CN109022568A - It is a kind of for the microRNA biomarker of diagnosis of rheumatoid arthritis and its application - Google Patents
It is a kind of for the microRNA biomarker of diagnosis of rheumatoid arthritis and its application Download PDFInfo
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Abstract
The invention discloses a kind of for the microRNA biomarker of diagnosis of rheumatoid arthritis and its application, belongs to technical field of biological.This 3 microRNA biomarkers provided by the present invention and its corresponding primer sets and probe can be used for preparing diagnostic kit, and it has excellent sensitivity and specificity when being applied to the diagnosis of rheumatoid arthritis of peripheral blood mononuclear cells sample.The AUC value that 3 kinds of microRNA of the present invention join together can achieve 0.745, and sensitivity and specificity are respectively 73.5% and 76.7%.This 3 kinds of microRNA can be used as the biomarker of the rheumatoid arthritis peripheral blood mononuclear cells sample diagnosis of people, be conducive to the development for pushing the early diagnosis of China's rheumatoid arthritis, predicted treatment, monitoring recurrence.
Description
Technical field
The present invention relates to a kind of for the microRNA biomarker of diagnosis of rheumatoid arthritis and its application, belongs to
Technical field of biological.
Background technique
Rheumatoid arthritis (rheumatoid arthritis, RA) is that one kind is showed with erosive arthritis to be main
Chronic generalized autoimmune disease.There are about 1% crowds to suffer from the disease at present in the whole world, wherein developed country's illness rate
For 0.5-1%, annual new cases are 5-50/10 ten thousand, and CONTINENTAL AREA OF CHINA illness rate is 0.2-0.36%, and illness sum is more than
5000000.It is clinical to show as symmetry, duration arthroncus and pain more, it is often accompanied by bone loss, bone erosion, articular cartilage
It loses, synovial membrane inflammation and joint capsule swelling.Patient then can gradually cause muscle not to be good for bone if do not controlled the state of an illness
Entirely, physical function decline and induction symbiosis disease, advanced stage disability rate is higher, seriously affects the quality of life of patient, and with heavy
The social economical burden of weight.Therefore the new biomarkers of screening RA, disclose its molecule pathogenic mechanism, establish early stage disease and examine
Disconnected model and preventive means are the particularly important strategies for controlling RA immune inflammation, preventing and slow down later period skeletal injury or deformity
Measure.
It is not yet clear about the nosogenesis of RA and mechanism at present, but generally believe that RA is one kind by environmental factor and heredity
The complex disease that factor regulates and controls jointly.Wherein inherent cause is the important factor in order of RA morbidity, the proportion in RA morbidity
About 50-60%.Previous genome-wide association study has found more than 100 tumor susceptibility gene relevant to RA, and wherein most
To it is immune related.But the molecular mechanism of related RA morbidity at present not yet studies maturation, it has been found that science of heredity susceptibility loci combine
Get up to explain RA heritability very little (15%), still there is a large amount of gene to remain to be discovered.Epigenetic modification is many diseases
Occur and the important factor in order of development, participation influence difference, motor fluctuations etc. between the evening hair of complex disease, gender.
Important component of the microRNA (miRNA) as epigenetic modification, plays a role to the multiple levels of genome, shows as
Remaining epigenetic factor such as regulating DNA methylation, and modified by recruiting specific albumen composition combination promoter
The expression of the gene of encoding histone.
Regulation researches show that in human body 1/3 gene by miRNA, it not only with the proliferation of normal cell, break up, wither
Die, stress, fat metabolism, the function controlling etc. of growth and development and heart it is closely related, while also with malignant tumour and painstaking effort
The occurrence and development of the complex diseases such as pipe disease are closely related.It has been reported and shows as a ring important in epigenetic modification,
MiRNA plays an important role in the pathogenesis of RA.It can participate in influencing a plurality of RA by the Targeted-control to gene
Relevant cytokine signalling pathways and immunization inflammatory reaction, or directly to CD4+The immunocytes such as T cell, synovium dimension
Cell or synovial tissue, joint bone etc. have an impact, and then cause the generation and development of RA disease.
Containing a series of and immune-related cell in peripheral blood mononuclear cells, such as T cell, B cell, monocyte
Deng, therefore the functional group researchs for RA most at present are with peripheral blood mononuclear cells for research target cell.In peripheral blood
The miRNA of unconventionality expression in RA is filtered out in mononuclearcell as biomarker, and develops corresponding auxiliary diagnostic
Strong early diagnosis, predicted treatment, the monitoring recurrence etc. for pushing China RA is had important clinical value by box.
Summary of the invention
In order to solve the above technical problems, providing a kind of peripheral blood mononuclear cells for diagnosis of rheumatoid arthritis
MicroRNA biomarker.There is provided one kind simultaneously can be used in early detection and has higher sensitivity and higher when detecting
The rheumatoid arthritis detection kit and its application of specificity.
The first purpose of the invention is to provide a kind of microRNA biological markers for diagnosis of rheumatoid arthritis
Object, the microRNA biomarker are in has-miR-99b-5p, has-miR-26b-5p or has-miR-7641
Any one or more than one combination.
In one embodiment of the invention, the nucleotide sequence of the hsa-miR-99b-5p such as SEQID NO:1 institute
Show, the nucleotide sequence of the has-miR-26b-5p is as shown in SEQ ID NO:2, the nucleotides sequence of the has-miR-7641
Column are as shown in SEQ ID NO:3.
In one embodiment of the invention, described has-miR-99b-5p, has-miR-26b-5p and has-
MiR-7641 derives from peripheral blood mononuclear cells.
A second object of the present invention is to provide a kind of microRNA primer sets for diagnosis of rheumatoid arthritis and
The combination of probe, including has-miR-99b-5p primer, probe, has-miR-26b-5p primer, probe or has-miR-7641
Any one or more than one combination in primer, probe.
