CN107937516B - A kind of circular rna marker and its application - Google Patents
A kind of circular rna marker and its application Download PDFInfo
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Abstract
The invention discloses a kind of circular rna marker and its application in the kit for diagnosing CTEPH disease is being prepared, which is hsa-circ-0046159.The invention also discloses a kind of kit, which contains the specific primer and probe for hsa-circ-0046159.The present invention can accurately and effectively diagnose CTEPH disease, improve the sensitivity and specificity of medical diagnosis on disease.
Description
Technical field
The invention belongs to biomedical inspection field, it is related to a kind of circular rna marker for diagnosing CTEPH and its spy
Specific primer further relates to application and relevant kit of the marker in diagnosis CTEPH.
Background technique
Chronic thromboembolic pulmonary hypertension (chronic thromboembolic pulmonary
Hypertension, CTEPH) be to have difficulty in breathing, out of strength and Activity Endurance lower a kind of disease mainly to show, mainly by
It in pulmonary embolism recurrent exerbation, cannot dissolve, and then lead to pulmonary artery remodeling, the raising of pulmonary vascular resistance progressive, lung
Arterial hypertension and right heart insufficiency.CTEPH is a kind of disease for seriously threatening human life, but by the trouble of Normalized Treatment
Its prognosis of person can be obviously improved, therefore in order to obtain better therapeutic effect, it should be accomplished the early diagnosis of disease, early be controlled
It treats.
There is an urgent need to search out good sensibility, high specificity, be easy to measure, stable by other variable biologies at present
The diagnostic biomarkers for CTEPH that factor influences.Whole blood, serum or blood plasma are most widely used clinical pathway samples
Source, search out provide early diagnosis information clinical biomarker object, can greatly assisted diagnosis and processing disease, for the disease
The effective foundation of offers such as clinical early diagnosis, disease parting, extent of disease severity, and then mentioned for precisely treatment and new drug development
For foundation.
Circular rna (circular RNA, circRNA) is in closed hoop, is a kind of without the end 5' cap and the end 3'
The special endogenous non-coding RNA for holding tail, is mainly made of exon transcription product, and minority derives from introne or introne
Segment is the new hot spot of current RNA research field.CircRNA is present in a variety of eucaryotes, at different groups of same organism
It is also widely present in knitting；Most circRNA have highly conserved sequence, only a small number of not guard in evolution；Most of positioning
In cytoplasm, minority is positioned in nucleus.Moreover, part circRNA has the function of miRNAsponge, it is mutual with miRNA
Effect, regulates and controls the expression of target gene；Most of circRNA can play regulating and controlling effect in transcription or post-transcriptional level, and minority can be
Transcriptional level plays a role.Some researchs of forefathers find that circRNA is in the hardening of artery congee, nerve problems, protein virus disease
It plays an important role in the diseases generating process such as disease, diabetes, tumour and cancer.Therefore, research tool is carried out to circRNA
There is important clinical meaning.Tissue specificity and the stability of circRNA is there is a possibility that circRNA becomes good biomarker
Object.
Summary of the invention
A kind of the technical problem to be solved in the present invention is to provide sensibility good, high specificity is easy to measure, stablize not by it
The biomarker for CTEPH diagnosis that his variable biological factor influences.
The invention solves another technical problem be to provide a kind of easy to operate, result and accurately diagnose CTEPH's
Kit.
To achieve the above object, the invention adopts the following technical scheme:
During studying CTEPH disease, it was found that a kind of circular rna marker for CTEPH diagnosis,
The marker is hsa-circ-0046159；Its in the genome be positioned as chr:chr17；start(hg19):
79523912；end(hg19):79575848；genomic length:51936,spliced seq length:3764；Its core
Nucleotide sequence is as shown in SEQ ID No.1.
The present invention also provides above-mentioned circular rna markers to prepare the purposes in the product for diagnosing CTEPH disease.
