CN106191058A - Ring-type circRNA 011235 gene that a kind of leukemia is relevant and application thereof - Google Patents

Ring-type circRNA 011235 gene that a kind of leukemia is relevant and application thereof Download PDF

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CN106191058A
CN106191058A CN201610549845.5A CN201610549845A CN106191058A CN 106191058 A CN106191058 A CN 106191058A CN 201610549845 A CN201610549845 A CN 201610549845A CN 106191058 A CN106191058 A CN 106191058A
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聂新民
张军华
桂嵘
黄蓉
江静
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Third Xiangya Hospital of Central South University
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Abstract

The invention discloses relevant ring-type circRNA 011235 gene of a kind of leukemia and application thereof, the present invention first confirms that the existence of ring-type circRNA 011235 gene, by this expression conditions in detection whole body irradiation patient, find that its expression is significantly raised.The marrow stromal cell of the adenovirus vector of process LAN ring-type circRNA 011235 gene will be transfected compared with the comparison marrow stromal cell having transfected empty carrier, find that its intracellular TGF β expressing quantity substantially increases, cause myelofibrosis, it is unfavorable for the going back to the nest of hematopoietic cell, self renewal, breeds and break up, and then affect hematopoietic stem cell transplantation curative effect, thus ring-type circRNA 011235 gene and expression product thereof are as the label of prediction hematopoietic stem cell transplantation curative effect, as target gene and the medicine of preparation treatment leukemia medicament.

Description

Ring-type circRNA-011235 gene that a kind of leukemia is relevant and application thereof
Technical field
The invention belongs to oncomolecularbiology field, be specifically related to a kind of ring-type circRNA-relevant to leukemia 011235 gene and application thereof.
Background technology
Leukemia is commonly called as leukemia, and because its cause of disease is not yet clear and definite, patient's survival period is short, and mortality rate is high, the health to people Cause grave danger.According to statistics, there is ten thousand leukaemic more than 400 in China, the most also can increase 40,000 many cases newly.Hematopoietic stem cell moves Planting (HSCT) is the preferred option effecting a radical cure Malignant hematologic diseases at present, and whole body irradiation (Total body Irradiation, TBI) it is one of necessary supplementary means of transplanting of hematopoietic stem cell (HSCs).The purpose of TBI treatment: one is to exempt from Epidemic disease suppresses, and mainly makes bone marrow can be accepted by receptor.Two is that the malignant cell eliminated in body reaches to treat white blood Sick purpose.Three is to kill hematopoietic stem cell in bone marrow to make pulp cavity space occur, is beneficial to hematopoietic stem cell and implants.
TBI before bone marrow transplantation the damage of mesenchymal stem cells MSCs (BMSCs) may be affected HSCs implantation and Transplanting curative effect, even result in graft failure, whether TBI can affect it to the damage of BMSCs is supported HSCs hemopoietic and effect machine thereof Make unclear.Specifying TBI is conducive to clinicist rationally to select therapeutic scheme the impact of BMSCs, it is achieved efficient, low toxicity Clinical efficacy has the highest guiding value.
Summary of the invention
It is an object of the invention to provide a kind of ring-type circRNA-011235 gene, this gene and expression product conduct thereof Prediction HSCT transplants the label of curative effect, selects rational therapeutic scheme to provide new thinking for clinician.
Present invention discover that leukaemic after TBI in BMSCs ring-type circRNA-011235 gene upregulation express up to 10.38 times, predicted that by circRNABase the response element of this gene is miR143-3p, have research to have turned out at BMSCs Middle miR143-3p suppression TGF-β is expressed, and our experimental result showed that TBI process after in BMSCs TGF-β expression obvious Increasing, miR-143-3p lowers and expresses substantially simultaneously, and therefore we propose scientific hypothesis according to document and previous work: after TBI In BMSCs, the circRNA-011235 of up-regulated expression will adsorb miR-143-3p by " sponge effect ", thus it is right to reduce it The negative regulation of TGF-β, makes the abnormal rising of the TGF-β protein level in BMSCs, and this may cause myelofibrosis, be unfavorable for making The going back to the nest of hemocyte, self renewal, breed and break up, and then affecting HSCs and transplant curative effect.
A first aspect of the present invention, it is provided that a kind of ring-type circRNA-011235 gene, its cDNA sequence is SEQ ID Shown in NO:1.
A second aspect of the present invention, it is provided that ring-type circRNA-011235 gene preparation detect this gene primer, Purposes in test kit.