Third object of the present invention is to provide a kind of kits for diagnosis of rheumatoid arthritis, comprising to periphery
In blood mononuclear cell in has-miR-99b-5p, has-miR-26b-5p or has-miR-7641 any one or it is a kind of with
On the combination of microRNA primer sets and microRNA probe that is detected of combined expression quantity.
In one embodiment of the invention, the microRNA primer sets include that the reverse transcription of microRNA is drawn
Primer after primer and PCR before object, PCR.
In one embodiment of the invention, the reverse transcription primer sequence of the hsa-miR-99b-5p such as SEQ ID
Shown in NO:4, primer sequence is as shown in SEQ ID NO:7 before PCR, and primer sequence is as shown in SEQ ID NO:10 after PCR；It is described
The reverse transcription primer sequence of has-miR-26b-5p is as shown in SEQ ID NO:5, primer sequence such as SEQ ID NO:8 institute before PCR
Show, primer sequence is as shown in SEQ ID NO:10 after PCR；The reverse transcription primer sequence of the has-miR-7641 such as SEQ ID
Shown in NO:6, primer sequence is as shown in SEQ ID NO:9 before PCR, and primer sequence is as shown in SEQ ID NO:10 after PCR.
It in one embodiment of the invention, further include the general components of qPCR augmentation detection, the qPCR amplification inspection
The general components of survey include reverse transcriptase, buffer, dNTPs, MgCl2、dd H2O, fluorescent dye, Taq enzyme, standard items and control
Object.
Fourth object of the present invention is to provide a kind of chip for diagnosis of rheumatoid arthritis, comprising for pair
Any one or more than one combination in has-miR-99b-5p, has-miR-26b-5p or has-miR-7641 is examined
The microRNA probe of survey.
In one embodiment of the invention, the nucleotide sequence of the microRNA probe and described
The overall length maturation microRNA complete complementary of the test object of microRNA probe.
Fifth object of the present invention is to provide the microRNA biomarkers to close in preparation detection rheumatoid
Save the application in scorching product.
MicroRNA biomarker of the invention in diagnosis of rheumatoid arthritis application include:
Measurement from subject peripheral blood mononuclear cells biomarker expression to obtain measured value,
The biomarker in has-miR-99b-5p, has-miR-26b-5p and has-miR-7641 any one or
More than one combination；By the measured value compared with reference value, if has-miR-99b-5p measured value is higher than reference value,
Has-miR-26b-5p and has-miR-7641 measured value is lower than reference value, it is determined that subject suffers from rheumatoid arthritis.
In one embodiment of the invention, the application specifically includes:
Extract the biomarker in the peripheral blood mononuclear cells of subject；
Primer sets corresponding with the biomarker and probe are provided；
And the measured value is measured by PCR detection method；Wherein, the reverse transcription of the hsa-miR-99b-5p is drawn
Object sequence is as shown in SEQ ID NO:4, and primer sequence is as shown in SEQ ID NO:7 before PCR, primer sequence such as SEQ ID after PCR
Shown in NO:10；Primer sequence is such as shown in SEQ ID NO:5, before PCR for the reverse transcription primer sequence of the has-miR-26b-5p
Shown in SEQ ID NO:8, primer sequence is as shown in SEQ ID NO:10 after PCR；The reverse transcription primer of the has-miR-7641
Sequence is as shown in SEQ ID NO:6, and primer sequence is as shown in SEQ ID NO:9 before PCR, primer sequence such as SEQ ID after PCR
Shown in NO:10.
In one embodiment of the invention, the application specifically includes:
Extract the biomarker in the peripheral blood mononuclear cells of subject；
Detection chip is provided, the detection chip is loaded with complete with the overall length maturation miRNA of the biomarker
The miRNA probe of full complementary series；
And the detected value is measured with the detection chip.
The beneficial effects of the present invention are: this 3 microRNA biomarkers provided by the present invention and its corresponding primer
Group and probe can be used for preparing diagnostic kit, and it is in the rheumatoid joint for being applied to peripheral blood mononuclear cells sample
When inflammation diagnosis, with excellent sensitivity and specificity.The AUC value that 3 kinds of microRNA of the present invention join together can achieve
0.745, sensitivity and specificity are respectively 73.5% and 76.7%.The rheumatoid that this 3 kinds of microRNA can be used as people closes
The biomarker for saving scorching peripheral blood mononuclear cells sample diagnosis, is conducive to that the early stage of China's rheumatoid arthritis is pushed to examine
Disconnected, predicted treatment, monitoring recurrence development.
Detailed description of the invention
Fig. 1 is microRNA (p < 0.05, difference are not less than 2 times) analysis of the significant difference expression generated by clustering
Result schematic diagram；
Fig. 2 is the ROC curve figure that microRNA of the present invention is used to distinguish patient with rheumatoid arthritis and normal control；Its
Middle A~C distinguishes 3 microRNA and is used alone, and D is that 3 microRNA are used in combination；
Fig. 3 is 3 miRNA of the present invention in 35 rheumatoid arthritis and 35 healthy human peripheral blood mononuclearcell samples
Expression in this changes schematic diagram (P < 0.05)；
Fig. 4 is the miRNA expression quantity after miR-99b-5p overexpressing cell system constructs successfully；
Fig. 5 is influence of the miR-99b-5p overexpression to the cell proliferation of Jurkat cell；
Fig. 6 is influence of the miR-99b-5p overexpression to the Apoptosis of Jurkat cell；
Fig. 7 is influence of the miR-99b-5p overexpression to the cell cycle of Jurkat cell；
Fig. 8 is influence of the miR-99b-5p overexpression to the cell activation of Jurkat cell；
Fig. 9 is influence of the miR-99b-5p overexpression to proinflammatory cytokine in T cell.
