CN109628602A - The new application of circular rna hsa_circ_0012152 - Google Patents

Abstract

Application the invention discloses circular rna hsa_circ_0012152 as therapy target in preparation treatment children acute myelocytic leukemia drug.

Description

The new application of circular rna hsa_circ_0012152

Technical field

The invention belongs to biomedicine technical fields, and in particular to the new application of circular rna hsa_circ_0012152.

Background technique

Acute leukemia is the most common malignant tumour of children, accounts for about the 35% of the period malignant tumour.Although children are anxious Acute myeloid leukemia (acute myeloid leukemia, AML) only accounts for the 20% of acute leukemia, but its mortality Account for 50% or more of acute leukemia.With answering for combined chemotherapy, optimization risk stratification and hematopoietic stem cell transplantation technology With the survival rate of AML infant is greatly taken on a new look, but prognosis is still very poor, and high recurrence rate is up to 30% or more.This is not only seriously endangered The physical and mental health of infant, while to patient home bringing serious financial burden and psychological burden.Therefore, AML is furtherd investigate The molecular mechanism of morbidity, explores the new therapy target of AML and implementation individualized treatment is of great significance.And for intractable For AML, targeted therapy may be the only way in future, this will carry out new hope to the treatment zone of children AML.

Circular rna (circular RNA, circRNA) is a kind of RNA family newcomer for being different from conventional linear RNA, Without the end 5' cap and the end 3' poly (A) tail, and with the non-coding RNA molecule of covalent bond formation ring structure.Closely Nian Lai, there are the circRNA that ten hundreds of sequences is highly conserved in cell, these circRNA are main for scientists discovery It is positioned in cytoplasm, the wide expression in human cell.Its main feature first is that circRNA gene expression abundance is high, part The gene expression abundance of circRNA is as many as 10 times of its linear isomers, and also has apparent tissue specificity, timing special Property and disease specific.Main feature second is that it is more more stable than linear rna, be not easy to be degraded by exonuclease.Compared to mRNA General half-life period be 10 hours, circRNA long half time intracellular was up to 48 hours.These features prompt circRNA is likely to It is played an important role in the life process of biology, illustrates its function and be possible to find new disease treatment target spot.

CircRNA research in recent years obtains great breakthrough, and researchers have identified thousands of CircRNA, have in them it is some be reported with important biological function, be likely to become recruit's marker of diagnosis of disease Or therapy target.But this is only merely tip of the iceberg, especially circRNA and the correlation of leukaemia and they in white blood Effect in disease is unclear, and the research to children AML is even more to have not been reported, therefore, it is necessary to in children AML Expression, function and its regulatory mechanism of circRNA is studied.

Early period, we using circRNA chip V1.0 technology to 8 children AML (1 M1,2 M2,2 M4,2 M5, 1 M7) 5396 circRNA in Bone Marrow of Patients are detected, and 5 healthy childrens are as control.We will be in AML The expression of circRNA is compared with Normal group, the results show that the circRNA that differential expression is statistically significant It is 506 total, wherein 382 of up-regulation are expressed, 124 for expressing downward.We have chosen differential expression most significant preceding 10 A circRNA is verified again, has found the most significant circRNA of difference --- hsa_circ_0012152. Hsa_circ_0012152 (chr1:44877652-44878394) is the 2nd exon 1 by gene RNF220 (1p34.1) Domain is individually cyclized, long 742bp.Currently, biological action and clinical meaning of the hsa_circ_0012152 in children AML It is totally unknown.

Summary of the invention

The present invention is directed to study the new application of circular rna hsa_circ_0012152, and in particular to circular rna hsa_ Application of the circ_0012152 as therapy target in preparation treatment children acute myelocytic leukemia drug.

In addition, the present invention is directed to study a kind of novel targets that can be used for preparing treatment children AML drug.It is a kind of to treat children The target site of acute myelocytic leukemia drug, the target site are the cyclisation site of circular rna hsa_circ_0012152. The target site sequence is as shown in SEQ ID NO.5.

The present invention is studies have shown that hsa_circ_0012152 overexpression in children AML surpasses compared with the control group Cross nearly 14 times.More ironically, in the children AML patient of complete incidence graph, hsa_circ_0012152 is anxious after treatment Speed reduces, and almost maintains an equal level with normal child's marrow.And the circular rna is equally expressed in not alleviated and recurrence children AML Up-regulation.This result implies that circular rna hsa_circ_0012152 is likely to can be used as the novel targets of children AML treatment, is expected to As a kind of improvement AML patient treatment and new strategy of surviving.

