleotides,
There is very high transfection efficiency to most of eukaryocytes.Its unique formula be directly added into it can in culture medium, serum
In the presence of will not influence transfection efficiency, and then PVT1 interference sequence rotated into intracellular.
(2) Opti-mem culture solution contains HEPES, 2400mg/l sodium bicarbonate, hypoxanthine, thymidine, pyruvic acid
Sodium, L-Glutamine, microelement, growth factor, and decrement are phenol red to 1.1mg/l, as the auxiliary material of transfection reagent,
It in itself to cell without any harm, and more preferably more effectively rotates into cell, to obtain expected purpose.
(3) PBS phosphate buffered saline solution (phosphate buffer saline) plays dissolution protection generally as solvent
The effect of reagent.It is the most widely used a kind of buffer in biochemical research, main component Na2HPO4,
KH2PO4, NaCl and KCl, due to Na2HPO4 and KH2PO4, they have second level dissociation, and the pH value range of buffering is very wide；And NaCl
It is to increase salt ionic concentration with KCl main function.Exclude itself influence to experimental subjects.
Tissue collecting
We have collected 52 pairs 2014 to 2015 in Jiangsu Prov. People's Hospital, healthcare hospital for women & children, Jiangsu Province receives to cut open palace
Produce operation, pregnant women placental tissue of the diagnosis with preeclampsia and the normal pregnancies placenta tissue without containing any underlying diseases.
And record clinical feature: including pregnant woman age, having non-smoking history, pregnant week number, systolic pressure, diastolic pressure is with albuminuria and tire
Youngster's weight.Tissue specimen collection is first time liquid nitrogen or is stored in -80 DEG C, until RNA is extracted.The research is by Nanjing
The approval of Ethics Committee, medical university.Obtain the written informed consent of all patients.
Cell line
It chooses trophocyte (HTR-8/SVneo) and Canadian empress university offer is provided.HTR-8/SVneo cell is used
RPMI1640 culture medium culture；The equal chain containing 5% fetal calf serum, the penicillin of 100U/ml and 100mg/ml in culture medium
Mycin.Routine culture in 37 DEG C of constant incubators of 5%CO2.Every 2-3 days replacement fresh cultures, when cell fusion degree reaches
It is passed on when 80%-90%.All cell lines are verified by the DNA analysis of short tandem repeat.
RNA is extracted and quantitative PCR analysis
According to the operation instruction of reagent, total serum IgE is separated with Trizol reagent.Reverse transcription reaction application TaKaRa Prime
Script kit (TaKaRa, Dalian, China).Reverse Transcriptase kit carries out reverse transcription to 1 μ g total serum IgE, and final volume is 20 μ
l.Interpretation of result: the specificity and amplification efficiency of primer are analyzed, the atopic of primer is judged according to solubility curve.According to expansion
Increase curve and obtain Ct value, the analysis of target gene relative expression quantity is carried out using relative measurement and internal reference GAPDH.The primer of PVT1
It is as follows: 5 '-TGAGAACTGTCCTTACGTGACC-3 ' of Primer F (SEQ ID NO:4), Primer R 5 '-
AAGGGCATTCACCAACTCC-3 ' (SEQ ID NO:5) calculation formula are as follows: 2^ (- △ Ct), △ Ct=Ct gene-Ct
control。
Plasmid construction
People's overall length PVT1cDNA is synthesized, and is inserted into carrier for expression of eukaryon pCDNA 3.1, building PVT1, which is overexpressed, to be carried
Above-mentioned plasmid is then converted Escherichia coli by constitution grain, is shaken bacterium, is cultivated, selects the culture amplification of monoclonal bacterium.
