CN101892235B - MicroRNA used for inducing leukemia cell differentiation - Google Patents

Abstract

The invention provides microRNA used for inducing leukemia cell differentiation and application thereof in the induction of erythroid differentiation of tumor cell lines K562. The microRNA not only has the advantages of cheapness, no toxic or side effect, long duration, high transfection efficiency and the like, but also has the characteristics of stronger action and effectiveness of various indexes for tumor cell differentiation compared with a conventional human leukemia cell erythroid differentiation siRNA inducer.

Description

Be used for the microRNA and the application thereof of inducing leukemia cytodifferentiation

Technical field

The present invention relates to a kind of double stranded rna molecule and application thereof, particularly a kind of can the inducing leukemia cell red system differentiation double stranded rna molecule and application thereof.

Background technology

(for example see C.T.Jordan according to tumor stem cell is theoretical; M.L.Guzman, M.Noble, Cancer Stem Cells; N.Engl.J.Med.355 (2006) 1253-1261) and the unusual progress of tumour cell dysdifferentiation; The new approaches of oncotherapy have been proposed in recent years: the tumor inducing differentiation therapy, that is, utilize differentiating inducer that tumour cell is broken up to the normal cell direction; Alleviate or the malignant phenotype of reversing tumor cell; Thereby treatment tumour (for example seeing M.Leszczyniecka etc., Differentiation therapy of human cancer:basic science and clinicalapplications, Pharmacol.Therapeut.90 (2001) 105-156).Wherein vitamin A acid and arsenical induction-differential therapy promyelocytic leukemia; Clinical remission rate to the first visit patient can reach 90% (for example seeing Tumour stem cell-targeted treatment:eliminationor differentiation such as C.Massard, Annal.Oncology 17 (2006) 1620-1624).

Although experimental study has found much to induce differentiation agent; But the existing differentiation agent of inducing mainly is that micromolecular compounds such as retinoids, DMSO 99.8MIN. and some cytokines (are for example seen D.Zhang etc.; A critical role for the co-repressor N-CoR in erythroid differentiation andheme synthesis, Cell Res.17 (2007) 804-814; F.Wolter etc., Resveratrolenhances the differentiation induced by Butyrate in Caco-2 colon cancer cells, J.Nutr.132 (2002) 2082-2086; B.Zochodne etc.; Epo regulates erythroidproliferation and differentiation through distinct signaling pathways:implication for erythropoiesis and Friend virus-induced erythroleukemia, Oncogene 19 (2000) 2296-2304).

Wherein, Micromolecular compound has certain toxicity to human body more; Have easy relapse and produce chemical sproof problem, for example all-trans-retinoic acid can cause the vitamin A acid syndromes, but the severe patient threat to life (is for example seen P.Montesinos etc.; Differentiation syndrome in patients withacute promyelocytic leukemia treated with all-trans retinoic acid andanthracycline chemotherapy:characteristics; Outcome, and prognostic factors, Blood 113 (2009) 775-783); Cancer therapy drug cytarabine commonly used in the clinical chemotherapy for example again; Have very strong bone marrow inhibition and (for example see M.C.Cha etc.; Low dose docosahexaenoicacid protects normal colonic epithelial cells from araC toxicity, BMCPharmacol.23 (2005) 7).

Except that micromolecular compound class medicine; But the differentiation of some cytokine inducing tumor cells (is for example seen B.Zochodne etc.; Epo regulates erythroid proliferation and differentiationthrough distinct signaling pathways:implication for erythropoiesis and Friendvirus-induced erythroleukemia; Oncogene 19 (2000) 2296-2304); But the cytokine price is very expensive, has greatly limited its application clinically.

In addition; But some genes also differentiation of inducing tumor cell (are for example seen D.C.Tomlinson etc.; FGFR1 promotes proliferation and survival via activation of the MAPKpathway in bladder cancer, Cancer Res.69 (2009) 4613-4620; J.H.Schulte etc.; Lysine-specific demethylase 1 is strongly expressed in poorlydifferentiated neuroblastoma:implications for therapy; Cancer Res.69 (2009) 2065-2071); But because the gene transfection efficient that gene therapy exists is poor, the time length short, the gene integration site waits problem still unresolved at random, thereby the gene therapy means also are difficult in clinical popularizing in a short time.

