CN101914536B - Locked nucleic acid ribozyme of targeted serine-threonine protein kinase aurora kinase A and application thereof - Google Patents
Locked nucleic acid ribozyme of targeted serine-threonine protein kinase aurora kinase A and application thereof Download PDFInfo
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- CN101914536B CN101914536B CN2010102215584A CN201010221558A CN101914536B CN 101914536 B CN101914536 B CN 101914536B CN 2010102215584 A CN2010102215584 A CN 2010102215584A CN 201010221558 A CN201010221558 A CN 201010221558A CN 101914536 B CN101914536 B CN 101914536B
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Abstract
The invention relates to locked nucleic acid ribozyme of targeted serine-threonine protein kinase aurora kinase A, wherein the nucleotide sequence thereof is shown as SEQ IDNO:1 in a sequence table, 15 deoxynucleotides positioned at the middle part in the nucleotide sequence form a catalytic structure domain, 8 nucleotides positioned at the left side of the catalytic structure domain form a first substrate recognition structure domain, 8 nucleotides positioned at the right side of the catalytic structure domain form a second substrate recognition structure domain, and both the first substrate recognition structure domain and the second substrate recognition structure domain contain two locked nucleic acids tL. The locked nucleic acid ribozyme of the targeted serine-threonine protein kinase aurora kinase A can be applied to preparing medicines for treating prostatic cancer.
Description
Technical field
The present invention relates to target silk-serine-threonine protein kinase aurora kinase A ribozyme and the application thereof of (being called for short Aurora A, in the following content of specification sheets) with " Aurora A " expression silk-serine-threonine protein kinase aurora kinase A.
Background technology
Prostate cancer is one of modal malignant tumour, does not still have the specific treatment means so far, and the molecular mechanism that prostate cancer takes place is not clear fully as yet, but discovering in recent years, and the generation development of prostate cancer and Aurora A have closely related property unusually.The Aurora A assignment of genes gene mapping is in human chromosome 20q13, and amplification often appears in this zone in human malignancies, so gene therapy is worth exploring.
The contriver of present patent application once designed the DNAzyme DNAzyme2 that has synthesized at Aurora A, and (nucleotide sequence of DNAzyme2 is seen Cancer Gene Ther.200815 (8): 518), experiment shows, described DNAzyme2 can effectively suppress the expression of Aurora A in the prostate cancer cell, and suppresses the prostate cancer cell growth and (see Cancer Gene Ther.200815 (8): 517-525).But in order to improve the curative ratio of prostate cancer, bring benefit to the mankind, be necessary to obtain more ribozyme at AuroraA.
Summary of the invention
The locked nucleic acid ribozyme that the purpose of this invention is to provide a kind of novel target silk-serine-threonine protein kinase aurora kinase A (is called for short " Aurora A-LNAzyme ", in the following content of specification sheets, locked nucleic acid ribozyme with " Aurora A-LNAzyme " expression target silk-serine-threonine protein kinase aurora kinase A), so that develop the medicine of the treatment prostate cancer of better efficacy.
Aurora A-LNAzyme of the present invention, its nucleotides sequence classify as in the sequence table shown in the SEQ ID NO:1.In the described nucleotide sequence, 15 deoxynucleotide ggctagctacaacga that are positioned at the middle part form catalyst structure domain, are positioned at 8 Nucleotide t in catalyst structure domain left side
Lt
LAacagg forms the first substrate recognition structure territory, is positioned at 8 Nucleotide cct on catalyst structure domain right side
LGaaat
LForm the second substrate recognition structure territory, two lock nucleic acid t are all contained in the first substrate recognition structure territory and the second substrate recognition structure territory
L
Aurora A-LNAzyme of the present invention adopts the solid phase phosphoramidite triester method synthetic on automatic dna synthesizer.
Experimental results show that: Aurora A-LNAzyme of the present invention has suppressed the expression of Aurora A in prostate cancer cell; Obviously suppress the growth of prostate cancer transplanted tumor in the athymic mouse body, tumour inhibiting rate can reach 61%.Therefore, AuroraA-LNAzyme of the present invention can use in the medicine of preparation treatment prostate cancer.
The present invention has following beneficial effect:
1, the present invention design and synthesized a kind of locked nucleic acid ribozyme of novel target silk-serine-threonine protein kinase aurora kinase A is for treatment of prostate cancer provides new medicine.
2, experiment shows that Aurora A-LNAzyme of the present invention can suppress the expression of Aurora A in prostate cancer cell; Can obviously suppress the growth of prostate cancer transplanted tumor in the athymic mouse body, compare with DNAzyme2, stronger to the inhibition ability of tumor growth, serum half-life is longer, is expected to become more excellent efficient, special, stable prostate cancer inhibitor.