Cell transfecting
Plasmid vector (pCDNA3.1-PVT1, si-PVT1, pCDNA3.1-TP53INP1 and unloaded constitution for transfection
Grain), extracted with the endotoxic plasmid extraction kit (DNA Midiprep kit, Qiagen) of removal.The interference of PVT1
Sequence, that is: si-PVT 1#GCUUGGAGGCUGAGGAGUUTT (SEQ ID NO:2) and si-PVT 2#
AACUCCUCAGCCUCCAAGCTT (SEQ ID NO:3), and out-of-order control (si-NC) are purchased from Invitrogen company
(Invitrogen company, CA, USA).By cell HTR-8/SVneo by 2 × 105, every hole cell kind in 6 well culture plates, to thin
After born of the same parents are adherent, 6h, which inhales, before transfecting abandons original culture medium, changes culture medium without double antibody into；10 μ L liposomes are taken to be diluted in 240 μ L's
In OPTI-MEM, mild piping and druming mixes is incubated for 5min at room temperature；Take 100pmol siRNA, si-NC or 4ug plasmid vector difference
It is diluted in 250 μ L OPTI-MEM, piping and druming mixes and is incubated for 5min at room temperature；The liposome being incubated for and siRNA or plasmid is dilute
Liquid mixing is released, mild piping and druming mixes.Continue to be incubated for 20min at room temperature；Said mixture is uniformly instilled and adds good 1.5mL in advance
In 6 well culture plates of OPTI-MEM, mix gently.37 DEG C, continues in 5%CO2 incubator after cultivating 6h, change complete medium.
36h after transfection, collects cell extraction RNA or albumen carries out real-time quantitative RT-PCR or immunoblotting assay.
Cell-proliferation activity detection
MTT experiment, by treated, cell presses 2000-3000, every hole cell inoculation in 96 well culture plates.To cell
After 80% is adherent, cell synchronization 12h discards original culture medium.6 multiple holes are arranged in each sample, and every hole total reaction volume is
200μl.The MTT reaction solution (5mg/ml is dissolved in PBS) of 20 μ l is added in every hole, and 37 DEG C are protected from light incubation 4h.Discard supernatant liquid, every hole
150 μ l dimethyl sulfoxides (DMSO) are added, shake 10min, microplate reader measures the absorbance at 490nm wavelength.
EdU experiment, will be in appropriate treated cell in 24 orifice plates, and 10 μM of EdU reagents are added in every hole.After 2h, use
4% paraformaldehyde fixes 30 minutes.Cleaning is dyed 30 minutes using Click-iTR Edu kit, then with DAPI dyeing 5
Minute, then using fluorescence microscope shooting (Olympus, Japan).Finally, being analyzed using Image-Pro Plus software.
Flow cytometry
Apoptosis detection, the HTR-8/SVneo cell after being transfected 48 hours with collected by trypsinisation, then according to FITC
Annexin V apoptosis detection kit (BD) and its operation instruction give Annexin V-FITC fluorescence probe and propidium iodide
(PI) it dyes.Flow cytomery and analysis.
Cell cycle detection gives PI using CycleTESTTM PLUS DNA kit (BD) according to specification and dyes,
Then analyzed with FACScan.
Cell migration and Matrigel
The cell Transwell of 8 μm of pore sizes is placed in 24 orifice plates.Cell invasion experiment, with 50mg/l BD
Matrigel1:6 dilution is coated with the upper chamber face of the cell Transwell bottom film, and the cell being coated with is put into 24 orifice plates, incubates
6h is incubated in case.Vitellophag, centrifugation discards culture solution after terminating digestion, is washed 1-2 times with PBS, with the free serum culture containing BSA
Base weight is outstanding.Cell density is adjusted to 3x105.Take 300 μ l of cell suspension that the cell Transwell is added.Room is added 700 under 24 orifice plates
Culture medium of the μ l containing 10%FBS is put into incubator routine culture for 24 hours.Cell migration assay, adjustment cell density to 3x104.It takes
The cell Transwell is added in 300 μ l of cell suspension.700 culture mediums of the μ l containing 10%FBS are added in room under 24 orifice plates, are put into incubator
Middle routine culture is for 24 hours.Cell is taken, wipes matrigel and upper indoor cell with cotton swab, with 0.1% crystal violet by cell outer bottom
Cell dyeing taken pictures counting using cell of the inverted microscope to the dyeing that room side above and below the cell Transwell counterdie is adhered to.
Sub-cellular orientation
It is thin using PARIS kit (Life Technologies, USA) separation HTR-8/SVneo according to operation instructions
The nucleus and cytoplasm of born of the same parents.Use the distribution of the detection of qPCR method PVT1, GAPDH and U1 in cytoplasm and nucleus.
GAPDH is cytoplasm reference, and U1 is nucleus reference.With total serum IgE percentage presentation PVT1, GAPDH and U1 in cytoplasm and carefully
Expression in karyon.