Obtain small segment RNA interfering (the small interfering RNA of broad research in recent years; SiRNA) and non-coding microRNA (microRNA; MiRNA) become the technological important member of genetic expression inhibition because of its specific efficient restraining effect to the expression of target gene of sequence homology; And opened up the frontier of utilizing microRNA to prepare nucleic acid drug and (for example seen S.M.Elbashir etc.; Duplexes of 21-nucleotide RNAs mediate RNA interference in culturedmammalian cells, Nature 411 (2001) 494-498.).

Compare with gene therapy with traditional compounds medicine; Utilize the microRNA pharmacy to have the following advantages: microRNA is discerned target gene with sequence complementary mode and is suppressed it and express, thereby thereby can inference can to act on the quantity of little RNA (effective little RNA) of target gene performance biological effect more than traditional compounds medicine; Genetic engineering technique capable of using and library technology are carried out extensive high flux screening to microRNA; Need rely on cell inner expression different with the external source htrb gene, microRNA can be obtained by multiple modes such as chemosynthesis, in-vitro transcription, cell inner expressions, and cost is lower; The external source htrb gene is different with expressing, and the molecule of microRNA is little, is easy to transfered cell and plays a role; MicroRNA is easy to carry out genetic engineering modified and chemically modified, can improves specificity, validity, stability, targeted cells specificity of its effect etc.Therefore microRNA demonstrates huge application potential in nucleic acid pharmacy and gene therapy.

That has reported at present is directed against the known sequences Design with K562 clone or the relevant siRNA of its corresponding erythroleukemia cell differentiation, comprises the siRNA to 3 kinds of gene PU.1, CAT1, BP1.Wherein, PU.1siRNA is that MEL has in the differentiation of red system effect (for example see M.Papetti etc. to friend's cell only; Reprogramming Leukemia Cells toTerminal Differentiation and Growth Arrest by RNA Interference of PU.1, Mol.Cancer Res.5 (2007) 1053-1062; R.Blaybel etc.; Downregulation of theSpi-1/PU.1 oncogene induces the expression of TRIM 10/HERF 1; A key factorrequired for terminal erythroid cell differentiation and survival; Cell Res.18 (2008) 834-845); This clone is isolated clone from the friend's cell that the Friend virus induction produces, because generation, development and performance and the human erythroleukemia of this MEL have than big-difference, so the still difficult expectation of its effect to the human leukemia treatment.

In addition; The CAT1 siRNA that has reported the only effect of the cell speed of growth of influential K562 (for example sees T.MAEDA etc.; Changes of Differentiation and Proliferation in K562Cells with Various Levels of Knockdown of Cationic Amino Acid Transporter1, Drag Metab.Pharmacokinet.23 (2008) 181-187).

BP1 siRNA only has the effect of rising beta-globin expression amount (for example to see O.P.Zoueva etc. to the K562 cell; Inhibition of beta protein 1expression enhances beta-globinpromoter activity and beta-globin mRNA levels in the human erythroleukemia (K562) cell line, Exp.Hematol.32 (2004) 700-708).

Simultaneously, because the known gene that can be used as disease targets is very limited, thereby be limited in the very narrow scope to the possibility of known sequences Design siRNA.

Therefore; Need provide such siRNA tumor inducing to divide chemoattractant molecule: can not only have siRNA cheapness, have no side effect, advantageous effects such as longer duration, transfection efficiency height, and the effect of comparing with existing siRNA inductor is strong, effective to the multiple index of tumour cell.

Summary of the invention

In order to solve the problems referred to above that existing differentiating inducer exists; Expansion can be used for treating the siRNA sequence of tumour such as white blood disease; The inventor utilizes genetic engineering technique (to see A universal plasmidlibrary encoding all permutations of small interfering RNA such as WO2004/001044 and M.Chen of the inventor in the siRNA library at random from large vol; Proc.Natl.Acad.Sci.102 (2005) 2356-2361, its full content is incorporated this paper into to quote mode) in filter out and can induce the siRNA of people's chronic myeloid leukemia K562 clone to red corpuscle direction differentiation (differentiation of red system).

