CN103233009B

CN103233009B - Application of substance capable of reducing expression of zinc finger protein CTCF to preparation of drugs for treating leukemia

Info

Publication number: CN103233009B
Application number: CN201310153407.3A
Authority: CN
Inventors: 郑胡镛; 鲍时来; 张寒; 朱琳; 刘潇
Original assignee: Beijing Childrens Hospital; Institute of Genetics and Developmental Biology of CAS
Current assignee: Beijing Childrens Hospital; Institute of Genetics and Developmental Biology of CAS
Priority date: 2013-04-27
Filing date: 2013-04-27
Publication date: 2015-04-08
Anticipated expiration: 2033-04-27
Also published as: CN103233009A

Abstract

The invention discloses application of a substance capable of reducing expression of a zinc finger protein CTCF to preparation of drugs for treating leukemia. In particular, the substance reducing expression of the zinc finger protein CTCF can be short hairpin RNA (shRNA) forming a stem-loop structure. The name of the shRNA is shRNA-3. One chain sequence of the stem in the stem-loop structure is SEQ ID No.1 and the other chain sequence of the stem in the stem-loop structure is SEQ ID No.2. The invention proves that the zinc finger protein CTCF is an anti-apoptosis factor and has the function of suppressing cell apoptosis in the ALL (acute lymphoblastic leukemia) cells. Apoptosis of tumor cells-leukemia cells can be promoted by interfering with expression of CTCF. The invention provides a new way for leukemia treatment. The zinc finger protein CTCF is expected to become a potential target for anti-leukemia treatment and has very extensive application prospect in the medical field.

Description

Reduce the application of material in preparation treatment leukemia medicament that zinc finger protein CTCF expresses

Technical field

The present invention relates to the application of material in preparation treatment leukemia medicament reducing zinc finger protein CTCF and express.

Background technology

Zinc finger protein CTCF also known as CCCTC binding factor, many zinc fingerses albumen of the high conservative be made up of 727 amino acid.It participates in various biological regulatory function, comprises transcriptional activation or suppression, chromatin insulation, Genomic Imprinting and x chromosome inactivation etc.

Research finds that CTCF participates in the generation evolution of multiple human tumor.People have found the tumour-specific sudden change of CTCF zinc fingers in mammary cancer, prostate cancer and the nephroblastoma.This sudden change makes CTCF lose the binding ability with downstream tumour target gene, causes these abnormal gene expressions, causes tumour thus.In addition, CTCF participates in the generation of tumour by epigenetics mechanism.Investigator finds that in the nephroblastoma, colorectal carcinoma and the rectum cancer CTCF binding site exists abnormal methylation phenomenon, and infers that can cause CTCF combined function thus loses, and then induced tumor.Visible CTCF genetics and epigenetics change play a key effect in tumour occurs.

To be hematopoietic stem/progenitor be obstructed and malignant proliferation and the Patients with Hematopoietic Malignancies: A that causes in different differentiation phases generation apoptosis obstacle, differentiation leukemia.This disease is Childhood modal malignant tumour and the modal cause of death.China newly sends out leukemia of children 1.5 ten thousand example left and right every year, and wherein children acute lymphoblastic leukaemia (AcuteLymphoblastic Leukemia, ALL) is modal type, accounts for 75% of leukemia of children.After the eighties in 20th century, due to the combined reinforced treatment of multiple medicines and strong support treatment, the curative ratio of children ALL reaches about 80%, but over nearly 30 years, ALL curative ratio is without significantly improving, and major cause is that leukemic pathogeny is failed to understand so far.In addition, leukemia relapse is also one of important factor.The recurrence rate of current leukemia of children, up to about 15%, has become the most critical factor affecting leukemia children rehabilitation, not only gives infant itself but also brings irremediable grievous injury humorous factor on good terms to family and society.

Therefore, the drug target of the leukemic pathogeny of further investigated, exploration leukemia of children will contribute to the diagnosis and treatment of leukemia of children.

Summary of the invention

A technical problem to be solved by this invention is to provide a kind of short hairpin RNA (shRNA, short hairpin RNA) and the relevant biological material thereof that promote apoptosis of leukemia.

The short hairpin RNA promoting apoptosis of leukemia provided by the present invention, name is called shRNA-3, form stem ring (Stem-loop) structure, in described loop-stem structure, a chain-ordering of stem (Stem) is SEQ ID No.1, and in described loop-stem structure, another chain-ordering of stem is SEQ ID No.2.