A third aspect of the present invention, it is provided that ring-type circRNA-011235 gene is in preparation treatment leukemia medicament Purposes.
A fourth aspect of the present invention, it is provided that for detecting the primer pair of ring-type circRNA-011235 gene, its sequence As described in SEQ ID NO:2, SEQ ID NO:3.
Within the scope of the present invention, each technical characteristic specifically described in the above-mentioned technical characteristic of the present invention and Examples below Can be combined with each other, thus constitute new technical scheme, as space is limited, repeat the most one by one.
The present invention confirms the objective reality of a new ring-type circRNA-011235 gene first, by detection whole body This expression conditions in irradiation patient, finds that its expression substantially reduces, this ring-type circRNA-011235 gene and Expression product can select rational therapeutic scheme to carry as the label of prediction hematopoietic stem cell transplantation curative effect for clinician Supply new thinking；Containing this ring-type circRNA-011235 gene and the test kit of primer pair thereof, may be used for Hematopoietic Stem thin Born of the same parents transplant Prognosis；Ring-type circRNA-011235 gene can also be as the target gene for the treatment of whole body irradiation medicine, in vain Significant role is played in disorders of blood treatment.
Accompanying drawing explanation
Circular rna chip results figure in Fig. 1 normal group marrow stromal cell；
Fig. 2 whole body irradiation process group marrow stromal cell circular rna chip results figure；
The cluster analysis figure of ring-type rna expression situation in Fig. 3 normal group and whole body irradiation process group marrow stromal cell；Figure Middle left side is the cluster analysis figure of ring-type rna expression situation in normal group marrow stromal cell, and right side is whole body irradiation process group The cluster analysis figure of ring-type rna expression situation in marrow stromal cell.
The real-time quantitative PCR amplification curve of Fig. 4 internal reference GAPDH gene；
Fig. 5 circRNA-011235 gene real-time quantitative PCR amplification curve；
In Fig. 6 normal group and process group, TGF-β protein level is abnormal rises figure.；Wherein 1 for having transfected process LAN In the marrow stromal cell of cricRNA-011235 gene adenovirus vector, TGF-β 1 is expressed, and 2 is the bone marrow base of transfection empty carrier In cell plastid, TGF-β 1 is expressed.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further detail, described in be explanation of the invention and not It is to limit, method used in the present invention and relevant reagent thereof, other can be had optional and replacement scheme, it is possible to reach phase With technical result.
Embodiment 1: expand ring-type circRNA-011235 gene primer pair
According to the cDNA sequence shown in ring-type circRNA-011235 genes of SEQ ID NO:1, design amplification primers, respectively The particular sequence of primer is shown in Table 1.
Form 1 ring-type circRNA 011235 gene amplification primer sequence
Primer Sequence (5 ' → 3 ') SEQ.ID
F AACAAGGACCGAAACTAAGAGG 2
R TGTATCCACCAGAATTACTCCC 3
After above-mentioned primer synthetic, as expanding ring-type circRNA-011235 gene cDNA sequence primer to use.
Embodiment 2: the preparation of total serum IgE in sample
According to TRIZOL method RNA extraction step, extract in the marrow stromal cell sample of matched group, whole body irradiation process group Total serum IgE.It is summarized as follows:
1, homogenate
Sample is according to TRIZOL method description, according to cell quantity, after adding TRIZOL reagent, and homogenate；
2, two-phase laminated flow
After sample after homogenate hatches 5 minutes at 15-30 DEG C, add according in the sample that the TRIZOL reagent of every 1ml is homogenized Enter the chloroform of 0.2ml, cover tightly lid.The most acutely vibration body is after 15 seconds, hatches 2 to 3 minutes for 15 to 30 DEG C.At 4 DEG C 12, 000 × g is centrifuged 15 minutes.Mix liquid after Li Xin and be classified into the red phenol chloroform phase of lower floor, core upper strata, intermediate layer colourless Aqueous phase.RNA is all distributed in aqueous phase.
3, RNA precipitate
Aqueous phase is transferred in new centrifuge tube.Aqueous phase mixes with isopropanol to precipitate RNA therein, adds the amount of isopropanol For adding the isopropanol now adding 0.5ml of 1ml TRIZOL reagent during each sample homogenization.10 are hatched for 15 to 30 DEG C after mixing After minute, in 4 DEG C 12,000 × g is centrifuged 10 minutes.
