CN1138784C - Antisense nucleic acid pointing at urokinase type plasminogen activating agent gene for inhibiting carcer - Google Patents
Antisense nucleic acid pointing at urokinase type plasminogen activating agent gene for inhibiting carcer Download PDFInfo
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Abstract
The present invention relates to a series of antisense nucteic acid with specific sequences synthesized by a chemical method, and the base array sequence is complementary with a section of sequences of mRNA of an urokinase type plasminogen activator (u-PA) in human bodies. The oligonucleotide can be specifically hybridized with the messenger ribonucleic acid (mRNA) of the urokinase type plasminogen activator (u-PA) of human bodies in cells, and thereby, the expression of u-PA proteins encoded by u-PA genes is blocked to achieve the action of inhibiting the growth, the infiltration and the transfer of tumor cells. The antisense oligonucleotide can be developed into medicines for inhibiting the growth, the infiltration and the transfer of tumor of human bodies.
Description
The present invention relates to suppress the oligonucleotide of human body tumour cell growth and transfer, relate to specifically suppress the human tumor growth, soak into and shift at the oligomeric deoxynucleotide of urokinase type plasminogen activator (u-PA) and contain the composition of described oligonucleotide, and described oligonucleotide is used to prepare the medicine that suppresses tumor growth, infiltration and transfer.
Oligonucleotide set forth in the present invention, the one section sequence complementation with human body u-PA gene that puts in order of its base, these oligonucleotide are hybridized with the messenger RNA(mRNA) (mRNA) of human body u-PA gene in cell specifically, thereby the u-PA protein expression that stops the u-PA genes encoding reaches the effect that suppresses growth of tumour cell, infiltration and transfer.
U-PA is a kind of serine proteinase enzyme like plasmin, and molecular weight is about 50-60KD, is a glycoprotein.Contain two disulfide linkage, the C end is the serineprotein kinase structural domain, and the N end contains a kringe domain and somatomedin structural domain.U-PA at first found (Gandolfo GM et al.Anticancer Res.1996 by Astedt etc. in 1976; 16:2155-2160).
U-PA is by the vascular endothelial cell expression-secretion, is transformed into activated double-stranded u-PA by strand non-activity uPA (pro-u-PA) through catalysis such as plasmin, cathepsin B, L and Hageman factor α, prostate specific antigen fracture peptide bond K158-I159.(Hoyer-Hansen?G，et?al，EurJ.Biochem.1997，243：21-26)。The concentration of u-PA is approximately 20pm in the blood plasma, and major part is and the formed mixture of its inhibitor (PAI-1) that small portion is the pro-u-PA form.The u-PA system component be expressed in regulation and control (Dear DE, et al.Fibrinolysis, 1995 that are subjected to hormone, somatomedin and division of cytoplasm element etc. to a great extent; 9:321-330).
Our selected antisense nucleic acid is by hybridizing with the mRNA of u-PA is complementary, suppressing mRNA by number of mechanisms and express u-PA albumen.Because antisense nucleic acid can specific combining target mRNA, can design a kind of antisense nucleic acid, require itself and tumour that the mRNA complementation of relevant u-PA takes place, the specificity selection of height also can make it not produce the toxic side effect that present other drug produces.
The present invention filters out the antisense nucleic acid special to u-PA in cell cultures, be incorporated on the mRNA of u-PA gene, demonstrates the inhibition to the mRNA marking protein of u-PA.
Recently studies show that u-PA is in the transfer of kinds of tumor cells, invading aspects such as tissue and capillary vessel generation has crucial effect.The transfer of tumour cell mainly depends on its infiltration capability to healthy tissues.At the bottom of nineteen ninety-five, in " key factor of cancer development and transfer " symposium that houston, u.s.a is held, name is devoted to explore that the expert of cancer metastasis is consistent thinks that the key of cancer metastasis is to break through " blockade " of basilar membrane surplus in the of 500.What this wherein mainly relied on is many enzymes that tumour cell produces, these enzyme hydrolyzable basilar membrane and extracellular matrixs, and it is thixotropic loose that cell-cell-cell one basilar membrane is connect, thereby strengthened the ability that tumour cell is invaded and shifted.In this process, u-PA plays crucial effect.(Rabbani?SA，In?vivo?98，12(1)：135-142)
In addition, u-PA can induce some cytokines to generate, as TGF-α, and VEGF, bFGF etc. bring out capillary vessel and generate, after tumour cell shifts, play an important role aspect the vasculogenesis (Hinsbergh VM, EXS.97,79:391-411).