Specific embodiment
The present invention will be further explained below with reference to the attached drawings and specific examples, so that those skilled in the art can be with
It more fully understands the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
In embodiment do not specify actual conditions experiment, usually according to normal condition as manufacturers instruction, experiment guide or
The condition of textbook content.
Before being described to example, it is necessary to some remarks explanations are provided: can be made using the reagent of different manufacturers, different batches
At the difference of experimental result, belong to normal phenomenon.
When carrying out small scale experiments, to guarantee the repeatability between parallel laboratory test, it is proposed that after configuration reagent, mix well simultaneously
Packing, to guarantee the homogeneity of each experiment reagent.
Embodiment 1: the cDNA microarray of differential expression microRNA (miRNA) in peripheral blood mononuclear cells
1, after obtaining patient's informed consent, 25 is collected from attached First People's Hospital of University Of Suzhou and is diagnosed as rheumatoid
The peripheral blood sample of property arthritic and 18 Healthy Peoples as normal control peripheral blood sample in sodium citrate anticoagulant tube.
2, patient with rheumatoid arthritis and PBMC of healthy people, tool are obtained by density-gradient centrifugation method
Steps are as follows for body:
(1) human peripheral blood sample is centrifuged, and makes its layering, 2000rpm × 2 minute.
(2) in leucosep separating pipe be added 15ml separating liquid, be centrifuged 5100g, 1 minute.
(3) supernatant of the peripheral blood sample after layering is focused on into 15ml centrifuge tube, is centrifuged 2500g, 15min.Then
Packing is stored in -80 DEG C of refrigerators to 2ml centrifuge tube.
(4) the supernatant amount being extracted is supplemented with the PBS of equivalent, then mixed liquor is concentrated in the centrifuge tube of 50ml.Then
The PBS of equivalent mixed liquor is added.
(5) mixed liquor is moved in the leucosep pipe in (2), be centrifuged 5300g, 20 minutes.
(6) supernatant abandoned in separating pipe is inhaled.Middle layer leucocyte is extracted to new 50ml centrifuge tube, PBS is added to whole body
Product be 15ml, be centrifuged 400g, 10 minutes.
(7) supernatant abandoned in centrifuge tube is inhaled.4ml PBS is added to mix, draws 10ul and carries out cell count, remaining cell liquid
Average mark is filled in 2ml cryovial, be centrifuged 2000rpm, 2 minutes.
(8) after draining centrifuge tube, wherein 3 are taken, is separately added into 1mlTRIzol to prevent RNA from degrading.Piping and druming is protected after mixing
It is stored in -80 DEG C of refrigerators.
3, single from the peripheral blood of patient with rheumatoid arthritis and normal healthy controls added with TRIzol in discovery experiment
In nucleus, mirVana is usedTMMiRNA Isolation Kit kit (ambion) extracts Total RNA, passes through full base
Because of a group expression of chip of expression spectrum detection miRNA.The chip (Affymetrix miRNA 4.0) covers Sanger
All miRNA maturation bodies of miRBase 20.0 version 2,03 species, including 2578 human mature miRNA.Chip is real
It is normalized to test step, can refer to www.thermofisher.com.
4, data are extracted and are analyzed
The fluorescent scanning image of chip through AGCC software ( CommandSoftware digital signal) is converted by picture signal, obtains the fluorescence signal intensity of each probe.Then
With Affimetrix Expression Console software Robust Multi-array Average (RMA) module into
Whether the pretreatment of row data, normalization, probe signals including initial data are significantly higher than the differentiation of background signal, by probe
Signal integration is probe groups signal.The expression value that index is converted to miRNA finally is carried out to pretreated data.
MiRNA Differential expression analysis is carried out to pretreated miRNA expression modal data.Analysis, which specifically includes that, to be passed through
Fold-change value (FC) method compares the miRNA differential expression of case group and control group；Using t checking computation case group with
Difference p value between control group.Screening conditions are that differential expression is not less than 2 times (FC>=2 or≤0.5) or p<0.05.Then use
The miRNA for the differential expression that Cluster3.0 obtains screening carries out clustering.
5, thin using peripheral blood single core of the miRNA chip to 25 patient with rheumatoid arthritis and 18 normal controls
Born of the same parents carry out Differential expression analysis, filter out the miRNA of 18 differential expressions altogether, are hsa-miR-101-3p, hsa-miR- respectively
1184、hsa-miR-1246、hsa-miR-142-3p、hsa-miR-142-5p、hsa-miR-195-5p、hsa-miR-26b-
5p、hsa-miR-29b-3p、hsa-miR-29c-3p、hsa-miR-3201、hsa-miR-3607-5p、hsa-miR-3613-
3p、hsa-miR-3651、hsa-miR-4448、hsa-miR-4668-5p、hsa-miR-7641、hsa-miR-8084、hsa-
miR-99b-5p.Compared with normal control, in addition to has-miR-99b-5p up-regulation, remaining miRNA is outside rheumatoid arthritis
Expression is lowered in all blood mononuclear cells (referring to Fig. 1).
Embodiment 2: the qRT-PCR experiment of miRNA in peripheral blood mononuclear cells
1, according to miRNA chip results, picked from the miRNA of 18 differential expressions by the following conditions 9 miRNA into
The verifying of row qRT-PCR.Actual conditions are as follows: (1) miRNA be not reported before this in RA crowd carry out RT-PCR test
Card；(2) miRNA possesses higher FC value；(3) miRNA possesses higher expression in the chips.Selected hsa-
miR-99b-5p、hsa-miR-26b-5p、hsa-miR-7641、hsa-miR-142-5p、hsa-miR-29c-3p、hsa-miR-
The sequence of 3607-5p, hsa-miR-3651, hsa-miR-4448, hsa-miR-1184 are shown in Table 1.Rheumatoid arthritis is suffered from
The peripheral blood mononuclear cells of person and normal control carries out the qRT-PCR detection of miRNA, implements stringent matter in entire research
Control, each sample continuously detect at least three times.