4, RNA cleans
Remove supernatant, the sample of every 1ml TRIZOL reagent homogenate adds 75% ethanol of at least 1ml, cleans RNA Precipitation.After vibration, 4 DEG C 7,500 × g is centrifuged 5 minutes.
5, RNA precipitate is again dissolved
Remove ethanol solution, air drying RNA precipitate 5-10 minute, be sure not traditional vacuum and be dried.Notice that RNA precipitate is not It is completely dried, otherwise will be substantially reduced the solubility of RNA.Partly soluble RNA sample A260/280Ratio will be less than 1.6.Molten When solving RNA, it is initially charged the water rifle without RNase and repeatedly blows and beats several times, then hatch 10 minutes for 55 to 60 DEG C.The RNA obtained is molten Liquid is stored in-70 DEG C.
6, total serum IgE quality testing
The total serum IgE extracted, usesND-1000 measures RNA concentration and purity, first with dissolving RNA before measurement DEPC water zeroing.
The total serum IgE quality of extraction, number quality should be able to meet the requirement of follow-up test.
Embodiment 3: the synthesis of total cDNA sequence
Through the total serum IgE that quality testing is qualified, method as described below carries out the synthesis of cDNA.Reagent used by synthesis And manufacturer is as follows:
RNase inhibitor (Epicentre)；SuperScriptTM III Reverse Transcriptase (Invitrogen)；5 × RT buffer (Invitrogen)；(dATP, dGTP, dCTP and dTTP are each for 2.5mM dNTP mixed liquor 2.5mM)(HyTest Ltd)；Primer (Ying Jun Bioisystech Co., Ltd)
CDNA synthesis thermal cycler is Gene Amp PCR System 9700 (Applied Biosystems).
Concrete operational approach is as follows:
1, preparation annealing mixture
RNA 800ng
0.5ug/ul Random(N9) 1μl
dNTPs Mix(2.5mM) 1.6μl
Add the H without RNase2O to cumulative volume 14.5 μ l；Mixed liquor, 65 DEG C of water-baths 5 minutes, is placed 2 minutes on ice.
2, of short duration centrifugal after, centrifuge tube is sequentially added into RT reactant liquor
Mix rear 37 DEG C of constant temperature 1 minute, then inhale gently with liquid-transfering gun and beat mix homogeneously several times.
3,50 DEG C of incubations 60 minutes.
4,70 DEG C of incubations make enzyme inactivate in 15 minutes.
5, total cDNA puts that ice bath is stand-by or-20 DEG C of preservations.
Embodiment 4: ring-type circRNA-011235 gene cDNA expands
In the present invention, ring-type circRNA-011235 gene cDNA amplification system is as follows:
Total cDNA sample of Example 3 synthesis, configures PCR reaction system, and reaction system is as follows:
The soft solution that makes mixes, and 5000rpm is of short duration centrifugal.The amplification of cDNA is carried out according to following procedure: 95 DEG C, 10min；40 PCR cycle (95 DEG C, 10 seconds；60 DEG C, 60 seconds).
Real-time fluorescence quantitative PCR signal collection is at 60 DEG C of PCR cycle, and 60 second stage was carried out.
Reaction terminates PCR primer with 100bp DNA Ladder in 2% agarose gel electrophoresis, ethidium bromide staining, inspection Survey whether PCR primer is single specificity amplified band.
Embodiment 5: ring-type circRNA-011235 gene detecting kit
A kind of circular rna gene detecting kit, including archaeal dna polymerase, magnesium ion, buffer, circular rna gene specific Property primer, water.
Concrete system is as follows:
During use, add the total cDNA sample obtained through reverse transcription, expand in following PCR amplification program: 95 DEG C, 10min；40 PCR cycle (95 DEG C, 10 seconds；60 DEG C, 60 seconds).
Embodiment 6: ring-type circRNA-011235 expression conditions in gene chips research whole body irradiation patient
Bone Marrow of Patients stromal cell after whole body irradiation of learning from else's experience process, according to the preparation of total serum IgE in sample in embodiment 2, The total serum IgE extracted carries out circular rna genechip detection.Specifically comprise the following steps that
1, the purity of total serum IgE and Concentration Testing
By purity and the concentration of the total serum IgE of NanoDrop ND-1000 Detection and Extraction, result is as follows:
The purity of form 2 total serum IgE and concentration quality testing
2, RNA labelling
Up-to-standard total serum IgE, is enriched with circular rna with Rnase.CircRNA after enrichment uses Arraystar after amplification Super RNA label probe (Arraystar, the Inc) labelling of company.