< 1>the invention provides a kind of double stranded rna molecule, its double-stranded complementary sequence is respectively the sequence that contains following sequence:

SEQ NO.1:5 ' ACAUGUACAUAGGGAUAUC3 '; With

SEQ?NO.2：5’GAUAUCCCUAUGUACAUGU3’，

Wherein 5 ' end and/or the 3 ' end at SEQ NO.1 and SEQ NO.2 is extended with 1～3 any Nucleotide.

< 2>another program of the present invention is a kind of double stranded rna molecule, and its double-stranded complementary sequence is respectively following sequence:

SEQ NO.1:5 ' ACAUGUACAUAGGGAUAUC3 '; With

SEQ?NO.2：5’GAUAUCCCUAUGUACAUGU3’。

< 3>the present invention also provides the as above application of < 1>or < 2>described double stranded rna molecule in preparing the medicine that inducing tumor cell is the differentiation of the red system of K562.

< 4>double stranded rna molecule of the present invention can make the CD235 of tumor cell line K562 express increases, ε-globin is expressed increases, γ-globin expression increases, beta-globin is expressed increases, GATA-2 expresses reduction and/or cell proliferation rate slows down.

< 5>the invention still further relates to like the application of < 1>or < 2>described double stranded rna molecule in the medicine of preparation treatment people chronic myeloid leukemia.

< 6>the present invention also provides the dna molecular of coding as < 1>or < 2>described double stranded rna molecule.

< 7>the present invention also provides the nucleic acid carrier that comprises like < 5>said dna molecular.

< 8>the present invention also provides like < 5>described dna molecular and/or as the application of < 6>described nucleic acid carrier in preparing the medicine that inducing tumor cell is the differentiation of the red system of K562.

< 9>dna molecular of the present invention and/or nucleic acid carrier can make the CD235 of tumor cell line K562 express increases, ε-globin is expressed increases, γ-globin expression increases, beta-globin is expressed increases, GATA-2 expresses reduction and/or cell proliferation rate slows down.

< 10>the present invention also provides like < 5>said dna molecular or as the application of < 6>described nucleic acid carrier in the medicine of preparation treatment people chronic myeloid leukemia.

Surprisingly, the people's of inducing chronic myelogenous leukemia K562 cell of the present invention does not have report at present as yet to the sequence of the clone-67 siRNA of red corpuscle direction differentiation (differentiation of red system).Clone-67siRNA of the present invention has and induces the effect of people's chronic myelogenous leukemia K562 cell to red corpuscle direction differentiation (differentiation of red system), and the K562 cell differentiation is increased, its rate of propagation that slows down, thus alleviate its malignant phenotype.

Clone-67siRNA of the present invention screens the siRNA library from large vol at random, does not have homology with known any gene, therefore is different from the siRNA of existing treatment white blood disease or inducing leukemia cells in vitro differentiation.

Description of drawings

To combine accompanying drawing that the present invention is specifically described below, in the accompanying drawing:

Fig. 1 shows the CD235 positive cell ratio through the K562 cell of 12 siRNA transfections of random choose;

Fig. 2 shows that the clone-67siRNA transfection increases the CD235 expression of K562 cell;

Fig. 3 shows that the clone-67siRNA transfection increases ε-globin, γ-globin and the beta-globin expression amount of K562 cell;

Fig. 4 shows the GATA-2 of K562 cell after the clone-67siRNA transfection and the change of GATA-1 expression amount;

Fig. 5. the rate of propagation of K562 cell behind the transfection clone-67siRNA.