1 primer sequence table of table
2, after obtaining patient's informed consent, 35 is collected from attached First People's Hospital of University Of Suzhou and is diagnosed as rheumatoid
The peripheral blood sample of property arthritic and 35 Healthy Peoples as normal control peripheral blood sample in sodium citrate anticoagulant tube.
3, it is obtained outside patient with rheumatoid arthritis and normal person using density-gradient centrifugation method in the same manner as in Example 1
All blood mononuclear cells.
4, the reverse transcription to extraction, miRNA added with TRIzol peripheral blood mononuclear cells progress sample total serum IgE and cDNA
Quantitative PCR.Routine experiment step has: remained in the extraction of sample rna, RNA the digestion of genomic DNA, reverse transcription reaction,
QPCR reaction.
5, data are analyzed: PCR amplification result is indicated with Ct value, i.e. when fluorescence signal reaches set threshold value in PCR reaction
Recurring number.The △ Ct value for calculating the miRNAs of differential expression, is standardized, with relative quantification as internal reference using RNU48Method calculates the differential expression of miRNA between case and control group.Comparison among groups use t-test comparing difference, and P < 0.05 is recognized
For with statistical difference.ROC curve analysis is carried out with 15.8 software of MedCalc, and calculates sensitivity and specificity.
6, data analysis result is shown, the difference table of hsa-miR-99b-5p, hsa-miR-26b-5p, hsa-miR-7641
Up to conspicuousness (Fig. 2, p < 0.05), and under the ROC curve of 3 miRNA, area (AUC) is as follows respectively: hsa-miR-99b-
5p, 0.632 (95% confidence interval, 0.503-0.748)；Hsa-miR-26b-5p, 0.660 (95% confidence interval, 0.532-
0.773)；Hsa-miR-7641,0.701 (95% confidence interval, 0.575-0.808) (Fig. 2A-Fig. 2 C).In optimal cutoff
Under value, the sensitivity and specificity of miRNA are as follows: hsa-miR-99b-5p, respectively 50.0% and 80.6%, hsa-miR-
26b-5p, respectively 55.9% and 96.8%, hsa-miR-7641, respectively 52.9% and 83.9%.This 3 microRNA connection
AUC value altogether can achieve 0.745, and (95% confidence interval, 0.621-0.846), sensitivity and specificity are respectively
73.5% and 76.7% (Fig. 2 D) is better than single miRNA.These results indicate that hsa-miR-99b-5p, hsa-miR-26b-
5p, hsa-miR-7641 join together to detect rheumatoid arthritis peripheral blood mononuclear cells, close to rheumatoid
The scorching diagnosis of section has very high sensitivity and specificity.
Embodiment 3: specific miRNA (miR-99b-5p) in Jurkat immunocyte to cell growth and inflammatory cell because
The effect of son
Above-described embodiment shows that hsa-miR-99b-5p, hsa-miR-26b-5p, hsa-miR-7641 close rheumatoid
It saves scorching peripheral blood mononuclear cells and detects diagnostic value with higher (highly sensitive and specificity), the present embodiment surrounds miR-
99b-5p carries out its association study between rheumatoid arthritis.
1. constructing miR-99b-5p in Jurkat cell using pCDH-CMV-MCS-EF1-copGFP carrier to be overexpressed carefully
Born of the same parents system
The building of 1.1 miR-99b-5p slow virus carriers
According to the complete sequence of hsa-miR-99b-5p and its primers of each 1000bp of upstream and downstream, target is prepared
The PCR product of sequence.Obtained PCR product is subjected to electrophoresis verifying.After verifying is correct, PCR product is purified
(Qiaquick PCR purification kit), digestion (EcoRI, XbaI), repurity (Qiaquick gel reclaims kit), and use
T4DNA ligase is even reacted with the enzyme of Lentiviral pCDH.After 16 DEG C of connections overnight, the slow virus of recombination is expressed and is carried
Body is converted, and picking monoclonal colonies expand culture, while carrying out sequence verification to monoclonal colonies.
The building of 1.2 miR-99b-5p slow virus cell lines
The plasmid extraction of miR-99b-5p Lentiviral is carried out to the bacterium solution for expanding culture after verifying.Then with slow
Viral related packaging plasmid (psPAX/pMD2G, SBI) carries out the packaging of slow virus.Virus is carried out to the slow virus that packaging is completed
Concentration, and titer determination is carried out to the slow virus after concentration using qPCR titre detection kit (abm).Followed by preparing
Slow virus the slow virus overexpressing cell system of miR-99b-5p is constructed in Jurkat cell, steps are as follows:
(1) the good Jurkat cell of growth conditions is taken, is inoculated in 96 orifice plates, 1 × 105Cell/ware, volume of culture
100ul/ ware, and set 3 multiple holes.Culture dish is placed in overnight incubation in 37 DEG C of cell incubators.
Jurkat cell quantity is long to about 30% in culture dish before (2) second days virus infections.It is diluted with 1ml culture medium
Culture medium of the 100ul containing polybrene is added in the polybrene of 1ul 7.5mg/ul, every hole.With the ladder of MOI=20,200,2000
Virus infection Jurkat cell is added dropwise in degree.It is subsequently placed in incubator and cultivates.
(3) after cultivating 48h, cell growth condition is observed with inverted fluorescence microscope.By the Jurkat after slow-virus infection
Cell, which is transferred to 12 orifice plates, to be continued to expand culture.