Hybridization in situ technique (FISH)
The characteristics of according to PVT1 gene transcripts, designs corresponding probe and (is closed by Shanghai Bo Gu biotechnology company
At), by HTR-8/SVneo cell seeding in containing in 15mm creep plate 6 orifice plates, when cell is to 80% or so, abandons culture medium and use
After PBS is cleaned twice, adds sample presentation after the fixed 30min of the methanol of 2ml, carried out by Shanghai Bo Gu biotechnology company next
Reason selects the silver-colored light microscope of inversion to take pictures, sub- positioning of the qualitative detection PVT1 in cell, further verifying caryoplasm separating experiment
As a result.
RNA sequencing
By cell seeding in six orifice plates, after cell it is long to the lip2000si-PVT1 for awarding 10ul after 80% or so and
Si-NC is handled, and is handled after 48h with Trizol and is collected cell, and sample presentation selects Illumina by Beijing genetic test mechanism for implementing
Subsequent experiment is carried out, obtain and handles corresponding data.
RNA immunoblotting (RIP)
HTR-8/SVneo cell is cracked to use for endogenous PRC2 compound immunoblot experiment.By cell conditioned medium and packet
EZH2, SNRNP70 are identified respectively and compare albumin A/G agarose magnetic bead of IgG in 4 DEG C of 6 hours of incubation.Then, it cleans
Magnetic bead is incubated for 30 minutes at 55 DEG C with 0.1%SDS/0.5mg/ml Proteinase K to remove removing protein.RNA is extracted to analyze for qPCR.
Chromatin immune is co-precipitated (CHIP)
Consolidate HTR-8/SVneo cell using 4% paraformaldehyde, is incubated for 10 minutes to generate DNA- protein-crosslinking.Ultrasound is split
Cell is solved to generate the chromosome fragment of 200-300bp, then with EZH2 and H3K27me3 specific antibody and control IgG antibody
It is incubated for precipitating.Restore chromatin dna, is then tested and analyzed using qPCR.
Protein immunoblotting (Westblotting)
The cell protein pyrolysis product being denaturalized is added to 10% pre-fabricated modacrylic acyl ammonia gel (SDS-
PAGE in well), albumen in separating sample.NC film is then switched into, and is incubated for specific antibody.Finally shone with ECL
Hydraulic exposure.GAPDH antibody is control, and TP53INP1 antibody is purchased from CST company, and ANGPTL4 antibody is purchased from Proteintech
Company.
Data processing
Experimental data all uses SPSS17.0 software to analyze, and is indicated with the average value ± standard error tested three times, group difference
With double tail Student ' s T inspection, rank sum test and Chi-square Test.In single factor analysis p < 0.05 then reuse it is multifactor
Analysis.
Detailed description of the invention
Fig. 1, PVT1 express downward in preeclampsia pregnant women placental tissue
1a PVT1 is expressed in preeclampsia pregnant women placental tissue (n=52) and is lowered compared with normal tissue
1b PVT1 is expressed in preeclampsia pregnant women placental tissue (n=52) and is lowered compared with normal tissue
The influence of Fig. 2, PVT1 to trophocyte HTR-8/SVneo ability of cell proliferation
2a-b strikes low PVT1 in HTR-8/SVneo cell and is overexpressed PVT1
2c-d strikes the proliferative capacity of low PVT1 expression inhibiting HTR-8/SVneo cell；It is overexpressed PVT1 and promotes HTR-8/
SVneo cell Proliferation
2e-f, which strikes low PVT1, can be such that HTR-8/SVneo Cell clonality weakens；Being overexpressed PVT1 can enhance
HTR-8/SVneo Cell clonality
2g-h, which strikes low PVT1, can reduce the proliferation of HTR-8/SVneo cell；HTR-8/ can be enhanced by being overexpressed PVT1
SVneo cell Proliferation
Influence of Fig. 3 PVT1 to HTR-8/SVneo cell cycle, apoptosis, migration and invasion
3a, which strikes low PVT1, can promote trophocyte HTR-8/SVneo apoptosis
3b strikes low PVT1 inducing cell cycle arrest in the G0/G1 phase；Being overexpressed PVT1 promotes G0/G1 phase to the S phase to convert.