Wherein, SEQ ID No.1 is made up of 19 ribonucleotides, and SEQ ID No.2 is made up of 19 ribonucleotides.

Ring (loop) in described short hairpin RNA makes described short hairpin RNA can produce the double-chain small disturbance RNA (siRNA) be made up of the single stranded RNA shown in the single stranded RNA shown in SEQ ID No.1 and SEQ ID No.2 as long as meet.In one embodiment of the invention, the nucleotide sequence of described short hairpin RNA is SEQ ID No.3.

Wherein, SEQ ID No.3 is made up of 47 ribonucleotides, and the 1-19 position of SEQ ID No.3 is identical with SEQ IDNo.1, and the 29-47 position of SEQ ID No.3 is identical with SEQ ID No.2.

The biomaterial that shRNA-3 provided by the present invention is relevant is 1) to 6) in any one:

1) DNA molecular of coding shRNA-3;

2) containing 1) expression cassette of described DNA molecular;

3) containing 1) recombinant vectors of described DNA molecular or containing 2) recombinant vectors of described expression cassette;

4) containing 1) recombinant microorganism of described DNA molecular or containing 2) recombinant microorganism of described expression cassette or containing 3) recombinant microorganism of described recombinant vectors;

5) containing 1) transgenic cell line of described DNA molecular or containing 2) transgenic cell line of described expression cassette or containing claim 3) transgenic cell line of described recombinant vectors;

6) siRNA, the nucleotide sequence of a chain is as SEQ ID No.1, and the nucleotide sequence of another chain is as SEQ ID No.2.

In the biomaterial that above-mentioned shRNA-3 is relevant, 1) DNA molecular of described coding shRNA-3 specifically can be 1) to 3) in the double chain DNA molecule of any one:

1) nucleotide sequence of a chain is SEQ ID No.4, another chain nucleotide sequence be SEQ ID No.5.

2) encoding sequence of described DNA molecular is the 1-47 position of SEQ ID No.4;

3) encoding sequence of described DNA molecular is the 12-58 position of SEQ ID No.5.

Wherein, SEQ ID No.4 is made up of 54 deoxyribonucleotides, and SEQ ID No.5 is made up of 58 deoxyribonucleotides.The 1-47 position of SEQ ID No.4 and the 12-58 position reverse complemental of SEQ ID No.5.

In the biomaterial that above-mentioned shRNA-3 is relevant, 2) described expression cassette, refer to the DNA that can express shRNA-3 in host cell, this DNA not only can comprise the promotor starting shRNA-3 genetic transcription, also can comprise the terminator stopping shRNA-3 genetic transcription.Further, described expression cassette also can comprise enhancer sequence.In one embodiment of the invention, described expression cassette is made up of Pol III terminator (sequence is 5 '-TTTTT-3 ') of the DNA molecular of U6 promotor (its sequence is as shown in SEQ ID No.8), coding shRNA-3 and the DNA molecular of termination coding shRNA-3.3) described recombinant vectors specifically can be the recombinant expression vector pDsU6-sh-3 replacing the expression shRNA-3 that fragment obtains between the Sac I of pDsU6 and Hind III with the DNA molecular of coding shRNA-3, or for replacing the recombinant expression vector pDsU6-GFP-sh-3 of the expression shRNA-3 that fragment obtains between the BamH I of pDsU6-GFP and Hind III with the DNA molecular of coding shRNA-3.PDsU6-GFP uses GFP(green fluorescent protein) gene replaces the recombinant expression vector of the expression GFP that fragment obtains between the Afl II of pDsU6 and BamH I.4) described recombinant microorganism specifically can be bacterium, yeast, algae and fungi.5) transgenic cell line described in does not comprise the reproductive material of animal and plant.

Another technical problem to be solved by this invention is to provide shRNA-3 and relevant biological material is treating and/or preventing the face application of leukemia side.

Any one being applied as in C1 to C4 provided by the present invention:

C1, treat and/or prevent leukemic product (as medicine), its activeconstituents comprises at least one in b1-b3:

b1、shRNA-3；

The DNA molecular of b2, coding shRNA-3;

The biomaterial that b3, above-mentioned shRNA-3 are relevant.

The product (as medicine) of C2, promotion apoptosis of leukemia, its activeconstituents comprises at least one in described b1-b3;

In C3, described b1-b3, at least one material treats and/or prevents the application in leukemic product (as medicine) in preparation;

In C4, described b1-b3, at least one material promotes the application in the product (as medicine) of apoptosis of leukemia in preparation.