Embodiment

The large vol that the present invention uses siRNA library at random is that the large vol that the inventor sets up (is seen WO2004/001044 in the siRNA library at random; With M.Chen etc.; A universal plasmid libraryencoding all permutations of small interfering RNA; Proc.Natl.Acad.Sci.102 (2005) 2356-2361; Its full content is incorporated this paper into to quote mode), in this siRNA library about 3x10 is arranged ⁷Individual different siRNA sequence.

The K562 clone (available from ATCC, article No. CCL-243) that the present invention uses is an isolated clone from people's chronic myeloid leukemia patient's tumour cell, belongs to erythroleukemia cell system, is the cell model commonly used of research leukemia cell differentiation.The K562 cell a series of characteristic can occur and change after red corpuscle direction differentiation (differentiation of red system): for example CD235, globin are expressed and are increased, and along with the propelling of differentiation process, the expression of GATA-2 reduces gradually, and the expression of GATA-1 increases gradually; The K562 cell is the propelling along with differentiation process to red corpuscle direction differentiation (differentiation of red system) back, and cell proliferation rate slows down gradually, and withdraws from the cell cycle the most at last after the entering whole end differentiation and no longer breeds.

Term among this paper " differentiation of red system " is meant the regulation process of the low tumour cell of differentiation degree through a series of related gene expressions, to the high noble cells transforming process of the no virulent with red corpuscle character.The differentiation of red system is to be usually used in estimating cell differentiation in this area tumour cell to be broken up one of index of regulating effect with medicine.

CD235 does not express in hemopoietic forebody cell, and one of K562 cell characteristic change that meeting occurs after red corpuscle direction differentiation (differentiation of red system) is that CD235 begins to express and increase gradually, and mature erythrocyte keeps high-caliber CD235 amount.

The generation of oxyphorase is one of important symbol of red system differentiation; Oxyphorase is made up of protoheme and globin; What form oxyphorase mainly is α-Zhu Danbai and beta-globin, and beta-globin divides 5 kinds: ε-globin, γ (A and G)-globin, δ-globin and beta-globin.One of characteristic change that the K562 cell can occur after red corpuscle direction differentiation (differentiation of red system) is that the expression of the globin of some kind increases.

GATA-2 and GATA-1 be red be transcription factor important in the atomization.The K562 cell is the propelling along with differentiation process to red corpuscle direction differentiation (differentiation of red system) back, and the expression of GATA-2 reduces gradually, and the expression of GATA-1 increases gradually.

Utilizing the screening method among the embodiment 1 is that object filters out the CD235 positive cells with the K562 cell the siRNA library from above-mentioned large vol at random; SiRNA encoding sequence to contained in these positive cells checks order; Can learn to cause that CD235 expresses the sequence of the siRNA that increases, further the effect behind these siRNA importings K562 cell identified again.

With aforesaid method; We are from screen a siRNA:clone-67 that can induce the K562 cell to red corpuscle direction differentiation (differentiation of red system) the siRNA library at random, and the dna encoding sequence of its two complementary strands is respectively (direction is from 5 ' to 3 '): ACATGTACATAGGGATATC and GATATCCCTATGTACATGT.

But; The present invention is not limited to the sequence of clone-67 itself; The present invention comprises that also the sequence of clone-67siRNA is the basis; The sequence of respectively adding 1～3 any Nucleotide at its sequence two ends; This is because the mechanism of action of siRNA is the regulation and control to homologous sequence, has shown that the action effect of siRNA depends primarily on its intermediary core sequence, and (has for example seen Q.Du etc. near the base sequence of both sides to the influence of its action effect is less; A systematic analysis of the silencingeffects of an active siRNA at all single-nucleotide mismatched target sites; Nucleic Acids Res.33 (2005) 1671-1677.), and add the action effect that 1～3 Nucleotide not only can not weaken this siRNA in the both sides of siRNA, the effect that also can strengthen siRNA (is for example seen S.D.Rose etc.; Functional polarity is introduced by Dicer processing ofshort substrate RNAs; Nucleic Acids Res.33 (2005) 4140-4156. and D-H Kim etc., Synthetic dsRNA Dicer sustrates enhance RNAi potency and efficacy, Nature Biotech.23 (2005) 222-226.).