3c-d, which is overexpressed PVT1, can promote the transfer ability of HTR-8/SVneo cell
3e-f, which is overexpressed PVT1, can promote the invasive ability of HTR-8/SVneo cell
Fig. 4 participates in the potential downstream target gene that PVT1 mediates trophocyte growth by sequencing detection
The correlation analysis of 4a-b cell sequencing and GO analysis
It is its potential target gene that 4c-d, which strikes low PVT1 and is overexpressed PVT1 verifying TP53INP1 and ANGPTL4,
Fig. 5 PVT1 inhibits the transcription of TP53INP1 and ANGPTL4 promoter region by combining EZH2 albumen
5a-b PVT1 is positioned at nucleus
PVT1 is in conjunction with EZH2 for 5c RIP experimental verification
5d-f protein immunization imprinting tests (CHIP), and verifying obtains PVT1 and inhibited by the binding with EZH2 albumen
The expression of TP53INP1 and ANGPTL4
Fig. 6 is overexpressed influence of the TP53INP1 to trophocyte growing multiplication
6a-c is overexpressed TP53INP1 and inhibits ability of cell proliferation, while also weakening Cell clonality
6d is overexpressed TP53INP1 and inhibits cell invasion ability
6e-f remedies the breeding that experimental verification TP53INP1 takes part in PVT1 regulation trophocyte
The expression of downstream target gene TP53INP1 is detected in Fig. 7 clinical samples
7a-b detection downstream target gene TP53INP1 expresses more normal pregnant women placental in sub-line pregnant women placental early period sample
Expression quantity obviously increases
Expression of the 7c with immunohistochemistry detection TP53INP1 in placenta, obtains TP53INP1 in sub-line pregnant woman's early period
More normal pregnant women placental expression quantity is expressed in placental samples obviously to increase.
Specific embodiment
The present invention will be further explained by examples below, but does not limit the present invention.
The experimental method of the dated actual conditions in end in embodiment is substantially all and writes according to Sambrook, J et al.
" Molecular Cloning:A Laboratory guide (the 3rd edition) " (MolecularCloning:ALaboratoryManual, 3rded. Huang Peitang etc.
Translate, Science Press .2002.8) described in condition and method or according to condition proposed by material supplier and method into
Row, it is well known standard method that other technologies being not described in, which correspond to for those skilled in the art,.
Material of the invention: the cell strain and culture medium referred in the application has supply of commodities or with other approach energy
For obtained by the public, they are only for example, to the present invention be not uniquely, can respectively with other suitable tool and biomaterial come
Instead of.
Embodiment 1
Detect expression of the PVT1 in tissue and cell
0.1g is taken to organize, liquid nitrogen grinding sufficiently (at powdered) or the cell of 1-5 × 107 abandons culture medium, the PBS rinse of pre-cooling
2 times.The Trizol lysate of 1ml is added, is blown and beaten and is mixed with no enzyme pipette tips, stands 5min, lysate immigration is marked in advance
The centrifuge tube without enzyme 1.5ml in.4 DEG C of 7500g are centrifuged 5 minutes, are taken supernatant that the chloroform of 1/5 volume is added, are mixed by inversion 30s,
Stand 2min.4 DEG C, 12000g centrifugation, 15min.Three layers (water phase-white precipitate-red organic matter) of solution point shifts aqueous layer
Into new 1.5ml centrifuge tube, try not to be drawn onto white precipitate.Isometric isopropanol is added, is gently mixed by inversion, places 5-
10min.4 DEG C, 12000g centrifugation, 10min.It inhales and abandons supernatant, the ethyl alcohol (now matching) of 1ml 75% is added, wash RNA precipitate.4 DEG C,
7500g centrifugation, 5min abandon supernatant.The alcohol of removal 75% as far as possible, dries, about 15min in room temperature.With no RNA enzyme water (20-
25 μ l) dissolution RNA precipitate.
