CN105821119A - Nucleic acid label and kit for auxiliary diagnosis of Kawasaki disease - Google Patents
Nucleic acid label and kit for auxiliary diagnosis of Kawasaki disease Download PDFInfo
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Abstract
The invention belongs to disease detection kit, and concretely relates to a kit for auxiliary diagnosis of Kawasaki disease by using miRNA as a biological label. The nucleic acid label is hsa-miR-223-3p, and the kit includes a primer subjected to a real-time quantitative polymerase chain reaction (RT-qPCR) for hsa-miR-223-3p. The kit can combine blood routine examination, high-sensitivity C reactive protein, erythrocyte sedimentation rate and cardiac ultrasonography, and is conducive to auxiliary diagnosis of Kawasaki disease and prediction damage of coronary artery.
Description
The invention belongs to disease detection test kit, particularly relate to miRNA as biological marker auxiliary diagnosis river
The test kit of rugged disease.
Background technology
Mucocutaneous lymphnode syndrome (Kawasaki disease, KD) is a kind of acute vascular inflammation syndrome, is mainly in infant, is child
Modal acquired heart disease, pathological changes mainly involves arteriolar, especially coronary artery, can be formed coronary artery expansion or
Coronary aneurysm, be grow up after one of the risk factor of ischemic heart desease.The cause of disease and the pathogenesis of KD are still not very clear,
Research tendency thinks that KD is on the basis of certain hereditary susceptibility at present, exogenous antigen trigger, cause immunity of organism merit
Can be disorderly caused, immune advanced activation and vasculitis are the basic pathology features of KD.The diagnosis of KD is at present also without special
The lab index of property, mainly makes diagnosis according to clinical manifestation, although the diagnosis of KD is played an important role by Cardiac ultrasound,
But still lack the most sensitive and specific diagnosis index.In untreated KD infant, 25% can to develop into coronary artery permanent
Property infringement, ultimately result in the formation of coronary aneurysm.Use intravenous immunoglobulin (the intravenous immune of heavy dose
Globulin, IVIG) (2g/kg) associating aspirin treatment become the standard care of KD, can make lesion of coronary artery
Incidence rate is down to about 5%, but still has part infant reactionless to IVIG treatment.Therefore, although people are to the understanding of KD and place
Reason deepens continuously, but how to alleviate KD vascular inflammation thus alleviate vascular endothelial injury, and how early diagnosis mucocutaneous lymphnode syndrome is also
Intervene in time, always have problem to be solved.
The invention of Application No. 201280024575.6 provides the compositions for diagnosing and treat mucocutaneous lymphnode syndrome and method.
More specifically, protein group enrichment sleeping protein A, the tenuin B and tenuin C of KD patient, described albumen is used as KD's
Biomarker (with potential therapy target).Therefore, the compositions provided in the present invention and method is used to detect these biological
Mark can be that the therapy being delivered to experimenter provides information.
The invention of Application No. 201280070975.0 provides the biomarker of mucocutaneous lymphnode syndrome (KD).In some aspects,
The method that present invention provide for detecting KD biomarker, such as the PDGFC expression for detecting rising.Similarly, this
The bright method describing the experimenter treating the biomarker with KD.
The invention of Application No. 201310217292.X provides PPP3CC gene or its expression product at treatment mucocutaneous lymphnode syndrome
In application.In particular it relates to a kind of calcineurin 3 catalytic subunit γ subtype protein (PPP3CC) gene, albumen or
The purposes of its accelerator, is used for preparing (i) and improves Isosorbide-5-Nitrae, the expression of 5 InsP3s 3 kinase c (ITPKC) and/or activity；
And/or the pharmaceutical composition of (ii) treatment mucocutaneous lymphnode syndrome (Kawasaki disease).The present invention is of value to sending out of understanding mucocutaneous lymphnode syndrome
Pathogenesis system and path, and develop the medicine for the treatment of mucocutaneous lymphnode syndrome.
The nucleic acid markers of Application No. 201410709423.0 disclosure of the invention quick diagnosis mucocutaneous lymphnode syndrome and test kit thereof,
This nucleic acid markers is 4 kinds of miRNAs (microRNA miR-1246, miR-4436b-5p, miR-197-3p and miR-671-
5p), containing the primer carrying out fluorescence quantitative PCR detection for these 4 kinds of miRNAs in described test kit；Diagnosis mucocutaneous lymphnode syndrome the most only needs
4 kinds of miRNA content of this in exosome in quantitative analysis serum, then specifically analyze the Ct value of these 4 kinds of miRNA
?.The present invention have draw materials conveniently, simple to operate, high specificity, time spend less, quick and precisely, the advantage such as result is stable,
Timely, quick, objective, the Accurate Diagnosis Patients with Kawasaki Disease of energy, especially by once experiment can be by mucocutaneous lymphnode syndrome with common confusing
The virus of symptom of confusing infects separately, has the advantage not available for traditional diagnosis method.Therefore, fast to Patients with Kawasaki Disease of the present invention
Speed diagnosis has great clinical value, for developing the quick diagnosis reagent kit used on Patients with Kawasaki Disease further
Provide directivity.