In the product of above-mentioned C1 and C2, activeconstituents also can be only at least one in b1-b3.

Another technical problem to be solved by this invention is to provide the purposes of the material reducing zinc finger protein CTCF genetic expression.

The purposes of the material of reduction zinc finger protein CTCF provided by the present invention genetic expression is a1 or a2:

The material of a1, reduction zinc finger protein CTCF genetic expression treats and/or prevents the application in leukemic product (as medicine) in preparation;

The material of a2, reduction zinc finger protein CTCF genetic expression promotes the application in the product (as medicine) of apoptosis of leukemia in preparation.

Wherein, the material of described reduction zinc finger protein CTCF genetic expression is at least one in described b1-b3.

In such use, the aminoacid sequence of described zinc finger protein CTCF is as shown in SEQ ID No.7, and the encoding sequence of described zinc finger protein CTCF gene is as shown in the 445-2628 position of SEQ ID No.6.

Wherein, SEQ ID No.7 is made up of 727 amino-acid residues, and SEQ ID No.6 is made up of 3946 deoxyribonucleotides, and its encoding sequence is 445-2628 position.

Above described leukemia cell can be the cell in body, also can be clone, as people's pre B cell acute lymphoblastic leukemia cell system NALM-6.

Experiment proves, with the mRNA of zinc finger protein CTCF for target spot, RNA is disturbed recombinant expression vector---the recombinant expression vector pDsU6-GFP-sh-3 expressing shRNA-3 proceeds to the leukemia reconstitution cell obtained in the leukemic clone NALM-6 of bone-marrow-derived lymphocyte, its zinc finger protein CTCF expression level is starkly lower than contrast, cell population apoptosis ratio is 28%, is 3.8 times of contrast.The present invention proves that zinc finger protein CTCF is an anti-apoptosis factor, has the effect of inhibited apoptosis in ALL cell, and the expression of interference CTCF can promote that apoptosis occurs tumour cell leukemia cell, and increases the susceptibility of leukemia cell to chemotherapeutics.The present invention is that leukemia treating provides a new approach, and zinc finger protein CTCF is expected to the potential target spot becoming leukemia treatment, has very wide application prospect at medical field.

Accompanying drawing explanation

Fig. 1 is the Western blot result of CTCF in RNA interference restructuring leukemia cell.Wherein, a upper row is with CTCF monoclonal antibody for primary antibodie detects zinc finger protein CTCF, and next row is for primary antibodie detects internal reference GAPDH with GAPDH monoclonal antibody.

Fig. 2 is Annexin V (+)/PI (-) cell proportion that flow cytomery RNA disturbs in restructuring leukemia cell.

Fig. 3 is restructuring leukemia cell early apoptosis ratio bar graph.

Sh-luc, sh-CTCF-1, sh-CTCF-2 and sh-CTCF-3 in Fig. 1-3 represent restructuring leukemia cell NALM-6/pDsU6-GFP-sh-luc, NALM-6/pDsU6-GFP-sh-1, NALM-6/pDsU6-GFP-sh-2 and NALM-6/pDsU6-GFP-sh-3 respectively.

Embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Experiment in following embodiment all arranges three repetitions, results averaged.

Carrier pDsU6: be documented in following document: Shilai Bao, Tao Lu, Xin Wang, Huyong Zheng, Li-E Wang, Qingyi Wei, Walter N Hittelman and Lei Li.Disruption of theRad9/Rad1/Hus1 (9 – 1 – 1) complex leads to checkpoint signaling and replicationdefects.Oncogene, 2004,23,5586 – 5593.The public can obtain from Beijing Children's Hospital, Capital Medical University, to repeat the application's experiment.

B-ALL clone NALM-6(pre B cell acute lymphoblastic leukemia cell system NALM-6) be documented in following document: Gao Huifang, Zhang Han, Pei Pei, Liu Yi, Li Zhigang, Jiang Jin, Zhang Ruidong, superb, Qi Yu, Zheng Huyong. the expression of splicing factor SF2/ASF in childhood leukemia cells. " Capital University of Medical Sciences's journal ", volume the 03rd phase June the 30th in 2009.The public can obtain from Beijing Children's Hospital, Capital Medical University, to repeat the application's experiment.