The characteristics of due to circular rna itself, the present invention have determined that hsa_circ_0012152 exists using PCR and PCR sequencing PCR Authenticity and validity.And it is directed to the cyclisation site (target site: CGGAATGACAGATGTCTTA of hsa_circ_0012152 (SEQ ID NO.5)), special interference hsa_circ_0012152 expression is devised, without influencing its parental gene RNF220's The small molecules interference RNA (si-0012152) of expression.The plasmid vector for being overexpressed hsa_circ_0012152 is constructed simultaneously (PCD3.1-0012152)(SEQ ID NO.6).By the way that si-0012152 or over-express vector are transfected (PCD3.1- 0012152) into cells such as people's Acute Myeloid Leukemia Cells Contributing system HL60, THP-1 and K562, interference or overexpression are had studied After hsa_circ_0012152, the variation of cells growth activity, differentiation and proliferation etc..The results show that working as hsa_circ_ When 0012152 expression increases, undesired cell proliferation, differentiation is obstructed；After hsa_circ_0012152, which is expressed, to be reduced, malignant Proliferation is substantially reduced, and the ability of differentiation and maturation is strengthened, and the ratio of cancer cell death is higher.Therefore, hsa_circ_ 0012152, which can be used as a potential target site, applies in the drug of preparation treatment children acute myelocytic leukemia.

Detailed description of the invention

Fig. 1 is the cyclisation site of sanger sequencing identification hsa_circ_0012152；

Fig. 2 is the expression that QPCR detects hsa_circ_0012152 in the marrow in children AML different onset stage；

Fig. 3 is to be overexpressed compliance test result after plasmid and interference small molecule transfection；

Fig. 4 is the influence that hsa_circ_0012152 breaks up form to human myeloid leukemia cell THP-1；

Fig. 5 is shadow of the hsa_circ_0012152 to human myeloid leukemia cell THP-1 Surface Differentiation GAP-associated protein GAP molecule It rings；

Fig. 6 is influence (red-label apoptotic body) of the hsa_circ_0012152 to K562 Apoptosis；

Fig. 7 is influence of the hsa_circ_0012152 to K562 cell Proliferation；

Fig. 8 is the influence after interfering hsa_circ_0012152 expression to HL60 cell Proliferation.

Specific embodiment

Illustrate embodiments of the present invention below by way of specific specific example.

Technology according to the present invention is molecular cloning conventional technical means, the enzyme being directed to, primer, reagent and Reaction condition can reasonably select in the case where not specified (NS) according to the experience of those skilled in the art, be directed to try Agent consumptive material belongs to commercially available common product, and the detection means and instrument being directed to also are well known to the skilled person And it skillfully grasps.

One, the cyclisation site of sequence verification circular rna hsa_circ_0012152 gene

1, cell collection and pretreatment

2 each 1ml of umbilical vein blood of Guangzhou Women and Children's Medical Center's pregnant woman are collected, participant, which signs, to be known Feelings letter of consent.Mononuclearcell is collected using lymphocyte separation medium (Ficoll) density-gradient centrifugation method.It is eventually adding RNAiso Plus reagent (TaKaRa), piping and druming mixes repeatedly, and -80 DEG C save for use.

2, RNA is extracted

According to TaKaRa RNAiso Plus reagent specification extract RNA, survey RNA concentration (A260/A280=1.8~ 2.1), put -80 DEG C it is spare.

3, cDNA reverse transcription

Using the PrimeScript of TaKaRa^TM RT reagent Kit with gDNA Eraser(Perfect Real Time cDNA reverse transcription) is carried out.

(1) according to the formula processing of table 1 to remove genomic DNA.42 DEG C of response procedures, 2 minutes.It 4 DEG C, places spare.

Table 1

Reagent	Usage amount
		5×gDNA Eraser Buffer	2.0ul
gDNA Eraser	1.0ul
		Total RNA	1ug
RNase Free dH2O	Polishing 10ul

(2) it is formulated according to table 2 and carries out reverse transcription reaction, 37 DEG C of response procedures, 15 minutes；85 DEG C, 5sec；It is 4 DEG C, spare.