MicroRNA (miRNA) is a kind of endogenous noncoding strand microRNA, about by 21～25 nucleotide groups
Become.MiRNA is tied by 3'-noncoding region (3'-untranslated region, the 3'-UTR) specificity with target gene mRNA
Close, it is possible to make mRNA degraded or suppress it to translate, thus play its suppression said target mrna, reach the effect of modulin level.When
Before think that miRNAs can regulate and control the gene of 30% mankind's coded protein, and a miRNA can regulate and control multiple target gene table
Reach, and same gene also can be regulated and controled by multiple miRNA.MiRNA participates in the various biological mistakes such as cell proliferation, differentiation, apoptosis
Journey, the change of its expression can cause the generation of multiple disease, including cardiovascular disease.In the past it is believed that miRNA conduct
The gene expression of level after intracellular endogenic RNA regulatory transcription.But, it has recently been demonstrated that miRNA can be in circulation
Blood is detected, and can as disease biological markers (see: Mitchell PS, Parkin RK, Kroh EM,
et al.Circulating microRNAs as stable blood-based markers for cancer
detection[J].Proc Natl Acad Sci USA.2008；105:10513–10518；Skog J,Wurdinger T,
van Rijn S,et al.Glioblastoma microvesicles transport RNA and proteins that
promote tumour growth and provide diagnostic biomarkers.[J].Nat Cell
Biol.2008；10:1470–1476).Further, miRNA is present in serum or blood plasma with a highly stable state, even if
It is to stand freeze/thaw repeatedly, it is possible to from the degraded of ribonuclease.MiRNA in blood plasma has special express spectra, at the heart
Angiopathy, such as cardiac damage, coronary artery disease, heart failure etc., the miRNA in its circulation has been found to.Mucocutaneous lymphnode syndrome is made
For modal posteriority Children with Cardiovascular Diseases, show as polyangitis and vascular endothelium dysfunction in general immunity,
But the expression of miRNA and mechanism of action thereof in its blood plasma, report is the fewest both at home and abroad.
Summary of the invention
It is an object of the invention to provide the nucleic acid markers of a kind of quick auxiliary diagnosis mucocutaneous lymphnode syndrome.
It is a further object of the present invention to provide the test kit of a kind of quick auxiliary diagnosis mucocutaneous lymphnode syndrome.
In order to realize foregoing invention purpose, the technical scheme is that a kind of nucleic acid marking assisting diagnosis mucocutaneous lymphnode syndrome
Thing, described nucleic acid markers is hsa-miR-223-3p, has the nucleotide sequence as shown in SEQ 6
TGGGGTATTTGACAAACTGAC。
Preferably in technical scheme, described nucleic acid markers hsa-miR-223-3p is the miRNA in blood plasma.
Therefore, the miRNA in claimed blood plasma is as the application of the nucleic acid markers of mucocutaneous lymphnode syndrome.
The present invention claims a kind of test kit assisting diagnosis mucocutaneous lymphnode syndrome simultaneously, and described test kit includes in blood plasma
Hsa-miR-223-3p carries out the reagent of detection by quantitative.
Preferably in technical scheme, described test kit includes hsa-miR-223-3p is carried out real-time quantitative polymerase chain
The primer of reaction (RT-qPCR).In preferred technical scheme, described primer as shown in SEQ 4,5 '
TGTCAGTTTGTCAAATACCCCA 3′。
Preferably in technical scheme, described test kit includes positive reference reagent and stealthy reference reagent.
Described in the present invention is simultaneously claimed, there is the miRNA of nucleotide sequence described in SEQ 6 and alleviate mucocutaneous lymphnode syndrome in preparation
Vascular inflammation reaction medicine in application.
Due to the application of technique scheme, compared to the prior art, the present invention has the following advantages:
1, the present invention contributes to mucocutaneous lymphnode syndrome and the discriminating of common febrile disease, by miR-223-in quantitative analysis blood plasma
The content of 3p, can timely, quick, objective, Accurate Diagnosis Patients with Kawasaki Disease, especially by once experiment can by mucocutaneous lymphnode syndrome with
The virus of common confusing symptom of confusing infects separately, has the advantage not available for traditional diagnosis method.Therefore, the present invention is to mucocutaneous lymphnode syndrome
The quick diagnosis of infant has a great clinical value, and for developing use on Patients with Kawasaki Disease fast further
Speed diagnostic kit provides directivity.
2, due to miRNA chip and RT-qPCR the result collectively show that: hsa-miR-223-3p is at KD acute stage plasma
Middle expression significance raises, and therefore, the nucleic acid markers of quick auxiliary of the present invention diagnosis mucocutaneous lymphnode syndrome combines routine blood test, Gao Min
C reactive protein, erythrocyte sedimentation rate, Cardiac ultrasound, contribute to auxiliary diagnosis mucocutaneous lymphnode syndrome and prediction lesion of coronary artery.
3, mucocutaneous lymphnode syndrome about 2 weeks is the high-incidence season of lesion of coronary artery in the course of disease, in early days in detection mucocutaneous lymphnode syndrome blood plasma
The content of miR-223-3p and IL-6, contributes to predicting whether concurrent lesion of coronary artery.
4, the present invention have draw materials conveniently, simple to operate, high specificity, time spend less, quick and precisely, result stable etc.
Advantage.
