CN103993015A - Peripheral white blood cell miRNA markers associated with onset of human preeclampsia and application of miRNA markers - Google Patents

Abstract

The invention belongs to the field of genetic engineering and clinical medicine. The invention aims to provide peripheral white blood cell miRNA markers associated with the onset of human preeclampsia. The invention also aims to provide an application of the miRNA markers. The miRNA markers can be many of has-miR-15a-3p, has-miR-31-3p, has-miR-451a and has-miR-122-5p. The peripheral white blood cell miRNA markers include has-miR-15a-3p, has-miR-31-3p, has-miR-451a and has-miR-122-5p. The peripheral white blood cell miRNA marker is applied to preparation of reagent for diagnosis or monitoring of human preeclampsia. The reagent is capable of determining the expression of the peripheral white blood cell microRNA marker in peripheral white blood cell. According to the invention, verifications are carried out on samples in line with preeclampsia and samples of a healthy control population to prove that the expressions of the markers are significantly different and the markers have stability and thus the results demonstrate that the markers have specificity and can be used as markers.

Description

There is relevant peripheral blood leucocyte miRNA mark and application thereof preeclampsia in the mankind

Technical field

The invention belongs to genetically engineered and clinical medicine domain, be specifically related to the mankind relevant peripheral blood leucocyte miRNA mark and application thereof occur preeclampsia.

Background technology

Preeclampsia (Preeclampsia, PE) be common many organ injuries of idiopathic disease of the Gestation period, it is characterized in that occurring hypertension, proteinuria and edema after pregnant 20 weeks, and then may cause the generation of the severe complications such as placental abruption, DIC, eclampsia, HELLP syndrome, renal failure, cerebrovascular accident and heart failure.PE Gestation period incidence is 2%～8% in the world, and is the major cause of current pregnant and lying-in women, tire baby M & M.The clinical manifestation of PE has obvious heterogeneity, and this makes clinical acquisition effectively treat difficulty obviously to strengthen.

Mainly contain at present two theories and explain that the physiopathology of PE changes.First be vasculitic pathology theory, comprise anoxia of placenta, oxidative stress, inflammatory reaction, the abnormal enhancing of platelet aggregation and general blood vessel endothelium dysfunction etc.Wherein the obstacle of function of vascular endothelium is considered to even can extend to several weeks in postpartum.Next is immunologic dysfunction theory, as cellular immune abnormality, female tire dysimmunity, protective immunity functional disorder etc.The reason that PE occurs may comprise the many aspects such as inherited genetic factors, immune factor and biotic factor.The explanation of this feature is not only not easy to obtain biomarker comparatively reliably in clinical practice, and is difficult to determine the effective therapy that had both been conducive to pregnant woman and ensures again Fetal Maturation.Although have some researchs to relevant cell factor in peripheral blood in patients circulation, as vascular endothelial growth factor (VEGF), solubility endothelium glycoprotein (sEng), soluble vascular endothelial growth factor receptor (sFLT1) and placenta growth factor (PlGF) etc., but all generations of the PE of early prediction preferably.

MiRNA studies a class endogenous non-coding microRNA the most widely at present, length range is at 18～25 Nucleotide (nt), be a high conservative on evolving, there is timing and the specific negative gene conditioning agent of the organ-tissue family of growth, can be combined with 3 '-UTR of the mRNA of proteins encoded, cause corresponding protein expression downward or suppress its expression completely.A large amount of results of study confirms, miRNA participates in the regulate several biological processes of body genesis and development, comprises the reaction of biology and abiotic environment to external world of the generation of differentiation, organ and phylogeny, signal conduction, immunoregulation, disease and cancer of stem cell and organism.The mechanism of action of miRNA comprise by translation suppress, mRNA degraded and miRNA directly three kinds of the mRNA adenosine acidifyings of induction transcribe rear retarding effect and increase or suppress target gene expression.Nearest research shows, peripheral blood leucocyte particularly mononuclearcell miRNA wherein participates in regulating the various immunologic processes of human body.In mankind's various autoimmune disease, comprise that the diseases such as Crohn's disease, lupus erythematosus, rheumatoid arthritis and tumour have been found that the differential expression of multiple miRNA.But about effect in this disease pathogenesis of placenta in preeclampsia peripheral blood leucocyte or mononuclearcell miRNA and there is not yet report as the reliability of biomarker and the research of usefulness, further investigation can be diagnosis and the treatment of preeclampsia provides new approach.