(4) miR-99b-5p negative control bacterial strain (NC) is prepared according to the method described above: raw with pCDH zero load slow-virus infection
The good Jurkat cell of long status takes and carries out cell infection with the consistent virus concentration of miR-99b-5p overexpressing cell, obtains
To negative control bacterial strain continue expand culture.
The miR-99b-5p after expanding culture is collected by centrifugation after 4 days and is overexpressed (OE) group and negative control NC group cell, if
Vertical Jurkat healthy cell is control group, is imitated by the transfection that stream type cell analyzer detects miR-99b-5pOE group and NC group
Rate.
The identification of 1.3 miR-99b-5p overexpressing cell systems
The miR-99b-5p that growth conditions are good and positive rate is high is taken to be overexpressed OE group cell, negative control NC group cell,
It is inoculated in 6 orifice plates, every hole cell concentration is about 5 × 105It is a.Culture is collected by centrifugation after 2 days, and it is mixed that 1ml Trizol piping and druming is added
It is even, the fluorescent quantitative PCR experiment of the subsequent extraction for carrying out total serum IgE, cDNA reverse transcription and miR-99b-5p.
1.3.1 the extraction of cell total rna
(1) it takes out and the cell that 1ml Trizol piping and druming mixes is added early period: miR-99b-5p OE group, NC group and normal
The cell of Jurkat group.
(2) chloroform (200ul) of 1/5 volume is added into above-mentioned lysate.15s is acutely shaken after covering tightly centrifuge tube lid,
It is stored at room temperature 2min.
(3) centrifugation 12000rpm, 4 DEG C, 15min.Be divided into 3 layers after centrifugation: the colourless water sample layer in upper layer (contains RNA), middle layer,
Lower layer's red organic phenol chloroform layer.Middle layer and organic phase are albumen and DNA.
(4) it carefully draws in upper strata aqueous phase 500ul to a new centrifuge tube, isometric isopropanol (500ul) is added,
Avoiding being drawn onto middle layer leads to genome pollution.10min is placed at room temperature for after being mixed by inversion.
(5) centrifugation 12000rpm, 4 DEG C, 10min.Gelatinous precipitate is formed in pipe side or tube bottom after centrifugation.
(6) it carefully discards supernatant, 75% ethyl alcohol that 1ml DEPC water configures is added.Abundant sluicing pipe lid and tube wall, and it is light
Bomb tube bottom allows precipitating to suspend.
(7) centrifugation 12000rpm, 4 DEG C, 3min.It carefully discards supernatant, prevents RNA precipitate from losing.
(8) it is placed at room temperature for 2min, is dried.30ulDEPC water is added, takes until completely dissolved on a small quantity, uses uv-spectrophotometric
Instrument detects light absorption value and concentration of the RNA sample at wavelength 260nm and 280nm.When OD260/OD280 ratio 1.8-2.0 it
Between then to show that RNA is extracted qualified.May there are albumen or other organic pollutions if ratio is less than 1.8, be said if ratio is greater than 2.2
Bright RNA may have been hydrolyzed.
1.3.2 the synthesis of RNA tailings reactions and the first chain cDNA
Experiment uses TransScript miRNA First-Strand cDNA Synthesis SuperMix reverse transcription
Kit, experimental procedure are as follows:
(1) cDNA reverse transcription system (20ul) is configured: x ul RNA；1ul TransScript miRNA RT Enzyme
Mix；10ul 2xTS miRNA Reaction Mix；9-x ul RNase-Free Water.
RNA is 2ug in system, obtains corresponding RNA volume and corresponding water volume according to RNA concentration calculation.
(2) it mixes gently, the mixture of template ribonucleic acid and primer is subjected to 37 DEG C of incubations 1h, 85 DEG C of heating 5s and inactivates RT
Enzyme Mix。
1.3.3 Real-Time qPCR
It configures qPCR reaction system (20ul): 1ul cDNA；0.5ul Forward Primer(10uM)；0.5ul
Universal miRNA qPCR Primer(10uM)；10ul GoTaq qPCR Master Mix, 2 ×；8ul ddH2O。
CDNA obtained by reverse transcription is used after diluting 5 times.Every group experimental setup 3 parallel.
Hsa-miR-99b-5p upstream primer is special primer, and downstream primer selects universal primer, selecting response U6 conduct
Internal reference Real-Time PCR reaction utilizes ABI QuantStudioTMThe amplification of 6Flex global function real-time fluorescence quantitative PCR system
Standardization program.The amplification curve and solubility curve of Real Time PCR are made after reaction, the expression value of gene is used
It indicates.As the result is shown in miR-99b-5p overexpressing cell the expression quantity of miR-99b-5p be negative control group 5.23 times (P <
0.01, Fig. 4).The miR-99b-5p recombined lentivirus vector of research and establishment can effectively infect Jurkat cell and significantly improve thin
The expression of miR-99b-5p intracellular.
2.miR-99b-5p is overexpressed the influence grown to Jurkat T cell
2.1 CCK-8 cell Proliferations are examined
The miR-99b-5p overexpressing cell and NC control cell that growth conditions are good and positive rate is high are taken, 96 holes are inoculated in
In plate (hole 100ul/), every hole cell concentration is about 5 × 104A, it is parallel that every kind of cell sets up 5 rows.Experiment sets gradually 0h/12h/
For 24 hours/time gradient group of 48h tetra-.Error caused by prevent culture medium from volatilizing is added PBS in the every hole in four side of experimental port and (does not make
For Indexs measure hole).Separately set up CCK-8 blank control group (containing only culture medium and CCK-8 without cell).