It in another embodiment of the present invention, is by sequence table SEQ the present invention also provides one group of oligonucleotides
The oligonucleotides of base sequence described in ID No.2 to sequence table SEQ ID No.4 forms；Wherein SEQ ID No.2 and SEQ ID
No.3 is respectively the forward primer and reverse primer of hsa-circ-0046159, and SEQ ID No.4 is hsa-circ-0046159
Probe.
In another embodiment of the present invention, the present invention also provides above-mentioned oligonucleotides to prepare for diagnosing
Purposes in the product of CTEPH disease.
In another embodiment of the present invention, the present invention also provides a kind of kits for diagnosing CTEPH disease, including
For measuring the reagent of hsa-circ-0046159 marker；
Wherein, the reagent includes the specific primer and probe for detecting hsa-circ-0046159, wherein hsa-
The nucleotide sequence of circ-0046159 forward primer is as shown in SEQ ID No.2, the nucleotide sequence of reverse primer such as SEQ
Shown in ID No.3；The nucleotide sequence of hsa-circ-0046159 probe is as shown in SEQ ID No.4.
The present invention has the following advantages compared with the prior art:
(1) circRNAs is a kind of new biomarkers in blood, is different from traditional biological marker, stable, minimally invasive
And it is quantitative accurate, organized specificity and good stability will greatly improve the sensibility and specificity of medical diagnosis on disease, such
The successful exploitation of microRNA biomarker facilitates the auxiliary diagnosis of chronic thromboembolic pulmonary hypertension, is other
The development of disease biomarkers is offered reference.
(2) by the expression of the circRNAs of detection blood sources come diagnosing human chronic thromboembolic pulmonary hypertension
The kit whether occurred is a kind of system, comprehensively diagnosis and monitoring reagent box, can be used for chronic thromboembolic pulmonary artery
The auxiliary diagnosis of high pressure patient facilitates the morbid state for reflecting chronic thromboembolic patients with pulmonary hypertension, for clinical doctor
It is raw quick and precisely to grasp conditions of patients, the therapeutic scheme of more personalized is taken to provide support in time；
(3) strict design and appraisement system are used, the present inventor's initial stage carries out circRNA using circRNA chip
Analysis, and the method enlarged sample of application QPCR is verified；The application of above method and strategy accelerates and ensure that in blood
The application of circRNAs biomarker and diagnostic kit is also the development providing method and plan of other diseases biomarker
Reference on slightly.
Detailed description of the invention
Fig. 1 is the expression that QPCR verifies peripheral blood hsa-circ-0046159；
Fig. 2 is ceRNA prediction result network；
Specific embodiment
Invention is further explained combined with specific embodiments below.
The design of 1 primer and probe of embodiment
According to the sequence of hsa-circ-0046159, design primer and probe sequence.Particular sequence is as follows:
The forward primer of hsa-circ-0046159: CTCCTGCTTCGGCTCTGT
The reverse primer of hsa-circ-0046159: GGATGTAGGCGTGGAAGG
The probe of hsa-circ-0046159: AAGCGCCTGAAGCAACCATTCGATGAGGAC
Above-mentioned primer is synthesized by " Jin Weizhi Biotechnology Co., Ltd "；Probe is limited by the rich brilliant allusion quotation biotechnology difficult to understand in Beijing
Company provides.
2 total RNA of embodiment is extracted
1. 0.75ml lysate RLS is added in every 0.25ml whole blood sample, with sample loading gun piping and druming fluid sample several times to help
Cell in lysate sample.0.75ml lysate RLS is at least added in every 5~10 × 106 cells.Lysate RLS and fluid sample
Final volume than always 3:1.
2. homogenised sample acutely to be shaken to mixing, 5 minutes are incubated under the conditions of 15-30 DEG C so that ribosome divides completely
Solution.
3. optional step: 12000rpm is centrifuged 10 minutes under conditions of 4 DEG C, and supernatant is carefully taken to be transferred to a new RNase
In the centrifuge tube of free.