The u-PA overexpression is the omen of many malignant tumours, as (Schmitt M.J.Obstet Gynaecol.1995,21 (2): 151-165) such as cancer of the stomach, lung cancer, mammary cancer, ovarian cancers.
In malignant tumour, the level of u-PA is significantly higher than the carcinoid level in healthy tissues or same source.And u-PA, u-PA-R, PAI-1, the level of PAI-2 has sizable variation and relevant with patient's prognosis in malignant tumour.In mammary cancer, its no knurl existence of the active high expression level person of u-PA is significantly shorter than the active low expresser of u-PA, and in all Prognosis in Breast Cancer factors, the u-PA antigen levels is the most significant.U-PA and special acceptor (u-PA-R) thereof and inhibition (PAI-1) also have very big dependency with hepatocellular carcinoma (HCC) infiltration, transfer and prognosis.Zheng Qi etc. (" Chinese tumour magazine " 1998,1) find that u-PA, u-PA-R and PAI-1 express obviously rising in HCC, and u-PA and u-PA-R and HCC soak into closely related, and u-PA and PAI-1 are positive, and HCC patient's prognosis is relatively poor.
U-PA, u-PAR density is relevant with the prostate cancer process, and density is low in the prostate gland of hyperplasia of prostate, and density is very high in the malignant prostate tissue, the density that has the prostate cancer of transfer more not shift is much higher, and (Miyake H, Int J.Oncol.1999 14 (3): 535-541).
One of purpose of the present invention is that a kind of antisense dna oligo (DNA) or Yeast Nucleic Acid (RNA) sequence can be provided, but it is characterized in that the different zones of specificity in conjunction with the mRNA of u-PA gene.
Another object of the present invention provides the pharmaceutical composition that contains antisense oligonucleotide of the present invention.
Another object of the present invention has provided the purposes that antisense oligonucleotide of the present invention is used to prepare antitumor drug.The method of using Northern Blotting has detected the content of u-PA mRNA among four kinds of clone: A549, MCF, C33A and U2-OS respectively.Wherein A549 is the human lung cancer cell line, and MCF is the sick clone of people's mammary gland fibrous capsule, and C33A is people's cervical cancer tumer line, and U2-OS is people's a osteosarcoma cell line.A549 and C33A all have higher u-PA mRNA content, and u-PA mRNA content is seldom among MCF and the U2-OS.This explanation lung cancer and cervical cancer are more suitable for the medicine in the present invention.Similarly, other u-PA mRNA content tumors of higher also is applicable to the medicine among the present invention.
The present invention has also designed the antisense nucleic acid molecule of different zones of the mRNA of a series of u-PA that can be incorporated into the u-PA gene, the antisense nucleic acid that screening suppresses u-PA genetic expression specificity in cell cultures.Cell cultures adopts the A549 cell, sets up a negative control PAC23, a positive control PAC40, and all antisense nucleic acid molecule length of screening are 20 Nucleotide.The selection result shows that wherein nine antisense nucleic acides all have the characteristic of inhibition human body tumour cell growth in various degree, they are named respectively be PAC130, PAC131, PAC132, PAC133, PAC134, PAC135, PAC136, PAC137, PAC138, wherein PAC136 can suppress the activity of human body tumour cell growth the biglyyest.