The concentration of determination of uv absorption method measurement RNA.RNA concentration and purity, measurement are measured using ultraviolet specrophotometer
It is preceding first to be returned to zero with the DEPC water of dissolution RNA.Readings 1 is to indicate 40ng/ μ l at 260nm, the A260/A280's of RNA solution
Ratio is used for the detection of RNA purity, and ratio range shows to meet the requirements 1.8 to 2.1.Agarose gel electrophoresis identifies RNA's
Integrality.Prepare 1% agarose gel.Agarose is dissolved by heating, it is cooling, 1 μ l ethidium bromide (EB, 10mg/ml) is added.It shakes up
The glue that falls afterwards is placed in electrophoresis tank after glue condensation, is dipped in 1 × TAE buffer and balances 10min, for use.Point sample.By 1:4 (v/
V) 5 × nucleic acid electrophoresis sample-loading buffer is mixed with sample, accurately the RNA that each sample contains 1 μ g is added in gel pore.80V
Constant pressure electrophoresis 50min.After electrophoresis, result is observed in gel imager.
Tris- acetic acid (TAE) buffer formulation (1L) 50 ×:
Real-time quantitative PCR
Preeclampsia pregnant women placental tissue and normal pregnancies placenta tissue sample, the total serum IgE of HTR-8/SVneo cell are inverse
Responsive transcription application TaKaRa PrimeScript kit (Dalian treasured bioengineering Co., Ltd).Reverse transcription reaction system is such as
Under:
Reverse transcription reaction condition is as follows: 37 DEG C of 15min (reverse transcription reaction)；(inactivation of reverse transcriptase is anti-by 85 DEG C of 5sec
It answers).According to Genebank provide gene order, design primer sequence,
QPCR applies 7300 PCR systems (Applied Biosystems, Warrington, UK).CDNA sample uses three
Portion's method PCR amplification standardization program.Reaction system:
Reaction condition:
Interpretation of result: the specificity and amplification efficiency of primer are analyzed, the atopic of primer is judged according to solubility curve.
Ct value is obtained according to amplification curve, the analysis of target gene relative expression quantity is carried out using relative measurement and internal reference GAPDH.It calculates
Formula are as follows: 2^ (- △ Ct), △ Ct=Ct gene-Ct control.
The primer of PVT1 is as follows:
5 '-TGAGAACTGTCCTTACGTGACC-3 ' of Primer F is shown in SEQ ID NO:4,
5 '-AAGGGCATTCACCAACTCC-3 ' of Primer R is shown in SEQ ID NO:5.
The result shows that lncRNA PVT1 lowers (Figure 1A) compared with normal tissue in preeclampsia pregnant women placental tissue expression.I
Have detected expression water of 52 pairs of preeclampsia pregnant women placental tissue expressions compared with PVT1 in normal tissue using real-time quantitative PCR
It is flat.As the result is shown compared with normal pregnancies placenta tissue, the preeclampsia pregnant women placental of the expression of PVT1 in 78.8% (41/52)
(multiple>1.5, P<0.05) (Figure 1B) is reduced compared with normal tissue expression in tissue.Prompt PVT1 may be in preeclampsia disease
Important role is play in diagnosis, occurrence and development and treatment.
The clinical data of Table 1 preeclampsia pregnant woman and normal pregnancy pregnant woman
Preeclampsia (N=52) normal pregnancy (N=52) P value
29.96 ± 5.639 34.69 ± 3.226 p > 0.05 of parent age (year)
± 10.885 72.28 ± 9.185 p > 0.05 of maternal body mass 74.75
It smokes 00 p > 0.05
162.51 ± 15.472 116.73 ± 7.728 p < 0.01 of systolic pressure (mmHg)
106.71 ± 11.155 74.59 ± 7.57 p < 0.01 of diastolic pressure (mmHg)
Albuminuria (g/day)>p<0.05 0.3g<0.3g
± 1013.032 3389.42 ± 387.72 p < 0.05 of neonatal weight (g) 2365.57
Embodiment 2.
In order to study influence of the PVT1 to normal trophocyte HTR-8/SVneo cell phenotype.