4. every 1ml lysate RLS adds 0.2ml chloroform.Sample tube cover is covered tightly, acutely vibrate 15 seconds and incubates it at room temperature
It educates 3 minutes.
5. being centrifuged 10 minutes in 4 DEG C of 12000rpm, sample can be divided into three layers: lower layer's organic phase, middle layer and upper layer are colourless
Water phase, RNA is present in water phase.The capacity of aqueous layer is about the 60% of added lysate RLS volume, and water phase is transferred to
In new pipe, next step operation is carried out.
6. 1 times of 70% ethyl alcohol of volume is added (please first to check whether and dehydrated alcohol has been added！), it is mixed by inversion and (at this time may
It will appear precipitating).Obtained solution and possible precipitating is transferred in adsorption column RA (absorption column sleeve is in collecting pipe) together.
7.10000rpm is centrifuged 45 seconds, discards waste liquid, adsorption column is recovered collecting pipe again.
8. adding 500 μ l protein liquid removal RE, 12000rpm centrifugation 45 seconds, waste liquid is discarded.
9. 700 μ l rinsing liquid RW are added (please first to check whether and dehydrated alcohol has been added！), 12000rpm is centrifuged 60 seconds, is abandoned
Fall waste liquid.
10. 500 μ l rinsing liquid RW, 12000rpm is added centrifugation 60 seconds, waste liquid is discarded.
11. adsorption column RA is put back in sky collecting pipe, 12000rpm is centrifuged 2 minutes, as far as possible removing rinsing liquid, in order to avoid rinsing
Residual ethanol inhibits downstream reaction in liquid.
12. taking out adsorption column RA, it is put into a RNase free centrifuge tube, according to expected RNA yield in adsorbed film
Intermediate position adds 50-80 μ l RNase free water (heating effect is more preferable in 65-70 DEG C of water-bath in advance), is placed at room temperature for 2
Minute, 12000rpm is centrifuged 1 minute.If necessary to more RNA, obtained solution can be rejoined in centrifugal adsorbing column, from
The heart 1 minute, or in addition add 30 μ l RNase free water again, it is centrifuged 1 minute, merges eluent twice.
3 sample RNA fluorescent marker of embodiment
1. reverse transcription synthesizes First strand cDNA
With Total RNA starting, the T7Random Primer containing T7 promoter sequence is primer, uses First
Strand Enzyme Mix synthesizes First strand cDNA.Reaction process is specific as follows:
1.1 take Total RNA 200-500ng, are added in the centrifuge tube of 0.2mL nuclease free.
1.2 the Agilent spike-in of respective volume is added according to following table.
1 spike-in volume of table corresponds to table
1.3 mix gently prepared reverse transcription Master Mix (being shown in Table 2, single reaction system dosage), it is of short duration from
The heart is placed on ice bath.5 μ L Master Mix are taken to be transferred in the 0.2mL centrifuge tube containing Total RNA sample respectively.
2 reverse transcription Master Mix component of table
| Ingredient names | Volume |
| First Strand Buffer Mix | 4μL |
| First Strand Enzyme Mix | 1μL |
1.4 mix gently solution, react 2 hours for 42 DEG C after brief centrifugation.Of short duration centrifugation and ice bath after reaction.Immediately
Carry out subsequent process steps.
Wherein, T7 Random Primer is synthesized by Sheng Gong bioengineering limited liability company.
The sequence of T7 Random Primer:
(V can be AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGCTTTTTTTTTTTTTT TTVN
G,A,C)
The Ambion WT Expression that First Strand Enzyme Mix is produced from Ambion producer
Kit, 30rxn kit.
2. synthesizing Second strand DNA:
Second Strand is converted by the RNA chain in DNA-RNA heterozygote with Second Strand Enzyme Mix
CDNA, synthetic dsdna.Reaction process is specific as follows:
2.1 prepare Second Strand Master Mix (being shown in Table 3, single reaction system dosage), are mixed gently,
Ice bath after of short duration centrifugation.The Second Strand Master of 20 μ L is added in the resulting each sample cell of above-mentioned steps 1.4
Mix。
3 Second Strand Master Mix component table of table
Wherein, the Ambion WT that Second Strand Enzyme Mix is produced from Ambion producer
Expression Kit, 30rxn kit.