It is as follows to have active PAC series antisense oligonucleotide based composition of the human body tumour cell growth of inhibition and order:
PAC130： 5’-CTC?GGT?GGC?CTG?CGG?CAG?GA-3’ 20mer
PAC131： 5’-AGC?AGG?GCT?CTC?ATG?GTG?GC-3’ 20mer
PAC132： 5’-GCA?CTC?TTG?GAC?AAG?CGG?CT-3’ 20mer
PAC133： 5’-AGG?CAG?ATG?GTC?TGT?ATA?GT-3’ 20mer
PAC134： 5’-GTG?ACT?TCA?GAG?CCG?TAG?TA-3’ 20mer
PAC135： 5’-CCA?TCT?GTG?CAG?AGC?CTA?TC-3’ 20mer
PAC136： 5’-ACA?AGT?TGC?TGG?TCA?GTA?AC-3’ 20mer
PAC137： 5’-CAG?CTC?TTA?CTC?ACA?CTT?AC-3’ 20mer
PAC138： 5’-CTG?AGA?CAG?TGC?TGG?TCA?CA-3’ 20mer
Set up one of positive control, PAC40, its sequence is as follows:
PAC40：5’-GAA?AAC?GTC?AGC?CAT?GGT?CC-3’20mer
Set up PAC23 of negative control, its sequence is as follows:
PAC23：5’-CAC?GGT?GCG?TCG?ACG?CAC?TA-3’20mer
More than designed antisense oligonucleotide all be that length is 20 Nucleotide and through the oligonucleotides of thio-modification, target of attack is the messenger RNA(mRNA) (mRNA) of human body u-PA gene.This section antisense nucleotide can combine with messenger RNA(mRNA) (mRNA) specificity of u-PA gene in tenuigenin, thereby the proteic expression of u-PA of blocking-up u-PA genes encoding can finally reach the effect that suppresses tumor growth and transfer like this.The designed antisense base sequences of the present invention is by bringing into play its distinctive biological characteristics with the specific hybrid of the mRNA of u-PA gene.At present, the hybridization avidity of RNA and mRNA has very high status than the hybridization avidity height of DNA and mRNA in the nucleic acid hybridization on the medical science pharmaceutical use.But because the artificial-synthetic DNA is again far away than low many of the cost of synthetic RNA, according to the needs of medical market and the cost requirement of clinical application, the antisense drug of using in clinical trial is DNA at present.The cost of synthetic RNA is high, and along with the continuous progress of science and technology and the improvement of technology, the cost of synthetic RNA will decrease in future, be expected to use clinically.Synthetic oligomerization Yeast Nucleic Acid (RNA) will be developed as new drug with the chimeric antisense nucleic acid that forms that links to each other of RNA (ribonucleic acid) monomer and thymus nucleic acid (DNA) monomer.
The antisense nucleic acid medicament of the present invention's design, it is active that its sequence has specific biological.For a certain gene locus complementary antisense nucleic acid and complementary length much relations are arranged, longer as complementary, then biologic activity is just higher, and the effect of curing the disease will be better, but the cost of the antisense nucleic acid of synthetic will increase, and does not so just meet antisense nucleic acid as medicinal purpose.The anti sense nucleotide sequence through 20 base length of screening is all adopted in this invention, increase or reduce an antisense nucleic acid that is complementary to same gene locus to several bases, equally also can have biologic activity in various degree, also can reach the effect of curing the disease in various degree.Being complementary near the homologous genes site antisense nucleic acid combining site simultaneously has in various degree overlapping, and these can think to have in various degree sequence homology, have identical biologic activity in various degree too.
Various chemical modification methods are a lot of in the antisense nucleic acid research now, why adopt the antisense nucleic acid of thio-modification, the preclinical studies such as pharmacology, pharmacokinetics, toxicology that mainly are the antisense nucleic acid of sulfo-are that research is the most comprehensive in the antisense nucleic acid of various chemically modifieds.The sulfo-antisense nucleic acid can prevent effectively that in vivo a large amount of exonucleases are cut Degradation to the enzyme of antisense nucleic acid in the human body, makes antisense nucleic acid keep its due biologic activity.Therefore the antisense nucleic acid of sulfo-also can excite the activity of RNA enzyme in addition, and degraded is the RNA chain of hybridization with it, selects for use the antisense nucleic acid of the particular design of thio-modification all to have in various degree biologic activity.
In sum, the antisense nucleic acid that is complementary to u-PA gene mRNA different zones has the human body tumour cell growth of inhibition and transfer character, treats tumour clinically and has using value very widely.Filter out the different lengths and the anti sense nucleotide sequence of homology in various degree through chemically modified, can develop into antitumor drug with clinical value.Antisense nucleic acid of the present invention can be combined with pharmaceutical carrier, be used for animal (comprising Mammals and people).Used pharmaceutical carrier can be phosphate buffered saline buffer (pH7.4) and other carrier and damping fluids commonly used.Marginal data:
Fig. 1: the content (Northern Blotting result) of u-PA mRNA in different clones.
The method of using Northern Blotting has detected the content of u-PA mRNA among four kinds of clone: A549, MCF, C33A and U2-OS respectively.A549 is the human lung cancer cell line, and MCF is the sick clone of people's mammary gland fibrous capsule, and C33A is people's cervical cancer tumer line, and U2-OS is people's a osteosarcoma cell line.Cultivate under suitable condition respectively, extract the total RNA of cell with RNAzol B then, content with every hole 5ug carries out electrophoresis, transfers on the nitrocellulose filter, carries out nucleic acid hybridization (Northern blot) with the u-PA probe of DIG mark and analyzes (as Fig. 1).