Embodiment

The screening of embodiment 1:12 positive siRNA

The K562 cell is with adding 10% foetal calf serum (available from Sigma; Article No. 12003C) IMDM substratum is (available from Thermo-Fisher; Article No. SH30228.01B) in the cell culture incubator (available from Sanyo) of 37 ℃ of 5% carbonic acid gas, cultivates (only if point out in addition; Below among each embodiment K562 cell cultures mode identical therewith), will comprise 3x10 ⁷The library mixing of the siRNA at random plasmid of individual different siRNA encoding sequences is transfected into 6.7x10 with Lipofectamine 2000 (available from Invitrogen, article No. 11668-019) ⁷Individual K562 cell, after the transfection 48 hours, in nutrient solution, add G418 (available from Merck, article No. 345810; Final concentration 800ug/ml) kills not by cells transfected; After 14 days, cell concn is adjusted into 1x10 ⁷Individual cell/ml is according to per 10 ⁶The ratio that individual cell adds antibody 1ug with anti-CD235 antibody (available from R&D, article No. MAB1228) in 4 ℃ of labeled cell half a hour, adding again

Pan Mouse IgG (available from Invitrogen, article No. 110-41) magnetic bead (that is, is washed cell once with the solution in the 10ml test kit 1, cell is resuspended in the solution 1 concentration 1x10 according to the method for magnetic bead specification sheets ⁷Individual cell/ml adds magnetic bead 1ml, and 4 ℃ of half a hour, the volume solution 1 that doubles, magnetic bead was inhaled 2 minutes, abandoned supernatant, washed magnetic bead 3 times with solution 1, abandoned supernatant, obtained the CD235 positive cells) filter out the CD235 positive cells.

DNA in the CD235 positive cell that filters out more than the extraction; Pcr amplification goes out siRNA encoding sequence wherein; Be cloned into the pCUH carrier, connect the product transformed into escherichia coli, 100 order-checkings of picking at random from transformant; Again from the siRNA encoding sequence that order-checking obtains at random 12 of pickings verify (among the embodiment 2 detail) separately; Promptly respectively with these 12 siRNA according to its encoding sequence chemosynthesis with it corresponding double-stranded siRNA (synthetic) by Invitrogen, to get rid of the non-specific influence that carrier maybe pair cell produces, induce the effect of red system differentiation to verify each siRNA.

An embodiment 2:12 siRNA expresses in various degree the CD235 of K562 cell to be increased

Except that 12 at random the synthetic siRNA, set up a negative control group (Con): only add transfection reagent HiPerFect Transfection Reagent during transfection, do not add the siRNA group.

Method with embodiment 1 is cultivated the K562 cell; And when reaching (about 60% o'clock of degree of converging) at cell; (available from Qiagen, Cat#301704) with the double-stranded independent transient transfection K562 cell respectively of these siRNA, the concentration of siRNA is 25nM during each transfection with HiPerFect Transfection Reagent; Carried out for the first time the transfection second time after the transfection in 60 hours; With 48 hours 72 hours collecting cells after the transfection second time, be used to detect the index of red system differentiation, thereby identify that these siRNA induce the effect of red system differentiation.

The CD235 that detects respectively with the K562 cell of 12 siRNA transient transfections expresses: each organizes 72 hours collecting cells after twice transfection of cell; With the anti-CD235 antibody of FITC mark (available from BD Pharmingen; Article No. 559943) 4 ℃ of dyeing is 1 hour, and PBS (phosphate buffered saline buffer) washes behind the cell 3 times the expression with the Accuri C6 flow cytometer detection CD235 of Accuri company.

Test result is as shown in Figure 1; Fig. 1 is the flow cytometer detection figure of CD235 positive cell ratio after each siRNA transfection; Therefrom can find out; Have behind 6 transient transfection K562 cells CD235 positive cell percentage than negative control (Con) was increased to 1.5 times or more in 72 hours among 12 siRNA; Wherein clone-67siRNA degree that positive cell percentage is raise is the highest, estimates therefore that clone-67siRNA has the K562 cell the strongest to induce red to be differentiation capability, to choose the detection that this clone-67 carries out following a plurality of red systems differentiation index.