2.2 soft mixings, 16 DEG C are reacted 1 hour, 65 DEG C of 10min.
Reaction tube is placed in continues synthetic reaction on ice after reaction by 2.3, or is frozen in -20 DEG C rapidly.
3. synthesis cRNA is transcribed in vitro
Using Second Strand cDNA as template, cRNA is synthesized using T7 Enzyme Mix.The detailed process of reaction is such as
Under:
Master Mix (being shown in Table 4, single reaction system dosage) is transcribed in vitro firstly, preparing, mixes gently, of short duration centrifugation
Solution is collected in tube bottom, 30 μ L Master Mix are added into the resulting each sample cell of above-mentioned steps 2.3.
Master Mix component table is transcribed in vitro in table 4
| Ingredient names | Volume |
| Nuclease-free Water | 4μL |
| T7Buffer Mix | 20μL |
| T7Enzyme Mix | 6μL |
It is soft to mix, 40 DEG C reaction 8-14 hours.After the reaction was completed, column purification cRNA is crossed.
Wherein, Ambion WT Expression Kit, 30rxn that T7Enzyme Mix is produced from Ambion producer
Kit.
4.cRNA purifying
Column purification cRNA is purified using RNA, except reagents such as salt, enzymes in dereaction, and cRNA is quantified.
5. reverse transcription cRNA:
Using cRNA as template, Random Primer is primer, and II enzyme of CbcScript carries out reverse transcription.Purification and recovery reversion
It records obtained cDNA and quantifies.Specific reaction process is as follows:
5.1 take 5 μ g of cRNA purified product, and adjustment volume to 7.5 μ L is added in 0.2mL nuclease free centrifuge tube, and is added
Enter 4 μ L Random Primer, mix, 65 DEG C 5 minutes, ice bath 5 minutes.
5.2 prepare cRNA reverse transcription Master Mix (being shown in Table 5, single reaction system dosage), soft to mix, of short duration centrifugation
Solution is collected in tube bottom.8.5 μ L Master Mix are added into the resulting each sample cell of above-mentioned 5.1 step.
5 cRNA reverse transcription Master Mix component table of table
| Ingredient names | Volume |
| 4×CbcScriptⅡBuffer | 5μL |
| 0.1M DTT | 2μL |
| CbcScriptⅡ | 1.5μL |
Wherein, the Ambion WT that Random Primer, CbcScript II's enzyme are produced both from Ambion producer
Expression Kit, 30rxn kit.
5.3 soft mixings are reacted 10 minutes for 25 DEG C after of short duration centrifugation, and 37 DEG C are reacted 1.5 hours.
5.4 are added 5 μ L of Terminate Solution after reaction, mix.
5.5 65 DEG C are reacted 10 minutes, are stored at room temperature 5 minutes.
5.6 are added 1 μ L of Neutralize Solution, mix, and prepare purifying.
6. label
Using the cDNA product of reverse transcription as template, Random Primer is primer, with Klenow Fragment enzymatic synthesis
CDNA complementary strand simultaneously mixes the dNTP (Cy3-dCTP) for having fluorophor, purifies simultaneously quantitative mark product.With fluorophor
DNA can be used to chip hybridization.