As seen from the figure, A549 and C33A all have higher u-PA mRNA content, and u-PA mRNA content is seldom among MCF and the U2-OS.
Fig. 2: antisense nucleic acid Pac series is to the influence (Northern Blotting result) of u-PA mRNA content
The A549 cell strain is divided into control group (negative and positive) and antisense nucleic acid treatment group, in containing 10% foetal calf serum HAM ' s S/F-12 nutrient solution, cultivate, being cultured to total cellular score is 100,000 o'clock, change cell culture fluid into serum-free medium, the not homotactic sulfo-antisense nucleic acid that adds the liposome of 20 μ g/ml and final concentration and be 800nM was handled 5 hours, change ordinary culture medium again into, recovered 20 hours, extract the total RNA of cell with RNAzol B then and carry out u-PA mRNA quantitative analysis.With the probe of the probe of u-PA gene and G3PDH (glyceraldehyde 3-phosphate dehydro-genase) gene with carry out nucleic acid hybridization (Northern blot) analysis through electrophoretic separation and the cell total rna sample transferred on the nitrocellulose membrane, as internal reference, the result as shown in Figure 2 with the mRNA of G3PDH gene.
No. 23 negative contrasts of sample among the figure, No. 40 is the antisense nucleic acid at PKC-α gene, as positive control, other are the antisense nucleic acid sample of different Pac series.Visible Pac series antisense nucleic acid is to the different inhibition activity of u-PA mRNA among the figure, and is the highest with the inhibition degree of Pacl36.
Embodiment 1: antisense nucleic acid is attacked the synthetic of determining of target position target and antisense nucleic acid
The 391-04 type DNA-RNA synthesizer that uses U.S. applying biological Account Dept of PE company (Applied Biosystems) to make.Antisense nucleic acid with phosphorous acid acid amides solid phase synthesis sulfo-.
Synthesis material is available from U.S. Glen company, and main raw material has:
Four kinds of di-isopropyl-cyanoethyl phosphorous acid amide monomers: Desoxyadenosine (dA), pancreatic desoxyribonuclease (dG), Deoxyribose cytidine (dC), deoxythymidine (dT).
Four kinds of control aperture glass powder (CPG).Solid phase synthesis post (A, C, G, T), scale is 10 micromoles.
Block reagent:
Block reagent A: diacetyl oxide, lutidine, tetrahydrofuran (THF)
Block reagent B: monomethyl imidazoles, tetrahydrofuran (THF)
Thio reaction reagent: Beaucage reagent, acetonitrile
Detritylation reagent: trichoroacetic acid(TCA), methylene dichloride
Liquid reagent: acetonitrile, trichloromethane
Synthetic scale is 10 micromoles, and the base monomer is dissolved in the anhydrous acetonitrile, and concentration is 100 mmoles, and other reagent concentration and consumption all carry out according to the instrument handbook.
Itself can use time variable control the building-up process instrument.
Synthetic product cuts down with strong aqua (28%) and is collected in the withstand voltage vial of sealing, handles various hydroxy-protective groups and the amido protecting group of sloughing on the antisense nucleic acid molecule in 15 hours in 55 ℃.After deprotection finishes; most of ammonia is removed in volatilization; freeze-drying gets the white powder crude product; again be dissolved in the damping fluid; carry out purifying with Pharmacia Source 30Q chromatography column and AKTA chromatographic system; purification of samples G-15 molecular sieve sephadex chromatography post desalination, freeze-drying gets the pure product of white cotton-shaped antisense nucleic acid, is stored in-30 ℃.The sample that takes a morsel carries out following analysis:
1, surveys its purity and molecular weight with capillary electrophoresis;
2, survey its purity with high performance liquid chromatography (HPLC);
3, the dna sequencing method is measured its sequence accuracy;
4, P
31NMR (nucleus magnetic resonance) method is measured its degree of thioation;
Capillary electrophoresis and high-efficient liquid phase technique purity check result show that the pure product purity of Pac series antisense nucleic acid is all greater than 90%.
Sequencing proof Pac series anti sense nucleotide sequence is identical with implementation sequence.
P
31NMR analyzes and shows that Pac series antisense nucleic acid sulfo-rate is greater than 98.5%.