Cell takes out after cultivating 0h/12h/24h/48h in incubator, and every hole is added 10ul CCK-8 solution, when sample-adding
It to avoid generating bubble and influencing absorbance when detecting as far as possible.Culture plate is then put back to incubator to continue to be incubated for 1.5h, is incubated
With the absorbance at enzyme-linked immunosorbent assay instrument Detection wavelength 450nm after educating.
The bis- dye Apoptosis detections of 2.2 Annexin V/PI
Growth conditions are good and positive rate is high miR-99b-5p is taken to be overexpressed OE cell, NC control cell and normal
Jurkat cell, is inoculated in 6 orifice plates that (cell concentration is no less than 1 × 106), it is parallel that every kind of cell sets up 3 rows.After culture for 24 hours, from
Heart 1000rpm × 5min abandons supernatant, then is washed 2 times with PBS.Finally with 1 × Annexin of 1ml V binding buffer weight
Outstanding cell.The preparation of dead cell: it takes 100ul Jurkat cell and 100ul NC cell to mix and 200ul Jurkat and NC is made
Mixed liquor therefrom takes 50ul mixed liquor, then is placed in 5min in 99 DEG C of thermostat water baths together with another 100ul Jurkat cell, cold
But dead cell is obtained to room temperature.Cell apoptosis assay design is as follows:
The design of 2 cell apoptosis assay of table
General indemnity and GFP compensation is arranged in compensation group, and Jurkat and NC (containing GFP) mixed liquor group and its is arranged in GFP compensation
Anyway double group, for adjusting influence of the GFP fluorescence to Apoptosis.
It is parallel that every kind of cell of detection group sets up three rows, is added 5ul PE and 5ul 7-AAD by design respectively, it is mild mix after
It is protected from light, is incubated at room temperature 15min.1 × Annexin of 400ul V binding buffer is added after incubation, after soft mixing
Apoptosis assay is carried out with flow cytometer.
2.3 PI staining cell periodic inspections
The miR-99b-5p overexpressing cell and NC control cell that growth conditions are good and positive rate is high are taken, 12 holes are inoculated in
(cell concentration is about 1 × 10 in plate5), it is parallel that 3 rows are arranged in every kind of cell.Preculture for 24 hours after, be centrifuged 1000rpm × 5min, precipitating
Cell.Then careful inhale abandons supernatant, and the PBS that 1ml pre-cooling is added washs cell, and 1.5ml centrifuge tube is transferred to after resuspension.Again from
It is carefully inhaled after the heart and abandons supernatant, flicked the appropriate cell dispersion of tube bottom, prevent cell agglomerating.Inhaling every time can suitably remain when abandoning supernatant
50ul supernatant avoids siphoning away loss cell.
70% ethyl alcohol of 1ml pre-cooling is added in the cell precipitation after washed, soft piping and druming is put in 4 DEG C of refrigerators after mixing
It is fixed to stay overnight.Take out centrifugation 1000rpm × 5min afterwards for 24 hours.After supernatant is abandoned in careful suction, cell is washed with the PBS that 1ml is pre-chilled, then
It is inhaled after secondary centrifugation and abandons supernatant, it is agglomerating to prevent cell to flick tube wall.
It configures propidium iodide stain liquid: containing 500ul dye solution, 25ul propidium iodide stain in 535ul end system
Liquid (20 ×), 10ulRNase A (50 ×).Mixed system as 500ul is added in every solencyte sample, is slowly sufficiently resuspended
After cell, 37 DEG C are protected from light warm bath 30min.It is subsequently placed at and stores on ice, carry out flow cytometer detection.
2.4 CD69/CD25 cytoactive detections
The miR-99b-5p overexpressing cell and NC control cell that growth conditions are good and positive rate is high are taken, 12 holes are inoculated in
(cell concentration is about 1 × 10 in plate5), it is separately added into the phytohemagglutinin (PHA of 5ug/ml；Beyotime
Biotechnology it) or is not added.It is parallel that 3 rows are arranged in every kind of cell.After cultivating 48h, collects and wash cell.Then it is added
3uLAPC- people CD69 or people CD25 antibody (BioLegend, USA).After being protected from light incubation 30 minutes, flow cytomery is used
The positive cell rate of CD69 or CD25.
The analysis of 2.5 data
Experimental result is shown, compared with negative control cell, the cell-proliferation activity of miR-99b-5p overexpressing cell is aobvious
It writes and is higher than control group (P < 0.05, Fig. 5), and apoptosis rate is substantially less than negative control group (P < 0.001, Fig. 6).In miR-99b-5p
Cell is stranded in G2/M phase (Fig. 7) more under overexpression state, and miR-99b-5p is prompted to have stimulated cell differentiation and proliferation.CD69 and
CD25 is the Activation marker of T lymphocyte early period and mid-term respectively.After PHA is stimulated, miR-99b-5p overexpressing cell
Two kinds of antibody positive rates of group and negative control group significantly increase (P < 0.01, Fig. 8), and overexpression group positive rate is significantly higher than
Negative control group (P < 0.05, Fig. 8).To sum up, miR-99b-5p is overexpressed the cell increasing that can effectively facilitate Jurkat T cell
It grows, inhibits Apoptosis, promote cell activation.
3.miR-99b-5p being overexpressed the influence to inflammatory cytokine
The miR-99b-5p that growth conditions are good and positive rate is high is taken to be overexpressed OE group cell, negative control NC group cell,
It is inoculated in 6 orifice plates, every hole cell concentration is about 5 × 105It is a.Culture is collected by centrifugation after 2 days, extracts cell RNA using TRIzol.
It is then cDNA (Promega GoScript reverse transcription system kit) by RNA reverse transcription, experimental procedure is as follows:
(1) take a certain amount of template ribonucleic acid that primer, reaction system (10ul): x ul RNA is added；1ul Random
Primers；1ul Oligo(dT)15Primer；8-x ul Nuclease-Free Water.