4 QPCR of embodiment verifies differential expression circRNA
1) Agilent circRNA chip data includes: disease group: 160039E&H-1,160039E-2,160039E&H-
3,160039G-1 and 160039E&H-4；Control group: 160039F-1,160039F-2,160039F-3,160039F-4 and
160039G-2。
2) pretreatment and variance analysis of chip: to the tiff lattice after obtained circRNA chip of expression spectrum hybrid screens
Formula image data is pre-processed using Feature Extraction software, then using GeneSpring GX software to core
Sheet data is normalized.Later, it is based on express spectra matrix file, sample is divided into two groups: experimental group vs control group.So
The t method of inspection is used afterwards, calculates differential expression circRNA.The screening threshold value of differential gene are as follows: | log2FC | > 1.0 and Pvalue
<0.05。
3) differential expression circRNA function be enriched with analysis: using DAVID online tool (Version6.8, https: //
David-d.ncifcrf.gov/), with default parameters (setting Classification Stringency=Medium) to difference
Allogene has carried out and the enrichment analysis of KEGG access, chooses hypergeometry and examines conspicuousness P.value < 0.05 as significant enrichment
Threshold value.
4) the miRNA forecast analysis of difference circRNA: circRNA can be targeted in conjunction with miRNA, indirect regulating mRNA
Translation.Using miRanda software, the target miRNA for the circular rna for selecting differential expression is predicted, selects SCORE > 140
As threshold value, the list of miRNA is obtained.
5) coexpression of difference circRNA and difference miRNA are analyzed: we to the expression spectrum matrix of difference circRNA with
The expression spectrum matrix of difference miRNA carries out coexpression analysis, uses Pearson correlation coefficients method.The model of Pearson correlation coefficients r
Enclosing is [- 1,1] because the regulation relationship between miRNA and circRNA be it is negatively correlated, select r < -0.5 as miRNA and
The coexpression relationship pair of circRNA.
6) ceRNA is analyzed: competitive endogenous RNA (ceRNA) hypothesis, it is known that microRNA can be by causing in conjunction with mRNA
Gene silencing, and ceRNA can be by competitively adjusting gene expression in conjunction with microRNA.CeRNA can pass through response
Element (microRNA response elements, MREs) influences gene caused by microRNA in conjunction with microRNA
Silencing.CeRNA can be circRNA or lncRNA.
It is generally believed that there are common miRNA regulation numbers to be more than or equal to 5 between circRNA and mRNA, then this
CircRNA be possible to be exactly this mRNA ceRNA.
7) network constructs: using cytoscape software to the cyberrelationship of acquisition to progress network building, gained net
Network figure is as shown in Figure 2.Hsa-circ-0046159 is obtained as ceRNA.
5 RT-PCR of embodiment verifying
Qualified sample is 10 samples after RNA is detected, and is divided into two groups, every group of 5 samples.Wherein, control group sample
Number: N1, N4/N4-1, N5, J-10；Experimental group sample number: N6,160750B-1,160039H-2, C-8, B-1；
Firstly, carrying out the reverse transcription reaction of circRNA, reaction system and parameter are as follows:
6 circRNA reverse transcription reaction system of table
Reaction condition:
37℃ 60min
85℃ 5s
Preparation is used for standard curve gradient dilution DNA profiling.
For gene hsa-circ-0046159 and house-keeping gene β-actin that needs measure, cDNA template is selected to carry out
PCR reaction, reaction system and response parameter are as follows:
7 pcr amplification reaction system of table
| Reagent | Volume |
| 2×Master Mix | 5μl |
| Sense primer, 10 μM | 0.5μl |
| Antisense primer, 10 μM | 0.5μl |
| cDNA | 2μl |
| Add water to total volume | 10μl |
It flicks tube bottom to mix solution, the of short duration centrifugation of 5000rpm, setting PCR reacts: 95 DEG C, 10min；40 PCR cycles
(95 DEG C, 10 seconds；60 DEG C, 60 seconds (collecting fluorescence)).
In 2% agarose gel electrophoresis, ethidium bromide staining detects PCR product by PCR product and 100bp DNA Ladder
It whether is single specificity amplified band.
PCR product is subjected to 10 times of gradient dilutions: setting PCR product concentration as 1, is diluted to 1 × 10 respectively-1, 1 × 10-2, 1 × 10-3, 1 × 10-4, 1 × 10-5, 1 × 10-6, 1 × 10-7The DNA of these gradient concentrations.