Embodiment 2: at the Pac series antisense nucleic acid of u-PA
The inhibition of in culturing cell, u-PA being expressed (Northern Blot)
Select for use human body lung cancer cell line A549 as the screening experiment cell strain, document (Miwa.W.et with reference to Miwa.W etc., al.1994 Biological Chemistry Lipopeseyler, 375 (10): 705-709), test is grouped into: negative control group, positive controls, antisense nucleic acid treatment group.
The A549 cell strain is cultivated in containing 10% foetal calf serum HAM ' s S/F-12 nutrient solution, being cultured to total cellular score is 100,000 o'clock, change cell culture fluid into serum-free medium, the not homotactic sulfo-antisense nucleic acid that adds the liposome of 20 μ g/ml and final concentration and be 800nM was handled 5 hours, change ordinary culture medium again into, recovered 20 hours, and extracted the total RNA of cell with RNAzol B then and carry out the quantitative analysis of u-PA gene mRNA.With the probe of the probe of u-PA gene and G3PDH (glyceraldehyde 3-phosphate dehydro-genase) gene with carry out nucleic acid hybridization (Northern blot) analysis through electrophoretic separation and the cell total rna sample transferred on the nitrocellulose membrane, with the mRNA of G3PDH gene as internal reference.The Molecular Cloning:A Loboratory Manual.2 that the Northern hybridization analysis is write with reference to Sambrook.J etc.
Nd.ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y..The result as shown in Figure 2.
No. 23 negative contrasts of sample among the figure, No. 40 is the antisense nucleic acid at PKC-α gene, as positive control, other are the antisense nucleic acid sample of different Pac series.Visible Pac series antisense nucleic acid is to the different inhibition activity of u-PA mRNA among the figure, and is the highest with the inhibition degree of Pac136.
Implement the structure of chemical composition of side 3:Pac136
With reference to Nucleic Acids Research.1995, Vol:24, the method for No:2.P4219-4223 is carried out sequencing to Pac136, and the result is as follows:
PAC136：5’-ACA?AGT?TGC?TGG?TCA?GTA?AC-3’ 20mer
Consistent with implementation sequence, with the molecular weight of capillary electrophoresis mensuration Pac136, the result is 6424, with theoretical molecular 6426 approximately equals.
Pac136 is the antisense nucleic acid that 20 deoxynucleosides link to each other with the phosphoric acid thioester bond, and the target target of its attack is the mRNA of u-PA gene.
Embodiment 4:Northern Blotting hybridizing method
Measure the content of u-PA mRNA in different clones
The method of using Northern Blotting has detected the content of u-PA mRNA among four kinds of clone: A549, MCF, C33A and U2-OS respectively.A549 is the human lung cancer cell line, and MCF is the sick clone of people's mammary gland fibrous capsule, and C33A is people's cervical cancer tumer line, and U2-OS is people's a osteosarcoma cell line.Cultivate under suitable condition respectively, extract the total RNA of cell with RNAzol B then, content with every hole 5ug carries out electrophoresis, transfers on the nitrocellulose filter, carries out nucleic acid hybridization (Northern blot) with the u-PA probe of DIG mark and analyzes (as Fig. 1).
As seen from the figure, A549 and C33A all have higher u-PA mRNA content, and u-PA mRNA content is seldom among MCF and the U2-OS.
Embodiment 5: the pharmaceutical composition that contains antisense nucleic acid
Antisense nucleic acid medicine composition as the animal vivo medicine-feeding is composed as follows:
Antisense nucleic acid Pac136 l0mg/ml
One hypophosphite monohydrate, one hydrogen sodium 1.73mg/ml
Seven hypophosphite monohydrate disodium hydrogen 14.33mg/ml
Sodium-chlor 4.4mg/ml
Water for injection is an amount of
Other several antisense oligonucleotides also adopt same prescription among the present invention.