Vascular endothelial cell damage is the important step of current generally acknowledged Attack of Preeclampsia.Once vascular endothelial cell damage occurs placenta in preeclampsia, can cause vascular permeability to increase, albumen and intravenous extravasation in blood vessel; Cause platelet aggregation, activate blood coagulation system, blood is concentrated, sluggish, and disease progression can cause DIC; Increase the synthetic of vasoconstrictive factor and discharge, reduce the synthetic of vascular relaxing factor and discharge, make vasoactive factors unbalance, vasoconstriction, thus cause a series of pathological change preeclampsia and clinical symptom.Wherein peripheral leukocytes in patients, monocyte produce cytokine, elastin and other proteolytic enzyme, further increase the weight of the damage of endotheliocyte, and oxyradical and lipid peroxide can suppress the generation of prostacyclin, increase TXA ₂, NO generation and change vascular permeability, and the change of vascular permeability causes patient clinically to show as oedema and proteinuria.In prior art, show the feasibility that serum miRNA has in placenta in preeclampsia diagnosis and prediction.Because sample size is less, the repeatability of research is poor, simultaneously due to the limitation of research method, fails to obtain the result that has clinical meaning.Also there is application miRNA biochip technology to detect the miRNAs of differential expression in patients with psoriasis vulgaris peripheral blood mononuclear cell and normal pbmc, and part otherness miRNAs is carried out to fluorescence real-time quantitative PCR checking.It is closely related that result of study prompting miRNA-146a, miRNA-99a level and PV skin damage severity of symptom, can be used as one of biological indicator judging the state of an illness and clinical efficacy.Research at present shows, the genetic expression of placenta in preeclampsia peripheral blood leucocyte and normal pregnancy exist very large difference, show that be a complicated pathophysiological process preeclampsia, relate to the extensive and multi-level variations such as cellular immunization, metabolism, cell cycle, signal conduction.More than research shows, the miRNA abnormal expression of the outer blood leukocytes of placenta in preeclampsia may become important symbol thing and the targeted therapy new way of diagnosis.

Summary of the invention

The object of this invention is to provide the mankind relevant peripheral blood leucocyte miRNA mark occurs preeclampsia.Another object of the present invention is to provide the application of above-mentioned miRNA mark.

The present invention is realized by following technical scheme: relevant peripheral blood leucocyte miRNA mark occurs preeclampsia the mankind, is selected from multiple in has-miR-15a-3p, has-miR-31-3p, has-miR-451a, has-miR-122-5p.Described peripheral blood leucocyte miRNA mark is made up of has-miR-15a-3p, has-miR-31-3p, has-miR-451a and has-miR-122-5p.Wherein the sequence of has-miR-15a-3p is SEQ ID No.1, and the sequence of has-miR-31-3p is SEQ ID No.2, and the sequence of has-miR-451a is SEQ ID No.3, and the sequence of has-miR-122-5p is SEQ ID No.4.

Described peripheral blood leucocyte miRNA mark preparation the mankind diagnose preeclampsia or monitoring reagent in application.Described reagent is the reagent that can measure these peripheral blood leucocyte microRNA mark expression amount in peripheral blood leucocyte.

The present invention is described in detail as follows:

Crowd's Back ground Information and clinical data that systematic collection is complete, gather standard compliant blood sample with Standard operation procedure SOP (SOP), and adopt TRIzol method to extract the total RNA of peripheral blood leucocyte, adopt miRNA high-throughput HiSeq2000 order-checking, Genbank database comparison result, or by synthetic total peripheral blood leucocyte RNA reverse transcription cDNA, adopt Real-time qPCR method to detect.

The experimental technique of research mainly comprises following components specifically:

One, research object is included in and is got rid of foundation

A group: experimental group, placenta in preeclampsia 5 people, without other general major diseases; B group: normal healthy controls group: 5 people, without other general major diseases; Analyze between organizing after having checked order for two groups.

1 group: normal age placenta in preeclampsia (PE) group 13 people, the mean age is 25.69 ± 1.32 years old; 2 groups: placenta in preeclampsia at advanced age (PA) group 13 people, the mean age is 34.84 ± 3.18 years old; 3 groups: have complication (PC) group 13 people preeclampsia, the mean age is 30.08 ± 2.65 years old; 4 groups: normal contemporaneously pregnant woman (N) control group 13 people, the mean age is 29.07 ± 2.59 years old.4 groups of synthetic cDNA of the total RNA reverse transcription of peripheral blood leucocyte, Real-time qPCR method detects post analysis result.

Two, the separation of peripheral blood leucocyte and pre-treatment

1. peripheral blood collection: gather participation patient 8ml blood and be loaded on respectively EDTA heparin tube, after collection, softly put upside down and mix 10 times, be then placed in 4 DEG C of refrigerators temporary;

2. the blood sample of pair collection is centrifugal, collect white corpuscle: 4 DEG C of centrifugal 15min of 3000rpm/min, centrifugal rear sample layering, the blood plasma on upper strata is sub-packed in the cryopreservation tube that 2mlDEPC-treated water processed, in heparin tube, retain a small amount of blood plasma, be close to liquid level with Dispette and draw middle leukocytic cream from top to bottom: in the EP pipe that the hemocyte layer in remaining upper strata blood plasma and the following 4mm of leukocytic cream is drawn together at 2ml;

3. remove red corpuscle: will in the EP pipe of the above-mentioned 2ml that white corpuscle sample is housed, add 1ml erythrocyte cracked liquid, turn upside down and mix, leave standstill 10min, then 4 DEG C, centrifugal 2 min of 12000～13000rpm/min, discard the red clear liquid in upper strata, collect white precipitate, if erythrocyte splitting is incomplete, repeat above-mentioned cracking process 1-3 time, until the precipitation color after centrifugal is white;

4. the supernatant liquor after centrifugal is outwelled, and sucked residual liquid with suction nozzle.Then add PBS solution 1ml to blow and beat and mix gently; 4 DEG C, the centrifugal 5min of 12000～13000rpm/min; Supernatant discarded, adds 1.5ml Trizol solution to blow and beat and mix gently, and room temperature is placed 5min, makes its abundant cracking; By DEG C preservation of sample-80 of cracking.