3, Array hybridization
CircRNA Yu Arraystar company circRNA chip (8*15K, Arraystar) after labelling divides in Agilent Sub-hybridization instrument is hatched 17 hours for 65 DEG C, hybridizes.
4, Array scanning
Fully after washing, chip Agilent scanner G2505C detects.
5, data are obtained with Agilent data processing software；
6, circRNAs expression analysis
Carry out a series of data including homogenization with R software kit to process.
7, the differential expression of CircRNA
Analyze differential expression in irradiation group and matched group respectively have statistics by multiple change cutoff value or volcano figure The circRNA of meaning.
8, annotation circRNA and microRNA interacts
Using Arraystar ' s forecasting software to analyze the interaction of circRNA Yu microRNA, all differences is expressed CircRNA annotates the microRNA interacted therewith the most in detail.
9, circular rna genechip detection result
Ring-type rna gene chip detection result such as Fig. 1,2 institutes in normal group and whole body irradiation process group marrow stromal cell Showing, the cluster analysis result of expression is as shown in Figure 3.
Ring-type circRNA chip detection result initial data is as shown in the table
Form 3 circRNA 011235 gene is raw florescent intensity in ring-type circRNA chip detection result
Group CircRNA-011235 fluorescence intensity
Matched group 8.21693054345939
TBI process group 11.5937732455632
Gained fluorescence intensity data, after processing with Agilent data processing software, obtains whole body irradiation process group bone marrow base In cell plastid, circRNA-011235 gene is than matched group up-regulated expression 10.3879762 times.
Utilize cytoscape bioinformatics software to carry out ceRNA analysis, cirRNA-011235 can be doped permissible The miR-143-3p regulation and control to TGF-β are regulated by sponge adsorption.
Utilize two kinds of bioinformatics software forecast analysis of TargetScan and miRanda, find that miR-143-3p is The response element of circRNA-001235.
Embodiment 7: ring-type circRNA-011235 gene and miR-in real-time quantitative PCR checking whole body irradiation patient samples 143-3p expression conditions
Sample Total RNAs extraction, total cDNA synthesizes, the parameter such as real-time fluorescence quantitative PCR reaction system composition, amplification such as enforcement Shown in example 1,2,3,4, real-time fluorescence quantitative PCR is used to detect circRNA-in matched group and experimental group marrow stromal cell respectively 011235 gene and the expression of miR-143-3p gene, the genes of interest of each sample and house-keeping gene carry out the most glimmering respectively Fluorescent Quantitative PCR reacts.According to the gradient dilution DNA standard curve drawn, the concentration knot of each sample genes of interest and house-keeping gene Fruit is directly generated by machine.The genes of interest concentration of each sample, divided by the concentration of its house-keeping gene, is this gene of this sample Correction after relative amount.
The real-time quantitative PCR amplification curve of internal reference GAPDH gene as shown in Figure 4, circRNA-011235 gene real-time Quantitative pcr amplification curve is as shown in Figure 5.Wherein the △ Rn in Fig. 4, Fig. 5 real-time fluorescence quantitative PCR amplification curve diagram is meant that Rn The standardization result obtained after deduction baseline, Rn (Normalized reporter) is that the fluorescent emission of fluorescent reporter group is strong The ratio of the fluorescent emission intensity of degree and reference dye.
CircRNA-011235 and miR-143-3p in matched group and experimental group marrow stromal cell, real-time quantitative PCR is tied Fruit correction is as follows:
Ring-type circRNA 011235 gene and miR 143 3p expression conditions in table 4 whole body irradiation patient samples Real-time fluorescence quantitative PCR result
After carrying out data process, the expression of gained is as shown in the table:
In table 5 whole body irradiation patient samples, ring-type circRNA 011235 gene and miR 143 3p gene are relative to interior Ginseng GAPDH expression conditions
Data compare scheme circRNA_011235/GAPDH miR-143-3p/GAPDH
Process group/normal group 1.60 0.48
Result shows, circRNA-011235 up-regulated expression in whole body irradiation Bone Marrow of Patients stromal cell, compared with normal Organizing high 1.60 times, miR-143-3p lowers and expresses substantially simultaneously, only 0.48 times of normal group.