Do not use superfluous words, antisense nucleic acid of the present invention to a great extent can by different chemically modifieds, adopt different length and in various degree the sequence of homology develop into antitumor drug with clinical value.It can use separately or unite use with other medicines and carry out tumor treatment, and these it seems it is conspicuous to one skilled in the relevant art.Sequence table (1), generalized case be the sequence sum (iii): 9 (1). No. 1 sequence situation
(i). sequence signature
(A). length: 20 bases
(B). type: nucleic acid
(C). link: strand
(D). configuration: linearity is (ii). and molecule type: picodna is (iv). antisense nucleic acid whether: be (xi). sequence explanation: No. 1 sequence PAC130
CTC GGT GGC CTG CGG CAG GA 20 (2). No. 2 sequence situation (i). sequence signature
(A). length: 20 bases
(B). type: nucleic acid
(C). link: strand
(D). configuration: linearity is (ii). and molecule type: picodna is (iv). antisense nucleic acid whether: be (xi). sequence explanation: No. 2 sequence PAC1 31
AGC AGG GCT CTC ATG GTG GC 20 (3). No. 3 sequence situation (i). sequence signature
(A). length: 20 bases
(B). type: nucleic acid
(C). link: strand
(D). configuration: linearity is (ii). and molecule type: picodna is (iv). antisense nucleic acid whether: be (xi). sequence explanation: No. 3 sequence PAC132
GCA CTC TTG GAC AAG CGG CT 20 (4). No. 4 sequence situation (i). sequence signature
(A). length: 20 bases
(B). type: nucleic acid
(C). link: strand
(D). configuration: linearity is (ii). and molecule type: picodna is (iv). antisense nucleic acid whether: be (xi). sequence explanation: No. 4 sequence PAC133
AGG CAG ATG GTC TGT ATA GT 20 (5). No. 5 sequence situation (i). sequence signature
(A). length: 20 bases
(B). type: nucleic acid
(C). link: strand
(D). configuration: linearity is (ii). and molecule type: picodna is (iv). antisense nucleic acid whether: be (xi). sequence explanation: No. 5 sequence PAC134
GTG ACT TCA GAG CCG TAG TA 20 (6). No. 6 sequence situation (i). sequence signature
(A). length: 20 bases
(B). type: nucleic acid
(C). link: strand
(D). configuration: linearity is (ii). and molecule type: picodna is (iv). antisense nucleic acid whether: be (xi). sequence explanation: No. 6 sequence PAC135
CCA TCT GTG CAG AGC CTA TC 20 (7). No. 7 sequence situation (i). sequence signature
(A). length: 20 bases
(B). type: nucleic acid
(C). link: strand
(D). configuration: linearity is (ii). and molecule type: picodna is (iv). antisense nucleic acid whether: be (xi). sequence explanation: No. 7 sequence PAC136
ACA AGT TGC TGG TCA GTA AC 20 (8). No. 8 sequence situation (i). sequence signature
(A). length: 20 bases
(B). type: nucleic acid
(C). link: strand
(D). configuration: linearity is (ii). and molecule type: picodna is (iv). antisense nucleic acid whether: be (xi). sequence explanation: No. 8 sequence PAC137
CAG CTCTTA CTC ACA CTT AC 20 (9). No. 9 sequence situation (i). sequence signature
(A). length: 20 bases
(B). type: nucleic acid
(C). link: strand
(D). configuration: linearity is (ii). and molecule type: picodna is (iv). antisense nucleic acid whether: be (xi). sequence explanation: No. 9 sequence PAC138
CTG?AGA?CAG?TGC?TGG?TCA?CA 20
Claims (3)
1, a kind of oligomerization antisense thymus nucleic acid (DNA) or Yeast Nucleic Acid (RNA), but it is characterized in that the different zones of specificity in conjunction with human body urokinase type plasminogen activator (u-PA) gene, and described oligonucleotide sequence is selected from:
1.PAC130：5’-CTC?GGT?GGC?CTG?CGG?CAG?GA-3’
2.PAC131：5’-AGC?AGG?GCT?CTC?ATG?GTG?GC-3’
3.PAC132：5’-GCA?CTC?TTG?GAC?AAG?CGG?CT-3’
4.PAC133：5’-AGG?CAG?ATG?GTC?TGT?ATA?GT-3’
5.PAC134：5’-GTG?ACT?TCA?GAG?CCG?TAG?TA-3’
6.PAC135：5’-CCA?TCT?GTG?CAG?AGC?CTA?TC-3’
7.PAC136：5’-ACA?AGT?TGC?TGG?TCA?GTA?AC-3’
8.PAC137：5’-CAG?CTC?TTA?CTC?ACA?CTT?AC-3’
9.PAC138：5’-CTG?AGA?CAG?TGC?TGG?TCA?CA-3’
2, contain anti sense nucleotide sequence in the with good grounds claim 1 and the medicine of pharmaceutically acceptable carrier.
3, be used to prepare the purposes of antitumor drug according to the antisense nucleic acid in the claim 1.
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