Table 2

Reagent	Usage amount
		The reaction solution of step 1	10.0ul
PrimeScript RT Enzyme Mix I	1.0ul
		RT Primer Mix	1.0ul
5×PrimeScript Buffer 2	4.0ul
		RNase Free dH2O	4.0ul
Total	20l

4, PCR reacts

Using the Premix Taq of TaKaRa^TMEnzyme is carried out according to formula as below.Circular rna hsa_circ_0012152 is set It is a pair of to count reverse primer, primer upstream sequence F:5 '-TGCTGTCTCTGGCCTCATTTC (SEQ ID NO.1)；Primer downstream sequence It arranges R:5 '-GGGAATCATTCCCTCCTAAGAC (SEQ ID NO.2).94 DEG C of response procedures, 2 minutes initial denaturations；94 DEG C of denaturation 30sec, 60 DEG C of annealing 30sec, 72 DEG C extend 2 minutes, continue 40 circulations；72 DEG C 10 minutes.(table 3)

Table 3

Reagent	Usage amount
		Premix Taq(Ex Taq Version 2.0plus dye)	10ul
CDNA template or gDNA	2ug
		Primer F(10uM)	0.8ul
Primer R(10uM)	0.8ul
		RNase Free dH2O	Polishing 20ul

5, carrier T, conversion, bacterium colony PCR, sequencing are connected

It is completed using the pGM-T Cloning Kit of TIANGEN.Carrier T 1ul and PCR product 1ul are gently mixed, room temperature Centrifuge tube is placed on ice by (20 DEG C~37 DEG C) reaction 5min after reaction.Using (the 42 DEG C of heat shocks of heat-shock transformed method 90s) carrier T is transformed into competent escherichia coli cell.50~100 μ L conversion is applied on ampicillin medium plate Liquid is inverted overnight incubation in 37 DEG C of incubators.

At normal temperature, with the single colonie in the random picking reformer plate of the toothpick of sterilizing, the toothpick for speckling with thallus is placed in dress Several lower rear taking-ups are washed in the PCR pipe for having PCR mixture reaction system.PCR reaction system and response procedures are same as above.It chooses positive Bacterium colony send company to be sequenced.The sequence of hsa_circ_0012152 is shown in NO.1；Sequencing result is shown in Fig. 1: clearly detecting cyclisation site.

Two, QPCR detects hsa_circ_0012152 in the marrow in children acute myelocytic leukemia different onset stage Expression

1, blood specimen collection and processing

New children AML marrow specimen 52 of Guangzhou Women and Children's Medical Center 2015.1-2018.11 month is collected, Recurrence 9, sample, normal child's control 32, marrow, the complete incidence graph case of pairing 5,.All participants, which sign, to know Letter of consent.EDTA anticoagulant tube acquires marrow specimen, records patient information.Leucocyte is collected by the way of erythrocyte splitting.Most RNAiso Plus reagent (TaKaRa) is added afterwards, piping and druming mixes repeatedly, and -80 DEG C save for use；

2, RNA is extracted: the same；

3, cDNA reverse transcription: the same；

4, QPCR amplification experiment

Hsa_circ_0012152 and GAPDH is expanded using QPCR.Using the TB GreenTM Premix Ex of TaKaRa (the Tli RNaseH Plus) kit of TaqTM II is completed.It is completed according to 4 experimental system of table.

Table 4

Response procedures are two-step method: PCR amplification standardization program: 95 DEG C of initial denaturation, 30sec；95 DEG C of denaturation 5sec of second step, 60 DEG C of extension 30sec, the step carry out 40 circulations.

Ct_PurposeFor target gene Ct value, Ct_{House keeper}For house-keeping gene Ct value.△ Ct=Ct_Purpose-Ct_{House keeper}, indicate each sample purpose Opposite Ct value of the gene with respect to house-keeping gene, △ △ Ct=(△ Ct)_Test-(△Ct)_Control, indicate processing group relative comparison group It is normalized, 2^-△△CtIt indicates the relative expression quantity of processing group relative comparison group, indicates target gene relative fold expression.Mesh Mark the relative expression quantity calculation formula of gene are as follows:5, result is shown in Fig. 2, as a result shows Show: hsa_circ_0012152 overexpression (Fig. 2A) in the marrow of the initial AML patient of children and patients with recurrent, and passing through Cross after chemotherapy that expression reduces (Fig. 2 B) in the infant marrow of complete incidence graph.These results suggest that hsa_circ_0012152 is in youngster It is played an important role in virgin AML treatment.