Embodiment 8: ring-type circRNA-011235 gene overexpression is abnormal with TGF-β protein level to be risen
The data of ring-type circRNA-011235 expression conditions in whole body irradiation patient samples in embodiment 6,7, Utilize cytoscape bioinformatics software to carry out ceRNA analysis, cirRNA-011235 can be doped and can pass through sponge The adsorption regulation miR-143-3p regulation and control to TGF-β.
For verifying this prediction, the marrow stromal cell of process LAN circRNA-011235 gene adenovirus vector will be transfected Compared with the comparison marrow stromal cell of transfection empty carrier, find that its intracellular TGF-β protein level is abnormal and rise, the most such as Under:
1, circRNA-011235 gene amplification
Ring-type circRNA-011235 amplimer is designed according to embodiment 1；Ring-type circRNA-is prepared according to embodiment 2 011235 gene amplification sample；The synthesis of total serum IgE gene cDNA sequence is carried out according to embodiment 3；Then according to embodiment 4 is entered Row ring-type circRNA-011235 gene cDNA amplification, and reclaim the cDNA of the circRNA-011235 gene of amplification.
2, pcDNA3-circRNA-011235 recombiant plasmid and the preparation of adenovirus
With taq enzyme, circRNA-011235 gene cDNA ring-type after amplification is linked on pcDNA3 carrier, and is coated In rAD-EGFP adenovirus, named rAD-4-circRNA011235 adenovirus.
3, cell transfecting
Before transfection, 24h is by 1 × 105Marrow stromal cell inoculates 6 orifice plate culture dishs, when cell length to 70%-80% merges Time transfection.Transfection discarded old culture medium the same day, washed 2 times with PBS, added serum-free, without dual anti-pure DMEN 1ml, this experiment Matched group is with 1 × 105RAD-EGFP adenovirus, process group is with 1 × 105RAD-4-circRNA011235 adenovirus infection bone 6 orifice plates are placed in 37 DEG C after transfection by marrow stromal cell, cultivate 6h, discard culture medium, change 2ml into and contain in the incubator of 5%CO2 The DMEN culture medium of 10% serum, cultivates to 48h, receives cell detection TGF-β protein expression level.
4, TGF-β expression detection
In this test control group and process group cell, TGF-β expression is as shown in Figure 6, has transfected process LAN circRNA- The marrow stromal cell of 011235 gene adenovirus vector with transfection empty carrier comparison marrow stromal cell compared with, TGF-β 1 egg White level is abnormal to be risen.
In the present invention, inventor find leukaemic after TBI in BMSCs circRNA-011235 up-regulated expression high Reaching 10.38 times, be miR143-3p by the response element of circRNABase prediction circRNA-011235, experimental result shows After TBI process, in BMSCs, TGF-β expression substantially increases, and miR-143-3p lowers and expresses substantially simultaneously, and this may cause bone marrow Fibrosis, is unfavorable for the going back to the nest of hematopoietic cell, self renewal, breeds and break up, and then affects HSCs and transplant curative effect.
Above detailed description of the invention is only the preferred embodiment of this creation, not in order to limit the present invention, all in the present invention Spirit and principle within any modification, equivalent substitution and improvement etc. done, should be included in protection scope of the present invention it In.

Claims (6)

1. a ring-type circRNA-011235 gene, it is characterised in that its cDNA sequence is as shown in SEQ ID NO:1.
Ring-type circRNA-011235 gene the most according to claim 1 answering in preparation diagnoses leukemic reagent With.
Ring-type circRNA-011235 gene the most according to claim 2 answering in preparation diagnoses leukemic reagent With, it is characterised in that this circular rna gene is as the label of hematopoietic stem cell transplantation curative effect.
The ring-type circRNA-011235 gene the most according to claim 1 application in preparation treatment leukemia medicament.
5. test right requires a ring-type circRNA-011235 gene detecting kit described in 1, mainly by archaeal dna polymerase, Buffer, water, amplimer are to composition, it is characterised in that described amplimer is to nucleotide sequence such as SEQ ID NO:2, SEQ Described in ID NO:3.
Ring-type circRNA-011235 gene amplification primer the most according to claim 5 is at preparation diagnosis leukemia reagent In application.
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CN109880825A (en) * 2019-02-25 2019-06-14 广州市妇女儿童医疗中心 A kind of circular rna hsa_circ_0012152 and its application
CN116064778A (en) * 2022-12-30 2023-05-05 中国辐射防护研究院 circRNAs molecule for identifying radon exposure early injury and application thereof

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