The RNA of embodiment 1, zinc finger protein CTCF disturbs recombinant expression vector to build

1, RNA disturbs the selection of target sequence

For the full length cDNA sequence (SEQ ID No.6) of zinc finger protein CTCF encoding gene CTCF, following three segment DNA sequences are selected to be the target sequence that RNA disturbs:

The 809-827 position (i.e. 5 '-TGACTGTACCTGTTGCTAC-3 ') of sh-1:SEQ ID No.6

The 1103-1122 position (i.e. 5 '-ATGTAGATGTGTCTGTCTAC-3 ') of sh-2:SEQ ID No.6

The 1398-1416 position (i.e. 5 '-TACTCGTCCTCACAAGTGC-3 ') of sh-3:SEQ ID No.6

In SEQ ID No.6,445-2628 position is open reading frame, the zinc finger protein CTCF shown in coding SEQ ID No.7.

2, siRNA (siRNA)

Design three kinds of siRNA(siRNA-1, siRNA-2 and the siRNA-3 of three kinds of target sequences for step 1 zinc finger protein CTCF respectively).The target sequence of the target sequence of siRNA-1 to be the target sequence of sh-1, siRNA-2 be sh-2, siRNA-3 is sh-3.

1）siRNA-1

siRNA-1-F：5’-ugacuguaccuguugcuac-3’；

siRNA-1-R：5’-guagcaacagguacaguca-3’；

2）siRNA-2

siRNA-2-F：5’-auguagaugugucugucuac-3’；

siRNA-2-R：5’-guagacagacacaucuacau-3’。

3）siRNA-3

siRNA-3-F：5’-uacucguccucacaagugc-3’（SEQ ID No.1）；

siRNA-3-R：5’-gcacuugugaggacgagua-3’（SEQ ID No.2）。

3, short hairpin RNA (shRNA)

According to three kinds of siRNA of step 2, design produces the shRNA-3 of the shRNA-1 of siRNA-1, the shRNA-2 producing siRNA-2, generation siRNA-3.

The sequence of shRNA-1, shRNA-2 and shRNA-3 is following (capitalization is ring sequence, and all the other sequences are stem sequence, forms inverted repeats):

shRNA-1：

5’-ugacuguaccuguugcuacUUCAAGAGAguagcaacagguacaguca-3’；

shRNA-2：

5’-auguagaugugucugucuacUUCAAGAGAguagacagacacaucuacau-3’；

shRNA-3：

5’-uacucguccucacaagugcUUCAAGAGAgcacuugugaggacgagua-3’（SEQ ID No.3）。

Meanwhile, by the shRNA-luc of reticent luciferase gene in contrast:

shRNA-luc：5’-gaagcgcauccaauaccagcUUCAAGAGAgcugguauuggaugcgcuuc-3’

Luciferase (luciferase) be one do not have in front B acute lymphoblastic leukemia cell system NALM-6 cell express albumen, so by with luciferase be the shRNA-luc of target spot in contrast.

4, the Design and synthesis of the DNA molecular of coding siRNA and shRNA

1) be that two single-stranded DNA sequence of double chain DNA molecule (name is called sh-CTCF-1) of expression shRNA-1 of target spot are as follows with sh-1:

CTCF-ssDNA809-1-F：

5’-tgactgtacctgttgctacTTCAAGAGAgtagcaacaggtacagtcacTTTTT A-3’；

CTCF-ssDNA809-1-R：

5’- AGCTTAAAAAgtgactgtacctgttgctacTCTCTTGAAgtagcaacaggtacagtca-3’。

2) be that two single-stranded DNA sequence of double chain DNA molecule (name is called sh-CTCF-2) of expression shRNA-2 of target spot are as follows with sh-2:

CTCF-ssDNA1103-2-F：

5’-atgtagatgtgtctgtctacTTCAAGAGAgtagacagacacatctacatcTTTTT A-3’；

CTCF-ssDNA1103-2-R：

5’- AGCTTAAAAAgatgtagatgtgtctgtctacTCTCTTGAAgtagacagacacatctacat-3’。

3) be that two single-stranded DNA sequence of double chain DNA molecule (name is called sh-CTCF-3) of expression shRNA-3 of target spot are as follows with sh-3:

CTCF-ssDNA1398-3-F（SEQ ID No.4）：

5’-tactcgtcctcacaagtgcTTCAAGAGAgcacttgtgaggacgagtacTTTTT A-3’；

CTCF-ssDNA1398-3-R（SEQ ID No.5）：

5’- AGCTTAAAAAgtactcgtcctcacaagtgcTCTCTTGAAgcacttgtgaggacgagta-3’。

4) be that two single-stranded DNA sequence of double chain DNA molecule (name is called sh-luc) of expression shRNA-luc of target spot are as follows with luciferase:

lucDNA-F：

5’-gaagcgcatccaataccagcTTCAAGAGAgctggtattggatgcgcttccTTTTTTT A-3’；

lucDNA-R：

5’- AGCTTAAAAAAAggaagcgcatccaataccagcTCTCTTGAAgctggtattggatgcgcttc-3’。

Above-mentioned 8 single stranded DNAs are synthesized by Invitrogen company, and underscore part is Hind III cohesive end sequence.

Respectively get 5 μ g(10ng/ μ l) complementary single stranded DNA mixing, 100 DEG C of thermal treatments are after 5 minutes, and annealing at room temperature, obtains double chain DNA molecule, get double chain DNA molecule 5 μ g and carry out phosphatizing treatment, obtain double-stranded DNA reactant sh-CTCF-1, sh-CTCF-2, sh-CTCF-3 and sh-luc.

Wherein, the reaction system (50 μ l) of above-mentioned phosphatizing treatment: double-stranded DNA 5 μ g, 10 × T ₄kinase Buffer5 μ l, ATP(10mM) 5 μ l, T ₄polynucleotide Kinase(10U/ μ l) 2 μ l, use ddH ₂o is supplemented to 50 μ l.

The reaction conditions of above-mentioned phosphatizing treatment: 37 DEG C of water-baths 1 hour, then 100 DEG C of slowly cool to room temperature after 10 minutes, obtain the double-stranded DNA reactant that can be used for construction recombination plasmid.

5, the RNA of zinc finger protein CTCF disturbs recombinant expression vector to build

1) RNA disturbs the structure of recombinant expression vector pDsU6-sh-1, pDsU6-sh-2, pDsU6-sh-3 and pDsU6-sh-luc

Get after carrier pDsU6 first cuts with Sac I enzyme, end-filling process is carried out by T4Polymerase Klenow fragment, the linearized fragment that gel reclaims uses Hind III digestions again, reclaim the skeleton fragment of pDsU6 carrier, the four kinds of double-stranded DNA reactant obtained with step 4 are respectively connected, obtain four kinds of RNA and disturb recombinant expression vector pDsU6-sh-1, pDsU6-sh-2, pDsU6-sh-3, pDsU6-sh-luc, confirm through order-checking, pDsU6-sh-1 is the recombinant expression vector replacing the expression shRNA-1 that fragment obtains between the Sac I of pDsU6 and Hind III with sh-CTCF-1, pDsU6-sh-2 is the recombinant expression vector replacing the expression shRNA-2 that fragment obtains between the Sac I of pDsU6 and Hind III with sh-CTCF-2, pDsU6-sh-3 is the recombinant expression vector replacing the expression shRNA-3 that fragment obtains between the Sac I of pDsU6 and Hind III with sh-CTCF-3, pDsU6-sh-luc is the recombinant expression vector replacing the expression shRNA-luc that fragment obtains between the Sac I of pDsU6 and Hind III with sh-luc.

2) RNA of GFP mark disturbs the acquisition of recombinant expression vector

The construction process of carrier pDsU6-GFP-sh-1, pDsU6-GFP-sh-2, pDsU6-GFP-sh-3, pDsU6-GFP-sh-luc: the RNA getting above-mentioned structure respectively disturbs the recombinant expression vector pDsU6-GFP of recombinant expression vector pDsU6-sh-1, pDsU6-sh-2, pDsU6-sh-3, pDsU6-sh-luc and expression GFP to carry out BamH I and Hind III double digestion, and postdigestive fragment reclaims through gel; The recovery fragment of recombinant expression vector is disturbed to be connected with pDsU6-GFP carrier segments respectively above-mentioned four kinds of RNA, thus obtain RNA interference recombinant vectors pDsU6-GFP-sh-1, pDsU6-GFP-sh-2, pDsU6-GFP-sh-3, pDsU6-GFP-sh-luc of expressing GFP, confirm that pDsU6-GFP-sh-1 is the recombinant expression vector replacing expression GFP and shRNA-1 that fragment obtains between the BamH I of pDsU6-GFP and Hind III with sh-CTCF-1 through order-checking; PDsU6-GFP-sh-2 is the recombinant expression vector replacing expression GFP and shRNA-2 that fragment obtains between the BamH I of pDsU6-GFP and Hind III with sh-CTCF-2; PDsU6-GFP-sh-3 is the recombinant expression vector replacing expression GFP and shRNA-3 that fragment obtains between the BamH I of pDsU6-GFP and Hind III with sh-CTCF-3; PDsU6-GFP-sh-luc is the recombinant expression vector replacing expression GFP and shRNA-luc that fragment obtains between the BamH I of pDsU6-GFP and Hind III with sh-luc.