Accompanying drawing explanation
Fig. 1 is that the scatterplot array that in embodiment one, single Fluorescence chip sample data compares two-by-two is depicted as matrix diagram and (dissipates
In point diagram, each point represents the probe points on chip, and this position in two dimensional surface is true by its X-axis coordinate and Y-axis coordinate
Fixed.X-axis: matched group data/experimental group data；Y-axis: experimental group data/matched group data.Fall in the graphic on y=x straight line
Point, represents this probe points standardization signal value (Fold change, FC) difference=1 in two chips；Fall bit line in the graphic
Point outside the line of 45 °, both sides, it represents this probe points signal value difference FC in two chips > 2)；
Fig. 2 is embodiment one gained volcano figure (Grey Point: FC absolute value ＜ 2, the miRNA of p ＞ 0.05.Red point: FC is exhausted
To value >=2, the miRNA of p≤0.05, these miRNA are significant difference miRNA.)；
Fig. 3 is the cluster analysis figure of differential expression miRNAs in embodiment one chips result；
Fig. 4 is hsa-miR-16 amplification curve and solubility curve in embodiment one；
Fig. 5 is hsa-miR-33b-3p amplification curve and solubility curve in embodiment one；
Fig. 6 is hsa-miR-223-3p amplification curve and solubility curve in embodiment one；
Fig. 7 is hsa-miR-765 amplification curve and solubility curve in embodiment one；
Fig. 8 is KD group and matched group hsa-miR-765, hsa-miR-223-3p, hsa-miR-33b-3p in embodiment one
Expression average ratio is relatively；
Fig. 9 is IL-6 concentration curve in embodiment two；
Figure 10 is hsa-miR-16 amplification curve and solubility curve in embodiment two；
Figure 11 is hsa-miR-223-3p amplification curve and solubility curve in embodiment two；
Figure 12 is that in embodiment two, each group blood plasma hsa-miR-223-3p relative expression quantity compares；
Figure 13 be in embodiment two KD acute stage relative with nCAL group blood plasma hsa-miR-223-3p with subacute stage CAL group
Expression compares；
Figure 14 is that in embodiment two, each group Association of Plasma IL-6 Level compares；
Figure 15 be in embodiment two KD acute stage compare with nCAL group Association of Plasma IL-6 Level with subacute stage CAL group.
Specific embodiment
The invention will be further described with embodiment below in conjunction with the accompanying drawings.Should be understood that these embodiments are merely to illustrate
The present invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to
Normal condition, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to the condition proposed by manufacturer.Used in embodiment
Various conventional chemical reagent, be commercially available prod.
Embodiment one, chooses acute stage KD infant 6 example that attached children's hospital of University Of Suzhou is medical, and healthy children 6 example
As comparison, extract fasting blood, separated plasma early morning, extract total serum IgE in blood plasma, use miRNA chip hybridization technology for detection river
The miRNAs of differential expression in rugged sick group and Normal group blood plasma.Choose acute stage KD infant and each 8 examples of healthy children again, adopt
By real-time quantitative PCR (real-time quantitative PCR, RT-qPCR) technical identification chip results.Utilize
Tri-data bases of TargetScan, PITA and microRNAorg carry out microRNA target prediction to difference miRNAs, use DAVID raw
Thing bioinformatics analysis software carries out GO and KEGG and analyzes common target gene, finds the target gene relevant to KD vascular inflammation.
1. the instrument related in embodiment
The key instrument that 1.1 blood plasma separate and preserve
1.2 genechip detections need key instrument
The 1.3 required key instruments of RT-qPCR detection
2. the main agents related in embodiment
2.1 genechip detections need main agents
The 2.2 required main agents of RT-qPCR detection and primers
Nuclease-free H2O (without RNase water) (company)
Cell Disruption Buffer (cell breakage buffer)
Phenol/chloroform
Dehydrated alcohol
miScript HiSpec Buffer
Nucleics Mix
MiScript Reverse Transcriptase Mix (miScript reverse transcriptase mixture)
By miRNA(http://www.mirbase.org/)Gene database obtains hsa-miR-16, hsa-miR-
765, the gene order of hsa-miR-33b-3p, hsa-miR-223-3p, uses the Primer Primier 5 software (U.S.
AppliedBiosystem Inc) carry out design of primers.MiRNA upstream specific primer sequence is as follows, see table.Downstream primer
Carried by Qiagen test kit.
Primer nucleic acid sequence table
| Gene Name | Forward primer sequence | Temperature (DEG C) |
| Hsa-miR-16 (internal reference) | SEQ 1 | 60 |
| hsa-miR-765 | SEQ 2 | 60 |
| hsa-miR-33b-3p | SEQ 3 | 60 |
| hsa-miR-223-3p | SEQ 4 | 60 |
3. microRNA target prediction software and data analysis software
MicroRNA target prediction software: TargetScan (http://www.targetscan.org/)
PITA(http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html/)
microRNAorg(http://www.microrna.org/microrna/home.do/)
Data analysis software: Venn (http://bioinfogp.cnb.csic.es/tools/venny/)
DAVID functional annotation gene chip bioinformatic analysis software (Functional Annotation
Bioinformatics Microarray Analysis, DAVID) it is a kind of biomolecule information database, incorporate number biology
According to and analytical tool, provide system to combine for large-scale gene or albumen list (hundreds and thousands of gene I/D or protein I D list)
The biological function annotation information closed；Can be from molecular function (molecular function, MF), biological process
(biological process, BP), cell composition (cellular component, CC) three aspects carry out function to gene
Analyze, we use DAVID (Http:// david.abcc.ncifcrf.gov/,Version:6.7) at Gene Ontology
The aspect such as (cell composition, molecular function, biological process), KEGG path carries out functional annotation to miRNA target gene.
4. clinical data
Clinical data used by 4.1 genechip detections
(1) KD group: acute stage KD infant 6 example that in May, 2013 in August, 2013, my institute accepted for medical treatment, all meets China and exists
In the KD symposium that 2006 hold revision diagnostic criteria, the maleest 2 examples, female 4 example, July at age to 3 years old October, in
2 years old age of position.
(2) Normal group: with age group health examination child 6 example.Man 3 examples, female 3 example, May 1 years old age to 3 years old January,
1 years old November of the median age.See table.
KD infant and matched group clinical data table
Clinical data used by 4.2 RT-qPCR detections
(1) KD group: in JIUYUE, 2013 is to acute stage KD infant 8 example that in October, 2013, my institute accepted for medical treatment, and diagnostic criteria is the same,
The maleest 5 examples, female 3 example, November at age to 4 years old August, 1 years old November of the median age.
(2) Normal group: with age group health examination child 8 example.Man 6 examples, female 2 example, February at age to 4 years old January, in
Position July 1 years old age.See table.