Embodiment 3:clone-67siRNA expresses the CD235 of K562 cell to be increased

The training method of CD235 cell and rotaring transfecting mode are with embodiment 2; The clone-67siRNA that in embodiment 2, obtains; All identification experiments all set up two negative control group (only if point out in addition; Below negative control among each embodiment identical therewith): only add transfection reagent HiPerFect Transfection Reagent during transfection, do not add siRNA group (Con group); The negative control Allstars negative control siRNA that transfection does not act on any gene (available from Qiagen, Cat#1027280) organizes (Allstars group).

Detection is expressed with the CD235 of the K562 cell of clone-67siRNA, Con and Allstars transient transfection: each organizes 72 hours collecting cells after twice transfection of cell; With the anti-CD235 antibody of FITC mark (available from BD Pharmingen; Article No. 559943) 4 ℃ of dyeing is 1 hour, and PBS (phosphate buffered saline buffer) washes behind the cell 3 times the expression with the Accuri C6 flow cytometer detection CD235 of Accuri company.Test sample preparation and measuring method: 5x10 in the 100ulPBS damping fluid ⁵Individual cell adds 0.5ug antibody, and flow cytometer is collected 20000 cells at the 525nm place with instrument setting program Protocol:525nm.PRO and analyzed.More than experiment is all independent repeats more than 3 times.

Test result is shown in Fig. 2 and table 1; Fig. 2 A is the flow cytometer detection figure of CD235 expression amount; The cell flow cytometer showed that wherein left figure is Con, middle graph are the cell flow cytometer showeds of Allstars, and right figure is the cell flow cytometer showed of clone-67siRNA; Fig. 2 B is the stacking diagram of above-mentioned 3 analytical resultss, and Fig. 2 C is the histogram that shows the per-cent of above-mentioned 3 groups CD235 positive cell.Mean+SD among the figure is the MV and the standard deviation of 3 independent repeated experiments.* represents significant t-test p＜0.01.Therefrom can find out with the Con negative control and compare; Can make the CD235 positive cell percentage be increased to average 61.46% in 72 hours behind the clone-67siRNA transient transfection K562 cell from average 16.57%; There were significant differences for statistical analysis; And the CD235 expression intensity of cell significantly increases, and the Allstars negative control does not have obviously change to the CD235 expression of K562 cell.

The per-cent of table 1.CD235 positive cell

Embodiment 4:clone-67siRNA increases the expression of ε-globin, γ-globin and the beta-globin of K562 cell

The training method of CD235 cell and rotaring transfecting mode are with embodiment 2; Detection is with the relative expression of ε-globin, γ-globin and the beta-globin of the K562 cell of clone-67siRNA, Con and Allstars transient transfection: detect the expression amount of globin with respect to the internal control gene beta-actin with the Taqman fluorescence quantitative PCR method, Taqman primer and probe (table 2) are by PrimerExpress 2.0 software designs of ABI company.Primer is synthetic by Invitrogen company, and probe is synthetic by Takara company.

Extract cell total rna with Trizol (available from Invitrogen, article No. 15596-026), process for extracting is: the petridish area by every 3.5cm diameter adds Trizol 1ml, fully the mixing lysing cell; Room temperature was placed 5 minutes, and every 1ml Trizol adds the 0.2ml chloroform, violent mixing, and room temperature was placed 3 minutes; Centrifugal 15 minutes of 4 ℃ of following 12000g get upper phase, add the 0.5ml Virahol by every 1ml Trizol amount, fully mixing; Room temperature was placed 10 minutes, and centrifugal 10 minutes of 4 ℃ of following 12000g inhale and abandon supernatant, wash RNA with 75% ethanol and precipitate once; The RNA deposition is dried, and with no RNase water dissolution, ultraviolet is quantitative.Getting the total RNA of 3ug is cDNA with