The construction process of above-mentioned carrier pDsU6-GFP is as follows: with pGFP-N1(purchased from Clontech) be template, at primer P5(5 '-AAGGATCCATTACCGCCATGCATTAG-3 ') and P6(5 '-CCTACGCCTTAAGATACATTG-3 ') guiding under pcr amplification GFP gene, amplified production is through BamH I single endonuclease digestion, and digestion fragment reclaims through gel; Get carrier pDsU6 and carry out Afl II single endonuclease digestion, carry out end-filling process by T4PolymeraseKlenow fragment, after gel reclaims, linearized fragment uses BamH I digestions again, and after digestion, fragment reclaims through gel; Reclaim fragments by these two kinds to connect, obtain carrier pDsU6-GFP, confirm that pDsU6-GFP use GFP(green fluorescent protein through order-checking) recombinant expression vector of the expression GFP that fragment obtains between the Afl II of gene replacement pDsU6 and BamH I.

Embodiment 2, RNAi gene knockout recombinant vectors transfection leukemia cell

1, the cultivation of transfection cell

24h before transfection, with the RPMI-1640 substratum containing 10% foetal calf serum (FBS) at 15ml culture dish, 37 DEG C and 5%CO ₂cultivate under condition when B-ALL clone NALM-6 reaches 75% ~ 90% and carry out transfection.

2, transfection

The RNA with GFP label embodiment 1 obtained disturbs recombinant expression vector pDsU6-GFP-sh-1, pDsU6-GFP-sh-2, pDsU6-GFP-sh-3 and carrier pDsU6-GFP-sh-luc(RNAi positive control, for reticent luciferase gene) difference transfection procedure 1 cultured cells, obtain the restructuring leukemia cell NALM-6/pDsU6-GFP-sh-1 disturbing recombinant expression vector pDsU6-GFP-sh-1 containing RNA, the restructuring leukemia cell NALM-6/pDsU6-GFP-sh-2 of recombinant expression vector pDsU6-GFP-sh-2 is disturbed containing RNA, the restructuring leukemia cell NALM-6/pDsU6-GFP-sh-3 of recombinant expression vector pDsU6-GFP-sh-3 and the restructuring leukemia cell NALM-6/pDsU6-GFP-sh-luc containing carrier pDsU6-GFP-sh-luc is disturbed containing RNA.

The concrete grammar of above-mentioned transfection is as follows:

The NALM-6 cell density that counting step 1 is cultivated, gets containing 2 × 10 ⁶the nutrient solution of individual cell, centrifugal 5 minutes of 90g, abandons substratum, adopts 100 μ l electricity to turn liquid (Nucleofector Solution, Lonza) re-suspended cell; Transitional cell suspension is managed to 1.5ml Eppendorf, adds 2 μ g DNA plasmids, refers to that abdomen flicks abundant mixing; Cell/DNA suspension is transferred in electric revolving cup, avoids bubble, cover bowl cover; Electric revolving cup is positioned on the electric swivel base (Nucleofector Device, Lonza) of electroporation, selects electric carryover sequence C-005(to be specially applicable to NALM-6 clone); The RPMI-1640 substratum (10%FBS) getting 2ml preheating mixes with the cell suspension in electric revolving cup, transfers in Tissue Culture Dish, avoids repeatedly aspirating cell suspension; The cell of transfection is placed in incubator, 37 DEG C, 5%CO ₂continue in cell culture incubator to cultivate, for detection and the experiment of following embodiment.