The PBS solution using in the present invention's experiment is from button because of Development Co., Ltd of WaSunChina, and Trizol solution is from Invitrogen company.

Three, the total RNA of peripheral blood leucocyte extracts

(1) the EP pipe of the TRIzol lysate processing at-80 DEG C of Refrigerator stores is taken out, at 15 DEG C～30 DEG C of room temperatures, place 5min; The amount that adds 0.2ml chloroform by every 1ml TRIzol adds chloroform, covers EP pipe lid, firmly shakes 15 seconds in hand, places after 2～3min 2 DEG C～8 DEG C centrifugal 15min of 12000rpm/min at 15 DEG C～30 DEG C of room temperatures; Get upper strata water and be placed in new EP pipe, repeat above-mentioned steps, add the extracting of 0.2ml chloroform;

(2) get upper strata water and be placed in new EP pipe, the amount that adds 0.5ml Virahol according to every 1ml TRIzol adds Virahol, at 15 DEG C～30 DEG C of room temperatures, places 10min, 2 DEG C～8 DEG C centrifugal 10min of 12000rpm/min;

(3) abandon supernatant, 75% ethanol that adds the preparation of 1ml DEPC water by every 1ml TRIzol washs, vortex mixed, and 2 DEG C～8 DEG C centrifugal 5min of 7500rpm/min, abandon supernatant, seasoning under the RNA room temperature of precipitation; Dissolving RNA with RNase-free water precipitates;

(4) Nanodrop2000 ultramicrospectrophotometer is surveyed RNA concentration and 260/280 ratio; Agarose gel electrophoresis detects total RNA quality.

Four, with miRNA high-throughput HiSeq2000 order-checking, Genbank database comparison result

Get the total RNA of peripheral blood leucocyte of extraction, make it be dissolved in DEPC-treated water (Ambion), concentration is controlled at 25-500ng/ul, preparation RNA library; Obtain cDNA sample by RNA reverse transcription reaction.

1. reverse transcription reaction system and condition: first add 0.5 μ l RT-Primer (l00 μ M), place l0min for 65 DEG C, centrifugally add successively again after being cooled to room temperature: 1. 5x first strand buffer 2.0 μ l; 2. 10mM dNTP 0.6 μ l; 3. l00mM DTT l μ l; 4. (40U/ μ is l μ l l) for Rnase OUT, mix rear centrifugal in 42 DEG C place 3 minutes, (l), cumulative volume is 20 μ l to 200U/ μ finally to add l μ l Superscript, mix a little from, 42 DEG C of reactions 1 hour 7 DEG C of sex change 15 minutes again.

2.RT-PCR amplification reaction system is in table 1, and RT-PCR amplification reaction condition is in table 2:

Table 1

Table 2

After reaction finishes, amplified production is stored in to 4 DEG C of refrigerators and reclaims purifying and detection for next step PCR product.

3.PCR product reclaims purifying

(1) 6%TBE page glue is placed in damping fluid, adds l0 μ l l0xloading dye in PCR product, prepares Marker, 200V electrophoresis 30min after loading, dyes glue 3min and takes pictures, and the band that cuts about 92bp is placed in centrifuge tube, the centrifugal 2min of room temperature 14000rpm, adds 200ul H ₂0 on thermomix 2h eluted dna;

(2) blob of viscose and elutriant are transferred in Spin-X pipe, the centrifugal 2min of 14000rpm, adds Pellet Paint l μ l, the 3M NaAC of the effluent volume of 1/10 times, the dehydrated alcohol (20 DEG C frozen) of 3 times of effluent volumes.Mix rear 4 DEG C of centrifugal 25min of 14000rpm;

(3) abandon supernatant, 75% ethanol 500ul washing precipitation is dried, and adds the DNA product that detects Small RNA after 15 μ l EB solution with Agilent2100, and general concentration is at l0ng/ μ l～l00ng/ μ l, and size is in l00bp left and right.

4. produce DNA bunch

(1) relation while adopting qPCR determine the concentration that template is initial and carry out amplified reaction between change in concentration, does accurately quantitatively DNA library template amount, ensures the best packing density of the quality of data and wandering cells;

(2) sample having diluted is added in FC, carry out bridge-type PCR and make unit molecule cluster, reaction system and condition are as follows: bridge-type pcr amplification reaction system is in table 3, and bridge-type pcr amplification reaction condition is in table 4.