Three, over-express vector building, RNA interfering design and transfection verifying

1, the building of plasmid vector is overexpressed (using the pcDNA3.1+CircRNA of Guangzhou Yong Nuo Biotechnology Co., Ltd Min i Vector)。

Building carries the overexpression plasmid (pcDNA3.1-0012152) of hsa_circ_0012152.Both ends band is designed first There is reverse complemental to match primer, expand the linear fragment of hsa_circ_0012152, then make its cyclisation, then turns amplified production Change to T3 carrier, collects purified plasmid.

2, RNA interfering (si-0012152) is designed for the cyclisation site of hsa_circ_0012152, to avoid to parent The influence of gene.

Sense:5'-AUGACAGAUGUCUUAGGAGdTdT-3'(SEQ ID NO.3)；

Antisense:3 '-dTdTUACUGUCUACAGAAUCCUC-5 ' (SEQ ID NO.4).

3, the transfection (Lipofectamine of Invitrogen of siRNA and overexpression plasmid^TM Stem Transfection Reagent)

(1) by people myeloid leukemia cell line THP-1, HL60 and K562 cell of logarithmic growth phase, (1.5ml cell is outstanding Liquid) six orifice plates are reached, cell confluency degree is in 70% -90% transfection.

(2) 100ul Opti-MEM and pcDNA3.1-0012152 (or si-0012152) is added into a sterile centrifugation tube 2ug, featheriness mix, this is A liquid.

(3) 5 μ l Lipofectamine are taken^TMStem Reagent reagent in another sterile centrifugation tube with 100ul Opti-MEM mixing, featheriness mix, this is B liquid.

(4) A liquid and B liquid 1:1 are mixed, is incubated 10 minutes at room temperature.Then mixed liquor is transferred to respectively THP-1, In the culture solution of HL60 and K562 cell, mix, in 37 DEG C, 5%CO₂Cell is received after cultivating 60 hours in cell incubator.

4, RNA is extracted: the same.

5, cDNA reverse transcription: the same；

6, QPCR amplification experiment: the same；As a result see Fig. 3, the results show that after transfecting pcDNA3.1-0012152, hsa_ Circ_0012152 expression is obvious to rise (Fig. 3 A)；And after si-0012152, expression is then remarkably decreased.This illustrates that we construct Over-express vector and the interference sequence of design be all effective (Fig. 3 B).

Four, it is overexpressed influence of the hsa_circ_0012152 to myeloid cell Leukemia Cell Lines THP-1 differentiation form (Wright-Giemsa decoration method)

1, by pcd3.1-0012152 plasmid transfection into THP-1 cell: the same (to be put into cell in each hole before transfection Creep plate).The PMA that 25ng/ml is added in the medium simultaneously is induced, and 37 DEG C, 5%CO₂It is cultivated in cell incubator.

2, respectively for 24 hours, tetra- time points of 48h, 72h, 96h former culture medium is sucked, washed one time with PBS, then by cell Creep plate, which takes out, to be spontaneously dried, and fixes 3min with methanol, and Wright-Giemsa working solution 300ul is added dropwise and covers creep plate, room temperature dyeing After 1min, be added dropwise 2ml phosphate buffer gently blow it is even, make its cover 10 minutes；Then with water elution is distilled, room temperature is dried； 100 under microscope × oil microscopic observation.

3, result is shown in Fig. 4.The results show that after THP-1 (under PMA stimulation) is overexpressed hsa_circ_0012152, cell Metamorphosis is smaller, and cell slows down to macrophage differentiation, and the characteristics of more maintain monocyte.

Five, hsa_circ_0012152 is overexpressed to the detection (stream of myeloid cell Leukemia Cell Lines THP-1 division guideline Formula cell art)

1, by pcd3.1-0012152 plasmid transfection into THP-1 cell: the same.25ng/ is added in the medium simultaneously The PMA of ml is induced, and 37 DEG C, 5%CO₂It is cultivated in cell incubator.