Description of drawings
Fig. 1 is the electrophorogram of Aurora A and the ribozyme electrophorogram to the external cutting of Aurora A, and among the figure, 1 is the electrophorogram (405bp) of Aurora A; 2 is through the electrophorogram of the Aurora A of contrast ribozyme processing, does not see the cutting band; 3 is the electrophorogram through the Aurora A of AuroraA-LNAzyme processing, visible 293bp and two cuttings of 112bp band.
Fig. 2 is Aurora A expressed proteic electrophorogram in prostate cancer cell PC3, and among the figure, 1 is Aurora A expressed proteic electrophorogram in the prostate cancer cell PC3 of untransfected ribozyme; 2 is Aurora A expressed proteic electrophorogram in the prostate cancer cell PC3 of transfection contrast ribozyme; 3 is Aurora A expressed proteic electrophorogram in the prostate cancer cell PC3 of transfection Aurora A-LNAzyme; β-actin is the internal reference of Western blot, shows among the figure its expression level unanimity in 1,2,3 to illustrate that Aurora A band has quantitative comparability.
Fig. 3 is the cell cycle distribution figure that flow cytometer detects, among the figure, 1 period profile figure for contrast ribozyme importing prostate cancer cell PC3, experiment shows, handle 12h, 24h, 48h, the period profile of prostate cancer cell PC3 is similar, is the period profile of prostate cancer cell PC3 when handling 48h among the figure; 2 for Aurora A-LNAzyme imports the period profile figure that prostate cancer cell PC3 handles the prostate cancer cell PC3 of 12h, shows G2/M phase cell showed increased among the figure; 3 for Aurora A-LNAzyme imports the period profile figure that prostate cancer cell PC3 handles the prostate cancer cell PC3 of 24h, shows G2/M phase cell showed increased among the figure; 4 for Aurora A-LNAzyme imports the period profile figure that prostate cancer cell PC3 handles the prostate cancer cell PC3 of 48h, shows G2/M phase cell showed increased among the figure, occurs obvious apoptosis peak (SubG1, arrow indication) simultaneously.
Embodiment
Embodiment 1:Aurora A-LNAzyme design is with synthetic
Aurora A-LNAzyme design: introduce lock nucleic acid, the present patent application contriver was once designed synthetic DNAzyme DNAzyme2 modify, be designed to Aurora A-LNAzyme of the present invention.The nucleotides sequence of Aurora A-LNAzyme of the present invention is classified as in the sequence table shown in the SEQ ID NO:1, in the described nucleotide sequence, forms the Nucleotide in the first substrate recognition structure territory, catalyst structure domain and the second substrate recognition structure territory and arranges as follows:
The first substrate recognition structure territory catalyst structure domain, the second substrate recognition structure territory
5’t
Lt
Laacagg ggctagctacaacga cct
Lgaaat
L3’
In order to verify the effect of Aurora A-LNAzyme of the present invention, also designed contrast ribozyme (Control), contrast the nucleotide sequence of ribozyme and arrange as follows:
The first substrate recognition structure territory catalyst structure domain, the second substrate recognition structure territory
5’t
Lt
Laacagg ggctaactacaacga cct
Lgaaat
L3’
Contrast ribozyme and the difference of Aurora A-LNAzyme are that the 6th Nucleotide in the catalyst structure domain is different, be that the 6th Nucleotide in the Aurora A-LNAzyme catalyst structure domain is " g ", and the 6th Nucleotide in the contrast ribozyme catalysis structural domain is " a ".
Aurora A-LNAzyme of the present invention and contrast ribozyme all adopt the solid phase phosphoramidite triester method (referring to " and nucleic acid: structure. function and synthetic volume two ", Wang Debao, Qi Guorong chief editor, Science Press, 1987.6), on automatic dna synthesizer, synthesize.From the nineties, synthetic all specialized, the commercialization of relevant nucleotide sequence both at home and abroad.Therefore, designed Aurora A-LNAzyme being transferred to relevant specialized company with the contrast ribozyme synthesizes.It is synthetic that Aurora A-LNAzyme of the present invention and contrast ribozyme are transferred to U.S. sigma company.