Firstly, choosing research object of the normal trophocyte HTR-8/SVneo cell line as this experiment, utilize
Lip2000 as carrier, transfect PVT1 interference sequence si-PVT 1#GCUUGGAGGCUGAGGAGUUTT (SEQ ID NO:2) and
Si-PVT2# AACUCCUCAGCCUCCAAGCTT (SEQ ID NO:3) is to strike the morbidity that preeclampsia is simulated in the expression of low PVT1
Process, MTT proliferation experiment detection discovery, transfects the interference sequence of PVT1 in HTR-8/SVneo cell, after striking low PVT1 expression
Inhibit cell growth.In contrast, PVT1 plasmid is transfected in normal trophocyte, further verifies PVT1 function, hair
The overexpression of existing PVT1 promotes HTR-8/SVneo cell Proliferation (Fig. 2A-D).In addition, Clone forming Test the result shows that, lower
HTR-8/SVneo Cell clonality weakens after PVT1, and PVT1 enhances HTR-8/SVneo cell clone after being overexpressed
Forming ability (Fig. 2 E and Fig. 2 F).In addition, EDU dyeing proves, the proliferation of reduction HTR-8/SVneo cell after low PVT1 is struck, and
HTR-8/SVneo cell Proliferation enhances (Fig. 2 G and Fig. 2 H) after being overexpressed PVT1.It follows that these statistics indicate that, PVT1 can
Promote the proliferative capacity of HTR-8/SVneo cell.
Embodiment 3
Influence of the PVT1 to trophoblast HTR-8/SVneo period and apoptosis
In order to examine whether PVT1 to the cell cycle conversion of the proliferative effect of HTR-8/SVneo cell, normally to nourish
Cell HTR-8/SVneo cell line transfects PVT1 interference sequence si-PVT using lip2000 as carrier as research object
1#GCUUGGAGGCUGAGGAGUUTT (SEQ ID NO:2) and si-PVT 2#AACUCCUCAGCCUCCAAGCTT (SEQ ID
NO:3) to strike the pathogenic process that preeclampsia is simulated in the expression of low PVT1.Utilize the flow cytometry cell cycle.As a result table
Bright, after HTR-8/SVneo cell transfecting PVT1 interference sequence, cell-cycle arrest is in G1/G0 after the expression of low PVT1 is struck in discovery
Phase further verifies PVT1 function, after discovery is overexpressed PVT1 on the contrary, transfecting PVT1 plasmid in normal trophocyte
Cell-cycle arrest ratio shared by the G1/G0 phase reduces (Fig. 3 B, P < 0.05) compared with control group.In addition, that We conducted streamings is thin
Born of the same parents' instrument analysis Apoptosis whether take part in PVT1 strike it is low after induce cell growth it is suppressed.As shown in Figure 3A, early apoptosis
(UR) it is struck in PVT1 with late apoptic rate (LR) and is higher than cellular control unit in low HTR-8/SVneo cell.PVT1 affects taste
Support period and the apoptosis of cell
Embodiment 4
PVT1 participates in HTR-8/SVneo cell migration and invasion
An importance in invasion of trophoblasts and transfer Attack of Preeclampsia mechanism.HTR-8/SVneo cell line
PVT1 interference sequence si-PVT 1# is transfected using lip2000 as carrier as research object
GCUUGGAGGCUGAGGAGUUTT (SEQ ID NO:2) and si-PVT 2#AACUCCUCAGCCUCCAAGCTT (SEQ ID NO:
3) to strike the pathogenic process that preeclampsia is simulated in the expression of low PVT1.Using transwells experimental study PVT1 strike it is low after
HTR-8/SVneo cell migration and invasive ability are influenced.The result shows that inhibiting after striking low PVT1 compared with cellular control unit
Trophocyte HTR-8/SVneo transfer ability inhibits invasive ability (Fig. 3 C and D).These the result shows that, strike low PVT1 expression
The phenotype for inhibiting trophocyte afterwards, hinders the migration and invasion of trophocyte HTR-8/SVneo.As it can be seen that the low expression of PVT1
Expression affects cell migration and the invasive ability of normal trophocyte HTR-8/SVneo, further affects the shallow of placenta
Bed, induces the generation of eclampsia premenstrual disorders, and administration PVT1 can treat preeclampsia disease.
It discusses
In past ten years, with the fast development of sequencing technologies and bioinformatics, more and more lncRNAs quilts
People's cognition.Including lncRNA in preeclampsia in unconventionality expression in a variety of human diseases.Such as in preeclampsia
The abnormal expression of lncRNA MEG3 influences the proliferation and apoptosis capacity of trophocyte.In addition, some, researches show that lncRNAs ratios
Protein coding gene has more tissue specificity, this illustrate lncRNA more likely as different tumours it is more sensitive biology mark
Will object.Thus, it is found that preeclampsia relevant lncRNAs, studies their clinical meaning and biological function, can promote lncRNA
The diagnosing and treating of the preeclampsia of guidance.