KD infant and matched group clinical data table
The concrete operation step of embodiment one includes:
1. the separation of blood plasma: all cases all in extraction ulnar vein blood 2ml on an empty stomach in early morning, proceed to rapidly ethylenediaminetetraacetic acid
In potassium (EDTA-K2) anticoagulant tube, fully mixing, in 2 hours, under the conditions of 4 DEG C, 820g is centrifuged 10min.Draw about 1ml supernatant
Going in the 1.5ml centrifuge tube of cleaning, under the conditions of 4 DEG C, 16000g is centrifuged 10min, in absorption supernatant to new centrifuge tube, be placed in-
80 DEG C of Refrigerator stores are standby.
2. the miRNAs of differential expression in Agilent miRNA 19.0 cDNA microarray blood plasma, comprises the following steps:
2.1 blood plasma Total RNAs extraction: use mirVanaTMRNA separating kit (Applied Biosystem p/n
AM1556) total serum IgE is extracted；
2.2 miRNA chip operation flow processs: taking 100ng total serum IgE and initiate, constant volume, to 2 μ L, then dephosphorylations, makes sample
Degeneration, linkage flag.
2.3 chip washings: take out chip and wash 5min in washing liquid 1；Chip is put into washing 5min (37 in washing liquid 2 again
℃)。
2.4 chip scannings: utilize in Agilent Scanner G2505C (Agilent Technologies) scanner
Select corresponding parameter scanning, obtain original image.
The result of above-mentioned blood plasma RNA sample detection and experimental evaluation shows:
A) sample total serum IgE utilizes NanoDrop ND-2000 quantitatively and to detect through Agilent Bioanalyzer 2100
RNA integrity, specimen quality used meets the requirements.
B) experimental result assessment, median CV (%) represents the repeatability probe coefficient of variation, and median CV (%) value is more
Little, experimental precision is the highest, and the %CV value repeating probe in chip should be less than 15%, and each sample repeatability is relatively good, see table.
KD group assesses table with matched group experimental result
| KD group | Chip number | CV (%) | Matched group | Chip number | CV (%) |
| 1 | 12003-2-2 | 11.3983 | 1 | 12003-2-1 | 9.72617 |
| 2 | 11920-1-1 | 9.04963 | 2 | 12003-2-3 | 10.4688 |
| 3 | 11920-1-2 | 8.97566 | 3 | 12003-2-4 | 7.96456 |
| 4 | 12885-2-4 | 8.44535 | 4 | 12885-2-1 | 8.36837 |
| 5 | 12887-1-1 | 8.29865 | 5 | 12885-2-2 | 8.69797 |
| 6 | 12887-1-2 | 7.79005 | 6 | 12885-2-3 | 10.4861 |
C) data analysis is then carried out:
I. Feature Extraction software (version10.7.1.1, Agilent Technologies) place is used
Reason original image extracts initial data.Followed by Genespring software (version 12.5；Agilent
Technologies) quantile standardization and subsequent treatment are carried out.The initial data of single Fluorescence chip through standardization,
After being converted into the log logarithm with 2 as the end, in a two-dimensional direct angle coordinate system plane, draw scatterplot, see Fig. 1.Chip data
Scatterplot be usually used in assessing two groups of data population distribution central tendencies.
II., before screening difference miRNA, probe filtration is first carried out, in the often group sample that compares at least one group
75% probe being labeled as Detected stays and carries out subsequent analysis.For there being the analysis repeated biology, T inspection is utilized to obtain
Significance of difference p value and the fold differences of normalized signal value, FC value screens, and standard is FC >=2.0 and p≤0.05.
Differential screening result, see table.
KD group and matched group differential screening result
III. the most produced differential expression situation being reflected in the figure of volcano, X-axis is that fold differences takes with 2 the end of for
The value of logarithm, Y-axis is the value that p value takes negative logarithm with 10 end of for.(statistical analysis method is i.e. used in the case of repetition biology setting
Analyze differential expression), provide volcano figure, see Fig. 2.
IV. difference miRNA is carried out non-supervisory hierarchical clustering.Calculate multiple sample distance between any two, constitute distance
Matrix, nearest two classes of combined distance are a new class, calculate new class and the most all kinds of distances, remerge, calculate, until only
Till one class, calculate the direct dependency of sample, in general, same class sample with the expression of difference miRNA selected
Product can be occurred in same bunch by cluster, gathers the miRNA in same bunch and is likely to be of similar biological function.With
Thermal map (Heatmap) is shown, sees Fig. 3.
3. the miRNAs of differential expression in RT-qPCR checking blood plasma, comprises the following steps:
3.1 RNA extract: extract total serum IgE with chip.
3.2 reverse transcriptions synthesis (cDNA the first chain): utilize miScript II Reverse Transcription Kit
RNA reverse transcription to be measured is become cDNA by (Qiagen, Germany).Reverse transcription system: total serum IgE, 1 μ g；5×miScript HiSpec
Buffer, 4 μ l；10×Nucleics Mix,2μl；miScript Reverse Transcriptase Mix,2μl；
Nuclease-free H2O, adds to 20 μ l.Response procedures: 37 DEG C of 60min, 95 DEG C of 5min.80 μ l are added after reverse transcription
Nuclease-free H2It is standby that O is stored in-20 DEG C of refrigerators.
(1) taking out RNA from-80 DEG C of refrigerators, thawed at room temperature, it is anti-that according to the form below prepares reverse transcription in 0.2ml PCR pipe
Answer system.
| Reaction system | Concentration | Volume (μ l) |
| Total serum IgE | Conc.μg/μl | 0.5μg/Conc[note] |
| MiScript HiSpec buffer | 5× | 2 |
| Nucleic acid mixed liquor | 5× | 1 |
| MiScript reverse transcriptase mixed liquor | - | 0.5 |
| Water without RNase | - | To 10 |
| Total measurement (volume) | - | 10 |
[note] if RNA insufficient total amount 0.5 μ g, then the system that takes can accommodate maximum volume (6.5 μ l)
(2), after in ABI 9700 type PCR instrument, 37 DEG C of insulation 60min make reverse transcription reaction completely, 95 DEG C of 5min terminate reaction.