The external cutting of embodiment 2:Aurora A-LNAzyme Aurora A
1, reagent and material
RT-PCR test kit: be Qiagen LongRange 2-step RT-PCR Kit (German Qiagen company);
PC3, LNCaP and Du145 prostate cancer cell: available from U.S. ATCC;
Plasmid pcDNA3.1 (+): available from American I nvitrogen company;
The EcoRI enzyme, XhoI enzyme and T4DNA ligase enzyme: available from precious biotech firm;
Aurora A-LNAzyme: embodiment 1 design is mixed with the solution for standby of 50 μ M/L with synthetic with physiological saline;
Contrast ribozyme: embodiment 1 design is mixed with the solution for standby of 50 μ M/L with synthetic with physiological saline;
T7/SP6 in-vitro transcription test kit: available from U.S. Roche company;
Digoxin chemoluminescence method detection kit: available from U.S. Roche company;
Methane amide: available from U.S. Sigma company;
Tetrabromophenol sulfonphthalein: available from U.S. Sigma company;
EDTA: available from U.S. Sigma company;
Polyacrylamide: available from U.S. Sigma company.
2, experimental technique
(1) clone Aurora A cDNA part fragment
From gene library (number of entering AF011468), obtain the encoding sequence of Aurora A, with the conservative segment of RT-PCR clone Aurora A.Extract total RNA of three prostate cancer cell PC3, LNCaP and Du145 respectively, merge with same ratio then, with it as template, synthetic article one cDNA chain, be that template is carried out pcr amplification with described cDNA again: after 6 minutes, enter the cyclic amplification stage 95 ℃ of pre-sex change (94 ℃, 1min; 58 ℃, 1min; 70 ℃, 90s), circulate 34 times.The primer sequence design is as follows: ' GCGAATTCC ATCATGGACCGATCTAA-3 ' has EcoRI restriction enzyme site and two protection bases to upstream primer 5; Downstream primer 5 '-GCCTCGAGTTCAGGTGCCGATGGCAG-3 ' has XhoI restriction enzyme site and two protection bases.Obtaining length behind the pcr amplification is the PCR product (seeing SEQ ID NO:2) of 341bp, it is after the gel electrophoresis purifying reclaims, cut through EcoRI and XhoI enzyme, and by the catalysis of T4DNA ligase enzyme, be connected between the EcoRI and XhoI restriction enzyme site of plasmid pcDNA3.1 (+), form recombinant plasmid pcDNA3-Aurora A-1.
(2) the external cutting of Aurora A
Cut recombinant plasmid pcDNA3-Aurora A-1 with the XhoI enzyme, make it line styleization.Requirement by T7/SP6 in-vitro transcription test kit specification sheets, with the T7/SP6 in-vitro transcription test kit Aurora A of Gaoxin-UTP mark synthetically, the Aurora A of this digoxin-UTP mark constitutes (seeing SEQ ID NO:3) by 405 Nucleotide, and wherein the ribozyme point of contact is between the 112 bit base a and 113 bit base c of SEQ ID NO3.The Aurora A of digoxin-UTP mark and Aurora A-LNAzyme are added to 20 μ l reaction buffer (50mmol/L Tris-Hcl, pH7.2,15mM Mgcl with 1: 100 mol ratio
2, 0.01%SDS) in, 40 ℃ hatch 2 hours after, add the methane amide of mass concentration 96%, the tetrabromophenol sulfonphthalein and the 20mmol/L EDTA stopped reaction of mass concentration 0.1%.The sex change 8 minutes in the methane amide of mass concentration 50% of the reaction product of above-mentioned reaction places cooled on ice, then at the polyacrylamide gel (containing 8mol/l urea) of mass concentration 8%, and 500V, electrophoresis 1.5h.
After electrophoresis finishes,, detect the cutting band with the digoxin chemoluminescence method by the requirement of digoxin chemoluminescence method detection kit specification sheets.
Adopt the aforesaid method external cutting of contrast ribozyme Aurora A, and detect the cutting band with digoxin chemoluminescence method detection kit.
3, experimental result
What the digoxin chemoluminescence method detected the cutting band the results are shown in Figure 1, and Fig. 1 shows that Aurora A-LNAzyme of the present invention can cut about 80% Aurora A by enzyme, and the contrast ribozyme can not cut Aurora A.
Embodiment 3:Aurora A-LNAzyme transfection prostate cancer cell PC3
1, reagent, material and equipment
FuGENE6 is available from U.S. Roche company;
Aurora A-LNAzyme: embodiment 1 design is mixed with the solution for standby of 50 μ M/L with synthetic with physiological saline;
Contrast ribozyme: embodiment 1 design is mixed with the solution for standby of 50 μ M/L with synthetic with physiological saline;
Prostate cancer cell PC3: available from U.S. ATCC;
The Coulter flow cytometer is available from Beckman company;
Data analysis software ModFitLT 2.0, U.S. Verity Software House product.