Fluorescent quantitative PCR, amplification reaction system and parameter are as follows:
8 pcr amplification reaction system of table
| Reagent | Volume |
| SYBR Premix EX Taq(2x) | 10μl |
| 10 μM of forward primer | 1μl |
| 10 μM of reverse primer | 1μl |
| CDNA (is diluted with water into consistent level) | 8μl |
Reaction condition
Melt Curve 60℃ to 95℃:Increment 0.5℃ for 10s Plate Read
Statistical analysis
All results are presented in the form of mean value ± SEM value, and table is made.The software that data statistic analysis uses is
SPSS22.0, mapping software are Graphpad prism 5 (Graphpad Software, San Diego, CA), P < 0.05 and P
< 0.01 is the screening criteria of significant difference and extremely significant sex differernce.Shown in the result is shown in Figure 1, verifying through RT-PCR is proved, and right
It is compared according to group, the expression quantity of experimental group hsa-circ-0046159 significantly raises.
6 hsa-circ-0046159 marker specificity experiments of embodiment
The present embodiment collects Beijing Chaoyang Hospital Attached to Capital Medical Univ.'s chronic thromboembolic pulmonary hypertension disease
Patient peripheral's blood sample 30, normal human sample 30 detects the expression and specificity of hsa-circ-0046159.
As a result, it has been found that the expression of hsa-circ-0046159 is apparently higher than Normal group, difference in Patient Sample A
With statistical significance (P < 0.01), hsa-circ-0046159 is as the specificity of chronic thromboembolic pulmonary hypertension
85%.These results suggest that the expression of hsa-circ-0046159 can be used for chronic thromboembolic pulmonary artery height in blood
The auxiliary diagnosis for pressing patient facilitates the morbid state for reflecting chronic thromboembolic patients with pulmonary hypertension.
Obviously, the above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be pair
The restriction of embodiments of the present invention.For those of ordinary skill in the art, may be used also on the basis of the above description
To make other variations or changes in different ways.Here all embodiments can not be exhaustive.It is all to belong to this hair
The obvious changes or variations that bright technical solution is extended out are still in the scope of protection of the present invention.
Claims (3)
1. one group of oligonucleotides, which is characterized in that the base as described in sequence table SEQ ID No.2 to sequence table SEQ ID No.4
The oligonucleotides of sequence forms；The forward direction that wherein SEQ ID No.2 and SEQ ID No.3 is respectively hsa-circ-0046159 is drawn
Object and reverse primer, SEQ ID No.4 are the probe of hsa-circ-0046159.
2. one group of oligonucleotides described in claim 1 is preparing the purposes in the product for diagnosing CTEPH disease.
3. a kind of kit for CTEPH diagnosis, which is characterized in that including for measuring hsa-circ-0046159 marker
Reagent；Wherein, the nucleotide sequence of the hsa-circ-0046159 marker is as shown in SEQ ID No.1；The reagent
Specific primer and probe including detecting hsa-circ-0046159, wherein the core of hsa-circ-0046159 forward primer
Nucleotide sequence is as shown in SEQ ID No.2, and the nucleotide sequence of reverse primer is as shown in SEQ ID No.3；hsa-circ-
The nucleotide sequence of 0046159 probe is as shown in SEQ ID No.4.
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Non-Patent Citations (4)
| Title |
|---|
| Microarray expression profile of circular RNAs in chronic thromboembolic pulmonary hypertension;Ran Miao等;《Medicine》;20170731;第96卷(第27期);e7354、第1-8页 * |
| Ran Miao等.Microarray expression profile of circular RNAs in chronic thromboembolic pulmonary hypertension.《Medicine》.2017,第96卷(第27期), * |
| 环状RNA研究进展;张进威等;《畜牧兽医学报》;20161231;第47卷(第11期);第2151-2158页 * |
| 登录号NM_017921.3;Riemer A等;《NCBI_GenBank》;20141225;序列信息 * |
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