(3) 90 μ l Nuclease-free H are added2O is diluted to 100 μ l and is stored in-20 DEG C of refrigerators, for subsequent experimental.
3.3 design of primers: the design of Shanghai Ou Yi biomedicine Science and Technology Ltd. is the most limited by Shanghai JaRa biological engineering
Company synthesizes.
3.4 quantitative fluorescent PCRs: utilize480 SYBR Green I Master test kits (Roche,
Swiss) existReact on 480 II type quantitative real time PCR Instruments (Roche, Swiss).System: 2 ×480 SYBR Green I Master, 5 μ l；10μM Universal primer,0.2μl；10μM
MicroRNA-specific primer, 0.2 μ l；CDNA, 1 μ l；Nuclease-free H2O, 3.6 μ l.PCR program: 95 DEG C
10min；95 DEG C of 10s, 60 DEG C of 30s, 40 circulations.Melting curve is utilized to detect product specificities after loop ends: to delay from 60 DEG C
Slowly being warming up to 97 DEG C, every DEG C gathers 5 fluorescence signals.
(1) preparation PCR reaction system
(2) quantitative fluorescent PCR cycling condition
In above-mentioned RT-qPCR checking blood plasma, the result of the miRNAs of differential expression shows:
A) total serum IgE quality inspection result: extract total serum IgE, utilize NanoDrop 2000 spectrophotometric determination concentration and OD260/
OD280, agarose gel electrophoresis detection RNA integrity.Specimen quality used meets the requirements, and can be used for RT-qPCR detection.
B) quantitatively judge: in RT-qPCR, each sample is repeated 3 times, average as Ct value, calculate each section respectively with SPSS
The Average Ct values of gene expression, uses relative expression quantity 2-ΔΔCtThe differential expression of the more each gene of method: Δ Ct=CtGenes of interest-
CtReference gene；Δ Δ Ct=Δ CtExperimental group sample-Δ CtControl sample。
C) hsa-miR-765, hsa-miR-223-3p, hsa-miR-33b-3p phase during RT-qPCR measures KD infant blood plasma
To expression.During RT-qPCR, with hsa-miR-16 (internal reference), hsa-miR-765, hsa-miR-223-3p, hsa-
MiR-33b-3p fluorescent value and period mapping, automatically derive amplification curve and solubility curve (Fig. 4,5,6,7).Gene gained melts
Solution curve is simple spike, and PCR specific amplification is preferable, and the repeatability of three technology repetitions is preferably.
D) comparison sheet of hsa-miR-765, hsa-miR-223-3p, hsa-miR-33b-3p relative expression quantity between two groups
Bright: KD group hsa-miR-223-3p, hsa-miR-33b-3p relative expression quantity apparently higher than Normal group (p < 0.05)；KD
Group hsa-miR-765 relative expression quantity compared with normal matched group no significant difference (p > 0.50), see table, Fig. 8.
The relative ratio of KD group miRNAs expression and Normal group ()
| hsa-miR-765 | hsa-miR-33b-3p | hsa-miR-223-3p | |
| KD group | 0.99±0.84 | 2.84±1.99a | 3.38±2.07a |
| P value | 9.71E-01 | 3.92E-02 | 1.40E-02 |
Note: a.KD group compares p < 0.05 with Normal group.
4. microRNA target prediction
Respectively with TargetScan, it is pre-that PITA, microRNAorg data base carries out target gene to the miRNA of differential expression
Surveying, the target gene predicting three data bases carries out Venny analysis and takes common factor, uses DAVID bioinformatic analysis software
Common target gene carries out GO and KEGG analyze.
MicroRNA target prediction result: respectively with TargetScan, PITA, microRNAorg data base is to difference hsa-miR-
223-3p carries out microRNA target prediction, and the target gene predicting three data bases carries out Venny analysis and takes common factor, found that in advance
The common target gene surveyed has 415, see table.
Hsa-miR-223-3p is via TargetScan, the part target gene of PITA, microRNAorg prediction gained
| Gene ID | Gene symbol | Gene ID | Gene symbol |
| 54522 | ANKRD16 | 136319 | MTPN |
| 158586 | ZXDB | 27347 | STK39 |
| 57659 | ZBTB4 | 6198 | RPS6KB1 |
| 5903 | RANBP2 | 7170 | TPM3 |
| 60682 | SMAP1 | 8899 | PRPF4B |
| 431707 | LHX8 | 10365 | KLF2 |
| 22828 | RBM16 | 3480 | IGF1R |
| 5997 | RGS2 | 65055 | REEP1 |
| 57600 | FNIP2 | 6602 | SMARCD1 |
| 54467 | ANKIB1 | 57151 | LYZL6 |
Target gene is carried out GO analysis, thus the function of this gene is described.GO includes three macroplates, biology
Process (BP), cell composition (CC) and molecular function (MF), so there being three class results.Add up target included in each GO entry
Gene number, and the significance of each GO entry target gene enrichment is calculated by the method for statistical test.The result calculated can be returned
Returning the p value of an enrichment significance, little p value represents that target gene occurs in that enrichment in this GO entry.Can analyze according to GO
Result combine biological significance thus select the gene for follow-up study, for above 415 target genes, utilize DAVID
Software, carries out GO analysis, these genes are projected to respectively on the three big application functions of GO (i.e. molecular function, biological process,
Cellular component), result display hsa-miR-223-3p prediction target gene set is enriched in transcriptional regulatory, transcriptional activation decile respectively
Subfunction, transcribes, on the cell such as biological process and aixs cylinder, inner membrance, plasma membrane such as the negative regulation of metabolic process, developmental process composition
(p < 0.05), see table.