Table 3

Table 4

5. genome analysis instrument checks order and analyzes:

GO enrichment is analyzed: Gene Ontology(is called for short GO) be the Gene Ontology of an International standardization, provide a set of standard vocabulary table dynamically updating (controlled vocabulary) to describe the attribute of gene and gene product in organism comprehensively.GO always has three ontology, describes respectively the bioprocess (biological process) of the molecular function (molecular function) of gene, residing cell position (cellular component), participation.

GO functional analysis for be target gene predict the outcome (hereinafter to be referred as " candidate's target gene ").This analysis can provide with reference to after genetic comparison, the GO function entry of significant enrichment in candidate's target gene, and filter out candidate's target gene and which biological function significant correlation.Analyze the main biological function that can determine that candidate's target gene is exercised by the enrichment of GO function significance.

KEGG path analysis: with GO analyze the same, KEGG path analysis for be also candidate's target gene.In vivo, different genes coordinates to exercise its biological function mutually, and the analysis based on pathway contributes to further to understand the biological function of gene.Analyze main biochemical metabolism approach and the signal transduction pathway that can determine that candidate's target gene participates in by the enrichment of path significance.

Finally confirm as the healthy pregnant women and the placenta in preeclampsia peripheral blood leucocyte miRNA that there are differences expression and comprise has-miR-15a-3p(SEQ ID No.1), has-miR-31-3p(SEQ ID No.2), has-miR-451a(SEQ ID No.3), has-miR-122-5p(SEQ ID No.4).Compared with Normal group pregnant woman, in preeclampsia PE group peripheral leukocytes in patients, SEQ ID No.3 and SEQ ID No.4 expression level significantly raise, and SEQ ID No.1 and SEQ ID No.2 expression level significantly reduce simultaneously, and difference has statistical significance.Placenta in preeclampsia is increased to subgroup analytical results and show, SEQ ID No.3 expression level raises, and it is stable that SEQ ID No.1 and SEQ ID No.2 expression level are reduced in each group of eclamptic patients performance simultaneously.

Beneficial effect of the present invention: the inventor is by separating and comparing normal control and placenta in preeclampsia peripheral leukocytes miRNA, in discovery peripheral blood leucocyte, there is the miRNA sequence that can be used for assessing specificity and the susceptibility of whether suffering from placenta in preeclampsia, thereby proposed the peripheral leukocytes miRNA of placenta in preeclampsia and the generation of placenta in preeclampsia peripheral blood vessel endothelial cell damage and oxidative stress, develop closely relatedly, can be used as the candidate markers of pregnancy period preeclampsia prediction.

The present invention adopts peripheral leukocytes miRNA to be as the superiority of the mark of evaluating preeclampsia:

1) peripheral leukocytes miRNA is a kind of new bio mark, be different from traditional biological mark, not only stable, Wicresoft, be easy to detect, and quantitatively accurate, susceptibility and the specificity of diagnosis preeclampsia will be improved greatly, the successful exploitation of such microRNA biomarker is to overturning taking albumen as main traditional biological mark, the control that merges syndromes is started to brand-new situation, for the development of other diseases biomarker is offered reference for gestation.

2) peripheral leukocytes miRNA mark provided by the invention can be used as diagnosis marker preeclampsia, can avoid invasive diagnosis, and can carry out in early days auxiliary diagnosis, thereby for the further testing in depth testing of clinician provides foundation, for quick and precisely grasp patient morbid state and coincident with severity degree of condition, take the scheme of preventing and treating of more personalized to provide support in time, delay and stop progression of disease.

3) the present invention adopts the sample that meets preeclampsia and normal healthy controls crowd to verify, proves that these several marker expression amounts exist significant difference and have stability, to illustrate that this mark has specificity, can be used as mark and uses.

Brief description of the drawings

Fig. 1 is the total RNA quality of sample of the present invention gel electrophoresis figure, in figure: M represents Marker; N represents that 4 groups is normal contemporaneously pregnant woman control group; PC represents that 3 groups is to have complication group preeclampsia, and PE represents that 1 group is normal age placenta in preeclampsia group; PA represents that 2 groups is placenta in preeclampsia group at advanced age, shows that sample quality is stable in figure; Fig. 2 is that 1-4 respectively organizes sample miRNA-451a relative expression quantity; Fig. 3 is that 1-4 respectively organizes sample miRNA-15a-3p relative expression quantity; Fig. 4 is that 1-4 respectively organizes sample miRNA-31-3p relative expression quantity; Fig. 5 is that 1-4 respectively organizes sample miRNA-122-5p relative expression quantity, and wherein asterisk shown in Fig. 2-Fig. 5 has shown each component differences size, and two asterisks represent that group difference is large, have the value of prediction.

Embodiment

The invention will be further elaborated by the following examples.

Embodiment 1: research object is selected and grouping foundation

Being chosen in and playing the pregnant woman who gives birth in hospital at Second Hospital of Shanxi Medical University's Obstetric and Gynecologic Department in January, 2014 in April, 2013 is research object.

A group: experimental group, placenta in preeclampsia 5 people, without other general major diseases; B group: normal healthy controls group: 5 people, without other general major diseases; Analyze between organizing after having checked order for two groups.