RNA is 2ug in system, obtains corresponding RNA volume and corresponding water volume according to RNA concentration calculation.
(2) mixture of template ribonucleic acid and primer is subjected to 70 DEG C, 5min initial denaturation, takes out be placed on ice after the completion.
(3) RT-Mix (10ul) is prepared: 1.6ul Nuclease-Free Water；4ul GoScriptTM5×
Reaction Buffer；2ul MgCl2(25mM)；1ul PCR Nucleotide Mix；0.4ul Recombinant
Rnasin Ribonuclease Inhibitor；1ul GoScriptTM Reverse Transcriptase。
(4) be arranged reverse transcription program, annealing (25 DEG C, 5min), extend (42 DEG C, 60min), reverse transcriptase inactivation (70 DEG C,
15min).CDNA is obtained after the completion of program.
QPCR reaction is carried out using Promega GoTaq qPCR Master Mix to the cDNA of synthesis, reaction system is such as
Under (15ul): 4.5ul Nuclease-Free Water；1ul upstream primer (10uM)；1ul downstream primer (10uM)；7.5ul
GoTaq qPCR Master Mix, 2 ×；1ul cDNA.
Every group of experimental setup 3 parallel, select GAPDH as reference gene, PCR reaction detection IL-1 β, IL-2, IL-
4, the expression of the cell factors such as IL-6, IL-8, TNF-α, IFN-γ.Real-Time PCR reaction utilizes ABI
QuantStudioTMThe amplification standardization program of 6Flex global function real-time fluorescence quantitative PCR system.Real is made after reaction
The expression value of the amplification curve and solubility curve of Time PCR, gene is indicated with 2- △ △ CT.The results show that proinflammatory cytokine
IL-2, IL-6, TNF-α and the IFN γ significantly high expression (P < 0.05, Fig. 9) in miR-99b-5p overexpressing cell prompt
MiR-99b-5p participates in the immunization inflammatory reaction in modulating T cell.
Embodiment 4: human rheumatoid arthritis's diagnosis qPCR kit
Above-described embodiment shows that hsa-miR-99b-5p, hsa-miR-26b-5p, hsa-miR-7641 close rheumatoid
It saves scorching peripheral blood mononuclear cells and detects diagnostic value with higher (highly sensitive and specificity), therefore hsa- can be based on
MiR-99b-5p, hsa-miR-26b-5p, hsa-miR-7641 producer's diagnosis of rheumatoid arthritis kit.The reagent
Box includes hsa-miR-99b-5p primer, hsa-miR-26p-5p primer, hsa-miR-7641 primer and general probe.Respectively
Primer specifically includes before reverse transcription primer, quantitative PCR primer after primer and general quantitative PCR, and draws after general quantitative PCR
Object is combined convenient to use with general probe premix.It should also include normal needed for corresponding PCR reaction in certain kit
Enzyme and reagent are advised, such as reverse transcriptase, buffer, dNTPs, MgCl2、dd H2O, fluorescent dye, Taq enzyme, standard items and reference material
Deng.The design of primer and probe is conventional technical means in the art, may be designed as other sequences.The value of this kit is
Peripheral blood mononuclear cells sample is only needed, without tissue, peripheral blood mononuclear cells sample, so that it may be used for rheumatoid
The arthritic diagnosis of property.
Embodiment 5: human rheumatoid arthritis, which diagnoses, uses chip agent box
Similarly, the miRNA detection based on chip can also be used for the diagnosis of rheumatoid arthritis peripheral blood mononuclear cells.
By making the chip of a small amount of probe, the table of hsa-miR-99b-5p, hsa-miR-26b-5p, hsa-miR-7641 can be detected
It reaches, so that Cytokines in Peripheral Blood Mononuclear sample carries out the diagnosis of rheumatoid arthritis.
1, the overall length maturation miRNA of the miRNA probe sequence of all designs detection corresponding with them is complete in miRNA chip
It is complementary.The miRNA of detection includes hsa-miR-99b-5p, hsa-miR-26b-5p, hsa-miR-7641.
2, the extraction of the total serum IgE of peripheral blood mononuclear cells TRIzol reagent, tiny RNA is with Ambion's
MiRNAIsolation Kit is extracted.Tiny RNA is marked using T4DNA ligase labelling technique, i.e., by the tiny RNA of 1 μ g
It is mixed with the FlashTag Ligation Mix Biotin (Genisphere) of 4uL, in T4DNA ligase (New England
Biolabs it is marked under) acting on.Label reaction carries out 30 minutes at 25 DEG C.It is taken out after incubation, brief centrifugation is collected molten
Liquid is mixed well after terminate liquid is added, is placed on ice in tube bottom.
3, chip hybridization liquid is initially positioned on constant-temperature metal bath, and 99 DEG C incubate 5 minutes, then 45 DEG C of incubations take out after five minutes,
Chip is injected in 13200rpm centrifugation after five minutes.Chip balance is placed in hybrid heater (hybridization oven 640) again
Middle 60rpm, 48 DEG C of hybridized overnights, hybridization time are 16 hours.
4, it is cleaned after chip hybridization with washing solution, then cleaning dyeing is carried out to chip with AGCC software, to cleaning
The chip of completion is scanned.After 3000 scanner of GeneChip Scanner (Affymetrix) scanning, with AGCC software
( Command Software) digital letter is converted by picture signal
Number, obtain the fluorescence signal intensity of each probe.Then with Affimetrix Expression Console software
Robust Multi-array Average (RMA) module carries out the pretreatment of data, normalization, probe including initial data
Whether signal is significantly higher than the differentiation of background signal, probe signals is integrated into probe groups signal.It is returned and is divided according to Logistic
Analysis equation diagnoses the human peripheral blood single nucleus cell sample of offer.To 35 rheumatoid arthritis and 35 Healthy Peoples
Hsa-miR-99b-5p, hsa-miR-26b-5p, hsa-miR-7641 in peripheral blood mononuclear cells carry out chip detection, knot
Fruit shows the expression of the miRNAs in two groups of human peripheral blood single nucleus cells, and there were significant differences (P < 0.05), such as Fig. 3.