Hsa-miR-223-3p prediction target gene GO biological process classification (part)
Hsa-miR-223-3p prediction target gene GO groups of cells constituent class (part)
Hsa-miR-223-3p prediction target gene GO molecular function classification
Utilize KEGG data base that the target gene that obtains of prediction carries out Pathway analysis, and by the method for statistical test
Calculate the significance of genetic enrichment in each Pathway entry.The result calculated can return the p value of an enrichment significance, little
P value represent gene in this Pathway, occur in that enrichment.Pathway analyzes the effect having prompting to experimental result, passes through target
The Pathway of gene analyzes, and can find the Pathway entry of enriched target gene, and the target gene finding different sample may be with
The change of which cell pathway is relevant.415 prediction target genes being carried out biological pathway enrichment analyze, result shows, at KEGG
In path, hsa-miR-223-3p significant enrichment is in tumor path, and in the Proteolytic enzyme signal path of ubiquitin mediation (p <
0.05), see table.
The KEGG pathway database enrichment of hsa-miR-223-3p prediction target gene is analyzed
Target gene to inflammation-related: application DAVID software analysis finds the target gene totally 7 relevant with inflammation, for
IL6ST、F3、EPHA3、SMAD1、HDAC9、NLRP3、HHEX.Its GO ID be (GO:0050727, GO:0050741, GO:
0002675、GO:0050729、GO:0002673、GO:0002526、GO:0006954、GO:0030948、GO:0010574、GO:
0030947, GO:0010575), its principal biological function includes regulation and control and supervision inflammatory reaction, and participating in activation acute inflammation is anti-
The plasma protein answered and modulating vascular endothelial cell growth factor (ECGF) signal pathway.Wherein IL6ST relate to wherein most merit biology
Energy.
Therefore, embodiment one shows: miRNA chip screens the miRNAs of 7 notable rises in KD acute stage plasma
(hsa-let-7b-5p,hsa-miR-223-3p,hsa-miR-4485,hsa-miR-4644,hsa-miR-4800-5p,hsa-
MiR-6510-5p and hsa-miR-765) and 3 notable downwards miRNAs (hsa-miR-33b-3p, hsa-miR-4443 and
hsa-miR-4515).In conjunction with RT-qPCR the result, find that hsa-miR-223-3p expresses significance and raises and chip results
Unanimously.MicroRNA target prediction analysis obtains 415 common target genes altogether, uses DAVID software analysis to find and inflammation-related
Target gene totally 7, respectively IL6ST, F3, SMAD1, HDAC9, NLRP3, EPHA3 and HHEX.In conjunction with microarray chip technology with
RT-qPCR technology we obtain the miRNAs of differential expression in acute phase of Kawasaki disease blood plasma.Hsa-miR-223-3p may participate in
The pathophysiological process of KD.
Embodiment two, with hsa-miR-223-3p as object of study, inquires into hsa-miR-223-3p at KD gamma globulin
Treating the expression in Plasma Before And After and the dependency with inflammatory cytokine IL-6 thereof, Primary Study hsa-miR-223-3p detects
Clinical meaning to KD.Select 30 example KD infants, wherein coronary artery injury person 10 example, non-coronary arterial injury person 20 example.Send out
Heat and each 12 examples of Normal group.Use hsa-miR-223-in RT-qPCR technology for detection KD acute stage and subacute stage blood plasma
The expression of 3p, uses the inspection of enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) method
Survey IL-6 level in blood plasma, relatively each group difference, and analyze the dependency of hsa-miR-223-3p Yu IL-6.
1. the instrument that embodiment relates to
Analysis software: Ascent software for Multiskan
2. the main agents related in embodiment
RT-qPCR detection is with the required main agents of Part I real time PCR detection and primer
Enzyme labelled antibody working solution (Enzyme Conjugate) (company)
Sample diluent (Sample Buffer)
Concentrated cleaning solution (Wash Buffer)
Standard substance (Standards)
Substrate working solution (TMB Solution)
First antibody working solution (Biotinylated Antibody)
Stop buffer (Stop Solution)
3. the clinical data related in embodiment
(1) KD group: KD infant 30 example that in August, 2013 in August, 2014, my institute accepted for medical treatment, all meets China and called together in 2006
The diagnostic criteria of revision, the maleest 18 examples, female 12 example, April at age to 7 years old June, the median age 1 in the KD symposium opened
June in year.Being divided into acute stage (before IVIG treatment, the course of disease the 1st～11 day) according to KD infant by the course of disease, (IVIG treats subacute stage
After, body temperature is normal, the course of disease the 11st～21 day).Two groups will be divided into the phase: merge CAL person according to heart Color Doppler Detection result
10 examples, without lesion of coronary artery (non-Coronary artery lesion, nCAL) person 20 example.KD coronary artery injury
(CAL) standard: (1) coronary artery expansion: coronary artery diameter<3 years old>2.5mm, 3～9 years old>3.5mm；(2) coronary aneurysm:
Difform coronary artery expansion, internal diameter > 4mm, or coronary artery differentially expanding and adjacent inner diameter ratio are more than 1.5 times；
(3) coronary endometrium Echoenhance.
(2) heating matched group: the same period is with age group acute stage heating Hospitalized Children 12 example, male 7 examples, female 5 example, August at age
To 6 years old JIUYUE, in 2 years old May of the median age, serum CA125 was normal, and gets rid of other connective tissue diseases the most juvenile idiopathic joint
Inflammation, polyarteritis nodosa etc..