2, experimental technique
The conventional prostate cancer cell PC3 that cultivates, after prostatitis adenocarcinoma cell PC370% merges, require respectively Aurora A-LNAzyme liquid to specifications by FuGENE6 with concentration 300nmol/L, the contrast ribozyme liquid transfection of concentration 300nmol/L is in prostate cancer cell PC3, collect above-mentioned transfected Aurora A-LNAzyme after 24 hours and 48 hours respectively in transfection, the prostate cancer cell PC3 of contrast ribozyme, detect the Aurora A albumen that does not change among the prostate cancer cell of the right ribozyme PC3 respectively with conventional Western trace method then, transfection Aurora A-LNAzyme and the contrast ribozyme prostate cancer cell PC3 in AuroraA albumen.
For measuring the influence of Aurora A expression level cell cycle process among the prostate cancer cell PC3, use flow cytometry to measure cell cycle distribution, measurement operation: require respectively AuroraA-LNAzyme liquid to specifications with concentration 300nmol/L by FuGENE6, the contrast ribozyme liquid transfection of concentration 300nmol/L is in prostate cancer cell PC3, in transfection (processing) 12,24, collect the prostate cancer cell PC3 of transfected Aurora A-LNAzyme and the prostate cancer cell PC3 of transfected contrast ribozyme after 48 hours respectively, use the PBS flushing of 0.1M then respectively, with Krishan damping fluid (the 0.1%sodium citrate that contains the 0.05mg/L propidium iodide, 0.2mg ml_1RNAase, 0.3%NP40) termination reaction, hatched 30 minutes at 4 ℃, continue after carry out check and analysis with the Coulter flow cytometer, carry out data analysis with ModFitLT 2.0 softwares.
3, experimental result
Western trace method detected result is seen Fig. 2, and Fig. 2 shows, to contrast among the prostate cancer cell PC3 of ribozyme the proteic expression level of Aurora A identical for the proteic expression level of Aurora A and untransfected among the prostate cancer cell PC3 of transfection contrast ribozyme; The prostate cancer cell PC3 of transfection Aurora A-LNAzyme compares with the prostate cancer cell PC3 of untransfected Aurora A-LNAzyme, and contained Aurora A albumen obviously reduces.Above-mentioned detected result shows that Aurora A-LNAzyme of the present invention has suppressed the expression of AuroraA in prostate cancer cell.
Flow cytometry the results are shown in Figure 3, and Fig. 3 shows, in the cell of Aurora A-LNAzyme treatment group, comes across G
2The cell percentage of-M phase was increased to about 18.23%, 24 hour and is 28.02%, 48 hour at 12 hours be 39.56%.In the cell of contrast ribozyme treatment group, G
2The cell percentage of-M phase all approaches 9.27% at 12 hours, 24 hours, 48 hours each time point.Compared to the cell of contrast ribozyme treatment group, some is in the inferior G in the cell cycle in the cell of Aurora A-LNAzyme treatment group
1Cell (18.33%), the prompting apoptotic cell obviously increase.Above-mentioned detected result shows that the Aurora A expression by inhibitation of AuroraA-LNAzyme mediation of the present invention can cause that prostate cancer cell PC3 is at G
2-M the phase assembles unusually until final apoptosis.
Embodiment 4:LNAzyme suppresses the growth of mouse prostate cancer transplanted tumor
1, material and laboratory animal
Prostate cancer cell PC3: available from U.S. ATCC;
Aurora A-LNAzyme: embodiment 1 design is with synthetic;
Contrast ribozyme: embodiment 1 design is with synthetic;
The BALB/C nude mice: Sichuan University experimentation on animals center provides.
2, experimental technique
12 of the female nude mices of BALB/C, 12 of BALB/C male nude mouses, 5 weeks of mean age, mean body weight 20g.All nude mices are divided into three groups, are made up of four female BALB/C nude mices and four male BALB/C nude mices for every group.First group is the physiological saline group, and second group is Aurora A-LNAzyme group, and the 3rd group is contrast ribozyme group.
With 1X10
7Prostate cancer cell PC3 is transplanted to every nude mice of each group through subcutaneous injection.After 5 weeks, the volume of tumor tissues is increased to about 65mm
3Be used for subsequent experimental.