2,48h carefully scrapes cell collection, centrifugation with cell scraper after transfecting.It discards supernatant, 1ml FACS Buffer is added It is resuspended after mixing and is centrifuged, 50ul mixing streaming antibody (including CD11b, CD14, CD206) is added.4 DEG C of incubation 20min；It is added 1ml FACS Buffer washes away unbonded antibody, and supernatant is removed after centrifugation, and 200ul FACS Buffer is added, and filters up flow type Pipe is expressed with flow cytomery cell surface molecule.

3, result is shown in Fig. 5.The results show that after THP-1 (under PMA stimulation) is overexpressed hsa_circ_0012152, cell The molecular marker on surface changes, and compared with the control group, CD 11b and CD14 expression reduce.This illustrates hsa_circ_ 0012152 can inhibit leukaemia cell to break up.

Six, it is (thin using green skies one-step method TUNEL to be overexpressed influence of the hsa_circ_0012152 to K562 Apoptosis Born of the same parents' apoptosis detection kit is completed)

1, by pcd3.1-0012152 plasmid transfection into K562 cell: the same.37 DEG C, 5%CO₂It is trained in cell incubator Support 60h.

2,10 are collected⁶A cell, centrifugation, PBS are washed 5 minutes.It is used again after fixing cell 30 minutes with 4% paraformaldehyde PBS washing.Cell is resuspended in the PBS of the X-100 containing 0.3%Triton, and is incubated at room temperature 5 minutes.PBS is cleaned 5 minutes.

3, according to each 5 μ l of sample TdT enzyme, the ratio of 45 μ l of fluorescent labeling solution prepares suitable TUNEL and detects liquid.Each Sample is added 50 μ l TUNEL and detects liquid, and 37 DEG C are protected from light incubation 60 minutes；After PBS wash 2 times, 5 minutes/time.Finally use 250PBS suspends.

4, in fluorescence microscopy microscopic observation after carrying out detection or smear with flow cytometer.The excitation wavelength of Cy3 is 550nm, launch wavelength are 570nm (red fluorescence).

5, almost without the fracture of DNA in the normal or cell that is being proliferated, and the disconnected of DNA can occur in the cell of apoptosis It splits, the DNA of these fractures can be labeled, to show the cell that apoptosis occurs.As a result see Fig. 6, as can be seen from Fig.: mistake After expressing hsa_circ_0012152, the quantity of K562 apoptosis is lower than control group.In other words, hsa_circ_ 0012152 can inhibit K562 Apoptosis to a certain extent.

Seven, it is overexpressed influence (CCK-8 kit) of the hsa_circ_0012152 to K562 cell Proliferation

1, by pcd3.1-0012152 plasmid transfection into K562 cell: the same.37 DEG C, 5%CO₂It is trained in cell incubator It supports.And by the cell after transfection according to 2 × 10⁴/ hole (100ul cell suspension) is distributed in 96 orifice plates.

2, respectively after transfection for 24 hours, 48h, 72h and 96 hours addition CCK-8 reagent 10ul, by culture plate in incubator Continue to be incubated for the light absorption value after 2h with microplate reader measurement at 450nm, 630nm is as reference wavelength.

3, result is shown in Fig. 7, as the result is shown: detecting by independent experiment three times, since for 24 hours, transfects pcd3.1- 0012152 K562 cell activity obviously rises compared with control group (PCD3.1).This prompt, after being overexpressed hsa_circ_0012152 With the ability (Fig. 7) for promoting tumour cell malignant proliferation.

Seven, the influence (CCK-8 kit) that detection si-0012152 is proliferated myelogenous leukemia cell lines HL60

1, si-0012152 microRNA is transfected into HL60 cell: the same.And by the cell after transfection according to 2.5 ×10⁴/ hole (100ul cell suspension) is distributed in 96 orifice plates.

2, respectively after transfection for 24 hours, 48h, 72h and 96 hours addition CCK-8 reagent 10ul, by culture plate in incubator Continue to be incubated for the light absorption value after 2h with microplate reader measurement at 450nm, 630nm is as reference wavelength.

3, result is shown in Fig. 8, as the result is shown: detecting by independent experiment three times, our results show each group after transfection Cell growth state gradually appears difference, since for 24 hours, transfects the HL60 cell activity of si-0012152 compared with control group (si- NC it) is decreased obviously, as time went on, the effect of Inhibit proliferaton is also significant.This prompt, targeting interference hsa_circ_0012152 There is the ability for inhibiting cancer cell multiplication afterwards.