(3) Normal group: with age group health examination child 12 example.Man 8 examples, female 4 example, age December to 7 years old, middle position
June 2 years old age.
Embodiment two specifically includes following steps:
1. the separation of blood plasma: all cases all in extraction 2ml ulnar vein blood on an empty stomach in early morning, proceed to rapidly ethylenediaminetetraacetic acid
In potassium (EDTA-K2) anticoagulant tube, fully mixing, in 2 hours, under the conditions of 4 DEG C, 820g is centrifuged 10min.Draw about 1ml supernatant
Going in the 1.5ml centrifuge tube of cleaning, under the conditions of 4 DEG C, 16000g is centrifuged 10min, in absorption supernatant to new centrifuge tube, is divided into
A, B group, is placed in-80 DEG C of Refrigerator stores standby, and A group detects for RT-qPCR, and B group detects for ELISA.
2. hsa-miR-223-3p expression in RT-qPCR detection blood plasma: method detects with RT-qPCR in embodiment one
The miRNAs of differential expression in blood plasma.
3. IL-6 level in ELISA method detection blood plasma: blood plasma thaws in mixture on ice, with anti-human IL-6 monoclonal antibody bag
By in ELISA Plate, every hole adds standard substance and blood plasma 100ul, Sptting plate fully mixes rearmounted 37 DEG C of 40min, with washing
Sptting plate is fully washed 4-6 time by liquid, and on filter paper, print is dry, and every hole adds distilled water and each 50ul of first antibody working solution is (empty
Except Bai), Sptting plate is fully mixed rearmounted 37 DEG C of 20min.Again with cleaning mixture, Sptting plate is fully washed 4-6 time, filter paper
Upper print is done, and every hole adds enzyme labelled antibody working solution 100ul.Sptting plate is put 37 DEG C of 10min.Again with cleaning mixture, Sptting plate is abundant
Washing 4-6 time, on filter paper, print is dry, and every hole adds substrate working solution 100ul, puts 37 DEG C of dark place reaction 15min, and every hole adds afterwards
100ul stop buffer sulphuric acid mixes.At 450nm, survey light absorption value by microplate reader in 30 minutes, record OD value, IL-6 concentration and OD value
It is directly proportional, by drawing standard curve (Fig. 9), calculates IL-6 concentration in blood plasma.
4. statistical analysis
Utilizing SPSS17.0 statistics software to carry out data process, measurement data is with mean ± standard deviation or median (model
Enclose) represent, compare employing T inspection or Kruskal-Wallis H rank test between group, compare employing Nemenyi inspection two-by-two,
Enumeration data uses X 2 test.Correlation analysis uses Spearman ' s rank correlation analysis.P < 0.05 represents that difference has statistics
Learn meaning.
5. result and analysis
1. ordinary circumstance compares: following table is the clinical data of each group of object of study, and through statistical analysis, KD group is right with heating
No significant difference (p > 0.05) in sex ratio, age structure according to group and Normal group.
KD infant and matched group clinical data
| Basic characteristics | KD acute stage | KD subacute stage | Heating matched group | Normal group | P value |
| Number of cases | 30 | 30 | 12 | 12 | |
| Age (moon) | 18(4-90) | 18(4-90) | 29(8-81) | 30(12-84) | P=0.898 |
| Sex (male/female) | 18/12 | 18/12 | 7/5 | 8/4 | P=0.31 |
2. KD infant acute stage and the relative expression quantity of subacute stage blood plasma hsa-miR-223-3p.
2.1 KD infants and the detection of matched group child's blood plasma hsa-miR-223-3p expression
2.1.1 total serum IgE quality inspection result
Extract total serum IgE, utilize NanoDrop 2000 spectrophotometric determination concentration and OD260/OD280, agarose gel
Electrophoresis detection RNA integrity.Specimen quality used meets the requirements, and can be used for RT-qPCR detection.
2.1.2 quantitatively judge
In RT-qPCR, each sample is repeated 3 times, and averages as Ct value, calculates each section of each gene expression with SPSS
Average Ct values, uses relative expression quantity 2-ΔΔCtThe differential expression of the more each gene of method: Δ Ct=CtGenes of interest-CtReference gene；ΔΔCt
=Δ CtExperimental group sample-Δ CtControl sample。
2.1.3 hsa-miR-223-3p expression during RT-qPCR measures KD infant blood plasma.
During RT-qPCR, map with hsa-miR-16 (internal reference), hsa-miR-223-3p fluorescent value and period,
Automatically derive amplification curve and solubility curve (Figure 10, Figure 11).Gene gained melting curve is simple spike, PCR amplifying specific
Property preferable, the repeatability that three technology repeat is preferably.
The comparison of hsa-miR-223-3p relative expression quantity between 2.2 each groups
KD group infant acute stage plasma hsa-miR-223-3p relative expression quantity is apparently higher than heating matched group (p < 0.05)
And Normal group (p < 0.05), difference is statistically significant；Blood plasma hsa-miR-between heating matched group and Normal group
223-3p expression no significant difference (p > 0.05).
KD group infant subacute blood plasma hsa-miR-223-3p relative expression quantity and heating matched group and Normal group between
No significant difference (p > 0.05).
KD-CAL group acute stage plasma hsa-miR-223-3p relative expression quantity is significantly lower than KD-nCAL group, and difference has system
Meaning (p < 0.05) learned by meter.KD-CAL group subacute stage blood plasma hsa-miR-223-3p relative expression quantity is less than KD-nCAL group, but
No significant difference (p > 0.05), is shown in the following table lattice and Figure 12, Figure 13.