1 group: normal age placenta in preeclampsia (PE) group 13 people, the mean age is 25.69 ± 1.32 years old; 2 groups: placenta in preeclampsia at advanced age (PA) group 13 people, the mean age is 34.84 ± 3.18 years old; 3 groups: have complication (PC) group 13 people preeclampsia, the mean age is 30.08 ± 2.65 years old; 4 groups: normal contemporaneously pregnant woman (N) control group 13 people, the mean age is 29.07 ± 2.59 years old.4 groups of synthetic cDNA of the total RNA reverse transcription of peripheral blood leucocyte, Real-time qPCR method detects post analysis result.

Embodiment 2: with the RNA order-checking of miRNA high-throughput HiSeq2000 Number of Peripheral Blood Leucocyte and Genbank database comparison result

Get the total RNA of peripheral blood leucocyte of extraction, make it be dissolved in DEPC-treated water (Ambion), concentration is controlled at 25-500ng/ul, preparation RNA library; Obtain cDNA sample by RNA reverse transcription reaction.

1. reverse transcription reaction system and condition: first add 0.5 μ l RT-Primer (l00 μ M), place l0min for 65 DEG C, centrifugally add successively again after being cooled to room temperature: 1. 5x first strand buffer 2.0 μ l; 2. 10mM dNTP 0.6 μ l; 3. l00mM DTT l μ l; 4. (40U/ μ is l μ l l) for Rnase OUT, mix rear centrifugal in 42 DEG C place 3 minutes, (l), cumulative volume is 20 μ l to 200U/ μ finally to add l μ l Superscript, mix a little from, 42 DEG C of reactions 1 hour 7 DEG C of sex change 15 minutes again.

2.RT-PCR amplification reaction system is in table 1, and RT-PCR amplification reaction condition is in table 2:

Table 1

Table 2

After reaction finishes, amplified production is stored in to 4 DEG C of refrigerators and reclaims purifying and detection for next step PCR product.

3.PCR product reclaims purifying

(1) 6%TBE page glue is placed in damping fluid, adds l0 μ l l0xloading dye in PCR product, prepares Marker, 200V electrophoresis 30min after loading, dyes glue 3min and takes pictures, and the band that cuts about 92bp is placed in centrifuge tube, the centrifugal 2min of room temperature 14000rpm, adds 200ul H ₂0 on thermomix 2h eluted dna;

(2) blob of viscose and elutriant are transferred in Spin-X pipe, the centrifugal 2min of 14000rpm, adds Pellet Paint l μ l, the 3M NaAC of the effluent volume of 1/10 times, the dehydrated alcohol (20 DEG C frozen) of 3 times of effluent volumes.Mix rear 4 DEG C of centrifugal 25min of 14000rpm;

(3) abandon supernatant, 75% ethanol 500ul washing precipitation is dried, and adds the DNA product that detects Small RNA after 15 μ l EB solution with Agilent2100, and general concentration is at l0ng/ μ l～l00ng/ μ l, and size is in l00bp left and right.

4. produce DNA bunch

(1) relation while adopting qPCR determine the concentration that template is initial and carry out amplified reaction between change in concentration, does accurately quantitatively DNA library template amount, ensures the best packing density of the quality of data and wandering cells;

(2) sample having diluted is added in FC, carry out bridge-type PCR and make unit molecule cluster, reaction system and condition are as follows: bridge-type pcr amplification reaction system is in table 3, and bridge-type pcr amplification reaction condition is in table 4.

Table 3

Table 4

5. genome analysis instrument checks order and analyzes:

GO enrichment is analyzed: Gene Ontology(is called for short GO) be the Gene Ontology of an International standardization, provide a set of standard vocabulary table dynamically updating (controlled vocabulary) to describe the attribute of gene and gene product in organism comprehensively.GO always has three ontology, describes respectively the bioprocess (biological process) of the molecular function (molecular function) of gene, residing cell position (cellular component), participation.

GO functional analysis for be target gene predict the outcome (hereinafter to be referred as " candidate's target gene ").This analysis can provide with reference to after genetic comparison, the GO function entry of significant enrichment in candidate's target gene, and filter out candidate's target gene and which biological function significant correlation.Analyze the main biological function that can determine that candidate's target gene is exercised by the enrichment of GO function significance.

KEGG path analysis: with GO analyze the same, KEGG path analysis for be also candidate's target gene.In vivo, different genes coordinates to exercise its biological function mutually, and the analysis based on pathway contributes to further to understand the biological function of gene.Analyze main biochemical metabolism approach and the signal transduction pathway that can determine that candidate's target gene participates in by the enrichment of path significance.

Finally confirm as the healthy pregnant women and the placenta in preeclampsia peripheral blood leucocyte miRNA that there are differences expression and comprise has-miR-15a-3p(SEQ ID No.1), has-miR-31-3p(SEQ ID No.2), has-miR-451a(SEQ ID No.3), has-miR-122-5p(SEQ ID No.4).Compared with Normal group pregnant woman, in preeclampsia PE group peripheral leukocytes in patients, SEQ ID No.3 and SEQ ID No.4 expression level significantly raise, and SEQ ID No.1 and SEQ ID No.2 expression level significantly reduce simultaneously, and difference has statistical significance.Placenta in preeclampsia is increased to subgroup analytical results and show, SEQ ID No.3 expression level raises, and it is stable that SEQ ID No.1 and SEQ ID No.2 expression level are reduced in each group of eclamptic patients performance simultaneously.