Sequence table

<110>Guangzhou Women and Children's Medical Center

<120>new application of circular rna hsa_circ_0012152

<141> 2019-02-25

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA

<213>artificial sequence (ABC)

<400> 1

tgctgtctct ggcctcattt c 21

<210> 2

<211> 22

<212> DNA

<213>artificial sequence (ABC)

<400> 2

gggaatcatt ccctcctaag ac 22

<210> 3

<211> 21

<212> DNA

<213>artificial sequence (ABC)

<400> 3

augacagaug ucuuaggagt t 21

<210> 4

<211> 21

<212> DNA

<213>artificial sequence (ABC)

<400> 4

ttuacugucu acagaauccu c 21

<210> 5

<211> 19

<212> DNA

<213>artificial sequence (ABC)

<400> 5

cggaatgaca gatgtctta 19

<210> 6

<211> 6244

<212> DNA

<213>artificial sequence (ABC)

<400> 6

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattccaa 960

agtgctgaga ttacaggcgt gagccaccac ccccggccca ctttttgtaa aggtacgtac 1020

taatgacttt ttttttatac ttcaggatat cgtcttagga gggaatgatt ccccagtaat 1080

attccctgcc ctgacccaaa gtgctggttg gcctccctcc cagggaagac tgcttcttgc 1140

gtaacgccgg ccacagaaag agactccgat ggacttacac cgggcagcct tcaagatgga 1200

gaactcatcc taccttccca accctctggc atccccagca ctgatggtcc tggcatccac 1260

ggctgaggcc agccgtgatg cttccatccc ttgtcagcag ccacgaccct ttggtgtacc 1320

tgtctcagtt gacaaggacg tgcatattcc tttcaccaac ggttcctata cctttgcctc 1380

tatgtaccat cggcaaggtg gggtgccagg cacttttgcc aatcgtgatt tccccccttc 1440

tctactacac ctccaccctc aatttgctcc cccaaatcta gattgcaccc caatcagtat 1500

gctgaatcat agtggtgtgg gggctttccg gccctttgcc tccaccgagg accgggagag 1560

ctatcagtca gcctttacgc cggccaagcg acttaagaac tgccatgaca cagagtctcc 1620

ccacttgcgc ttctcagatg cagatggcaa ggaatatgac tttgggacac agctgccatc 1680

tagctccccc ggttcactaa aggttgatga cactgggaag aagatttttg ctgtctctgg 1740

cctcatttct gatcgggaag cctcatctag cccagaggat cggaatgaca gatgcggccg 1800

ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc 1860

cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 1920

actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 1980

attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 2040

catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct 2100

agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 2160

cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct 2220

tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta 2280

gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt 2340

tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg 2400

ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat 2460

tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt 2520

taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt 2580

ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 2640

ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 2700

agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt 2760

ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg 2820

cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt 2880

gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag 2940

gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg 3000

agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt 3060

tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc 3120

tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt 3180

gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag 3240

tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg 3300

ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag 3360

cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg 3420

atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc 3480

gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca 3540

tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc 3600

gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg 3660

ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct 3720

atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc 3780

gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg 3840

cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct 3900

ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa 3960

tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 4020

caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc 4080

gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 4140

catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 4200

attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 4260

ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 4320

ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 4380

aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 4440

aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 4500

gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 4560

gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 4620

tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 4680

ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 4740

ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 4800

tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 4860

tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 4920

ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 4980

aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtt tttttgtttg 5040

caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 5100

ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 5160

aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 5220

tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 5280

agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 5340

gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 5400

accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 5460

tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 5520

tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 5580

acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 5640

atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 5700

aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 5760

tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 5820

agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc 5880

gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 5940

ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 6000

atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 6060

tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 6120

tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 6180

tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga 6240

cgtc 6244

Claims (3)

1. the new application of circular rna hsa_circ_0012152, characterized in that circular rna hsa_circ_0012152 conduct Application of the therapy target in preparation treatment children acute myelocytic leukemia drug.

2. a kind of target site for treating children acute myelocytic leukemia drug, the target site is circular rna hsa_circ_ 0012152 cyclisation site.

3. target site as claimed in claim 2, which is characterized in that the target site sequence is as shown in SEQ ID NO.5.