In this research, we have found a new lncRNA PVT1 and under proving that it is expressed in preeclampsia tissue
It adjusts.The PVT1 expression of downward has close relationship with Attack of Preeclampsia mechanism.In addition, inhibiting cell to increase after striking low PVT1
It grows, migrates and invade, promote trophoblast apoptosis.In addition, existing it is considered that striking the trophocyte growth inhibition that low PVT1 is mediated
The expression of TP53INP1 and ANGPTL4 is depended on to a certain extent.Herein, we confirm PVT1 in mankind's trophocyte for the first time
Middle enforcement correlation function is the expression by trophocyte the inhibiting factor TP53INP1 and ANGPTL4 of inhibition.LncRNAs is logical
The expression that various mechanism adjust target gene is crossed, chromatin is such as recruited and adjusts enzyme to target gene and cis or trans adjusting transcription, make
For scaffold combination relevant molecule original part or absorption miRNA.In this research, we have found that in HTR-8/SVneo cell
PVT1 can recruit them to TP53INP1 and ANGPTL4 promoter region and inhibit its transcription in conjunction with EZH2.
The core cell of EZH2-PRC2, is a histone methyltransferase, and the H3K27 for being catalyzed target gene is tri-methylated；
In this experiment, we further demonstrate that, TP53INP1 and ANGPTL4 is transcribed jointly in trophocyte HTR-8/SVneo, can quilt
The EZH2 that PVT1 is raised inhibits.TP53INP1 is one of the important member of a cytostatic factor big family, has been confirmed as
One new cell balance adjuster.Our result also indicated that trophocyte HTR- can be inhibited by raising its expression
8/SVneo grows and induces cell apoptosis, and weakens migration and the invasive ability of cell.In summary, our result of study table
Bright, lncRNA PVT1 expression reduces the shallow implantation that may cause placenta, induces the occurrence and development of preeclampsia.
Our research is found for the first time, is lowered in preeclampsia pregnant women placental tissue and cell PVT1 expression.Before eclampsia
Phase, pregnant women placental trophocyte HT-8/SVneo struck the expression of low PVT1, show cell inhibit function, show as cell Proliferation,
Migration is reduced and Apoptosis increases.Furthermore.Our discovery can further enrich Attack of Preeclampsia mechanism, and promote
The diagnosing and treating of lncRNA guidance.
SEQUENCE LISTING
<110>Jiangsu Prov. People's Hospital
<120>a kind of long non-coding RNA and its application in diagnosis/in treatment preeclampsia
<160> 5
<170> PatentIn version 3.3
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ggcaccttcc agtggatttc cttgcggaaa ggatgttggc ggtccctgtg acctgtggag 180
acacggccag atctgccctc cagcctgatc ttttggccag aaggagatta aaaagatgcc 240
cctcaagatg gctgtgcctg tcagctgcat ggagcttcgt tcaagtattt tctgagcctg 300
atggatttac agtgatcttc agtggtctgg ggaataacgc tggtggaacc atgcactgga 360
atgacacacg cccggcacat ttcaggatac taaaagtggt tttaagggag gctgtggctg 420
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gaatgtaaga ccccgactct tcctggtgaa gcatctgatg cacgttccat ccggcgctca 600
gctgggcttg agctgaccat actccctgga gccttctccc gaggtgcgcg ggtgaccttg 660
gcacatacag ccatcatgat ggtactttaa gtggaggctg aatcatctcc cctttgagct 720
gcttggcacg tggctccctt ggtgttcccc ttttactgcc aggacactga gatttggaga 780
gagtctcact ctgtggtcca ggctgaagta cagtggcatg atcccaggtc actgcaaccc 840
ccacctcccg ggttcaagtg atcctcctgc ctcagcctcc cgagtagctg gtattacagg 900
cgtgtgccac aaagcctggc taagttttgt atttttagta gagacggggt ttcaccatgt 960
tggccaggtt ggtctcgaac tcctgacctc aagtgatcca ctcactttgg cctttcaacg 1020
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gcactctgga cggacttgag aactgtcctt acgtgaccta aagctggagt attttgagat 1200