Embodiment 3, Western Blot detect the expression of zinc finger protein CTCF in restructuring leukemia cell

Four kinds of restructuring leukemia cell NALM-6/pDsU6-GFP-sh-1 in Example 2 after transfection 72h, NALM-6/pDsU6-GFP-sh-2, NALM-6/pDsU6-GFP-sh-3 and NALM-6/pDsU6-GFP-sh-luc, extract total protein respectively, with GAPDH(glyceraldehyde-3-phosphate dehydrogenase) be internal reference, carry out Western blot detection, the primary antibodie detecting zinc finger protein CTCF is CTCF (molecular weight 83KDa) monoclonal antibody (purchased from Millipore), the primary antibodie detecting internal reference GAPDH is GAPDH(molecular weight 34KDa) monoclonal antibody (purchased from Chinese Shanghai Kang Cheng company), result as shown in Figure 1, result shows: the restructuring leukemia cell NALM-6/pDsU6-GFP-sh-1 disturbing recombinant expression vector pDsU6-GFP-sh-1 containing RNA, disturb the restructuring leukemia cell NALM-6/pDsU6-GFP-sh-2 of recombinant expression vector pDsU6-GFP-sh-2 containing RNA and disturb containing RNA in the restructuring leukemia cell NALM-6/pDsU6-GFP-sh-3 of recombinant expression vector pDsU6-GFP-sh-3, zinc finger protein CTCF expression level is all lower than the restructuring leukemia cell NALM-6/pDsU6-GFP-sh-luc of contrast containing carrier pDsU6-GFP-sh-luc, wherein RNA disturbs recombinant expression vector pDsU6-GFP-sh-1 and RNA to disturb the interference effect of recombinant expression vector pDsU6-GFP-sh-3 better.

The concrete grammar of said extracted total protein is as follows:

90g collects restructuring leukemia cell in centrifugal 5 minutes, washes twice, add 150 ~ 300 μ l RIPA damping fluid [20mM Tris pH7.5,50mM NaCl, 2mM Na with the PBS of precooling ₃vO ₄, 10mM NaF, 1mM EDTA, 0.1%TritonX-100 and proteinase inhibitor (Roche)], cracking 30min on ice, collecting cell.8V voltage ultrasound 10 seconds × 2 times, 40 seconds, interval; 4 DEG C, the centrifugal 30min of 12000rpm; Draw supernatant, utilize Brandford method to carry out protein quantification; Respectively get 20 μ g total proteins and carry out Western blot detection.

The concrete grammar that Western blot detects is as follows:

1) electrophoresis and transferring film: carry out SDS-PAGE electrophoresis to protein sample to be measured, first electrophoresis 10-30 minute under voltage 80V, enters after separation gel until dye front, voltage is brought up to 160V and continues electrophoresis about 1 hour, until tetrabromophenol sulfonphthalein arrives the bottom of separation gel.After electrophoresis terminates, the protein sample of separation is transferred to nitrocellulose filter (NC film) by electricity consumption transfer method, 400mA(100V), 1 hour (or 350mA(95V), 1.5 hours).

2) closing membrane: wash (Nacl8.0g, 20ml1M Tris-Hcl, Tween-202ml, adding distil water 950ml demarcate pH value to 7.6, then be settled to 1L) NC film 10-15 minute with TBS-T solution.NC film is put into the TBS-T solution containing 5% skim-milk, room temperature closes 1 hour (or 4 DEG C of 12-24 hour).

3) immuning hybridization:

A, primary antibodie are hatched: be diluted in the TBS-T solution containing 5% skim-milk by CTCF monoclonal antibody or GAPDH monoclonal antibody by 1:2000, hatch together with NC film is on decolorization swinging table under room temperature about 1 hour (40 minutes to 1 hour 20 minutes), wash NC film 10 minutes with TBS-T solution 50mL, repeat 3 times.

B, two resists hatches: be diluted in the TBS-T solution containing 5% skim-milk by the goat anti-rabbit igg antibody in conjunction with horseradish peroxidase by 1:3000, hatch together with NC film is on decolorization swinging table under room temperature about 45 minutes (40 minutes to 1 hour 20 minutes), wash NC film 10 minutes with TBS-T solution 50mL, repeat 3 times.

4) ECL reagent colour development: use ECL protein hybridization detection kit (Amersham company of Sweden), carries out NC film color reaction with reference to operation instructions.

The apoptosis of embodiment 4, restructuring leukemia cell detects

Choose good recombinant expression vector pDsU6-GFP-sh-1 and pDsU6-GFP-sh-3 of RNA interference effect and carry out apoptosis detection.Three kinds of restructuring leukemia cells NALM-6/pDsU6-GFP-sh-1, NALM-6/pDsU6-GFP-sh-3 and NALM-6/pDsU6-GFP-sh-luc in Example 2 after transfection 72h, carry out statistical study with flow cytometer to percentage of cerebral apoptosis respectively, result as shown in Figures 2 and 3.