Embodiment 3: the real-time quantitative PCR checking of placenta in preeclampsia peripheral blood leucocyte part differential expression miRNA

The miRcute miRNA First-Strand cDNA Synthesis Kit test kit and the PCR instrument that use TIANGEN company, operate according to test kit additional disclosure book:

1. in the reaction tubes of precooling RNase Free, add following reagent to cumulative volume 20ul (finally adding E.coli Poly (A) Polymerase) on ice, as shown in table 5.

Table 5:miRNA 3' end adds Poly (A) to be processed

2. pipettor mixes the reaction solution of above-mentioned preparation gently, of short duration centrifugal after 37 DEG C reaction 60min; Prepare reaction solution on ice and carry out reverse transcription reaction, reverse transcription reaction system is in table 6:

The miRNA that table 6:Poly (A) modifies carries out reverse transcription reaction

3. pipettor mixes the reaction solution of above-mentioned preparation gently, of short duration centrifugal after 37 DEG C reaction 60min; Synthetic cDNA reaction solution can be positioned over-20 DEG C of preservations.

4. real-time quantitative PCR (Real-Time qPCR)

Dilution cDNA is for subsequent use after product cDNA is diluted to 10 times; Real-time quantitative polymerase chain reaction, reaction system is as table 7; What use is the miRcutemiRNA fluorescence quantitative detection kit of TIANGEN company, and wherein reverse primer is that test kit carries, and forward primer is the business primer that TIANGEN company buys, in table 8.

Table 7: fluorescent quantitation reaction system preparation table

Table 8:miRNA detects primer

Pcr amplification system and condition are set after upper machine, and quantitative pcr amplification reaction programming is in table 9.

Table 9: quantitative pcr amplification reaction programming

Each sample all does 3 multiple holes, after reaction finishes, makes melting curve analysis (temperature 60 C-90 DEG C).

5. statistical procedures:

Adopt SPSS 18.0 statistical softwares to store and analytical data.Measurement data represents with mean soil standard deviation (mean soil SD).Three groups of samples that each miRNA is corresponding adopt one-way analysis of variance ANOVA to test, and P<0.05 is for there being statistical significance.

Result shows:

1. between couple A, two groups of pregnant woman's groups of B, miRNA horizontal expression situation is carried out the demonstration of difference analysis result, and Probability is greater than 0.70, log2Ratio and is greater than 1.3 or be less than-1.4 miRNA and add up to and have 10 kinds, and that wherein raises has 6 kinds, downward have 4 kinds.Concrete title refers to table 10.

table 10: the miRNA cartogram that preeclampsia and normal pregnancies two group differences are expressed

2. couple two groups of pregnant woman miRNA that comparing difference is expressed between two carry out cluster analysis, and result and statistics software analysis result are similar, and wherein the express spectra of hsa-miR-15a-3p and hsa-miR-31-3p is adjacent, belongs to the similar miRNA of adjusting function.

3. the total RNA quality control of the research object sample of 4 of couple 1-4 groups and amplification quality control

Each research object peripheral blood leucocyte RNA detects with NanoDrop2000 ultramicrospectrophotometer respectively, in each group sample, total RNA detects and takes out of complete and clear at 28S, 18S and 5S bar through agarose gel electrophoresis, can think that total RNA quality control is better, can carry out follow-up experiment.Result as shown in Figure 1.

4. between preeclampsia PE group and normal pregnancies group, compare

Compared with Normal group pregnant woman, in preeclampsia PE group peripheral leukocytes in patients, miR-122-5p and miR-451a expression level significantly raise, and miR-15a-3p and miR-31-3p expression level significantly reduce simultaneously, and difference has remarkable statistical significance.Above result is consistent with research first part high-flux sequence result.Refer to table 11 and Fig. 2-Fig. 5.

The relative expression quantity (2 of different microRNA molecules in table 11:4 group ^{-Δ Δ C т}) comparison (means standard deviation)

A: with relatively P<0.05 of N group; B: with relatively P<0.01 of N group;

C: with relatively P<0.05 of PE group; D: with relatively P<0.05 of PC group

Preeclampsia PA group, PC group and normal pregnancies between organizing between two relatively

(1) compared with Normal group pregnant woman, preeclampsia, PA organized miR-451a expression level rising (P > 0.05) in peripheral blood leucocyte, and miR-122-5p, miR-15a-3p and miR-31-3p expression level significantly reduce (P < 0.01) simultaneously.

(2) compared with Normal group pregnant woman, in preeclampsia PC group peripheral blood leucocyte, miR-451a expression level raises (P < 0.01), miR-122-5p(P < 0.05 simultaneously), miR-15a-3p(P < 0.01) and miR-31-3p(P < 0.01) expression level significantly reduces.