Each nude mice in first group is accepted 10 μ l/ physiological saline injection every day.Each nude mice in second group is injected tumor tissues by multidigit point injection in the knurl with Aurora A-LNAzyme liquid every day, and the injection volume of each nude mice every day is: 8 μ g AuroraA-LNAzyme are dissolved in the Aurora A-LNAzyme liquid of 10 μ l physiological saline formation.Each nude mice in the 3rd group will contrast ribozyme liquid by multidigit point injection in the knurl and inject tumor tissues every day, and the injection volume of each nude mice every day is: 8 μ g contrast the contrast ribozyme liquid that ribozyme is dissolved in the formation of 10 μ l physiological saline.Inject and put to death every group of nude mice after 14 days.Measure each nude mice body weight and tumour weight in wet base and volume, data are represented with (mean number ± standard deviation).Calculate tumour inhibiting rate (tumour inhibiting rate=heavy X100% of (physiological saline group knurl weight-ribozyme treatment group knurl is heavy)/physiological saline group knurl), carry out statistical analysis by variance analysis, P＜0.05 has been considered as significant difference.
3, experimental result
Experimental data sees Table 1.
Nude mice body weight during table 114 day, transplanted tumor volume and weight (n=8)
* Aurora A-LNAzyme group compares P＜0.05 with physiological saline group and contrast ribozyme group
Experimental data shows that Aurora A-LNAzyme of the present invention has obviously suppressed the growth of human prostata cancer at the intravital transplanted tumor of athymic mouse, and the contrast ribozyme does not show the effect that suppresses tumor growth.
The comparison of embodiment 5:Aurora A-LNAzyme and DNAzyme2
1, serum half-life relatively
(1) Aurora A-LNAzyme serum half-life is measured
1. reagent and instrument
Phenol, chloroform, poly-propionic acid amide denaturant gel: available from U.S. Sigma company;
FH2408 automatic scaler: available from the Beijing Nuclear Instrument Factory;
Aurora A-LNAzyme: embodiment 1 design is with synthetic.
2. measuring method
Aurora A-LNAzyme's
32P mark reference literature [Sambrook J.Molecular Cloning:a LaboratoryManual, 2nd.New York:Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989] carries out.DeR is as follows: (counts per minute is 2 * 10 cpm) to get the per minute umber of pulse
5Mark Aurora A-LNAzyme, 20 μ l human serums, the unmarked Aurora A-LNAzyme of 0.9nmol, adding water to cumulative volume is 100 μ l.Mixing is put 37 ℃ of water-baths.Respectively get 20 μ l samples respectively at reaction back 1h, 2h, 4h, 8h, 16h, after phenol and the chloroform extracting, 20% poly-propionic acid amide denaturing gel electrophoresis,-20 ℃ of compressing tablet 8h, on X line viewbox, downcut corresponding band gel, the FH2408 automatic scaler is counted the CPM value, is calculated as follows the degradation rate of Aurora A-LNAzyme, degradation rate=degraded band CPM/ (the band CPM+ that do not degrade degraded band CPM) * 100%.
3. measurement result
AuroraA-LNAzyme is 12.3% at 16 hours degradation rate.
(2) the DNAzyme2 serum half-life is measured
The serum half-life of DNAzyme2 is measured with above-mentioned method, and measurement result is: the DNAzyme2 serum half-life is 4 hours.
2, the comparison of tumour inhibiting rate
The tumour inhibiting rate of Aurora A-LNAzyme is 61% (seeing embodiment 4), and the tumour inhibiting rate of DNAzyme2 is 53% and (sees CancerGene Ther.200815 (8): 523).
Above-mentioned data show, compare with DNAzyme2, Aurora A-LANzyme of the present invention is stronger to the inhibition ability of tumor growth, and serum half-life is longer, so Aurora A-LNAzyme is expected to become more excellent efficient, special, stable prostate cancer inhibitor.
Claims (2)
1. the locked nucleic acid ribozyme of a target silk-serine-threonine protein kinase aurora kinase A, the nucleotides sequence that it is characterized in that it is classified as in the sequence table shown in the SEQ ID NO:1, in the described nucleotide sequence, 15 deoxynucleotide ggctagctacaacga that are positioned at the middle part form catalyst structure domain, are positioned at 8 Nucleotide t in catalyst structure domain left side
Lt
LAacagg forms the first substrate recognition structure territory, is positioned at 8 Nucleotide cct on catalyst structure domain right side
LGaaat
LForm the second substrate recognition structure territory, two lock nucleic acid t are all contained in the first substrate recognition structure territory and the second substrate recognition structure territory
L
2. the application of the locked nucleic acid ribozyme of the described target silk-serine-threonine protein kinase aurora kinase A of claim 1 in the medicine of preparation treatment prostate cancer.
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| Draper.K等.登录号：DL158535.《GenBank》.2008, * |
| Silvia Galardi等.miR-221 and miR-222 Expression Affects the Proliferation Potential of Human Prostate Carcinoma Cell Lines by Targeting p27Kip1..《THE JOURNAL OF BIOLOGICAL CHEMISTRY》.2007, * |
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