In conclusion by above-mentioned technical proposal, this 3 microRNA biomarkers provided by the present invention and its phase
Primer sets and probe are answered to can be used for preparing diagnostic kit, and it is in the rheumatoid for being applied to peripheral blood mononuclear cells sample
Property arthritis diagnosis when, with excellent sensitivity and specificity.Further, inventor is confirmed by research, this 3 kinds
The AUC value that microRNA joins together can achieve 0.745, and sensitivity and specificity are respectively 73.5% and 76.7%.This 3 kinds
MicroRNA can be used as the biomarker of human rheumatoid arthritis's peripheral blood mononuclear cells sample diagnosis, and combines and examine
Disconnected sensitivity and specificity is higher than the sensitivity and specificity of single microRNA diagnosis, is conducive to push China's rheumatoid
Arthritic early diagnosis, the development of predicted treatment, monitoring recurrence.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention
It encloses without being limited thereto.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in the present invention
Protection scope within.Protection scope of the present invention is subject to claims.
Sequence table
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Claims (10)
1. a kind of microRNA biomarker for diagnosis of rheumatoid arthritis, which is characterized in that described
MicroRNA biomarker be has-miR-99b-5p, has-miR-26b-5p or has-miR-7641 in any one or
More than one combination.
2. microRNA biomarker according to claim 1, which is characterized in that the core of the hsa-miR-99b-5p
For nucleotide sequence as shown in SEQID NO:1, the nucleotide sequence of the has-miR-26b-5p is described as shown in SEQ ID NO:2
The nucleotide sequence of has-miR-7641 is as shown in SEQ ID NO:3.
3. microRNA biomarker according to claim 1, which is characterized in that the has-miR-99b-5p,
Has-miR-26b-5p and has-miR-7641 derives from peripheral blood mononuclear cells.
4. a kind of kit for diagnosis of rheumatoid arthritis, which is characterized in that comprising in Cytokines in Peripheral Blood Mononuclear
Any one or more than one combined expression in has-miR-99b-5p, has-miR-26b-5p or has-miR-7641
Measure the combination of the microRNA primer sets and microRNA probe that are detected.
5. kit according to claim 4, which is characterized in that the microRNA primer sets include microRNA's
Primer after primer and PCR before reverse transcription primer, PCR.
6. kit according to claim 5, which is characterized in that the reverse transcription primer sequence of the hsa-miR-99b-5p
As shown in SEQ ID NO:4, primer sequence is as shown in SEQ ID NO:7 before PCR, primer sequence such as SEQ ID NO:10 after PCR
It is shown；The reverse transcription primer sequence of the has-miR-26b-5p is as shown in SEQ ID NO:5, primer sequence such as SEQ before PCR
Shown in ID NO:8, primer sequence is as shown in SEQ ID NO:10 after PCR；The reverse transcription primer sequence of the has-miR-7641
As shown in SEQ ID NO:6, primer sequence is as shown in SEQ ID NO:9 before PCR, primer sequence such as SEQ ID NO:10 after PCR
It is shown.
7. kit according to claim 5, which is characterized in that it further include the general components of qPCR augmentation detection, it is described
The general components of qPCR augmentation detection include reverse transcriptase, buffer, dNTPs, MgCl2、dd H2O, fluorescent dye, Taq enzyme, mark
Quasi- product and reference material.
8. a kind of chip for diagnosis of rheumatoid arthritis, which is characterized in that comprising for has-miR-99b-5p,
The microRNA that any one or more than one combination in has-miR-26b-5p or has-miR-7641 is detected is visited
Needle.
9. chip according to claim 8, which is characterized in that the nucleotide sequence of the microRNA probe with it is described
MicroRNA probe test object overall length maturation microRNA complete complementary.
10. the described in any item microRNA biomarkers of claims 1 to 3 are in the production of preparation detection rheumatoid arthritis
Application in product.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109486942A (en) * | 2018-12-26 | 2019-03-19 | 苏州大学 | A kind of biomarker and its application for diagnosis of rheumatoid arthritis |
| CN111378742A (en) * | 2020-04-16 | 2020-07-07 | 嘉兴程瑞医药科技有限公司 | MicroRNA biomarker and application thereof in preparation of autoimmune disease detection kit |
| CN111549113A (en) * | 2020-04-17 | 2020-08-18 | 中山大学 | Rheumatoid arthritis diagnostic marker and application thereof |
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2018
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| 朱晓炜: "类风湿性关节炎全基因组miRNA调控网络及功能机制研究", 《中国博士学位论文全文数据库》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486942A (en) * | 2018-12-26 | 2019-03-19 | 苏州大学 | A kind of biomarker and its application for diagnosis of rheumatoid arthritis |
| CN109486942B (en) * | 2018-12-26 | 2021-04-06 | 苏州大学 | Biomarker for rheumatoid arthritis diagnosis and application thereof |
| CN111378742A (en) * | 2020-04-16 | 2020-07-07 | 嘉兴程瑞医药科技有限公司 | MicroRNA biomarker and application thereof in preparation of autoimmune disease detection kit |
| CN111549113A (en) * | 2020-04-17 | 2020-08-18 | 中山大学 | Rheumatoid arthritis diagnostic marker and application thereof |
| CN111549113B (en) * | 2020-04-17 | 2022-10-14 | 中山大学 | Rheumatoid arthritis diagnostic marker and application thereof |
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