Between 3 groups of placenta in preeclampsias relatively

(1) compared with preeclampsia PE group patient, in preeclampsia PA group and PC group peripheral blood leucocyte, miR-122-5p expression level all reduces (P < 0.05), the while miR-451a, miR-15a-3p and miR-31-3p expression level without significant difference.

(2) compared with preeclampsia PA group patient, in preeclampsia periphery PC group blood leukocytes and miR-122-5p expression level raise that (P < 0.05, miR-451a, miR-15a-3p and miR-31-3p expression level are without significant difference simultaneously.

Interpretation of result: existing bibliographical information:

MiR-122-5p gene be positioned at chr18:56118306-56118390[+], belong to intergenic sequence.Its target protein comprise G protein-coupled receptor kinase interactor 1 (GIT1), glycogen synthase 1 (muscle) (GYS1), erythropoietin (EPO), MAP/microtubule affinity-regulating kinase 1 (MARK1), Sp2 transcription factor (SP2) etc.Wherein GIT1 mainly regulates the activity of endothelial cell nitric oxide synthase (eNOS) by transcribing rear translation.The activation of this enzyme and NO's is synthetic relevant.GIT1 level declines and causes sinusoidal endothelial cell impaired.Again expressing the endotheliocyte of liver injury after GIT1 is repaired.The nearest research evidence such as Majumder shows, GIT1 crosses vasculogenesis and the metastases expressed with increase and is associated, and increasing of GIT1 level is that endotheliocyte directional migration and tumor-blood-vessel growth process are requisite.The researchs such as Wang show, VEGF is the most important tyrosine kinase receptor TKR of endotheliocyte (ECs), and for cell survival, migration and vasculogenesis are absolutely necessary.GIT1 plays a significant role in via PLCgamma approach VEGF induction EC podosome formation and cell migration.The important regulating effect that has of immunologic function, function of vascular endothelium, cellular metabolism function and the tumour of miR-122 to patient is described, further investigation has important clinical meaning to the pathogenesis that discloses blood vessel injury preeclampsia.This result of study shows, placenta in preeclampsia miR-122-5p horizontal expression obviously raises, may be closely related with the effect of its target protein, cause vascular endothelial damage, immunologic dysfunction, and the damage of liver blood sinus endothelium is caused to dysfunction of liver, the generation of the pathologies such as HELLP syndrome.

The gene of miR-451a is positioned at chr17:27188387-27188458 [-], belongs to gene extron subsequence.Adjacent gene cluster has hsa-mir-144 (chr17:27188551-27188636 [-]), hsa-mir-4732 (chr17:27188673-27188748 [-]), hsa-mir-451b (chr17:27188389-27188456 [+]), the mature sequence of miR-451a is hsa-miR-451a(17-aaaccguuaccauuacugaguu-38), its target protein comprises (G-rich RNA sequence binding factor 1, GRSF1), (GDP dissociation inhibitor 1, GDI1), (tuberous sclerosis 1, TSC1), (myelin basic protein MBP, epiregulin EREG), (glutamate decarboxylase-like 1, GADL1), (activating transcription factor 2, ATF2).The results of study such as Bruchova show, miR-451 crosses that expression may regulate and control potential IE gene UBE2H and ARPP-19 causes Patients with Polycythemia Vera hemogram abnormity.In gliomatous correlative study, find that miR-451 increasing expression suppresses LKB1/AMPK path, thereby invasion and attack and the multiplication capacity of tumour cell are strengthened, infer that thus miR-451 raises also likely by the similar fashion target gene that regulates and controls to be correlated with, and causes the infringement of many organs such as blood samples of patients system, nerve and system.The prompting of fourth timely rain result of study, miR-126 and miR-451 expression level significantly raise and the remarkable reduction of miR-142-3p expression level may be got involved closely related with the many organs of SLE patient.This result of study shows that placenta in preeclampsia peripheral blood leucocyte miR-451a level obviously increases may directly or indirectly be affected the activity of immunocyte and cause the generation of preeclampsia.

The gene of miR-15a is positioned at chr13:50623255-50623337 [-], belongs to gene intron and exon multidigit point sequence, and adjacent gene cluster has hsa-mir-16-1(chr13:50623109-50623197[+]).The mature sequence of miR-15a is hsa-miR-15a-5p(13-uagcagcacauaaugguuugug-35) and hsa-miR-15a-3p(51-caggccauauugugcugccuca-72), the target protein of miR-15a-3p has caspase10, BCL2 and Connective Tissue Growth Factor (connective tissue growth factor, CTGF).Wherein the mitogen of being secreted by vascular endothelial cell by the protein of this genes encoding of CTGF.Coded albumen is in the effect of the cell adhesion of chondrocyte's propagation, differentiation and many cell types, and closely related with platelet-derived somatomedin.Caspase10 and BCL2 affect lymphocytic apoptosis, relevant with diseases such as bone-marrow-derived lymphocyte leukemia.Zampetaki etc. find that by real-time quantitative PCR analysis diabetes B patient blood plasma presents low-level miRNA-20b ,-21 ,-24 ,-15a ,-126 ,-191 ,-197 ,-223 ,-320 and-486, but miRNA-28-3p slightly raises.Wherein miRNA-15a ,-29b ,-126 ,-223 express reduce and miRNA-28-3p express increase, and before there is symptom in diabetes the several years can detect.Wherein the miRNA-126 in endotheliocyte source and diabetes relation are the closest.The adjusting that miR-15a/16-1 expresses by the BCL-2 in Gene Handling cell cycle progression.Research finds that miR-15a/16-1 bunch of Microrna function is as tumor suppressor gene, and with gene BCL2, as its target, specifically, miR-15a/16-1 lowers the expression of BCL-2.The downward of this result of study prompting placenta in preeclampsia miR-15a-3p level may cause the increase of the expression of BCL-2 to affect leukocytic normal apoptotic and cause response to oxidative stress to increase.Finally cause blood vessel endothelium injury.