II Reverse Transcriptase (available from Invitrogen) method reverse transcription to specifications; Reaction volume is 20ul; From product, take out 1ul and carry out bifluorescence quantitative PCR reaction (instrument parameter) at the iQ5 of Biorad company quantitative real time PCR Instrument with iQ Multiplex Powermix (available from Bio-Rad); Quantitative fluorescent PCR reaction parameter: 95 ℃ of the first steps 3 minutes; Second the step 95 ℃ 15 seconds; 55 seconds 57 ℃ of the 3rd steps, second, third step repetition 40 circulations.The expression amount of globin is that standard is carried out stdn with the internal control gene beta-actin earlier; The stdn globin expression amount of transfection clone-67siRNA group is that standard is carried out stdn with the stdn globin expression amount (being set at 1.0) of Con group further again, and the result is expressed as relative expression's multiple of globin.More than experiment is all independent repeats 3 times, and the result is expressed as the mean+SD of 3 experimental datas.

Table 2 is used for the Taqman primer and the probe sequence of quantitative PCR experiment

Primer and probe sequence are 5 ' → 3 '

Test result is as shown in Figure 3; A has shown the relative expression quantity of above-mentioned 3 groups ε-globin; B has shown the relative expression quantity of above-mentioned 3 groups γ-globin, and C has shown the relative expression quantity of above-mentioned 3 groups beta-globin, all is expressed as means standard deviation (n=3).* represents significant t-test p＜0.01.Compare by scheming can know with the Con negative control, can make the expression of ε-globin, γ-globin and the beta-globin of cell increase by 1.9,1.6 and 2 times respectively behind the clone-67siRNA transient transfection K562 cell in 72 hours, there were significant differences for statistical analysis.The Allstars negative control is expressed not have obviously to the globin of K562 cell and is changed.

Embodiment 5:clone-67siRNA makes the GATA-2 of K562 cell express reduction

The training method of CD235 cell and rotaring transfecting mode detect GATA-2 and GATA-1 expression with the K562 cell of clone-67siRNA, Con and Allstars transient transfection with embodiment 2.Detect according to standard Western method, wherein anti-GATA-2 and GATA-1 polyclonal antibody and anti-beta-actin monoclonal antibody are all available from Santa Cruz (article No. sc-9008 and sc-13053).Two anti-are Goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP (all available from SantaCruz, article No. sc-2004 and sc-2005).The colouring reagents box is ECL Plus Western BlottingDetection Reagents (available from Amersham&Phamacia, article No. RPN2132).Concrete experimental procedure is: conventional polyacrylamide gel electrophoresis, electrophoresis finished the back and utilize and change film appearance (available from Bio-Rad, article No. 170-3940) and by changeing film appearance specification sheets electrophoresis albumen is gone on the pvdf membrane, with skimmed milk confining liquid room temperature closing membrane 1 hour;, with a dilution in anti-1: 400 film is immersed in the anti-diluent with Incubating Solution, 4 ℃ are spent the night; Wash film 3 times with TBS washing lotion room temperature, each 15 minutes, resist dilution in 1: 4000 with two with Incubating Solution; Film is immersed in the two anti-diluents, and room temperature 1 hour is washed film 3 times with TBS washing lotion room temperature; Each 15 minutes, film is taken out, press ECL Plus Western BlottingDetection Reagents test kit specification sheets; With the ECL reagent mulch film in the test kit, room temperature was placed after 1 minute, exposure X-ray sheet.The concrete prescription of confining liquid, Incubating Solution and TBS washing lotion that above Western blot is used disposes according to the molecular biology textbook.More than experiment is all independent repeats 3 times.

Detected result is as shown in Figure 4, and A shows the influence to the expression amount of GATA-2, and B shows the influence to the expression amount of GATA-1.From figure, can know; Compare with the Con negative control; Can make the GATA-2 of cell express reduction behind the clone-67siRNA transient transfection K562 cell in 72 hours, and the expression of GATA-1 does not have obvious change, the Allstars negative control does not all have obviously change to the GATA-2 and the GATA-1 expression of K562 cell.