Result shows: the total apoptosis ratio of cell of restructuring leukemia cell NALM-6/pDsU6-GFP-sh-1, NALM-6/pDsU6-GFP-sh-3 and contrast (restructuring leukemia cell NALM-6/pDsU6-GFP-sh-luc) is respectively 7.5%, 28% and 7.4%, illustrate that RNA disturbs recombinant expression vector pDsU6-GFP-sh-3 can effectively reduce/suppress the expression of CTCF in cell, NALM-6/pDsU6-GFP-sh-3 cell population apoptosis ratio is 3.8 times of contrast (restructuring leukemia cell NALM-6/pDsU6-GFP-sh-luc).

The concrete grammar of above-mentioned flow cytometer statistics percentage of cerebral apoptosis is as follows:

300g collects restructuring leukemia cell in centrifugal 5 minutes, washes twice with the PBS of precooling, uses two transfect cell apoptosis detection kit (purchased from American BD company, the BD Pharmingen556547) labeled cell of AnnexinV-APC/PI.Adopt flow cytometer (U.S. company BD, FACSAria II) to collect the cell of 10000 GFP positives, statistical study is carried out to the apoptosis per-cent of cell, observe the Variation Features that leukemia cell ties up to apoptosis aspect.

Embodiment 3,4 proves, zinc finger protein CTCF is an anti-apoptosis factor, in ALL cell, have the effect of inhibited apoptosis, the expression of interference CTCF can promote tumour cell---there is apoptosis in leukemia cell, and increases the susceptibility of leukemia cell to chemotherapeutics.

Claims

1. form the short hairpin RNA of loop-stem structure, it is characterized in that: in described loop-stem structure, a chain-ordering of stem is SEQ ID No.1, in described loop-stem structure, another chain-ordering of stem is SEQ ID No.2.

2. short hairpin RNA according to claim 1, is characterized in that: the nucleotide sequence of described short hairpin RNA is SEQ ID No.3.

3. the DNA molecular of short hairpin RNA described in coding claim 1 or 2.

4., with the relevant biological material of short hairpin RNA described in claim 1 or 2, be 1)-5) in any one:

1) expression cassette containing DNA molecular described in claim 3;

2) recombinant vectors containing DNA molecular described in claim 3 or containing 1) recombinant vectors of described expression cassette;

3) recombinant microorganism containing DNA molecular described in claim 3 or containing 1) recombinant microorganism of described expression cassette or containing 2) recombinant microorganism of described recombinant vectors;

4) transgenic cell line containing DNA molecular described in claim 3 or containing 1) transgenic cell line of described expression cassette or containing claim 2) transgenic cell line of described recombinant vectors;

5) siRNA, the nucleotide sequence of a chain is as SEQ ID No.1, and the nucleotide sequence of another chain is as SEQ ID No.2.

5. treat and/or prevent leukemic product, the activeconstituents of described product comprises at least one in b1-b3:

B1, profit require the short hairpin RNA described in 1 or 2;

B2, DNA molecular according to claim 3;

B3, relevant biological material according to claim 4.

6. promote the product of apoptosis of leukemia, the activeconstituents of described product comprises at least one in b1-b3:

B1, profit require the short hairpin RNA described in 1 or 2;

B2, DNA molecular according to claim 3;

B3, relevant biological material according to claim 4.

7. reduce the purposes of the material of zinc finger protein CTCF genetic expression, described purposes is a1 or a2:

The application that a1, the material reducing zinc finger protein CTCF genetic expression treat and/or prevent in leukemic product in preparation;

The material of a2, reduction zinc finger protein CTCF genetic expression promotes the application in the product of apoptosis of leukemia in preparation; The material of described reduction zinc finger protein CTCF genetic expression is at least one in b1-b3:

B1, profit require the short hairpin RNA described in 1 or 2;

B2, DNA molecular according to claim 3;

B3, relevant biological material according to claim 4.

8. purposes according to claim 7, is characterized in that: the aminoacid sequence of described zinc finger protein CTCF is as shown in SEQ ID No.7, and the encoding sequence of described zinc finger protein CTCF gene is as shown in the 445-2628 position of SEQ ID No.6.