The gene of miR-31 is positioned at chr9:21512114-21512184 [-], belongs to gene intron sequence.The mature sequence of miR-31 is hsa-miR-31-5p(8-aggcaagaugcuggcauagcu-28) and hsa-miR-31-3p(44-ugcuaugccaacauauugccau-65), the target gene of miR-31-3p comprises IL-15 (interleukin 15), TDP1 (tyrosyl-DNA phosphodiesterase 1), PIK3CG (phosphoinositide-3-kinase, catalytic gamma polypeptide), IMPG1 (interphotoreceptor matrix proteoglycan 1), MASP1 (mannan-binding lectin serine peptidase 1), OSTM1 (osteopetrosis associated transmembrane protein 1) etc.Kalantar etc. study discovery, contrast and compare with non-hypertensive pregnant woman, placenta in preeclampsia peripheral blood Serum Level of Tumor Necrosis Factor-α (TNF-α), interleukin 15 (IL-15) and interleukin-10 (IL-10) level obviously raise, and prompting patient Th1 type immune response enhancing is relevant with Attack of Preeclampsia.The researchs such as Baradie show, contrast and compare with normotensive contemporaneously pregnant woman, and in placenta in preeclampsia peripheral blood serum, IL-15, IL-16 and β-chorionic-gonadotropin hormone level significantly increase, and closely related with the severity of disease.Aspect diagnosis serious preeclampsia, serum IL-15 level is more valuable than β-chorionic-gonadotropin hormone.Bachmayer etc. have studied NK cell levels in placenta in preeclampsia peripheral blood, and cultivate in vitro monocyte and give after monokine stimulates and find, with normal non-pregnant woman with compare with pregnant all normal pregnancies, placenta in preeclampsia NK cell levels occurs abnormal relevant with the stimulation of IL-15 in blood plasma.The downward of this result of study prompting placenta in preeclampsia miR-31-3p level may cause the increase of the expression of IL-15 the immune response of Th1 type is strengthened and cause immunologic injury abnormal reaction, finally causes blood vessel endothelium injury, relevant with Attack of Preeclampsia.

Sequence table

<110> Mountain Western Medicine S University

There is relevant peripheral blood leucocyte miRNA mark and application thereof preeclampsia in the <120> mankind

<160>2

<170> PaUentIn Version 3.5

<210> 1

<211> 22

<212> RNA

<213> native sequences, derives from people (human) peripheral blood leucocyte

<223> microRNA:has-miR-15a-3p

<400> 1

51-caggccauau ugugcugccu ca-72 22

<210> 2

<211> 22

<212> RNA

<213> native sequences, derives from people (human) peripheral blood leucocyte

<223> microRNA: has-miR-31-3p

<400> 1

44-ugcuaugcca acauauugcc au-65 22

<210> 3

<211> 22

<212> RNA

<213> native sequences, derives from people (human) peripheral blood leucocyte

<223> microRNA: has-miR-451a

<400> 1

17-aaaccguuac cauuacugag uu-38 22

<210> 4

<211> 22

<212> RNA

<213> native sequences, derives from people (human) peripheral blood leucocyte

<223> microRNA: has-miR-122-5p

<400> 1

15-uggaguguga caaugguguu ug-36

Claims (4)

1. there is relevant peripheral blood leucocyte miRNA mark preeclampsia in the mankind, is selected from multiple in has-miR-15a-3p, has-miR-31-3p, has-miR-451a, has-miR-122-5p.

2. there is relevant peripheral blood leucocyte miRNA mark preeclampsia in the mankind according to claim 1, it is characterized in that: described peripheral blood leucocyte miRNA mark is made up of has-miR-15a-3p, has-miR-31-3p, has-miR-451a and has-miR-122-5p.

3. there is relevant peripheral blood leucocyte miRNA mark preeclampsia in the mankind according to claim 1 and 2, it is characterized in that: the sequence of has-miR-15a-3p is SEQ ID No.1, the sequence of has-miR-31-3p is SEQ ID No.2, the sequence of has-miR-451a is SEQ ID No.3, and the sequence of has-miR-122-5p is SEQ ID No.4.

Peripheral blood leucocyte miRNA mark according to claim 1 and 2 preparation mankind preeclampsia determine or monitoring reagent in application.