Embodiment 6:clone-67siRNA slows down the rate of propagation of K562 cell in soft agar and liquid nutrient medium

The training method of CD235 cell and rotaring transfecting mode be with embodiment 2, soft agar colony growth experiment: according to CytoSelect ^TMThe specification sheets of 96-Well Cell Transformation Assay kit (available from CellBiolabs) experimentizes.Every group of (every group of 3 holes) cell all is inoculated on 96 orifice plates by the density in 3000/hole, cultivates after 8 days under 37 ℃ of 5%CO2 conditions the cell count in the colony is carried out detection by quantitative with the multi-functional microplate tester of FLUOstar (available from BMG) in 485/520nm (excitation/emission) wavelength.

The detection of cell proliferation rate in the liquid nutrient medium: the specification sheets according to CellTiter 96 aqueousone-solution cell proliferation assay kit (available from Promega) experimentizes, and measures light absorption value at the 492nm place with the multi-functional microplate tester of FLUOstar (every group of 3 holes) (available from BMG).More than experiment is all independent repeats 3 times.

Experimental result is as shown in Figure 5, and A respectively organizes the rate of propagation of cell on soft agar, and B is the rate of propagation of respectively organizing in the cell liquid medium within.Therefrom can find out, compare with the Con negative control, clone-67siRNA transient transfection K562 cell after 48 hours the rate of propagation of cell on soft agar slow down, statistical analysis is variant; Rate of propagation in the liquid medium within also slows down, and there were significant differences for statistical analysis.The result is expressed as the mean+SD of 3 experimental datas.* represents significant t-test p＜0.01, and * represents significant t-test p＜0.05.

Those skilled in the art will recognize that; Can easily design the basis that other is equal to embodiment according to disclosure and embodiment in the above description; Thereby realize the purpose identical with the present invention; For example the sequence with clone-67siRNA is the basis, carries out respectively adding 1～3 any Nucleotide at its sequence two ends and also can obtain the same differentiation effect of inducing, and this is because the mechanism of action of siRNA is the regulation and control to homologous sequence; The action effect that has shown siRNA depends primarily on its intermediary core sequence; And less near the base sequence of both sides to the influence of its action effect, and add the action effect that 1～3 Nucleotide not only can not weaken this siRNA in the both sides of siRNA, also can strengthen the effect of siRNA.One skilled in the art will further recognize that this embodiment that is equal to does not deviate from the spirit and scope of the invention of illustrating in the appended claims.

Claims (9)

1. double stranded rna molecule, its double-stranded complementary sequence is respectively following sequence:

SEQ NO.1:5 ' ACAUGUACAUAGGGAUAUC3 '; With

SEQ?NO.2：5’GAUAUCCCUAUGUACAUGU3’。

2. the application of double stranded rna molecule as claimed in claim 1 in preparing the medicine that inducing tumor cell is the differentiation of the red system of K562.

3. application as claimed in claim 2, wherein said double stranded rna molecule are expressed the CD235 of tumor cell line K562 to be increased, ε-globin is expressed increases, γ-globin expression increases, beta-globin is expressed increases, GATA-2 expresses reduction and/or cell proliferation rate slows down.

4. the application of double stranded rna molecule as claimed in claim 1 in the medicine of preparation treatment people chronic myeloid leukemia.

5. the encode dna molecular of double stranded rna molecule as claimed in claim 1.

6. the nucleic acid carrier that comprises dna molecular as claimed in claim 5.

7. the application in preparing the medicine that inducing tumor cell is the differentiation of the red system of K562 of dna molecular as claimed in claim 5 and/or nucleic acid carrier as claimed in claim 6.

8. application as claimed in claim 7, wherein said dna molecular and/or said nucleic acid carrier are expressed the CD235 of tumor cell line K562 to be increased, ε-globin is expressed increases, γ-globin expression increases, beta-globin is expressed increases, GATA-2 expresses reduction and/or cell proliferation rate slows down.

9. the application in the medicine of preparation treatment people chronic myeloid leukemia of dna molecular as claimed in claim 5 and/or nucleic acid carrier as claimed in claim 6.

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