tggagaatta agagccagtc ttggtgctct gtgttcacct ggttcatctg aggagctgca 1260
tctaccctgc ccatgccata gatcctgccc tgtttgcttc tcctgttgct gctagtggac 1320
atgagaagga cagaataacg ggctcccaga ttcacaagcc ccaccaagag gatcacccca 1380
ggaacgcttg gaggctgagg agttcactga ggctactgca tcttgagact caggatgaag 1440
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tctatgggaa tcactactga ccttgcagct tattatagac ttatatgttt tttgcatgtc 1680
tgacacccat gactccacct ggaccttatg gctccaccca gaagcaattc agcccaacag 1740
gaggacagct tcaacccatt acgatttcat ctctgcccca accactcagc agcaagcacc 1800
tgttacctgt ccacccccac cccttccccc aaactgcctt tgaaaaatcc ctaacctatg 1860
agctttgaat aagatgagta cgaacttcat cgcccacgtg gcgtggccgg cctcgtgtct 1920
attaaattct ttttctacta aaaaaaaaaa aaaaaaa 1957
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aacuccucag ccuccaagct t 21
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tgagaactgt ccttacgtga cc 22
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Claims (2)
1. detecting application of the reagent of long non-coding RNA PVT1 in preparation diagnosis preeclampsia reagent.
2. inhibiting application of the inhibitor of PVT1 in preparation treatment preeclampsia drug, be typically characterized by: inhibitor is
A kind of siRNA interfering PVT1, as shown in sequence 2,3.
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| PCT/CN2017/080715 WO2018094955A1 (en) | 2016-11-28 | 2017-04-17 | Long non-coding rna and application thereof in diagnosing/treating preeclampsia |
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| WO2018167790A1 (en) * | 2017-03-16 | 2018-09-20 | Ramot At Tel-Aviv University Ltd. | Methods for identifying and monitoring pregnant women at risk of preeclampsia |
| CN108220434A (en) * | 2017-12-13 | 2018-06-29 | 南京医科大学第二附属医院 | A kind of application of the long-chain non-coding RNA and combinations thereof in diagnosis/treatment cholangiocarcinoma |
| CN108186665B (en) * | 2018-01-02 | 2020-03-20 | 深圳市第二人民医院 | Reagent for interfering expression of long-chain non-coding RNA PVT1 and application thereof |
| US11225666B2 (en) * | 2018-03-16 | 2022-01-18 | Research Foundation Of The City University Of New York | Plasmid vector for expressing a PVT1 exon and method for constructing standard curve therefor |
| CN109750038B (en) * | 2018-12-29 | 2021-08-31 | 烟台毓璜顶医院 | Long non-coding RNA and application thereof in preparation of medicines for diagnosing preeclampsia and treating target spot |
| CN111321148A (en) * | 2020-01-09 | 2020-06-23 | 宁夏医科大学总医院 | Marker gene for preeclampsia clinical risk assessment and application thereof |
| CN111172161B (en) * | 2020-01-15 | 2023-04-21 | 江苏省人民医院（南京医科大学第一附属医院） | Long non-coding RNA and application thereof in diagnosis/treatment of preeclampsia |
| WO2023215761A1 (en) * | 2022-05-03 | 2023-11-09 | Tacit Therapeutics, Inc. | Localization of trans-splicing nucleic acid molecules to and within the cellular nucleus |
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| CA2817256A1 (en) * | 2010-11-12 | 2012-05-18 | The General Hospital Corporation | Polycomb-associated non-coding rnas |
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| CN106047880A (en) * | 2016-08-18 | 2016-10-26 | 暨南大学 | PVT1 siRNA-1055 for inhibiting blood tumor cell proliferation and application thereof |
Non-Patent Citations (2)
| Title |
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| Amplification of PVT-1 is involved in poor prognosis via apoptosis inhibition in colorectal cancers;Y Takahashi等;《British Journal of Cancer》;20131105;第165页材料与方法部分 |
| Homo sapiens Pvt1 oncogene (non-protein coding) (PVT1), long non-coding RNA;Cui M等;《GenBank NCBI Reference Sequence: NR_003367.3》;20161008;全文 |
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