KD acute stage, subacute stage and the matched group blood plasma hsa-miR-223-3p expression phase with Normal group of generating heat
Reduced value ( )
| KD acute stage | KD subacute stage | Heating matched group | |
| hsa-miR-223-3p | 5.10±5.11a | 1.01±0.85b | 1.29±0.63c |
Note: a.KD acute stage compares p < 0.05 with Normal group, heating matched group；B.KD acute stage and subacute stage ratio
Relatively p < 0.05；C. not statistically significant p > 0.05 is compared between two matched groups.
CAL group and nCAL group blood plasma hsa-miR-223-3p relative expression quantity in KD ()
Note: A represents CAL group, and B represents that nCAL group, a. acute stage KD-CAL group compare p < 0.05 with KD-nCAL；B. sub-anxious
Property phase KD-CAL group and KD-nCAL comparing difference not statistically significant p > 0.05.
3. KD infant acute stage and subacute stage Association of Plasma IL-6 Level and and Normal group, heating matched group between ratio
Relatively KD group infant acute stage plasma IL-6 level is apparently higher than heating matched group (p < 0.05) and Normal group (p < 0.05), poor
Different statistically significant；Association of Plasma IL-6 Level no significant difference (p > 0.05) between heating matched group and Normal group.
The subacute Association of Plasma IL-6 Level of KD group infant and the no significant difference (p > 0.05) between matched group and Normal group that generates heat.
KD-CAL group acute stage plasma IL-6 level apparently higher than KD-nCAL group, difference statistically significant (p < 0.05).KD-CAL group
Subacute stage Association of Plasma IL-6 Level is higher than KD-nCAL group, but no significant difference (p > 0.05), be shown in the following table lattice and
Figure 14, Figure 15.
KD each phase and two matched group Association of Plasma IL-6 Levels (x ± s)
| KD acute stage | KD subacute stage | Heating matched group | Normal group | |
| IL-6 | 184.01±38.09a | 85.75±18.26b | 71.30±24.78c | 65.86±26.94 |
Note: a.KD acute stage compares with Normal group, heating matched group, p < 0.05；B.KD acute stage and subacute stage
Relatively p < 0.05；C. not statistically significant, p are compared between two matched groups > 0.05.
In KD, CAL group compares (x ± s) with nCAL group Association of Plasma IL-6 Level
Note: A represents CAL group, and B represents that nCAL group, a. acute stage KD-CAL group compare with KD-nCAL, p < 0.05；B. sub-anxious
Property phase KD-CAL group and KD-nCAL comparing difference not statistically significant, p > 0.05.
4. hsa-miR-223-3p Yu IL-6 correlation analysis
KD acute stage plasma uses between hsa-miR-223-3p expression and Association of Plasma IL-6 Level Spearman ' s order
Correlation analysis, result display hsa-miR-223-3p expression is negative correlation with IL-6 level, r=-0.563, p=0.089.
Embodiment two is by having carried out hsa-miR-223-3p and IL-6 in KD acute stage and subacute stage blood plasma point
Analysis, found that the expression of hsa-miR-223-3p is significantly raised in acute stage KD infant blood plasma, difference has statistics to anticipate
Justice；No significant difference between the expression of hsa-miR-223-3p and two matched groups in subacute stage KD infant blood plasma.KD-CAL
Group acute stage plasma hsa-miR-223-3p expression is significantly lower than KD-nCAL group, and difference is statistically significant；KD-CAL group is sub-
Acute stage plasma hsa-miR-223-3p expression is less than KD-nCAL group, but no significant difference.Prompting hsa-miR-
223-3p is likely to be of protective effect in KD vascular inflammation, and acute stage low-level hsa-miR-223-3p is probably crown dynamic
The high risk factor of arteries and veins infringement.
IL-6 level is significantly raised in acute stage KD infant blood plasma, and difference is statistically significant, with document[31-32]Report
Unanimously；No significant difference between IL-6 level and two matched groups in subacute stage KD infant blood plasma.KD-CAL group acute stage plasma
IL-6 level is apparently higher than KD-nCAL group, and difference is statistically significant.KD-CAL group subacute stage Association of Plasma IL-6 Level is higher than
KD-nCAL group, but no significant difference.Too much expressing of prompting IL-6 may take part in KD infant vascular inflammation damage machine
System.
Correlation analysis finds that in KD infant acute stage plasma, hsa-miR-223-3p expression and IL-6 level exist negative
Relevant.Therefore, it is presumed that, hsa-miR-223-3p may be after acute stage, may be by suppression the transcribing of target gene IL6ST
Express, further the activation of suppression IL-6/STAT signal path, thus alleviate the vascular inflammation reaction of KD.
Claims (7)
1. the nucleic acid markers assisting diagnosis mucocutaneous lymphnode syndrome, it is characterised in that described nucleic acid markers is hsa-miR-223-
3p, has the nucleotide sequence as shown in SEQ 6.
The nucleic acid markers of auxiliary diagnosis mucocutaneous lymphnode syndrome the most according to claim 1, it is characterised in that described nucleic acid markers
Hsa-miR-223-3p is the miRNA in blood plasma.
3. assisting a test kit for diagnosis mucocutaneous lymphnode syndrome, described test kit includes carrying out the hsa-miR-223-3p in blood plasma determining
The reagent of amount detection.
The test kit of auxiliary diagnosis mucocutaneous lymphnode syndrome the most according to claim 3, described test kit includes hsa-miR-
223-3p carries out the primer of realtime quantitative inspection.
The test kit of auxiliary diagnosis mucocutaneous lymphnode syndrome the most according to claim 4, described primer has the nucleotide shown in SEQ 4
Sequence.
6. there is the miRNA of nucleotide sequence described in SEQ 6 as the application of the nucleic acid markers of mucocutaneous lymphnode syndrome.
7. there is the miRNA of nucleotide sequence described in SEQ 6 in preparation alleviates the medicine of vascular inflammation reaction of mucocutaneous lymphnode syndrome
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Application publication date: 20160803 |